GB2518928A

GB2518928A - Method of depilation

Info

Publication number: GB2518928A
Application number: GB1408588.0A
Authority: GB
Inventors: Richard Edmonds
Original assignee: NEW ZEALAND LEATHER AND SHOE RES ASS
Current assignee: NEW ZEALAND LEATHER AND SHOE RES ASS
Priority date: 2013-05-15
Filing date: 2014-05-14
Publication date: 2015-04-08
Anticipated expiration: 2034-05-14
Also published as: AU2014202616B2; NZ624685A; AU2014202616A1; GB201408588D0; GB2518928B

Abstract

A method for removing wool/hair from a raw skin/hide having a flesh side and a grain side, the method comprising: a) contacting the skin/hide with a composition comprising a protective agent, preferably tetrakis(hydroxymethyl)phosphonium sulphate; b) contacting the skin/hide with a composition comprising a depilatory enzyme that is active at an alkaline pH greater than about 8, preferably an alkaline protease such as subtilisin; c) contacting the skin/hide with an activating composition comprising a base, preferably calcium hydroxide and/or sodium hydroxide and a deswelling agent, preferably sodium sulfate, wherein the base activates the depilatory enzyme in b) to act on the depilatory active structures in the skin/hide, and d) removing the wool or hair from the skin/hide. These methods use enzymes wholly without sodium sulfide or with levels of sodium sulfide normally considered too low for successful unhairing. Also disclosed are compositions for use in the methods.

Description

Intellectual Property Office Application No. GB1408588.0 RTM Date:28 January 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: Tetrapol (registered) Intellectual Property Office is an operating name of the Patent Office www.ipo.govuk

METHOD OF DEPILATION

TECHNICAL FIELD

The present invention relates generally to methods for removing wool or hair from hides or skins using enzymes wholly without sodium sulfide or with levels of sodium sulfide normally considered too low for successful unhairing. The invention also relates generally to compositions for use in the methods.

BACKGROUND OF THE INVENTION

The leather industry has long been interested in enzyme depilation of skins and hides with comprehensive reviews of enzyme depilation appearing every few decades (Green, 1952), (Yates, 1972), (Brady et al., 1989), (Cantera, 2001). Continued interest in the subject (Sivasubramanian et al,, 2008), (Wang et al, 2009) supports the assertion that there is a widespread desire to use enzymes as the means of depilating skins or hides. Enzymes are desirable for use as an alternative to conventional lime and sodium sulfide processing because conventional lime and sodium sulfide processing is hazardous and unpleasant. Conventional processing is used to generate high quality leather. Accordingly, alternatives to conventional lime and sodium sulfide processing needs to provide leather of the same or similar quality, or will not be generally acceptable.

Wholly enzyme based depilation processes have been described that purport to be successftil (Macedo et al,, 2005), (Sivasubramanian et al., 2008), (Saravanabhavan et al,, 2005), (Rose et al., 2007). However, problems have been reported when ostensibly successful methods have been trialled under temperate conditions (10-20°C) (Lowe, 1999), (Lowe, 2000), (Cooper & Lowe, 1997), (Cooper, 1997). Several patents are directed to enzyme-based, sulfide-free depilation methods, including W02005/049871 and US 7,198,647. However, the quality of the leather produced by the methods disclosed in W02005/049871 and US 7,198,647 is not discussed, Moreover, the methods disclosed in W02005/049871 and US 7,198,647 differ from the methods of the present invention in several important respects.

No enzyme depilation system has yet achieved a level of performance approaching that of the traditional lime and sodium sulfide process (Edmonds, 2012). Briefly; the major problems with presently used conventional methods are: incomplete depilation requiring additional sulfide based processing; damage to the grain surface resuhing in a nubuck effect; and looseness, Looseness is a fault in the leather in which the internal structure appears to lose its elasticity, and manifests itself as surface creasing when the leather is folded inwardly (Coul son, 1969).

Although enzymes are known that are able to depilate sheep skins (Yates, 1972), the resulting skins lack the quality of conventionally processed skins and the process is not used commercially.

The mechanisms of depilation are partially understood (Cmtera, 200 Ia), (Cantera, 2001b), (Cantera, 2001c), (Cantera, 2001d), (Brady et al., 1989b). However, as described above, the unsatisfactory effects of the enzymes used in known enzymatic depilation regimes results in unwanted damage to skins and ultimately, low quality leather (Wang et al., 2005),(Felicjaniak, B., 1985),(Tandt, H., et al, 1997),(Defect processing BLC leather technologies centre, 2001),(Hawkins, S., and J. Wassenberg, J., 1997)There is a need to develop enzyme based depilation methods, particularly methods that can be used industrially, that result in a consistent high quality leather, It is an object of the invention to go at least some way towards avoiding the above disadvantages and/or that at least provides the public with a useful choice.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art,

SUMMARY OF THE INVENTION

In a first aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a protective agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH greater than about 8, c) contacting the skin or hide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active stmctures in the skin or hide, and d) removing the wool or hair from the skin or hide.

In one embodiment the method further comprises e) neutralizing the base on and/or in the skin or hide.

In one embodiment the method further comprises f) washing the skin or hide with water between step a) and step b).

In one embodiment of the method removing the wool or hair from the skin or hide is carried out after neutralizing the base on and/or in the skin or hide.

In one embodiment the method further comprises g) removing a neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.

In one embodiment the method further comprises h) washing the skin or hide in water after step g).

In one embodiment the method further comprises i) processing the skin or hide using conventional deliming and then pickling techniques to produce pickled skin or pickled hide.

In one embodiment the method comprises j) as an alternative to (i) the steps of degreasing the skin or hide followed by processing the skin or hide directly to wet blue without a pickling step.

In one embodiment the method further comprises k) processing the pickled or wet blue skin or hide into leather.

In a second aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a tetrakis (hydroxymethy) phosphine (THP) compound, a tetrakis (hydroxymethyl) phosphonium salt (a THP salt), a THP condensate, or oxazolidine, b) contacting the skin or hide with a composition comprising an alkaline serine protease in a substantially inactive state, c) contacting the skin or hide with an activating composition comprising hydrated calcium oxide, thioglycolic acid, sodium sulfate and sodium hydroxide to activate the alkaline serine protease in b), and d) removing the wool or hair from the skin or hide.

In one embodiment the method further comprises e) contacting the skin or hide with a neutralizing agent comprising sucrose and calcium chloride to reduce the pH on and/or in the skin or hide.

In one embodiment the method comprises 17) washing the skin or hide with water between step a) and step b).

In a third aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide each having a flesh side and a grain side, the method compnsing a) contacting the skin or hide with a composition comprising a protein cross-linking agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is substantially inactive at the pH of the composition, c) contacting the skin or hide with an activating composition comprising an agent that activates the depilatory enzyme in b), and a deswefling agent, and d) removing the wool or hair from the skin or hide, In one embodiment the method comprises e) washing the skin or hide with water between step a) and step b).

In one embodiment the contacting in c) is contacting the flesh side of the skin or hide with the activating composition.

In a fourth aspect the invention relates to a method for removing wool or hair from at least one skin or hide, the skin or hide each having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a protein cross-linking agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme under conditions that maintain the enzyme in a substantially inactive state, c) contacting the flesh side of the skin or hide with an activating composition comprising an activating agent to activate the depilatory enzyme in b), and d) removing the wool or hair from the skin or hide.

In one embodiment the method comprises e) washing the skin or hide with water between step a) and step b).

In one embodiment of the method the activating composition in c) comprises a deswelling agent.

In a fifth aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with an composition comprising a protective agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH, c) contacting the flesh side of the skin or hide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active structures in the skin or hide, and d) removing the wool or hair from the skin or hide, wherein the method either i) does not use a sulfide, or ii), uses sodium sulfide at concentrations of less than about 1% Na2S (8Na2S/gSkin material).

In one embodiment the further comprises e) washing the skin or hide with water between step a) and step b).

In one embodiment of the method, ii) uses sodium sulfide at a concentration of about 0.5 to about 0.9% gNa2S/gSkin material, preferably about 0.7% 8Na2S/gSkin material.

In one embodiment the method of any of the first through fourth aspects either i) does not use sulfide or ii) uses sulfide at a concentration of less than about 1% Na2S (gNa2S/gSkin material).

In a sixth aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising about 2% tetrakis (hydroxymethyl) phosphonium sulfate (THPS) (wtTF-IPS/wtSkin material) for about three hours, b) contacting the skin or hide for about 2 hours with a composition comprising about 2% wtlvol of a depilatory enzyme subtilisin having an activity of 0,SATJ/min, the composition having a pH of about 7, c) contacting the skin or hide for about 2 to about 16 hours with an activating composition comprising about 5% hydrated calcium oxide (wt/vol), about 3% thioglycolic acid (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 10% sodium hydroxide (wt/vol) to activate the subtilisin, d) removing the wool or hair from the skin or hide.

In one embodiment the method of the sixth aspect further comprises the step of neutralizing the base on and/or in the skin or hide by contacting the skin or hide for about 2 hours with a neutralizing agent comprising sucrose and calcium chloride.

In one embodiment the method of the sixth aspect further comprises the step of removing a neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.

In one embodiment the method of the sixth aspect further comprises the step of washing the skin or hide in water between steps a) and b) or after step g) or both.

In one embodiment the method of the sixth aspect further comprises the step of processing the skin or hide using conventional deliming and then pickling techniques to produce pickled skin or pickled hide.

In one alternative embodiment the method of the sixth aspect comprises the step of degreasing the skin or hide followed by processing the skin or hide directly to wet blue without a pickling step.

In one embodiment the method of the sixth aspect further comprises the step of processing the pickled or wet blue skin or hide into leather, In one embodiment the method of the sixth aspect either i) does not use sulfide or ii) uses sodium sulfide at a concentration of about 0,7% (gNa2S/gSkin material).

In one embodiment the method of any of the preceding aspects comprises, before a), contacting the skin or hide with a composition comprising an acid. Preferably the acid is an organic acid, preferably a carboxylic acid, preferably acetic acid, preferably formic acid.

In one embodiment the composition comprising an acid further comprises a penetration enhancer. Preferably the penetration enhancer is a terpene or terpenoid, preferably limonene or menthol, preferably D-limonene or L-menthol, preferably D-limonene. In one embodiment, the skin or hide is contacted before a) with a composition comprising formic acid and D-limonene, preferably about 0.3% formic acid and about 1% D-limonene based on skin weight. In one embodiment, contacting with a composition comprising an acid and a penetration enhancer is for about 15 minutes to about 120 minutes, preferably for about 30 minutes to about 90 minutes, preferably for about 45 minutes to about 75 minutes, preferably for about 60 minutes.

Various embodiments of the method of the first, second, third, fourth, fifth and sixth aspects of the invention are set out below in the detailed description of the invention, but the invention is not limited thereto.

Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only arid with reference to the drawings in which: Figure 1 shows a comparative demonstration of a depilation method according to the invention.

Figure 2 shows the grain surface of an unprotected, conventional lime sulfide depilated ovine skin after processing to crust leather. Bar = 100 jim.

Figure 3 shows the grain surface of an unprotected enzyme depilated ovine skin after processing to crust leather. Bar = 00jim.

Figure 4 shows the grain surface of a protected conventional lime sulfide depilated ovine skin after processing to crust leather. Bar = I OOrim.

Figure 5 shows the grain surface of a protected enzyme depilated ovine skin after processing to crust leather. Bar= 100j.tm.

Figure 6 shows a comparative demonstration of alternative protective agents.

Figure 7 shows a comparative demonstration of alternative depilation enzymes.

Figure 8 shows a comparative demonstration of different reducing agents.

Figure 9 shows the grain surface of a protected enzyme depilated hide skin after processing to crust leather. Bar = 100i.tm.

Figure 10 shows the grain surface of a protected enzyme depilated deer skin after processing to crust leather. Bar = I DORm.

DETAILED DESCRIPTION OF THE INVENTION

Definitions Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in tanning, and hide and skin processing can be found in International Council Of Tanners, (1999). International Glossary Of Leather Terms (2nd Edition). Northampton.

It is also believed that general practice of the present invention can be performed using standard tanning, and skin or hide processing protocols and procedures as known in the art, and as described, for example in Sharphouse 1971, Bienkiewicz, K., Ed. (1983); Covington, A. (2009) The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

The term "protective agent" as used herein refers to any suitable agent that renders a protein impervious to enzymatic hydrolysis. In one non limiting embodiment, a "protective agent" is a molecule that is capable of inducing a chemical bond between proteins. For example, a molecule useful in the invention that is capable of inducing a chemical bond between proteins may be a "cross linking agent". Typically the cross linking agent will be a chemical which induces protein cross links, In one non limiting embodiment, the cross-linking agent is a molecule that is capable of inducing disulfide bonds between coflagen fibrils. In addition, additional protective agents that may be useful in the invention may be reagents which inhibit enzyme proteolysis but do not induce cross-links.

The term "activating agent" as used herein refers to an agent that, when contacted to a skin or hide, will penetrate the skin or hide, preferably fully penetrate the skin or hide, and activate the depilatory enzyme present /n s/tu in the skin or hide. ii

The term "deswelling agent" as used herein refers to an agent that will act to reduce the looseness of a depilated skin or hide when the skin or hide has been processed in a condition of high pH( >9) by a method of the invention.

The key to enzymatic depilation is the detachment of the dermal epidermal junction and the subsequent breakdown of the cells of the IRS and the ORS of the hair (Yates, 1968).

Accordingly, the term depilatory active stmctures" as used herein refers to the specific structures which attach the epidermis to the dermis and the substance of the outer root sheath (ORS) and the inner root sheath (IRS).

Quantities given as wt/wt herein refer to the given weight of an additive based on the given weight of a skin or hide material to be treated, For example 1% Na2S (gNa2S/gSkin material) refers to Ig Na2S per bOg Skin material.

The term "g/Skin material" as used herein refers to the weight in grams of a "green fleshed skin" or "green fleshed hide" as known and used in the art. For example, a green fleshed sheep skin is a freshly flayed sheep skin that has had adhering flesh and fat scraped from the flesh side and has not been salted or pickled.

The term "based on skin weight" refers to the weight of a "green fleshed skin" or "green fleshed hide" as described above, The term "acidic" as used herein with reference to a pH range refers to a pH of about 1,0 to about 6.5.

The term "neutral" as used herein with reference to a pH range refers to a pH of about 6.6 to about 8, The term "alkaline" as used herein with reference to a pH range refers to a pH of about 8.1 to about 14, The term "ultra low amount(s) of sodium sulfide" as used herein refers to a concentration of sodium sulfide that would be considered in the art to be too low for successful unhairing. For example, below about 40g/m2 (60% active sodium sulfide flake per square meter of hide or skin) or below a concentration in the drum of 1.2% sodium sulfide Na2S), but not limited thereto.

The term "alkaline protease" (AP), as used herein refers to an enzyme or enzyme mix comprising a broad spectrum of endopeptidase activity active in the alkaline pH range (8-14) The term "sugar reagent" as used herein refers to a sugar that will react with calcium hydroxide to form calcium sucrate or an analogous calcium sugar salt. Examples of sugar reagents include, but are not limited to, monosaccharides such as glucose, glyceraldehyde and galactose, disaccharides such as sucrose, lactose and maltose, and other sugars, What is important is that the "sugar reagent" has the ability to react with calcium hydroxide to form calcium sucrate under the conditions specified in the method.

The term "neutralizing agent" as used herein refers to any agent that will neutralize the pH in and/or on the skin or hide after treatment of the skin or hide with the neutralizing agent.

Preferably the neutralizing agent will reduce the alkalinity on and/or in the skin or hide, but not limited there to, For example, a neutralizing agent may, in some embodiments neutralize acidity.

The term "an agent that reduces the free alkalinity" refers to any agent or process that will reduce free alkalinity. Preferably an agent that reduces the free alkalinity of a processing bath reduces the concentration of free alkaline salts to less than about <1% wt/wt, In Ii The term "substantially inactive" as used herein with reference to enzyme activity refers to the activity of the enzyme which will, at the given p11, have either no activity or only residual activity such that any residual activity will have no material effect on the skin or hide. For an endopeptidase or alkaline protease used according to the invention this means that "an enzyme that is substantially inactive" or an enzyme that is in a "substantially inactive state" is an enzyme that will have no peptidase or protease activity or only residual peptidase or protease activity at the given pH such that any peptidase or protease activity of the enzyme will have no material effect on the skin or hide. For example, a "substantially inactive enzyme" or an enzyme in a "substantially inactive state" as used herein refers to an enzyme that shows less than about 1% of its normal activity at the tested pH when compared to its normal activity at its optimal pH, The term "deliming" as used herein refers to deliming processes as known in the art that are carried out on a skin or hide for completely neutralizing the residual alkali present in a skin or hide following an alkaline depilation, that allow tanning to properly proceed (Harris Li (Ed.) Dictionary of the terms used in the hides, skins, and leather trade. 1974 Washington D.C.: US department of Agriculture.) The term "pickling" as used herein refers to pickling processes as known in the art that are carried out on an unhaired or depilated skin or hide to reduce the pH of the unhaired or depilated slcin or hide to below pH 3.0 in the presence of ample salt. Normally 3°'b salt is based on the total weight of water used in the pickling process (Harris, D. 1974 Supra) The term "processed into leather" as used herein refers to skin or hide material which has been tanned to as to be imputrescible, impervious to and insoluble in water (Harris, D. 1974 Supra).

The term "wet blue" as used herein refers to leather, which after the chrome tanning process has not been further processed and is sold in the wet state (International Council Of Tanners, 1999).

The term "degreased" as used herein refers to hides or skins which have been treated to remove a substantial proportion of the natural fats present in the skin <1% wtlwt (dry matter basis) (Harris, ft. 1974).

"NOVOLIME®" is a proprietary enzyme composition comprising subtilisin and lipase.

The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or daim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

Description

The present invention relates generally to an enzymatic process for removing wool or hair from hides or skins using enzymes. The inventor has determined that the process described herein can be carried out in the absence of sodium sulfide, or in the presence of ultra ow amounts of sodium sulfide, to produce skins or hides having minimal to no grain damage. Skins or hides produced by the inventive methods disclosed herein have a higher grain quality as compared to skins or hides processed by other-enzyme based depilation methods.

The inventor believes that they are the first to show that the pre-treatment of raw skins or hides with a protective agent comprising a TI-IP compound, particularly a tetrakis (hydroxymethyl) phosphonium salt, particularly tetrakis (hydroxymethyl) phosphonium sulfate (THPS), acts to fully protect the skin or hide from degradation during a subsequent enzyme based depilation process. In particular, the inventor has shown that without the use of a protective agent before enzymatic depilation, a skin or hide that is processed by standard enzymatic depilation as may be known in the art, is badly damaged by the enzyme, thereby greatly reducing the quality of the leather produced from such a skin or hide.

In the inventive process disclosed herein, a depilatory enzyme in an inactive or substantially inactive state is applied to the skin or hide that has been pre-treated with a protective agent.

The enzyme is then activated in situ in the skin or hide. The inventor has determined that the pre-treatment of the skin or hide with the protective agent followed by in situ activation of an alkaline active depilation enzyme is particular effective at producing skins or hides of superior quality for ifirther processing.

In the method described herein the protective agent is applied to the raw skin or hide and allowed to fully penetrate the skin or hide such that the full thickness of the skin or hide contains the protective agent. An alkaline active enzyme is then applied to the pre-treated skin or hide at a neutral pH and allowed to penetrate the skin or hide to reach the region of depilatory activity around the follicle root, The enzyme is then activated in situ using an activating composition applied to the skin or hide. Preferably the activating composition is applied to the flesh side of the skin or hide. The activating composition may be in the form of an activating paint, but is not limited there to. The activating composition also contains a small amount of lime (hydrated calcium oxide), a reducing agent to ensure complete depilation of the skin or hide and a deswelling agent. The activating composition may contain a penetration enhancer that enhances penetration of the constituents of the activating composition into the skin or hide, Surprisingly, the inventor has found that a skin or hide of superior quality may be produced when the process described herein employs as a protective agent, a cross linking agent that is capable of inducing disulfide bonds between collagen fibrils. Particularly effective cross-linking agents include tetrakis(hydroxymethyl)phosphine compounds (THP), tetrakis(hydroxymethyl)phosphonium salts (THP salts), tetraki s(hydroxymethyl)phosphine condensates (THP condensates), and oxazolidine. Preferably the THP salt is tetrakis(hydroxymethyl)phosphonium salt. Preferably the tetrakis(hydroxymethyl)phosphonium salt is a sulfate, chloride, phosphate, nitrate, bromide, or fluoride salt. Preferably the THP salt is tetrakis(hydroxymethyl)phosphonium sulfate (THPS) or tetrakis(hydroxymethyl)phosphonium chloride (THPC), although the invention is not limited thereto. Preferably, the THP salt is tetrakis(hydroxymethyl)phosphonium sulfate (THPS).

In one embodiment, the method of the invention comprises an initial step of acidifying the raw skin or hide before contacting with a protective agent or cross-linking agent according to the methods described herein. Without wishing to be bound by theory, the inventor believes that acidification of the skin or hide before treatment with a protective agent or protein crosslinking agent as described herein may allow the protective agent or protein crosslinking agent to achieve better penetration of the skin or hide according to a method of the invention, allowing for a more "even" or less "astringent" tanning reaction. Leather produced from a skin or hide treated as described has less "draw", Accordingly, in various embodiments, a method of the invention comprises, before contacting the skin or hide with a protective agent or a protein crosslinking agent, acidifying the skin or hide by contacting the skin or hide with a composition comprising an acid. In some embodiments the composition comprising the acid frirther comprises a penetration enhancer. Preferably the acid is an organic acid, preferably a carboxylic acid, preferably acetic acid, preferably formic acid, and the penetration enhancer is a terpene or terpenoid, preferably limonene or menthol, preferably D-limonene or L-menthol, preferably D-limonene.

The applicant believes that they are the first to successfully demonstrate that following a method of the invention of pre-protecting a raw skin or hide before enzyme based depilation, provides a depilated skin or hide of superior quality for further processing.

Accordingly, in one aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method compnsing a) contacting the skin or hide with a composition comprising a protective agent, b) contacting the washed skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH greater than about 8, c) contacting the skin or hide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active structures in the skin or hide, and d) removing the wool or hair from the skin or hide.

In one embodiment the method comprises f) washing the skin or hide with water between step a) and step b).

In one embodiment the water further comprises an additional reagent.

In one embodiment of the method, removing the wool or hair from the skin or hide is carried out after contacting the skin or hide with the neutralizing agent. In one embodiment removing the wool or hair comprises pulling, but is not limited thereto, It is believed that various ways of removing the wool or hair can be employed in the method of the invention and that such ways are known to a person of skill in the art (Covington AD. 2009).

In one embodiment the method further comprises g) removing a neutralizing agent that has been used to neutralize the base on and/or in the skin or hide, In one embodiment the method further comprises h) washing the skin or hide in water after g).

In one embodiment the water in h) comprises an additional reagent.

In one embodiment the method further comprises the step of i) processing the skin or hide using conventional deliming and then pickling techniques to produce a pickled skin or pickled hide, In one embodiment the method comprises j) as an alternative to (i) the steps of degreasing the skin or hide followed by processing the skin or hide directly to wet blue without a pickling step.

It is believed that contacting the skin or hide according to the method of the invention can be carried out by the person of skill in the art according to standard methodology in the art, and as described herein (e.g., Carrie, 1960, supra).

In one embodiment the method comprises an initial acidification of the raw skin or hide before a) by contacting the skin or hide with a composition comprising an acid. In one embodiment the composition comprising an acid comprises about 0.01% to about 1% acid based on skin weight, preferably about 0.1% to about 0.5% acid based on skin weight, preferably at about 0.3% acid based on skin weight. Preferably the acid is an organic acid, preferably a carboxylic acid, preferably acetic acid, preferably formic acid, Other suitable acids are contemplated herein, the use of which will be readily apparent to the skilled worker.

In one embodiment, the composition comprising an acid further comprises a penetration enhancer, Preferably the penetration enhancer in the composition comprising an acid is a terpene or terpenoid, preferably limonene or menthol, preferably D-limonene or L-menthol, preferably D-limonene. In one embodiment the penetration enhancer is present at a concentration of about 0.1% to about 5%, preferably about 0.5% to about 3%, preferably about 0.75% to about 2%, preferably about 1% based on skin weight. Preferably the penetration enhancer is D-limonene and is present at a concentration of about 1% based on skin weight.

In one embodiment, the initial acidification of the skin or hide comprises contacting with the skin or hide with the composition comprising an acid or the composition comprising an acid and a penetration enhancer, for about 1 minute to about 120 minutes, preferably for about 10 minutes to about 90 minutes, preferably for about 30 minutes to about 75 minutes, preferably for about 60 minutes, Tn one embodiment, the contacting in a) is carried out by adding the protective agent to the composition comprising the acid or the composition comprising the acid and the penetration enhancer.

In one embodiment contacting the skin or hide with the composition in a) is contacting in a processing bath. Preferably the processing bath is comprised in a processing paddle, processing drum, or challenge cook processor, but not limited thereto. Preferably the skin or hide is wholly or partially immersed in the processing bath. Preferably the skin or hide is wholly immersed in the processing bath.

In one embodiment the contacting in a) is contacting by painting as known in the art. For example, paint application to skins is well described in the art (Carrie et al, 1960).

In one embodiment the composition in a) is an aqueous solution that comprises the protective agent. In one embodiment the protective agent is a cross linking agent that cross-links collagen fibrils, In one embodiment the contacting in a) is for a time sufficient to allow the protective agent to fully penetrate the hide or skin, Preferably the full thickness of the hide or skin should contain the protective agent. Preferably the full thickness of the hide or skin contains a protective agent that is a cross linking agent that has reacted with the collagen in the skin or hide.

In one embodiment the contacting in a) is from about 2 to about 16 hours, preferably from about 2.5 to about 8 hours, preferably about 3 to about 4 hours, preferably about 3 hours.

In one embodiment the protective agent is a cross linking agent that is a THP compound, THP salt or THP condensate, Preferably the THP salt is selected from the group consisting of a tetrakis (hydroxymcthyl) phosphonium salt of the formula THP"X' where "X' is an anion selected from the group consisting of snlfate, chloride, phosphate, bromide, nitrate, and fluoride. Preferably the anion is sulfate or chloride, preferably the compound is tetrakis (hydroxymethyl) phosphonium sulfate (11-lIPS).

In one embodiment the cross linking agent is a THP compound, THP salt or TJ-IP condensate or oxazolidine and is present in a) at a concentration of about 0.25% to about 20%, preferably about 0.5% to about 15%, preferably about 0,75 to about 10%, preferably about 1% to about 5%, preferably about 1.5% to about 3%, preferably about 2% wt/wt (gTHP, gTHP salt or gTHP condensate/gSkin material). Preferably the cross-linking agent is a TI-IP salt selected from the group consisting of a tetrakis (hydroxymethyl) phosphonium salt of the formula THP"X" where "X" is an anion selected from the group consisting of sulfate, chloride, phosphate, bromide, nitrate, and fluoride, Preferably the anion is sulfate or chloride, preferably the compound is tetrakis (hydroxymethyl) phosphonium sulfate (THPS).

In one embodiment the washing between a) and b) comprises washing with 300% w/vol of water.

In one embodiment the washing between a) and b) comprises washing the skin or hide in a processing bath that comprises water. Preferably the processing bath is comprised in a processing drum, Preferably the skin or hide is wholly or partially immersed in the processing bath, Preferably the skin or hide is wholly immersed in the processing bath.

In one embodiment the washing between a) and b) results in the removal of substantially all the un-reacted protective agent from the skin or hide.

In one embodiment, the composition in b) optionally comprises a deswelling agent.

In one embodiment the contacting in b) is comprises contacting the skin or hide in a processing bath that comprises a depilatory enzyme in an aqueous solution. Preferably the processing bath is comprised in a processing drum, Preferably the skin or hide is wholly or partially immersed in the processing bath, Preferably the skin or hide is wholly immersed in the processing bath, In one embodiment the contacting in b) is carried out by painting or spraying the flesh side of the skin or hide with an aqueous solution comprising a depilatory enzyme, or by at least partially immersing the skin or hide in an aqueous solution comprising a depilatory enzyme.

Preferably the contacting in b) is carded out by at least partially immersing the skin or hide in the aqueous solution.

In one embodiment the contacting in b) is carried out at an acidic or neutral pH.

In one embodiment the contacting in b) is carried out at a pH of at least 4 to about 8.

Preferably the pH is at least 5 to about 8. Preferably the pH is about 6 to about 8. Preferably the pHis about 6.5 to 7,5, Preferably the pHis about 7.

In one embodiment the contacting in b) is carried out in the absence of sodium sulfide.

In one embodiment the contacting in b) is carried out in the presence of ultra low amounts of sodium sulfide. Preferably an ultra low amount of sodium sulfide is an amount of sulfide that is normally considered in the art to be too low for successful unhairing. Preferably the contacting in b) is carded out with sodium sulfide concentration of less than about 40g/m2 (60% active sodium sulfide flake per square meter of hide or skin) or below a concentration in the dmm of 1.2% sodium sulfide (Na2S).

In one embodiment the ultra low amount of sodium sulfide is sulfide at a concentration of less than about 1%, preferably about 0.05 to about 0.95%, preferably about 01 to about 0.8%, preferably about 0.1 to about 0.7%, preferably about 0.7% wt/wt (gNa2S/gSkin material).

In one embodiment the enzyme in b) is an enzyme having a broad spectmm of endopeptidase activity at an alkaline pH. nfl Li

In one embodiment the enzyme in b) is an alkaline protease that comprises a broad spectrum endopeptidase activity. Preferably the alkaline protease is selected from the group consisting of an animal alkaline protease, plant alkaline protease or microbial alkaline protease. Preferably the alkaline protease is an alkaline serine protease. Preferably the alkaline protease is a microbial alkaline protease, preferably subtilisin, but not limited thereto.

In one embodiment the enzyme is present in b) at a concentration of about 0.25% to about 6%, preferably about 0.5% to about 5%, preferably about 0,75% to about 4%, preferably about 1% to about 3%, preferably about 2% or about 3% wt/vol of an enzyme with an activity of 0.5AU/min as assessed by the method of Mozersky and Bailey 1992. In one embodiment the enzyme is added in a 10 volume float.

In one embodiment the composition in b) optionally further comprises at least one additional enzyme. Preferably the at least one additional enzyme is a lipase.

In one embodiment the composition in b) fbrther comprises a penetration enhancer. Preferably the penetration enhancer is a terpene or terpenoid, preferably limonene or menthol, preferably D-limonene or L-menthol, preferably D-limonene. Without wishing to be bound by theory, the inventor believes that the addition of a penetration enhancer to the composition in b), particularly limonene or menthol, is a possible means to improve the quality of the depilation of the skin or hide.

In one embodiment the contacting in b) is for a time sufficient to allow the enzyme or enzymes in the solution to penetrate into the skin or hide, Preferably the contacting in b) is for a time sufficient to allow the enzyme to reach the region of depilatory activity around the follicle root, Preferably penetration of the enzyme or enzymes is penetration of the enzyme or enzymes into the wool or hair follide of the skin or hide.

In one embodiment contacting in b) is contacting for about 1 hour to about 20 hours, preferably about 1-16 hours, preferably about 1-12 hours, preferably about 1-8 hours, preferably about 2 to 4 hours, preferably about 2 hours.

In some embodiments the method comprises an additional step of removing excess composition contacted to the skin or hide in b) before contacting the skin or hide with the activating composition in c).

In one embodiment contacting the skin or hide with the activating composition in c) is for a time sufficient to activate the enzyme present on and/or in the skin or hide after contacting in b).

In one embodiment contacting with the activating composition in c) is for a sufficient time to allow the base comprised in the activating composition to raise the pH from an acidic or neutral pH to an alkaline pH and to allow the enzyme to act on the depilatory active structures in the skin or hide. Preferably the time sufficient to allow the base to raise the pH from an acidic or neutral pH to an alkaline pH and to allow the enzyme to act on the depilatory active structures in the skin or hide is from about 10 minutes to about 24 hours, preferably about 15 minutes to about 22 hours, preferably about 20 minutes to about 20 hours, preferably about 30 minutes to about 1 8 hours, preferably about 1 hour to about 17 hours, preferably about 2 hours to about 16 hours, preferably about 16 hours.

In one embodiment the pH is raised from a pH of about 4 to about 6, or from a pH of about 6 to about 8, to a pH of about 9 to about 12, preferably from a pH of about S to about 7 to a pH of about 10.5 to greater than about 3, preferably to a pt-I of at least about 13.

In one embodiment the pH is raised to a pH of a least 13 in the presence of about 8% to about 14% NaOH (wt/vol), preferably about 10% NaOH (wtlvol) to about 13% NaOH (wt/vol), preferably about 125% NaOH (wt/vol), In one embodiment contacting with the activating composition in c) comprises direct application of the composition to the flesh side of the skin or hide. Preferably direct application is by a direct application selected from a group consisting of partially immersing, pouting, painting or spraying or a combination thereof Preferably direct application is by spray coat or curtain coat.

In one embodiment the base in the activating composition in c) is provided at a concentration sufficient to activate the enzyme in the skin or hide.

In one embodiment the base is selected from the group consisting of calcium hydroxide and sodium hydroxide. Preferably the base is a combination of calcium hydroxide and sodium hydroxide, but is not limited thereto.

In one embodiment the base in the activating composition in c) is present at a concentration of about 1% to about 40%, preferaNy at about 3% to about 35%, preferably at about 7% to about 30%, preferably at about 7% to about 25%, preferably at about 8% to about 20%, preferably at about 9?/'b to about I 5%, preferably at about I 0% (w/vol), In one embodiment the base is a combination of calcium hydroxide and sodium hydroxide wherein thc calcium hydroxidc is prcscnt at a concentration of about to about 6%, preferably about 5?/ and the sodium hydroxide is present at a concentration of about I 5% to about 25%, preferably about 20% wt/vol, In one embodiment the deswelling agent present in the activating composition in c) acts to repress the swelling of the skin or hide at an alkaline pH, Preferably the deswelling agent is a sodium salt that acts to repress the swelling of the skin or hide at alkaline pH. Preferably the sodium salt is sodium sulfate.

In one embodiment the deswelling agent is present in the activating composition in c) at a concentration of about 0.1% to about 10%, preferably about 0.3% to about 9%, preferably about 05% to about 8%, preferably about 075% to about 7%, preferably about 0.1% to about 6%, preferably at about 5% w/vol, In one embodiment the contacting in c) further comprises contacting the skin or hide with a further agent that assists in depilation. Preferably the further agent provides a source of calcium ions, preferably the further agent is a calcium salt. Preferably the further agent is hydrated calcium oxide.

In one embodiment the further agent is present in the activating composition in c). Preferably the frirther agent is present in the activating composition at a concentration of about 1% to about 10%, preferably about 2% to about 8%, preferably at about 3% to about 6%, preferably at about 5% w/vol, In one embodiment the contacting in c) ftirther comprises contacting the skin or hide with a reducing agent. In one embodiment the reducing agent is present in the activating composition in c). In one embodiment the reducing agent is a sulfur-based organic reducing agent.

Preferably the sulfur based organic reducing agent comprises a thiol group. Preferably the sulfur based organic reducing agent is selected from the group consisting of sodium thioglycolate, thioglycolic acid, and 2-mercaptoethanol. Preferably the reducing agent is thioglycolic acid.

In one embodiment the reducing agent in the activating composition in c) is present at a concentration of about 1% to about 6%, preferably at about 2% to about 4%, preferably at about 3% w/vol, preferably at about 1% w/vol, In one embodiment the reducing agent is present in a molar concentration that is equivalent to the molar concentration of 0.7% wt/wt sodium sulfide.

In one embodiment the activating composition in c) is an aqueous solution comprising about 5?/ hydrated calcium oxide, about 3% thioglycollic acid, about 5% sodium sulfate and about 10% sodium hydroxide wt/vol.

In one embodiment the activating composition in c) is an aqueous solution comprising about 1% hydrated calcium oxide, about 1% sodium thioglycolate, about 5% sodium sulfate and about 12,5% sodium hydroxide wt/vol, Preferably the activating composition in c) comprises no sodium sulfide.

In one embodiment the neutralizing in e) comprises processing the skin or hide to reduce the pH on and/or in the skin or hide to an acidic or neutral p1-I. In one embodiment the process removes a sufficient amount of the base on and/or in the skin or hide to reduce the pH on and/or in the skin or hide to a pH of about 9 to about 12, preferably of about 10 to about 11, preferably of about 10,5, In one embodiment the neutralizing in e) comprises contacting the skin or hide with a neutralizing agcnt that neutralizes thc frcc alkali to a pH of 10.5.

In one embodiment the neutralizing agent is comprised in an aqueous neutralizing solution.

In one embodiment neutralizing in e) comprises contacting the skin or hide with a neutralizing agent for a time sufficient to neutralize at least some of the free alkali present in and/or on the skin or hide. In one embodiment, contacting the skin or hide is for a time sufficient to neutralize all, or substantially all, of the free alkali in and/or on the skin or hide.

In one embodiment contacting for a time sufficient to neutralize at least some, substantially all, or all, of the base is from about 10 minutes to about 12 hours, preferably about 20 minutes to about 10 hours, preferably about 25 minutes to about B hours, preferably about 30 minutes to about 4 hours, preferably about 30 minutes to about 1 houi preferably about 30 minutes.

In one embodiment neutralizing the skin or hide in e) comprises contacting the skin or hide with the neutralizing agent in a processing bath that comprises the neutralizing agent in an aqueous solution, Preferably the skin or hide is wholly or partially immersed in the processing bath, Preferably the skin or hide is wholly immersed in the processing bath. Preferably the processing bath is comprised in a processing drum, Tn another embodiment neutralizing the skin or hide in e) comprises directly applying a neutralizing agent to the flesh side of the skin or hide. Preferably directly applying the neutralizing agent is by a direct application selected from a group consisting of pouring, painting or spraying or a combination thereof Preferably direct application is by roller, sponge or brush or by a combination thereof In one embodiment the neutralizing agent comprises a sugar reagent that reacts with free calcium hydroxide, preferably to form calcium sucrate, and an agent that reduces the free alkalinity of the processing bath.

In one embodiment the sugar reagent is a monosaccharide or a disaccharide. Preferably the sugar reagent is selected from the group consisting glucose, glyceraldehyde, galactose, sucrose, lactose and maltose. Preferably the sugar reagent is sucrose.

In one embodiment the sugar is present in the neutralizing agent at a concentration of about 0.1% to about 8?/b, preferably about 1% to about 7%, preferably about 2% to about 6%, preferably about 3% to about 6%, preferably about 4% to about 5%, preferably about 5%.

In one embodiment the agent that reduces the free alkalinity in the processing bath is present in the neutralizing agent at a concentration of 6-14% w/vol, Preferably the concentration of the agent that reduces the free alkalinity in the processing bath is 10% wt/vol. Preferably the agent that reduces the free alkalinity in the processing bath is ammonium chloride or calcium chloride, preferably calcium chloride, In one embodiment the neutralizing agent further comprises a deswelling agent, preferably the deswelling agent is a sodium salt, preferably sodium sulfate.

In one embodiment the deswelling agent is present in the neutralizing agent in e) at a concentration of about 0,25% to about 6%, preferably at about 0,5% to about 5%, preferably at about 0.75% to about 4%, preferably at about 1% to about 3%, preferably at about 2%. In one embodiment the deswelling agent is sodium sulfate and is present at a concentration of about 1% to about 6%, preferably at about 5%, preferably at about 4%, preferably at about 3%, preferably at about 2%.

In one embodiment the neutralizing agent is removed from the skin or hide by washing.

Removal of the neutralizing agent by washing may be carried out by a person of skill in the art according to standard procedures known and used in the art (Covington AD. Supra; Sharpehouse JR., S;qn'a).

In one embodiment the neutralizing agent is removed from the skin or hide by washing in water from about ito about 10 times, preferably about 2-9 times, preferably about 3-8 times, preferably about 3-5 times, preferably about 3-4 times, preferably about 3 times.

In a second aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide each having a flesh side and a grain side, the method comprising a) contacting the skin or hide with an aqueous solution comprising a tetrakis (hydroxymethyl) phosphine (TE-IP) compound, a tetrakis (hydroxymethyl) phosphonium salt (a TI-IP salt), a TE-IP condensate or oxazolidine, b) contacting the skin or hide with an aqueous solution comprising an alkaline serine protease in a substantially inactive state, c) contacting the skin or hide with an activating composition comprising hydrated calcium oxide, thioglycolic acid, sodium sulfate and sodium hydroxide to activate the enzyme in b), and d) removing the wool or hair from the skin or hide.

In one embodiment the method further comprises e) contacting the skin or hide with a neutralizing agent comprising sucrose and calcium chloride to neutralize the pH on and/or in the skin or hide.

In one embodiment the method comprises a) washing the skin or hide with water between step a) and step b).

In one embodiment the method of the second aspect comprises an initial acidification of the skin or hide before a) as described in detail above for the first aspect of the invention.

In one embodiment of the method of the second aspect, the contacting in a) is carried out by adding a tetrakis (hydroxymethyl) phosphine (THP) compound, a tetrakis (hydroxymethyl) phosphonium salt (a THP salt), a THP condensate or oxazolidine to the composition comprising the acid or the composition comprising the acid and the penetration enhancer.

In a third aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide each having a flesh side and a grain side, the method compnsing a) contacting the skin or hide with a composition comprising a protein cross-linking agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is substantially inactive at the pH of the composition, c) contacting the skin or hide with an activating composition comprising an agent that activates the depilatory enzyme in b), and a deswelling agent, and d) removing the wool or hair from the skin or hide.

In one embodiment of the third aspect, the activating agent comprises a base in a concentration sufficient to activate the depilatory enzyme.

In one embodiment of the third aspect, the enzyme is abroad spectmm endopeptidase, preferably an alkaline serine protease, preferably subtilisin.

In one embodiment of the third aspect the method further comprises b) neutralizing the base on and/or in the skin or hide.

In one embodiment of the third aspect, the conditions that maintain the enzyme in a subst2mtially inactive state are conditions of acidic or neutral pH on and/or inside the skin or hide. Preferably the conditions are neutral pH on and/or in the skin or hide.

In one embodiment of the third aspect, the contacting in c) is contacting the flesh side of the skin or hide th the activating composition.

In one embodiment the method of the third aspect comprises an initial acidification of the skin or hide before a) as described in detail above for the first aspect of the invention.

In one embodiment of the method of the third aspect, the contacting in a) is carried out by adding protein cross-linking agent to the composition comprising the acid or the composition comprising the acid and the penetration enhancer.

In one embodiment the method comprises fin ii e) washing the skin or hide with water between step a) and step b).

In one embodiment of the fourth aspect the activating composition in c) comprises a desweffing agent.

In one embodiment of the fourth aspect the activating agent comprises a base in a concentration sufficient to raise the pH on and/or in the hide to an alkaline pH.

In one embodiment of the fourth aspect, the enzyme is a broad spectrum endopeptidase, preferably an alkaline serine protease, preferably subtili sin.

In one embodiment the method of the fourth aspect comprises an initial acidification of the skin or hide before a) as described in detail above for the first aspect of the invention.

In one embodiment of the method of the fourth aspect, the contacting in a) is canied out by adding a pmtein cross-linking agent to the composition comprising the acid or the composition comprising the acid and the penetration enhancer.

In a fifth aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with an composition comprising a protective agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH, c) contacting the flesh side of the skin or bide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active structures in the skin or hide, and d) removing the wool or hair from the skin or hide, wherein the method either i) does not use a sulfide, or ii), uses sodium sulfide at concentrations of less than about 1% Na2S (gNa2S/gSkin material).

In one embodiment of the fifth aspect, ii) uses sodium sulfide at a concentration of about 0.5 to about 0.9% gNa2S/gSkin material, preferably about 0.7% gNa2S/gSkin material.

In one embodiment the method of any of the first through fourth aspects either i) does not use sulfide or ii) uses sodium sulfide at a concentration of less than about 1% Na2S (gNa2S/gSkin material).

In one embodiment the method of the fifth aspect comprises an initial acidification of the skin or hide before a) as described in detail above for the first aspect of the invention.

In one embodiment of the method of the fifth aspect, the contacting in a) is carried out by adding a protective agent to the composition comprising the acid or the composition comprising the acid and the penetration enhancer.

In a sixth aspect the invention relates to a method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide th a composition comprising about 2% tetrakis (hydroxymethyl) phosphonium sulfate (THPS) (wtTHPS/wtSkin material) for about three hours, b) contacting the skin or hide for about 2 hours with a composition comprising about 2% wt!vol of a depilatory enzyme subtilisin having an activity of 0,5AU/min, the composition having a pH of about 7, c) contacting the skin or hide for about 2 to about 16 hours with an activating composition comprising about 5% hydrated calcium oxide (wt/vol), about 3% thioglycollic acid (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 10% sodium hydroxide (wt/vol) to activate the subtilisin, and d) removing the wool or hair from the skin or hide.

In one embodiment of the method of the sixth aspect, the composition in b) comprises about 3% wt/vol of a depilatory enzyme subtilisin having an activity of 0.SAU/min, the composition having a pH of about 7.

In one embodiment of the method of the sixth aspect, the activating composition in c) comprises about 1% hydrated calcium oxide (wt/vol), about 1% sodium thioglycolate (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 12,5% sodium hydroxide (wt/vol).

In one embodiment the method of the sixth aspect further comprises the step of processing the pickled or wet blue skin or hide into leather.

In one embodiment the method of the sixth aspect either i) does not use sulfide or ii) uses sodium sulfide at a concentration of about 0.7% (gNa2S/gSkin material).

In one embodiment the method of the sixth aspect either i) does not use sulfide or ii) uses sodium sulfide at a concentration of about 0,7% (gNa2S/gSkin material) or less.

In one embodiment the method of the sixth aspect comprises an initial acidification of the skin or hide before a) as described in detail above for the first aspect of the invention.

In one embodiment the method of the sixth aspect comprises an initial acidification before a) as described above, wherein the initial acidification is carried out using a composition comprising about 0.3% acid and about 1% penetration enhancer based on skin weight. Preferably the initial acidification is carried out for about 30 to about 90 minutes, preferably for about 60 minutes. Preferably the acid is formic acid. Preferably the penetration enhancer is D-limonene.

In one embodiment of the method, following an initial acidification as described above, the contacting in a) is carried out by adding tetrakis (hydroxymethyl) phosphonium sulfate (THPS) to a concentration of about 2% (wtTHPS/wtSkin material) into the composition comprising the formic acid and the D-limonene, the concentration of enzyme in b) is about 3%, and the activating composition in c) comprises about 1% hydrated calcium oxide (wt/vol), about % sodium thioglycolate (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 12.5% sodium hydroxide (wtlvol).

Following depilation according to a method of the invention, the depilated skin or hide can be processed to good quality leather by the person of skill in the art following standard methods as known and used in the art of felimongering and tanning (Carrie eta!. 1960, Supra; Sharphouse 1971, Szqra; Covington 2009; Suprci).

The invention will now be described by way of the following representative methods and examples which are provided to further illustrate the subject matter to which the invention relates. The use of any and all examples, or exemplary language (e.g., "such as" or "including") provided herein, is intended solely for the purposes of better describing the invention, The presence of examples and the use of exemplary language does not limit the scope of the invention as disclosed herein unless specifically otherwise indicated. No language used in the entirety of the disclosure of this application should be interpreted as indicating that any particular element or feature pertaining to the invention and as disclosed herein is essential to the practice of the invention, unless explicitly stated. For example, the skilled worker will be able to modify the quantities of reagents and processing times in the methods and processes as exemplified, as known in the art, according to the inventive concepts disclosed herein, Such modifications are considered to be within the scope of the present invention, Moreover the skilled worker will appreciate, when considering the entirety of the description of the invention described herein, that the various embodiments and preferences set out for various different aspects of the invention may be used together with various other embodiments and preferences, and in various other aspects of the invention to carry out a method of the invention as contemplated herein.

EXAMPLES

General Experimental Protocols

Example 1

Enzyme depilation of green sheep skins to produce a pickled skin In the following example all processing steps were carried out at 25°C.

Pre-treatinent with the protective agent Apre-fleshed sheep skin was initially processed in a processing drum containing two volumes of water (200% of skin weight) and 2% protective agent, tetrakis hydroxyphosphonium sulfate (THPS) (% wi/vol in solution). The skin was agitated by rotation for 3 hours at 5 rpm, allowing the protective agent to fully penetrate the raw skin, After penetration of the protective agent, the dmm was drained and the protected skin was washed with 300% of skin weight of water for 15 minutes at 5 rpm to remove any residual protective agent. The wash water was drained completely.

Enzyme app I/cation The pre-protected skin was treated in the processing drum with an aqueous solution comprising 100% of skin weight of water and 5% of skin weight of NOVOLIME® (an enzyme composition comprising subtilisin and lipase) (Novozymes A/S, Denmark). Enzyme activity in NOVOLB'IE is 0.5AU/min as assessed by the method of Mozersky and Bailey 1992. The skin was agitated by rotation for 2 hours at 5 rpm to allow the enzyme to fully penetrate into the wool follicle root of the skin. The processing drum was then drained completely and the skin made ready for application of the activating solution, Specifically the skin is drained of excess liquid by stacking in a pile overnight.

In situ enzyme activation The depilatory enzyme was activated in situ in the skin by painting the flesh side of the skin with an activating composition or "paint" comprising i) 5% hydrated calcium oxide ii) 1.7% sodium sulfide iii) 20% sodium hydroxide The above concentrations in the paint are given as % wt/vol and correspond to less than 1% g Na2S / gSkin material since much less than a 1:1 ratio of paint to skin weight is applied. For example, with reference to sodium sulfide, 1.7% sodium sulfide (60?/b active) applied at 600g/m2 results in about 0.3% g sodium sulfide! gSkin material, The activating paint also comprised 5% sodium sulfate as a deswelling agent to reduce looseness in the final product. The activating paint as applied to the skin by spraying, rolling or brushing the flesh side of the skin. Specifically in this case the paint was applied by brush.

After the activating paint was applied to the skin, the skin was held statically overnight (about 16 hours) to allow enzyme activation and subsequent proteolysis in situ, at the depilatory active structures of the skin. The wool was then removed from the skin manually by "pulling" or IVeufralizing The depilated skin was reweighed and processed with an aqueous neutralising solution (80% water, 104% calcium chloride and 52% sucrose percentages based on skin weight) for 1 hour at 5 rpm. The aqueous neutralizing solution was drained completely and the skin washed in 200% skin weight water for 15 minutes at 5 rpm. The skin was then washed 3 further times for minutes each in 200% skin weight water at 5 rpm with constant agitation. Mter the last wash, the skin was further treated with an aqueous solution containing 100% skin weight water, 2% ammonium chloride, for 20 minutes at 5 rpm with constant agitation. This lafter treatment is known in the art as the "deliming" stage. After 2 minutes, 2% hydrogen peroxide was added and the skin processed for an additional 75 minutes at 5 rpm with intermittent agitation.

After deliming, the pt-I of the skin was determined to be less than 9.0 by phenolphthalein staining of a neck cross section, The skin was washed an additional 3 times in 100% skin weight water for 20 minutes each with agitation at 12 rpm. After the final wash was drained, the skin was treated in an aqueous solution comprising 90% skin weight water, 20% sodium chloride and 0.1% UNISLIP FW'' (Union Specialties, Mass, USA) is a "slip agent" composed of minerals and polymeric modifiers that stops the skins from tangling. The use of UNISLIP FWTh1 is known in the art and the use of this slip agent is common throughout the industry. In particular, UNISLIP FWTM is effective at stopping the skins from tangling in the drum, The skin was processed for 10 minutes with agitation at 12 rpm, After 10 minutes, a pre-mixed composition comprising 0.06% fungicide (thiocyanomethylthio) benzothiazole (TCMTB), 10% skin weight water and 2% sulphuric acid was added and the skin processed for an additional 1,5 hours with agitation at 12 rpm. The skins were then processed at 5rpm for 16 hours with constant agitation then fully drained. The skin product (pickled skin) could then be processed to wet blue and then crust leather using conventional standard processes know in the art, for example, as described in Sharphouse 1971.

Proof of concept -enzyme depilation o/JHI'Spre-profecledsk/ns A two factor two level factorial experiment was carried out as a "proof of concept" to demonstrate the superiority of the depilation method described herein as compared to other enzyme based or conventional depilation methods. The overall results of the experiment are shown in Figure 1.

Four different skin treatment groups were tested comparatively as follows: skin treatment either with or without pre-protection with THPS, and skin treatment either with or without the addition of enzyme. The process used was that described in example I with the exception that the groups without THPS (not protected) had 0% THPS added at the "pre-treatment with protective agent' stage and the groups without enzyme (without enzyme) had 0% enzyme added at the "enzyme application" stage. After pickling the skins were processed to cmst leather using standard conventional processes as known in the art, For each treatment group the overall depilation was ranked, and the skins assessed for grain surface damage against a semi quantitative scale (Figure).

The results show that enzyme based depilation after pre-protection of the skin using THPS as described in example 1 had a positive influence on the depilation, resulting in a better quality grain surface (Figure 5), In contrast, the results of this comparative trial show that enzyme based depilation without pre-protection results in a marked reduction in quality of the grain surface (Figure 3), Overall, the use of a protective agent as described in example I had a positive influence on the grain surface particularly for the skins treated with the enzyme (Figures 3 and 5). Photomicrographs of the different skin treatments are shown in Figures 2-5.

Figure 2 shows the grain surface of crust leather produced from an un-protected (i.e., no THPS pre-treatment) skin that was conventionally depilated; Figure 3 shows the grain surface of crust leather produced from an un-protected skin that was enzyme depilated; Figure 4 shows the grain surface of ernst leather produced from a protected (i.e., pre-treated with THPS) skin that was conventionally depilated and Figure 5 shows the grain surface of ernst leather produced from a protected skin that was enzyme depilated.

Alternative protective agenL To examine the efficacy of alternative protective agents, the process as described above in example 1 was carried out using a range of different regents in place of THPS at the "pretreatment with protective agent" stage. After pickling the skins were processed to ernst leather using standard conventional processes. For each treatment group the overall depilation was ranked and the skins assessed for grain surface damage against a semi quantitative scale (Figure 6). The skin in some treatment groups was too damaged after depilation to continue processing and so no grain assessment of the skins from these treatment groups was possible.

The only protective agents used in the enzyme based depilation process that provided protection to the skin grain after processing to crust leather were THPS, biocide (potassium dimethyldithiocarbamate), gluteraldehyde, and chromium sulphate. The results demonstrate that protection of the skin with TI-IPS resulted in a highly depilated skin with high grain quality after processing to ernst leather as compared to the other protective agents trialled (Figure 6), Treatment with THPS was significantly better than other treatments with a score of 4, which corresponds only to some minor distortion to the grain pattern whereas the next best treatments, those of biocide orglutaraldehyde had scores around 3 which corresponds to major distortion 4j of the grain surface with regions of the fine grain structure know to the art as "enamel" completely destroyed exposing the fibrous material beneath, Alternative enzymes To examine the efficacy of different enzymes for depilation of skins according to the methods disclosed herein, a range of different protective agents were trialled following the process of example 1. In particular, a different commercial enzyme was used in place of NOVOLIMETM at the "enzyme application" stage. After pickling the skins were processed to crust leather using standard conventional processes.

For each treatment group the overall degree of depilation was ranked, the skins in each treatment group were assessed for grain surface damage against a semi quantitative scale, the strength of the skins in each treatment group was assessed using the standard method for double edge tear TSO//BS EN 3377-2:2002, and the looseness of the leathers produced from the skins in each treatment group was measured by non parametric comparison between skins against a qualitative sale of looseness (Figure 7). The primary selection criteria in this work was to find a depilatory enzyme which when used in the method of the invention generated the least damage in the form of grain damage or looseness while still providing some degree of depilation. The results show that the two best enzymes in terms of looseness were NUE and NOVOLIIVIETM. While Enzidase had a high score for grain quality it was not significantly better than either NUE or NOVOLIMIE''.

Alternative sharpenitig agents' To examine the effect of different sharpening agents, particularly as alternatives to sodium sulfide, a range of reducing agents were trialled using the process described in example 1.

Specifically, a different sharpening agent was used in place of sodium sulfide at the "in-situ enzyme activation" stage. After pickling the skins were processed to crust leather using standard conventional processes.

For each treatment group the overall depilation of the skin was ranked, the skins were assessed for grain surface damage against a semi quantitative scale, the strength of the treated skins was assessed using the standard method for double edge tear ISO/lBS EN 3377-2:2002, and the looseness of the leathers produced was measured by non parametric comparison between differently treated skins against a qualitative sale of looseness. The results of this comparative trial are shown in Figure 8.

In some cases the treated skins were insufficiently depilated to enable continued processing and so no results for grain surface, strength, or looseness were possible. The resuhs show that a skin processed without any sharpening agent had an extremely poor pull (i.e., poor depilation), indicating that a sharpening agent such as sodium sulfide, is required. The two best alternatives to sodium sulfide were the sulphur containing compounds, thioglycolic acid and 2-mercaptoethanol, The use of thioglycolic acid resulted in a better quality leather as compared to the use of 2-mercaptoethanol. The use of 3% thioglycolic acid in place of the sodium sulfide demonstrates that the sulfide free depilation of skins following the enzyme depilation protocol set out in example 1 can provide high quality leather.

Example 2: Enzyme depilation of green sheep skins to produce a wet blue skin All processing steps in this example were carried out at 25°C.

I're-treat;nent i.vith the protective agent Process carried out as per example I Enzyme application Process carried out as per example I In situ enzyme activation Process carried out as per example 1 Neutralizing, degreasing, and tanning As is typical for hide and deerskin processing percentages are given on the basis of the weight of the hide or skin material. ?/bwt/wt g reagent/g skin material.

The depilated skin was reweighed and processed with an aqueous neutralising solution (80?/o water, 10.4% calcium chloride, 2% sodium sulphate and 5.2% sucrose percentages based on skin weight). For 75 minutes at 5 rpm. The aqueous neutralizing solution was drained completely and the skin washed in 200?/ skin weight water for 15 minutes at 10 rpm. The wash was then drained completely and the skin processed in a degreasing wash comprising 100% water, 2?/b ammonium chloride, 4% nonyl phenyl ethoxylate (Tetrapol LTN), and 0,2% Hydrogen peroxide for 60 minutes at 45°C 10 rpm. The degreasing wash was then drained completely and the skin was processed with 150% water at 45°C for 15 minutes 10 rpm. A further 150% water was added with 1% sodium dihydrogen phosphate and 1% nonyl phenyl ethoxylate for 60 minutes at 45°C 10 rpm. The wash was then drained completely A further three 150% water washes were then carried out for 15 minutes each at 45°C, 10 rpm with complete draining after each wash, A further 150% water wash was carried out at 20°C for 15 minutes 10 rpm. This final was was then drained completcily.

A cross section of the neck of the skin was taken and the pH of the skin checked using the indicator phenolphthalein to ensure that the pH was below 9 throughout the entire thickness of the skin, Next 100% water 0,1% unislip, 1% disodium phthalate was added and the skin processed for 10 minutes at 10 rpm. Following this, 4,5% chromium sulphate was added and the skin was further processed for 30 minutes at 10 rpm. The skin was then processed overnight at 40°C at 5rpm. The resulting skin product (wet blue skin) was then ready to be processed to crust leather using conventional standard processes know in the art as described in Sharphouse 1972.

Example 3: Enzyme processing of hides or deerskins to produce a wet blue product In the following example, all processing steps were carried out at 25°C unless otherwise speci-fied.

Pre-treatinent with the protective agent A pre-fleshed hide or deerskin was initially processed in a processing drum containing two vol- umes of water (200% of hide or deerskin weight), 0.2% of a nonyl phenol ethoxylate degreas-ing agent (Tetrapol LTN) and 3% protecive agent, tetrakis hydroxyphosphonium sulfate (THPS). The hide or deerskin was agitated by rotation for 3 hours at 2 rpm, allowing the pro-tective agent to thIly penetrate the raw hide or deerskin.

After penetration of the protective agent, the drum was drained and the protected hide or deer-skin was washed with 100% of hide or deerskin weight of water for 30 minutes at 10 rpm to remove any residual protective agent. The wash water was drained completely.

Enzyme app! ication The pre-protected hide or deerskin was treated in the processing drum with an aqueous solution comprising 100% of hide or deerskin weight of water and 2% of hide or deerskin weight of NOVOLIME® (an enzyme composition comprising subtilisin and lipase). Enzyme activity in NOVOLIME is 0.SAU/min as assessed by the method of Mozersky and Bailey 1992. The hide or deerskin was agitated by rotation for 3 hours at 2 rpm to allow the enzyme to fully penetrate into the hair follicle root of the hide or deerskin, The processing drum was then drained com- pletely and the hide or deerskin made ready for application of the activating solution. Specifi-cally the hide or deerskin was processed with and aqueous solution comprising 100% hide or deerskin weight of water and 2% sodium sulphate. The drum was then drained completely.

In situ enzyme activation The depilatory enzyme was activated in situ in the hide or deerskin by processing in the drum with an aqueous solution comprising i) 100% water ii) 2% sodium sulphate iii) 1% hydrated calcium oxide iv) 10% sodium hydroxide v) 1.2% thioglycollic acid The hides or deerskins were then processed overnight intermittently; alternating between proc-essing and static holding as follows: processing at 2rpm for t minute each hour, and holding statically for 59 minutes each hour, Neutralizing, degreasing, and tanning After completely draining the depilatory process liquor the depilated hides or deerskins were processed with an aqueous neutralizing solution having (100% water and 2% sodium sulphate based on hide or deerskin weight). Processing was carried out at 4rpm for 10 minutes. The aqueous neutralizing solution was completely drained and the hide or deerskin washed with 100% water at 4rpm for 10 minutes. The processing drum was then completely drained.

Deliming was carried out by processing the hide or deerskin in an aqueous deliming solution (500% water and 0.5% ammonium chloride based on hide or deerskin weight). Deliming was carried out for 15 minutes at 10 rpm. This initial deliming solution was completely drained from the processing drum. A frirther deliming process was carried out in aqueous solution (35% water and 0,5% hydrogen peroxide based on hide or deerskin weight. The hider or deer- skin was processed at 30°C at 10rpm for 15. 2.5% ammonium chloride and 0.2% nonyl phe-nol ethoxylate degreasing agent (Tetrapol LTN) based on hide or deerskin weight was then added to the drum, Further processing was carried out at 30°C at 10rpm for 90 minutes.

A cross section of the neck of the hide or deerskin was taken and the pH of the hide or deerskin checked using a phenolphthalein indicator to ensure the pH was below 9 throughout the thick-ness of the hide or deerskin, The processing drum was completely drained and the hide or deerskin washed twice with water (tOO% water based on hide or deerskin weight) with process-ing at 35°C, at 10rpm for 20 minutes.

The processing drum was drained and the hide or deerskin processed for tO minutes at 10 rpm in 60% water and 8% sodium chloride based on the weight of the hide or deerskin. 1.2% sul-phuric acid (based on the weight of hide or skin) was added after being pre-diluted 10 fold in water and the hide or deerskin processed for t20 minutes to reduce the pH of the processing so-lution to between 2,8-3,0, 6% chromium sulphate was then added to the processing solution and the hide or skin processed for 180 minutes at 16rpm. 0.35% magnesium oxide was then added and the hide or deerskin was processed overnight at 5rpm at 40°C. The hide or deerskin product (wet blue) processed as above could then be further processed to crust leather using conventional standard processes know in the art as described in Sharphouse t972, The skilled person will appreciate that the invention as set forth and described herein is not limited solely to the aspects, embodiments, and examples as described, but also encompasses within the scope of the invention, those variations and modifications of the invention as would be obvious to the person of skill in the art (including the person of ordinary skill in the art) in view of the disclosures provided herein and the common general knowledge. As wou'd be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention (as set out in the accompanying claims).

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Example 4

Enzyme depilation of green sheep skins to produce a pickled skin -pre-acidification step In the following example all processing steps were carried out between 20°C and 25°C.

Pre-acidi/icaiton A pre-fleshed sheep skin was initially processed in a processing drum containing 10 volumes of water (1000% of skin weight), and 0.3% formic acid and 1% D-limonene for about 60 minutes at about 20°C (pH of about 4 or less).

ileatment with the protective agent About 3% protective agent, tetrakis hydroxyphosphonium sulfate (TFWS) (% wt/vol in solution) was added. The skin was agitated by rotation for 105 minutes at 5 rpm, allowing the protective agent to fully penetrate the raw skin.

After penetration of the protective agent, the drum was drained and the protected skin was washed with 300% of skin weight of water for 15 minutes at 5 rpm to remove any residual protective agent. The wash water was drained completely. After washing the skin was left overnight as a matter of convenience. We have achieved the same results using skins or hides processed directly after washing.

Enzyme application The pre-protected skin was treated in the processing drum with an aqueous solution comprising 1000% of skin weight of water and 3% of skin weight of NOVOLITvIE® (an enzyme composition comprising subtilisin and lipase) (Novozymes A/S, Denmark). Enzyme activity in NOVOLIIVIIE is O5AU/min as assessed by the method of Mozersky and Bailey 1992. The skin was agitated by rotation for 5 hours at 5 rpm to allow the enzyme to fully penetrate into the wool follicle root of the skin. The processing drum was then drained completely and the skin made ready for application of the activating solution. Specifically the skin is drained of excess liquid by stacking in a pile overnight.

In situ enzyme ac//vat ion The depilatory enzyme was activated in situ in the skin by painting the flesh side of the skin with an activating composition or "paint" comprising: i) 08% hydrated calcium oxide ii) 1.3% sodium thioglycolate iii) 10% sodium hydroxide iv) 2% pre-gelatinised starch The activating paint also comprised 4% sodium sulfate as a deswelling agent to reduce looseness in the final product.

The activating paint as applied to the skin by spraying, rolling or brushing the flesh side of the skin. Specifically in this case the paint was applied by brush. After the activating paint was applied to the skin, the skin was held statically overnight (about 16 hours) to allow enzyme activation and subsequent proteolysis in situ, at the depilatory active structures of the skin. The wool was then removed from the skin manually by "pulling" or wiping off the wool with hands.

Neutralizing The depilated skin was reweighed and processed with an aqueous neutralising solution (80% water, 10.4% calcium chloride, 5.2% sucrose and 2% sodium sulfate, percentages based on skin weight) for 1 hour at 5 rpm. The aqueous neutralizing solution was drained completely and the skin washed in 200% skin weight water for IS minutes at 5 rpm. The skin was then washed 3 further times for 15 minutes each in 200% skin weight water at 5 rpm with constant agitation.

After the last wash, the skin was further treated with an aqueous solution containing 100% skin weight water, 2% ammonium chloride, for 20 minutes at 5 rpm with constant agitation. This latter treatment is known in the art as the "deliming" stage. After 2 minutes, 2% hydrogen peroxide was added and the skin processed for an additional 75 minutes at 5 rpm th intermittent agitation.

After deliming, the pH of the skin was determined to be less than 9.0 by phenolphthalein staining of a neck cross section, The skin was washed an additional 3 times in 100% skin weight water for 20 minutes each with agitation at 12 rpm. After the final wash was drained, the skin was treated in an aqueous solution comprising 90% skin weight water, 20% sodium chloride and 0.1% LTNISLIP FWTh' (Union Specialties, Mass, USA) is a "slip agent" composed of minerals and polymeric modifiers that stops the skins from tangling. The use of UNISLIP FWN is known in the art and the use of this slip agent is common throughout the industry. In particular, UNISLIP FWTM is effective at stopping the skins from tangling in the drum.

The skin was processed for 10 minutes th agitation at 12 rpm. After 0 minutes, a pre-mixed composition comprising 0.06% fungicide (thiocyanomethylthio) benzothiazole (TCMTB), 10% skin weight water and 2% sulphuric acid was added and the skin processed for an additional 1.5 hours with agitation at 12 rpm, The skins were then processed at 5rpm for 16 hours with constant agitation then fully drained. The skin product (pickled skin) could then be processed to wet blue and then crust leather using conventional standard processes know in the art, for example, as described in Sharphouse 1971.

The overall results achieved following the protocol in example 4 (not shown) were at least as good, if not better than the results shown in Figure 1,

REFERENCES

(The following references are each incorporated by reference herein in their entireties) Sharphouse, S. H. (1971). Leather technician's handbook. London: Vernon Lock.

Green, 0. H. (1952). "Unhairingby means of enzymes (A review of literature)." Journal of the Society of Leather Technologists and Chemists 36: 127-134.

Yates, J. R. (1972). "Studies in depilation X. The mechanism of the enzyme depilation process." Journal of the Society of Leather Technologists and Chemists 56(5): 158-177.

Brady, D., J. R. Duncan, et al. ( 989). "Enzyme depilatory studies part I. Review of enzyme C,, depilation" LIRI Research Bulletin 978.

Cantera, C. S. (2001). "Hair-saving unhairing process Part 1. Epidermis and the characteristics ofbovine hair," Journal of the Society of Leather Technologists arid Chemists 85(1): 1- 5.Sivasubramanian, S., B. Murali Manohar, et at (2008). "Mechanism of enzymatic dehairing of skins using a bacterial alkaline protease." Chemosphere 70: 1025-1034.

Wang, R., C. Mi et a]. (2009), "Enzyme unhairing -An eco-friendly biotechnological process." Journal of the Society of Leather Technologists and Chemists 93(2): 51-55.

Macedo, A, J., W. 0. Beys de Silva, et al, (2005), "Novel Keratinase from Bacillus subti/is Sl4 exhibiting remarkable dehairing capabilities." Applied and environmental microbiology 71(1): 594-596.

Saravanabhavan, P., P. Thanikaivelan, et al. (2005). "An enzymatic beamhouse process coupled with semi-metal tanning arid eco-benign post tanning leads to cleaner leather production," Journal of the American Leather Chemists Association 100(5): 174-186, Rose, C,, L. Suguna, et al, (2007), Process for lime and sulphide free unhairing of skins or hides using animal and/or plant enzymes. US, Council of scientific and industrial research.

Lowe, E. K. (1999). Enzyme depilation of ovine skins using NTIS 0.6 MPX. Palmerston North, New Zealand Leather arid Shoe Research Association.

Lowe, E. Ic. (2000). Evaluation of SPIC depilatory enzyme under New Zealand operating conditions. Palmerston North, New Zealand Leather and Shoe Research Association, Cooper, S. M. and E. K, Lowe (1997), Enzyme depilation of ovine skins Part t. Palmerston North, New Zealand Leather and Shoe Research Association.

Cooper, S. M. (1997). Assessment of the dewooling potential of three enzymes. Palmerston North, New Zealand Leather and Shoe Research Association.

Edmonds, R. (2012). Part 1-Suiphide free enzyme depilation -towards solving the key issues.

Proceedings of the 63rd LASRA Conference. Wellington, New Zealand Leather and Shoe Research Association. 63: 98-119.

Coulson, R. Ii. (1969). "The SATRA Break/Pipiness Scale." Journal of the American Leather Chemists Association 64: 648.

Cantera, C. S. (20W), "Hair-saving unhairing process Part 2. Immunisation phenomenon." Journal of the Society of Leather Technologists and Chemists 85(2): 47-51.

Cantera, C. S. (2001). "Hair-saving unhairing process Part 3. "Cementing substances" and the basement membrane." Journal of the Society of Leather Technologists and Chemists 85(3): 93-Cantera, C, 5, (20W), "Hair-saving unhairing process Part 4. Remarks on the evolution of the investigations on enzyme unhairing." Journal of the Society of Leather Technologists and Chemists 85(4): 125-132.

Cantera, C. S., L. Goya, et al. (2003). "Hair saving unhairing process. Part S characterisation of enzymatic preparations applied in soaking and unhairing processes," Journal of the Society of Leather Technologists and Chemists 87(2): 69-77 Brady, D., J. R, Duncan, et al. (989), "Enzyme depilatory studies part III. A model for proteolytic depilation of skins," LIRI Research Bulletin 982, Wang, Y,, Z, Li, et al. (2005), "Study on changes of collagen fibril structure in pigskin tissue after enzyme treatment." Journal of the Society of Leather Technologists and Chemists 89(2): 47-56.

Felicjaniak, B. (1985). "Studies in enzymatic unhairing: Part I. The influence of pigskin alkalisation on destruction of skin grain during depilation with pancreatic enzymes." Journal of the Society of Leather Technologists and Chemists 69(6): 160-163.

Tandt, H., A. E. Russell, et al, (997), Inhibition of proteolytic activity causing grain enamel in leather. 24th HJT..TCS Congress proceedings.

Defect processing BLC leather technologies centre, (200]). "Keep your grain on: Tanners' di-lemma." Leather International 203(4710): 114-115.

Hawkins, S. C. and J. Wassenberg (1997). Endogenous enzyme fellmongering -skin structure and properties. 24th IULTCS Congress proceedings, London.

Covington, A. D. (2009). Tanninu chemistry, The science of leather. Cambridge, The Royal Society of Chemistry, Bienkiewicz, K., Ed. (1983). Physical chemistry of leather making. Malabar, Florida, Robert E. Krieger Publishing Company.

Carrie, M. S. and F. W. Woodroffe (1960). Fellmongers handbook, Wellington, RE. Owen, Government printer.

ISO/lBS EN 3377-2:2002: Physical and mechanical tests -Determination of tear load -Part 2: Double edge tear

Claims

Claims 1. A method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a protective agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH greater than about 8, c) contacting the skin or hide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active structures in the skin or hide, and d) removing the wool or hair from the skin or hide.
2. The method of claim 1, wherein the method further comprises e) neutralizing the base on and/or in the skin or hide.
3. The method of claim t or claim 2, wherein the method further comprises 0 washing the skin or hide with water between a) and b).
4. The method of claim 2 or daim 3, wherein removing the wool or hair from the skin or hide is carried out after neutralizing the base on and/or in the skin or hide.
5. The method of any one of claims 2 to 4, wherein the method further comprises g) removing a neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.
6. The method of claim 5, wherein the method further comprises h) washing the skin or hide in water after g),
7. The method of any one of claims I to 6, wherein the method further comprises i) processing the skin or hide using conventional deliming and then pickling techniques to produce a pickled skin or a pickled hide.
8. The method of any one of claims I to 6, wherein the method further comprises j) degreasing the skin or hide followed by processing the skin or hide directly to wet blue without pickling.
9. The method of claim 7 or claim 8, wherein the method further comprises k) processing the pickled or wet blue skin or hide into leather.
10. A method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a tetrakis (hydroxymethyl) phosphine (THP) compound, a tetrakis (hydroxymethyl) phosphonium salt (a THP salt), a THP condensate, or oxazolidine, b) contacting the skin or hide with a composition comprising an alkaline serine protease in a substantially inactive state, c) contacting the skin or hide with an activating composition comprising hydrated calcium oxide, thioglycolic acid, sodium sulfate and sodium hydroxide to activate the alkaline serine protease in b), and d) removing the wool or hair from the skin or hide.
ii. The method of claim 10, wherein the method further comprises e) contacting the skin or hide with a neutralizing agent comprising sucrose and calcium chloride to reduce the pH on and/or in the skin or hide.
12. The method of claim 10 or claim 11, wherein the method comprises Q washing the skin or hide with water between a) and b).
13. A method for removing wool or hair from at least one raw skin or hide, the skin or hide each having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a protein cross-linking agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is substantially inactive at the pH of the composition, c) contacting the skin or hide with an activating composition comprising an agent that activates the depilatory enzyme in b), and a deswelling agent, and d) removing the wool or hair from the skin or hide.
14, The method of claim 13, wherein the method comprises e) washing the skin or hide with water between a) and step b).
15. The method of claim 13 or claim 14, wherein the contacting in c) is contacting the flesh side of the skin or hide with the activating composition.
16. A method for removing wool or hair from at least one skin or hide, the skin or hide each having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising a protein cross-linking agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme under conditions that maintain the enzyme in a substantially inactive state, c) contacting the flesh side of the skin or hide with an activating composition comprising an activating agent to activate the depilatory enzyme in b), and d) removing the wool or hair from the skin or hide.
17. The method of claim 17, wherein the method comprises e) washing the skin or hide with water between a) and b).
18. The method of claim 16 or claim 17, wherein the activating composition in c) further comprises a deswelling agent.
19. The method of any one of claims I to 18, wherein the method does not use sulfide.
20. The method of any one of claims ito 18, wherein the method uses sulfide at a concentration of less than about 1% Na2S (gNazS/gSkin material).
21. The method of claim 20, wherein the method uses sodium sulfide at a concentration of about 0.7% gNa2S/gSkin material or less.
22. A method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with an composition comprising a protective agent, b) contacting the skin or hide with a composition comprising a depilatory enzyme that is active at an alkaline pH, c) contacting the flesh side of the skin or hide with an activating composition comprising a base and a deswelling agent, wherein the base activates the depilatory enzyme in b) to act on the depilatory active stmctures in the skin or hide, and d) removing the wool or hair from the skin or hide, wherein the method either i) does not use a sulfide, or ii) uses sodium sulfide at a concentration of less than about 1% Na2S (g Na2S /gSkin material).
23. The method of claim 22, wherein the method further comprises e) washing the skin or hide with water between a) and b).
24. The method of claim 22 or claim 23, wherein the method uses sodium sulfide at a concentration of about 0.5 to about 09% gNa2S /gSkin material.
25. The method of claim 22 or claim 23, wherein the method uses sodium sulfide at a concentration of about 0.7% g Na2S /gSkin material or less.
26. A method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising about 2% tetrakis (hydroxymethyl) phosphonium sulfate (THPS) (wtTHPS/wtSkin material) for about three hours, b) contacting the skin or hide for about 2 hours with a composition comprising about 2% wt/vol of a depilatory enzyme subtilisin having an activity of 0,5ATJ/min, the composition having a pH of about 7, c) contacting the skin or hide for about 2 to about 16 hours with an activating composition comprising about 5% hydrated calcium oxide (wt/vol), about 3% thioglycolic acid (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 10% sodium hydroxide (wt/vol) to activate the subtilisin, d) removing the wool or hair from the skin or hide.
27. The method of claim 26, wherein the method further comprises e) washing the skin or hide with water between a) and b).
28. The method of claim 26 or claim 27, wherein the method further comprises neutralizing a base on and/or in the skin or hide by contacting the skin or hide for about 2 hours with a neutralizing agent comprising sucrose and calcium chloride.
29. The method of claim 28, wherein the method further comprises removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.
30. The method of claim 29, wherein the method further comprises washing the skin or hide with water after removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.31 The method of claim 29 wherein the method further comprises washing the skin or hide with water between a) and b), and washing the skin or hide with water after removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.32. The method of any one of claims 26 to 31, wherein the method further comprises processing the skin or hide using conventional deliming and then pickling techniques to produce pickled skin or pickled hide.33. The method of any one of claims 26 to 31, wherein the method comprises degreasing the skin or hide followed by processing the skin or hide directly to wet blue without pickling.34. The method of claim 33, wherein the method further comprises processing the pickled or wet blue skin or hide into leather.35, The method of any one of claims 26 to 34, wherein the method does not use sulfide.36. The method of any one of claims 26 to 34, wherein the method uses sodium sulfide at a concentration of about 0.7% (gNa2S /gSkin material) or less.37. A method for removing wool or hair from at least one raw skin or hide, the skin or hide having a flesh side and a grain side, the method comprising a) contacting the skin or hide with a composition comprising about 3?. tetrakis (hydroxymethyl) phosphonium sulfate (TI-IPS) (wtTHPS/wtSkin material) for about one to about three hours, b) contacting the skin or hide for about two to about five hours with a composition comprising about 3% wt/vol of a depilatory enzyme subtilisin having an activity of 0,SAU/min, the composition having a pH of about 7, c) contacting the skin or hide for about 2 to about 16 hours with an activating composition comprising about i?' hydrated calcium oxide (wt/vol), about 1% sodium thioglycolate (wt/vol), about 0.5% sodium sulfate (wt/vol) and about 12.5% sodium hydroxide (wt/vol) to activate the subtilisin, d) removing the wool or hair from the skin or hide.38. The method of claim 37, wherein the method further comprises e) washing the skin or hide with water between a) and b).39. The method of claim 37 or claim 38, wherein the method thither compiises neutralizing a base on and/or in the skin or hide by contacting the skin or hide for about 2 hours with a neutralizing agent comprising sodium sulfate, sucrose and calcium chloride.40. The method of claim 38, wherein the method further comprises removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.41. The method of claim 40, wherein the method further comprises washing the skin or hide with water after removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.42. The method of claim 40 wherein the method further comprises washing the skin or hide with water between a) and b), and washing the skin or hide with water after removing the neutralizing agent that has been used to neutralize the base on and/or in the skin or hide.43. The method of any one of claims 37 to 42, wherein the method further comprises processing the skin or hide using conventional deliming and then pickling techniques to produce pickled skin or pickled hide.44. The method of any one of claims 37 to 42, wherein the method comprises degreasing the skin or hide followed by processing the skin or hide directly to wet blue without pickling.45. The method of claim 44, wherein the method fUrther comprises processing the pickled or wet blue skin or hide into leather.46. The method of any one of claims 37 to 45, wherein the method does not use sulfide.47. The method of any one of claims I to 46, wherein the method comprises before a), contacting the skin or hide with a composition comprising an acid and a penetration enhancer.48. The method of claim 47, wherein the acid is formic acid.49. The method of claim 47, wherein the penetration enhancer is D-limonene.50. The method of claim 48, wherein the concentration of formic acid is about 0.3% based on skin weight.51. The method of claim 49, wherein the concentration of D-limonene is about 1% based on skin weight.52. The method of claim 47, wherein the composition comprises about 0.3% formic acid based on skin weight and about 1% D-limonene based on skin weight.53, The method of any one of claims 47 to 52 wherein the contacting with the composition comprising the acid and the penetration enhancer is for about 30 to about 90 minutes.34. The method of claim 53 wherein the contacting with the composition comprising the acid and the penetration enhancer is for about 60 minutes.35. The method as claimed in any one of claims to 54 substantially as herein described with reference to any example thereof 56. A skin or hide from which wool or hair has been removed according to a method of any one of claims ito 54.