WO2010114938A1

WO2010114938A1 - Soluble hydrolyzed keratin production

Info

Publication number: WO2010114938A1
Application number: PCT/US2010/029513
Authority: WO
Inventors: Steven Geoffrey Aitken; Gregory D. Ellis
Original assignee: Keraplast Technologies, Ltd.
Priority date: 2009-03-31
Filing date: 2010-03-31
Publication date: 2010-10-07

Abstract

Keratin peptides and processes of production are described The processes use a reduced number of steps to produce the product peptides with a size predominantly less than 50kDa. The process may be used to produce peptides with a similar amino acid profile to the keratin source or can be manipulated to produce high or low sulfur amino acid profiles

Description

SOLUBLE HYDROLYZED KERATIN PRODUCTION

Field The present application is directed Io soluble hydrolyzed keratin production.

Background

Keratin proteins are well known in the art and are found in a number of sources comprising wool, leathers and hail. Keratin fibers consist of a complex mix ol related proteins that are all part of the keratin family. These proteins can bo grouped according to their structure and role wilhiπ the fiber in to the following groups:

• The intermediate filament proteins (IFP) which are fibrous proteins found mainly in the fiber cortex;

• High sulfur proteins (HSP) which ore globular proteins found in the matrix of the fiber cortex, as well as in the cuticle, • High glycine-tyrosine proteins (HGTP), found mainly in the fiber cortex.

The ultra structure of keratin fibers is well known in the art and is discussed in detail by R.C. Marshall et al., Structure and Biochemistry of Mammalian Hard Karatin, Electron Microscopy Reviews, (1991) 4, 47.

Keratin proteins are used in a wide variety of applications, including their use in personal care formulations, wound care applications, as orthopedic materials, as nutritional supplements and in the production of polymer films.

The protoins perform a number of functions including conditioning, film forming, as humeclants and as emollients.

Keratin proteins are characterized by a high amount of the amino acid cysteine, which imparts a high degree of cross linking to keratin proteins through disulfide links. Keralin proteins are also highly ordered proteins providing a fundamental structural tole to many biological tissues.

The most commonly used keratin proteins are hydiolyzed in order to impart sufficient solubility to facilitate inclusion in a formulation. Keratin protoins are inherently insoluble duo Io the cross links associated with the characteristically high degree ol cysteino present in the protein. A problem in the art is that as hydrolysis is a vigofous piocess, many ot the desiiuble proportion of the keratin protuins are lost such as functionality. Numerous examples of the use ot hydrolyzed pioteins, including keratins, in peistsnal care formulaiions aie known in the ait.

US 4,948,876 describes on S-sulphocysteine keratin peptide produced by enzymatic hydrolysis for use as an auxiliary in the dyeing of wool and hail. Enzymatic digestion Is used by lhe authors to prepaie low molecular weight peptides and achieve the dosiicd solubility The '876 patent only produces a polymeric product with substantially identical amino, acid composition as the keratin substance from which it is prepared. This is limiting in that it is often the case that the amino acid levels in the product are tailored for certain applications (or example depending on the degree ol inoistuio retention and cost of the end product In addition, the '876 patent teaches of soaking in hydrochloric acid as a means Io remove copper However, this introduces additional reagents into the process and creates handling issues particularly on a large scale Finally, a furthor draw back of the process taught in the '876 patent is use of copious' amounts of water which on a large scale is resource intensive and potentially expensive

US 5,262,307 describes a process for hydrolyzing keratin maintaining its cystine (disulfide) content This is achieved by oxidative sulfitolysis of keratin followed by enzymatic hydrolysis and then subsequent heating at a pH of 2 to 5 to convert cysteine s-sulfonate back to cystino in the keratin peptides produced The 307 patent does not consider ways to produce and manipulate sulfur amino acid content in the final product Also, sulfitolysis taught in the '307 patent is at an elevated temperature (6O⁰C to 100^uC) which experience has shown is detrimental to desired product functionality as this temperature results in significant conversion of cystine / cysteine to cysteic acid Finally, the '307 patent does not consider removal of heavy metals from the product which is essential for nutritional / cosmetic product applications US 6,270 791 describes a process for forming keratin peptides by oxidation This process irreversibly oxidises the cystino / cysteine residues to cysteic acid and therefore reduces or eliminates functionality vital for efficacy of the material in hair caro and nutπlional supplement applications

US 7 148 327 describes a process for manufacture of sulfonated keratin peptides obtained from specific fractions The process requires fractionation Io obtain the amino acid profile required in the product Alternatively, the method of achieving a specific amino ϋcid profile via the fractionation process would require thdt each fraction was obtained separately dnd then mixed in the correct ratio prior to hydrolysis This would result in a large amount of "waste" / unuttlι?ed keratin feedstock (wool, feathers etc)

It would therefore be desirable to provide an alternative process (or the manufacture of solubilized keratin proteins and in particuldr peptides, particularly where that process is more economical to use especially in larger scale production runs

Summary

According to embodiments disclosed herein, S-sulfonated keratin peptides can bo produced bya more efficient One pot process which can also be manipulated to tailor the sulfur amino acid profile of the S- sulfonated keratin peptides produced The process utilizes foui key steps, which include oxidative sulfitolysis digestion liquor separation a hold step and enzyme hydrolysis The resulting S-sulfoπated keratin peptide product is of a size less than 5OkD Tho process can bo tailored to pioducc S-sulfonated keraϋn peptides with an amino ncid sulfur profile ol betwβαπ 4 and 20 mol% based on the cysteine content of the peptide amino aciri profile Tin¹ fust embodiment is directed to ,i procov. foi preparing S sulfonated keratin peptides comprising the steps of;

(a) reacting a keratin source via oxidative sulfitolysis to produce solids comprising S-sulfoπated keratin fractions in digestion liquor;

(b) separating the solids (rom the digestion liquor; (c) mixing the solids with water and holding for a predetermined time to allow the S-sulfonated keratin lractions to migrate into the water and form a S-sulfonated koratin fraction mixture;

(d) reacting the solids and the S-sulfonated keratin traction mixture via enzyme hydrolysis to produce S- suifonatcd keratin peptides.

In the first embodiment, the S-sulfonated keratin peptides produced can either have the same amino acid profile as the starting keratin source or can be manipulated during processing. As used herein, the term amino acid profile means the amino acid composition of the protein / hydrolysed protein.

Manipulations may be to form S-sulfonated keratin peptides with a high sulfur amino acid profile (termed hereafter as 'high sulfur peptides') where the cysteine content of the S-sulfonated keratin peptides is greater than 8 mol%. Alternatively, manipulations may be to form S-sulfonated keiatin peptides with a low sulfur amino acid profile (termed hereafter as 'low sulfur peptides') where the cysteine content of the S-sulIonated keratin peptides is less than or equal to 8 mol%.

In the fust embodiment the keratin source may bo processed in the same vessel during steps (a) to (d). The second embodiment is directed to a process for preparing high sulfur amino acid profile S- sulfonated keratin peptides comprising the steps of:

(a) reacting a keratin source via oxidative sulfitolysis to produce solids comprising S-sullonated keratin fractions in digestion liquor;

(b) separating solids from the digestion liquor;

(c) mixing the solids with water and holding tor a predetermined time to allow tho S-sulfonated keratin fractions to migrate into the water and form a S-sulfonated keratin fraction mixture;

(d) leading the solids and Ihυ S-sulfonated keiatin lraction mixture via enzyme hydrolysis to produce S- sulfonatfKl keratin peptides:

(e) acidifying ttic S-sϋlfonated keratin peptides to produce a supernatant comprising S-sulfonntcd keratin peptides having high sulfur amino ucid profiles and a precipitate comprising partially or completely uπhydrolyzed S-sulfonated keraln fractions;

(0 separating tho precipitate from the supernatant.

In the second embodiment, the peptides produced have a higher sulfur amino acid profile compared to the starting keratin source which occurs through manipulation during processing. Manipulations completed form S-sulfoπated keratin peptides with a high sulfur amino acid profile (termed hereafter as 'high sulfur peptides') where the cysteine content of the S-sulfonated keratin peptides is greater than 8 mol%

In the second embodiment the keratin source may be processed in the same vessel during steps (a) to 5 (d)

The third embodiment is directed to a process for preparing low sulfur ammo acid S-sulfonated keratin peptides compπsing the steps of

(a) reacting a keratin source via oxidative sulfitolysis to produce solids comprising S-sulfonαted keratin fractions in digestion liquor 0 (b) separating solids from the digestion liquor,

(c) mixing the solids with water and holding for a predetermined time to allow trie S-sulfoπated keratin lractions to migrate into the water and form a S-sulfonated keratin fraction mixture.

(d) reacting the solids and the S-sulfoπated koratin fraction mixture via enzyme hydrolysis to produce S- sulfoπated keratin peptides 5 In the third embodiment, the S sulfonated keratin peptides produced have a lower sulfur amino acid profile compared to the starting keratin source which occurs through manipulation during processing

Manipulations completed form S sulfonated peptides with a low sulfur amino acid profile (termed hereafter as 'low sulfur peptides^') where the cysteine content ol the S-sulfonated keratin peptides is less than oi equal to 8 mol%. In the third embodiment the keratin suuicυ may be processed in the same vessel during steps (a) to

(d)

Brief Description of the Drawings

Further aspects ol the embodiments described herein will become apparent from the following descnption which is given by way of example only and with reference to the accompanying drawings in which Figure 1 shows a flow diagram illustrating a process for producing keratin peptides

Dutailed Description of the Preferred Embodiments.

In the first embodiment, a process for manufacturing soluble keratin proteins is disclosed wherein !he t process compribus ptepaπng S-sulfonated keratin peptides comprising the steps of

(a) reacting a keintin source via oxidative sulfitolysis to produce solids comprising products-sulfonated keratin fractions in digestion liquoi,

(b) completing a partial to complete separation of the solids from the digestion liquor

(el mixing the solids with wdtei and hυldinμ lor a predetermined tιπιι> to allow the S sulfonated kciatm¹^ tractions Io migrate into the water and form <i S-sulfonated kei.itin fraction mixture. (d) reacting the solids and the S-sulfonated keratin fraction mixture via enzyme hydrolysis to produce S- sulfonated keratin peptides.

S-sulfoπated keratin peptides produced by the process are predominantly less than 5OkDa in size. Typically, the S-sulfonated keratin peptides are predominantly loss than 3OkOa in size. In one embodiment, the S-sulfonated keratin peptides produced are less than 1 OkDa in size.

S-sulfonated keratin peptides produced by the process ore soluble.

S-sulfonated keratin peptides produced by the process are reversibly S-sulfonated and the functionality of the S-sulfonated keratin peptides is retained.

In the first embodiment, the S-sulfunated keratin peptides produced can either have the same amino acid profile as the starting keratin source or (he amino acid profile can be manipulated during processing. By amino ncid profile, it is meant the amino acid composition of the material.

Manipulations may be to form S-sulfoπated keratin peptides with a high sulfur amino acid profile (termed hereafter as 'high sulfur peptides') where the cysteine content of the S-sulfonated keratin peptides is greater than 8 rnol%. Alternatively, manipulations may be to form S-sulfonated keratin peptides with a low sulfur amino acid profile (termed hereafter as 'low sulfur peptides^") where the cysteine content of the S- sulfonated keratin peptides is less than or equal to 8 mol%. Reference is made to cysteine content although other sulfur containing amino acids may be present to form the S-sulfonated keraliπ peptides. Cysteine is used as this is the predominant sulfur-containing amino acid In tho S-sulfonated keratin peptides produced by the process and therefore provides o useful marker as to sulfur amino acid profile. High sullur peptides produced by the process may have cysteine content ol between 10 mol% and 20 mol%. In a further embodiment, tho high sullur peptides may have a cysteine content may be between 12 mol% and 16 mol%.

Low sulfur peptides produced by the process may h;ivo cysteine content of between 4 mol% and 8 mol%. In a further embodiment, the low sulfur peptides may have a cysteine content of between 4 rπol% and 7 mol%.

The keratin source used typically is made up of various protein fractions which influence the amino acid profile of the S-su!fonated keratin peptides produced from the process. For reference, keratin protein fractions may be classified into distinct groups fium within the keratin protein family, and include but arc not limited to intermediate filament proteins (IFP), high sulfur proteins (HSP) and high glycine-tyrosine proteins (HGTP). Intermediate filament proteins are described in detail by Orwin et al. {Structure and Biochemistry of

Mammalian Hard Keratin, Electron Microscopy Reviews.4.47, 1991) and also referred Io as low sulfur proteins by Gillespie (Biochemistry and physiology of the skin, vol 1 , Ed. Goldsmith Oxford University Press, London. 1983, pp. 475-510). Key characteristics of intermediate filament protein family are molecular, weight in the fungc 40-60 kDa and a cysteine content (measured as half cystine) ol around 4 πiol7o. The high sulfur protein family is also well described by Orwiπ and Gillespie in tho same publications referenced above. This protein family has a large degree of heterogeneity, but can be characterized as having a molecular weight in the range 1-30 kDa and a cysteine content of greater than 10 mol%. A subset of this family is the ultrahigh sulfur proteins, which can have a cysteine content of up to 34 moi%. The high glycine-tyrosine protein family is also well described by Orwin and Gillespie in the same publications referenced above. This family is also referred to as the high tyrosine proteins and has characteristics of a molecular weight less than 10 kDa, tyrosine content typically greater than 10 mol% and glycine content typically greater than 20 mol%.

It should be appreciated from the above desciiptjαπ that the S-sulfonated keratin peptides produced from the processes described can either be derived frorrvtfie high sulfur- proteins comprising but not limited to HSP (to produce high sulfur peptides) and / cr fiorn lower sultur proteins comprising but not limited to IFP (to produce low sulfur peptides).

In the above embodiment, tho keratin source material may be processed in the same vessel ('termed herein is 'one-pot') during steps (a) to (d) This has the advantage that processing occurs with minimal labor requirements and avoids the need to separate thu keratin material during steps (a) to (d).

As noted above, step (a) comprises an oxidative sulfitolysis reaction to form S-sulfonated keratin fractions. S-sulfonated keratin refers to keratin protein that undergoes a sulfitαlysis process wherein the disulfide bonds between cystine amino acid in keratin protein are reveisibly modified to create polar functional groups that allow for controlled re-introduction of the natural disulfide crosslinks originally present in the keratin protein. S-sulfonated keratins have cysteine/cystine present predominantly in the form of S-sulfocysteine. This highly polar group imparts a degree of solubility to proteins. Whilst being stable in solution, the'S-sulfo group is a labile cysteine derivative, highly reactive towards thiols, such aa cysteine, and other reducing agents. Reaction with reducing agents leads to conversion of the S-sulfo cysteine group back to cystine. S-sulfo cysteine is chemically different from cystcic acid, although both groups contain the SO_$ group. Cysteicaάd is produced irreversibly by the oxidation of cysteine or cystine and once formed cannot form disulfide crosslinks back to cystine. S-sulfocysteine is reactive towards cysteine and readily forms disulfide crosslinks

In one embodiment, the oxidative sulfitolysis of step (a) is conducted at a temperature of approximately 20^uC to 30^*C to ensure that the final product has a suitable amino acid profile. Any other temperatures may also be used. The time period over which the oxidative sullitυlysis of step (a) occurs may be varied dependent on the desired amino acid profile of tho S-sulfoπateιJ koraliπ peptides product and therefore whether a high sulfur peptide or low sulfur puptide is produced. Any suitable time period of oxidative sulfitolysis may be used In one embodiment, the time period ranges from 2 to 5 hours, which produces a high sulfur peptide with a cysteine content of approximately 12 to 14 mol%. Alternatively, the time penod may be longer than 6 hours to increase thu ieaction time and hence produce a low sulfur peptide, i.e. a S-sulfoπated kerntin peptide with loss than or equal to 8 mol% cysteine. In a further alternative, the time period may be shorter than 3 hours to decrease the reaction time and hence produce a high sulfur peptide with cysteine content greater than 14 mol%. It should be noted that the above times arc given by way of example and that, if the temperature or chemical concentration is varied the reaction kinetics will also be altered influencing the time periods as well. The high sulfur fraction of keratin undergoes oxidative sulfitolysis much quicker lhan the lower sulfur fraction of keratin. Therefore, the S-sulfonated keratin peptide produced can bo "sulfur enriched" by adjusting the oxidative sulfitolysis conditions, such as those described above and below, so that the mixture ot S- sulfonated keratin fractions is sulfur enriched (more high sulfur lraction than lυwer sulfur fraction).

The oxidative sulfitolysis of step (a) may be completed according to any oxidative sulfitolysis process well known to those of ordinary skill in the art. In one embodiment, lhe oxidative sulfitolysis cuniprises mixing the keratin source with a cupric ammonium complex, water, sulfuric acid and sodium sulfite. A number of other chemical methods aie available to achieve oxidative sulfitolysis of proteinacous disulfide bonds. Alternative reactions conditions are reviewed in R. C. Marshall and A. S. lnglis in "Practical Protein Chemistry — A Handbook" (Publisher A. Darbre) 1986, pages 49-53. In one embodiment, the cupric ammonium complex may be formed from a mixture of ammonium hydroxide and copper sulfate pentahydratc.

The concentration of the reagents used in the oxidative sulfitolysis of step (a) may be varied dependent on the desired amino acid profile of the S-sulfoπated keratin protein peptide product and therefore whether a high sulfur peptide or low sulfur peptide is produced. Any suitable concentration may be used. For exampie, when using 135 to 180 kg of wool as the keratin source, 26 -35kg of 0.91 g/cc ammonium hydroxide 24-32!<g copper sulfate pentahydrate, 61 -82kg anhydrous sodium sulfite dissolved in water, and 66-68kg ot^'2M sulfuric acid produces high sulfur peptides with cysteine contents of approximately 12 mol% to 14 ιnol%. Alternatively, the reagent concentrations may be increased to increase the reaction rate and hence produce a low sulfur peptide, i.e a S-sulfonated keratin peptide with less than or equal to 8 mol% cysteine. In a further alternative, the reagent concentrations may be reduced to decrease the reaction rate and hence produce a high sulfur peptide with cysteine content greater than 14 mol%. It should be noted that the above concentrations are given by way of example and that, if other parameters such as temperature and time are varied the overall reaction kinetics will also be altered influencing the reagent concentrations used as well.

Tt)C keratin source used in the oxidative sulfitolysis of step (a) is in one embodiment wool but may also comprise either in mixture with wool or alone: hail, horns, hooves, feathers, nails and/or scales.

The keratin source uocd in the oxidative sυlfitolysis of step (a) may be either whole fiber or chopped fiber. Where whole fiber is used, the fibers may be chopped before tho enzyme hydrolysis ol step (d) commences. Onr; method of chopping is use- of a high shea; pump. It has been found that whether the libers uro whole or chopped into fragments does not appreciably alter the reaction kinetics of the sulfitolysis stop mid subsequent liquor removal (stop (b)) and hold (step (C)) stops. Digestion liquor from oxidative sulfitolysis in step (a) is removed in step (b). Any suitable method of removing the digestion liquor may be used. In one embodiment, the digestion liquor is removed by decanting off the liquid from the solids. This step considerably reduces the need to do extensive further processing to separate heavy metals including copper from the S-sulfonatcd keratin peptides. It has been found that removal of 65% to 80% of the digestion liquor results in approximately 40% to 50% of the copper being removed.

The mixing of the solids with water in step (c) may bo performed by any suitable method for mixing. In one example, the mixing may occur by adding water to the vessel in which the solids are located after the digestion liquor has been separated in step (b). The amount of water mixed with the solids may be an equivalent amount to the a'mount of digestion liquor removed. An aim of the hold poriυd of step (c) is Iu allow sulfitolysis to finish whilst simultaneously allowing oxidation of any residual sulfite compounds to sulfate compounds.

The holding period of step (c) may comprise agitating the S-sullonated keratin fraction mixture during tho predetermined hold time period.

The holding period of step (c) may comprise holding the S-sulfonated keratin fraction mixture at a temperature of between 5°C and 80°C during the predetermined hold time period. In one embodiment, the temperature may be 20"C to 30°C during the predetermined hold time period.

The holding period of step (c) may comprise aerating the mixture during hold time period. Any suitable aeration method may be used. One method of aeration may comprise the addition of compressed air added at a controlled pressure and flow rate. The predetermined time period for step (c) may bo varied dependent on the desired amino acid profile of the S-sulfonated keratin peptide product and therefore whether a high sulfur peptide or low sulfur peptide is produced. For example, a time period of 16 to 20 hours produces high sulfur peptides with cysteine contents of approximately 12 mol% to 14 mol%. Alternatively, the time period may be increased to produce a low sulfur peptide, i.e. a S-sulfonated keratin peptide with less than or equal to 8 mol% cysteine. In a further alternative. the time period may be reduced to produce a high sulfur peptido with cysteine content greater than 14 mol%. It should bo noted that the above limes are given by way uf example and that if the hold temperature is also varied the overall reaction kinetics will also be altered influuncing the hold time period as well

The enzyme hydrolysis of step (d) may be completed using any enzyme hydrolysis procedure known to those of ordinary skill in the art. In one embodiment, the enzyme hydrolysis comprises using at least one type of protease enzyme, preferably at a temperature and pH commensurate with preferred enzyme characteristics. One method may be to add bacterial alkaline protear.c at a rote of approximately 200,000-1 ,200.000 DU/kg solids at a temperature of approximately 40°C. To reach the pieferred alkalinity, pH adjusters (for example, sodium hydroxide) may be added to maintain the pH at the cwyme optimum, in this illustration being 9-10.

The time peiiod for the enzyme hydrolysis of step (d) may be varied dependent υn tho desired amino acid profile of the S-sulfonated keratin peptide product and therefore whether a high sulfur peptide or low sulfur peptide Is produced. Any suitable time period may be used. For example, a time period of approximately 20 hours produces high sulfur peptides with cysteine contonts of approximately 12 mol% to 14 rnol%. Alternatively, the time period may be increased beyond 20 hours to produce a low sulfur peptide, i.e. a S-sulfoπated keratin peptide with less than or equal to 8 mol% cysteine. In a further alternative, the time period may be reduced below 20 hours to produce a high sulfur peptide with cysteine content greater than 14 mol%. It should be noted that the above times are given by way of example and that if the hold temperature or enzyme type and enzyme reaction kinetics are also varied the overall reaction kinetics will also be altered influencing the enzyme hydrolysis of step (d) time period as well.

Step (d) results in S-sulfoπated keratin peptide production where any keratin protein or polypeptide fractions are cleaved into smaller peptide sub-units. Smailor size is preferred because in order for the S- sulfonated keratin peptides to impart desired biological activity in-vivo it is important that the S-sulfonated keratin peptides are able to penetrate to the biological site of action. S-sulfonated keratin peptides have better penetration properties than whole proteins.

By changing various processing parameters of the enzyme hydrolysis, such as those described above, the ratio of the protein fractions which are converted through to peptides may be changed. Generally speaking, the high sulfur fraction undergoes hydrolysis quicker and is not acid precipitatable, whereas the lower sulfur fraction is slower to react and "unroacted^* tower sulfur protein / large peptide can be acid precipitated. As such the S-sulfonatod keratin peptide produced can be substantially "sulfur enrich" by choosing the enzymatic reaction conditions. The resulting S-sulfonaled kciutiii peptide product fiorn step (d) may be further piocessec! to further manipulate the amino acid profile of the S-sulfonated keiatin peptides. One example of further processing to manipulate the sulfur profile is to also use an acidification step.

The resulting S-sulfonated keratin peptide product from step (d) may also be further processed to clarify, concentrate, and/or purify the S-sulfoπated keratin peptides in the resulting product. The resulting soluble S-sulfonated keratin peptide is typically presented as a formulation although it can be dried by techniques such lyophilisation, spray drying or drum drying. The formulation may comprise the S- sulfonated keratin peptides as well as other suitable formulation ingredients. By way of illustration, for a personal care or cosmetic formulation, the formulation may contain water, preservatives and soluble S- sulfonated keratin peptides from 0.001 to 75% by weight. In the second embodiment, a procoss of producing a soluble keratin derivative is disclosed wherein the process comprises preparing high sulfur amino acid profile S-sulfoπated keratin peptides comprising the steps of:

(π) reacting a keratin source via oxidative sulfilolysis to produce solids comprising S-sulfonated keratin fractions in digestion Hηuor; (b) separating lho solids from the digestion liquor; (C) mixing the solids with water and holding for a predetermined time to allow the S-sulfonated keratin tractions to migrate into tho water and form a S-sulfonated keratin fraction mixture;

(d) reacting the solids and the S-sulfonated keratin fraction mixture via enzyme hydrolysis to produce S- sulfonated keratin peptides; (e) acidifying the S-sulfonated keratin peptides to produco a supernatant comprising S-sulfonated keratin peptides having a high sulfur amino acid profile and a precipitate comprising partially to completely unhydrolyzed S-sulfonated keratin fractions; and

(0 separating the precipitate from the supernatant.

As in the first embodiment, S-sulfonated keratin peptides produced by tho process are predominantly less than 5OkDa in size Typically, the S-sulfoπated keratin peptides are less than 3OkDa in size. In one embodiment, the S-sulfonated keratin peptides produced are predominantly less than 1OkDa in size.

S-sulfonated keratin peptides produced in the second embodiment are also soluble.

Further, S-sulfoπated keratin peptides produced by the second embodiment process are reversibly S- sulfonated and the functionality of the S-sulfonated keratin peptides is retained. In the second embodiment, the amino acid profile of the S-sulfoπated keratin peptides produced is manipulated during processing.

Manipulations completed are to form S-sulfoπaled keratin peptides with a high sulfur amino acid profile (termed hereafter as 'high sulfur peptides') where the cysteine content of the S-sulfonated keratin peptides is greater than 8 mol%. Reference is made to cysteine content although other sulfur containing amino acids may be present to form the S-sulfonatcd keratin peptides Cysteine is used as this is the predominant sullur containing amino acid In tho S-sulfonated kuratin peptides produced by the process and therefore provides a useful markfir as to sulfur amino acid profile.

High sulfur peptides produced by the process may have cysteine content of between 10 mol% and 20 mol%. In a further embodiment, the high sulfur peptides may have a cysteine content between 12 mol% aπd 16 molVβ.

The keratin source used typically is made up of various protein fractions which influence the amino acid profile of the S-sulfonated keratin peptides produced from tho process. In the second embodiment, the protein fractions are predominantly from the HSP fraction although other protein sources may be iπcorpoiated into the process. In the second embodiment, the keratin sourcυ mateiial may bo processed in the same vessel (termed herein is One-pot ) during steps (a) to (e). This has the advantage that processing occurs with minimal labor requirements and avoids the need to sepaiate the keratin material during steps (a) to (e).

Step (a) oxidative sulfitolysis proceeds in a similar manner to that of the first embodiment.

Preferably, to maximize high sulfui peptide production during step (a), the time period over which step (a) oxidative sulfitolysis occurs may be shorter than 3 hours in order to decrease the reaction time and hence produce a high sulfur peptide with cysteine content greater than 8 mol% It should be noted that the above times are given by way of example and that if the temperature oi chemical concentration is varied the reaction kinetics will also be altered influencing the time periods as well

Preferably, also to maximize high sulfur peptide production during stop (a) the concentration of the reagents used in step (a) oxidative sulfitolysis may be reduced In one embodiment, when using 180 kg ol wool as the keratin source, the reagent concentrations are equal to or less than 2Ckg of 0 91g/cc ammonium hydroxide, 24kg copper sulfate pentahydrate, 61 kg anhydrous sodium sulfite dissolved in water, and 66kg of 2M sulfuric acid to produce peptides with greater than 8 mol% cysteine content It should be noted that the above concentrations are given by way ot example and that, if other parameters such as temperature and time are varied the overall reaction kineticb will also be altered influencing the reagent conccntrattoπs used as well

Digestion liquor from oxidative sulfitolysis in step (a) is removed in stop (b) in the second embodiment in a similar manner to the first embodiment

Holding in step (c) of the second embodiment occurs largely in a similar manner to the first embodiment, except that the time period ol holding is varied to increase the production of high sulfur peptides For example a time period of less than ?0 hours may be used in order to produce peptides with cysteine content greater than 8 mol% It should be noted that the above lime is given by way of example and that if the hold temperature is also varied the overall reaction kinetics will also be altered influencing the hold time peπod as well

The time peπod for step (d) enzyme hydrolysis in the second embodiment may also be varied to maximize high sulfur peptide production For example, a time period ol less than approximately 20 hours produces high sulfur peptides with cysteine contents greater than 8 mol% It should be noted that the above time is given by way of example and that if the temperature pH or enzyme type are varied, the enzyme reaction kinetics may βlso be varied changing the overall reaction kinetics and hydrolysis time period

The second embodiment comprises a further step (e) o' acidification The aim of this step is to precipital^p out low sulfur protein or peptide fractions in the mixture, thereby increasing the sulfur content of the remaining S-sulfonated Keratin peptides One process for completing acidification is to reduce the pH of the mixture ot S sulfonated keratin peptides to appioximately 3 to 4 and then holding lhf mixture of S sulfonated keratin peptides at this pH for ά period of time More specifically the proi-ess may comprise jdriiπg sufficient sulfuric acid to the mixture of S-sulfonjtod keratin peptides Io achieve <) oH ol 3 5 and then hold the mixture of S sulfonated kerdtin peptides at this pH for a time peiiod oM to 3 hours Optionally <i preservative may dlso be added to the mixture of S sulfonated kt>ratιπ peptides at this stage Any suitable preservative m^py he used One preservative used may be potassium sorbate The precipitate resulting lrom the acidification may include fully unhydroly7od S-sulfonatod koratin fractions ami/or partly hydrolyzed S sulfonated keratin acid precipi'atdble fractions The acidified S-sulfonated keratin peptide lrom step (e) may also be further processed to clarify, concentrate, and/or purify the S-sulfonated keratin peptides in the resulting product.

The resulting soluble high sulfur keratin peptide is typically formulated as a solution although it can be dried by techniques such lyophilisation, spray drying and drum drying. The solution may comprise the S- sulfonated keratin peptides as well as other suitable formulation ingredients. By way of illustration, for a personal care or cosmetic formulation, the solution may contain water, preservatives and soluble S-sulfonated keratin peptides from 0.001 to 75% by weight.

In the third embodiment, a process to produce a soluble keratin protein derivative is disclosed wherein the process comprises preparing low sulfur amino acid S-sulfonated keratin peptides comprising the steps of: (a) reacting a keratin source via oxidative stilfitolysis to produce solids comprising S-sulfoπated keratin fractions in digestion liquor;

(b) separating the solids from the digestion liquor;

(c) mixing the solids with water and holding for a predetermined time to allow the S-sulfonated keratin fractions to migrate into the water and form a S-sulfonated keratin fraction mixture; (d) reacting the solids and the S-sulfonated keratin fraction mixture via enzyme hydrolysis to produce S- sulfonated keratin peptides.

As in the first and second embodiments, S-sulfonated keratin peptides produced by the process are predominantly less than 5OkDa in size. Typically, the S-sulfonatod keratin peptides are less than 3OkDa in size. In one embodiment, the S-sulfonated korotin peptides produced are predominantly less than 1 OkDa in size. S-sulfonated keratin peptides produced in the third embodiment are also soluble.

Further, S-sulfonaled keratin peptides produced by the third embodiment process are reversibly S- sulfonated and the functionality of the S-sullonated keratin peptides is retained.

In the third embodiment, the rtmino acid profile of the S-sulfonated keratin peptides produced is manipulated during processing. Manipulations completed are to form S-sulfonated keratin peptides with n low sulfur amino acid profile

(termed hereafter as 'low sulfur peptides') where the cysteine content of the S-sulfonatcd keratin peptides is less than or equal to approximately 8 mol%. Reference is made to cysteine content although other sulfur containing amino acids may be present to form the S-sulfonated keratin peptides. Cysteine is used as this is the predominant sulfur containing amino acid in the S-sulfonated keratin peptides produced by the process and therefore provides a useful marker as to sulfur amino acid profile.

Low sulfur peptides produced by the process may havo cysteine content of between 4 mol% and 8 mol% In a further embodiment, the low sulfui peptides may have a cysteine content of between 4 mol% and 7 mol%.

The keratin source used typically is made up o1 various ptotein fractions which influence tho ammo acid profile of the S-sulfonatβd keratin peptides produced from the process. In the third embodiment, Hie protein fractions are predominantly from the IFP fraction although other protein sources may be incorporated into the process.

In the third embodiment, the keratin source material may be processed in the same vessel ('termed herein is 'one-pot') during steps (a) to (d). This has the advantage that processing occurs with minimal labor requirements and avoids the need to separate the keratin material during stops (a) to (d).

Preferably, for low sulfur peptide production during step (a), the time period over which step (a) oxidative sulfitolysis occurs may be longer than 5 hours in order to increase the reaction time and hence produce a low sulfur peptide with a cysteine content loss than or equal to 8 mo!%. It should be noted that the above time is given by way of example and that, if the temperature or chemical concentration is varied the reaction kinetics will ϋlso bo altered influencing the time periods as well.

Preferably, for low sulfuY peplide production during step (a), the concentration of the reagents used in step (a) oxidative sulfitolysis may be increased, In one embodiment, when using 135 kg of wool as the keratin source, the reagent concentrations are greater than 35kg of 0.91 g/cc ammonium hydroxide, 32kg copper sulfate pentahydrate, 82kg anhydrous sodium sulfite dissolved in water, and 68kg of 2M sulfuric acid to produce S-sulfonated keratin peptides with less than or equal to B mol% cysteine content. It should be noted that the above concentrations are given byway of example and that, if other parameters such as temperature and time are varied the overall reaction kinetics will also be altered influencing the reagent concentrations used as well.

Digestion liquor from oxidative sulfitolysis in step (a) is removed in step (b) in the third embodiment in a similar manner to the first and second embodiments.

Holding in step (c) of the third embodiment occurs in a similar manner to the first embodiment, except that the time period of holding is varied to produce low sulfur peptides. For example, a time period of greater than 20 hours may bu used in order tu produce S-sulfoπated keratin peptides with cysteine content less than or equal to 8 mol%. It should bo noted that the above time is given by way of example and that if the hold temporature is also varied the overall reaction kinutics will also be altered influencing the hold time period as well.

Tho time period for stop (d) enzyme hydrolysis in the third embodiment may also be varied Io produce low sulfur peptides. For example, a time poriod of greater than approximately 20 hours produces low sullur peptides with cysteine contents loss lhan or equal to 8 mol%. It should be noted that the above time is given by way of example and that if the temperature, pH or enzyme type are varied, the enzyme reaction kinetics may also be varied changing the overall reaction kinetics and hydrolysis time period.

The third embodiment does not utilize an acidification step as described in the second embodiment. The S-sulfonated keratin peptide product from step (d) may also be further processed to clarify, concentrate. and/oι puiify the S-sulfonated keratin peptides in the resulting product. The resulting soluble low sulfur keratin peptide is typically formulated as a solution although it can be dried by techniques such lyophilisalion, spray drying or drum drying. The solution may comprise the S- sulfoπation keratin peptides as well as other suitable formulation ingredients. By way of illustration, for a personal care or cosmetic formulation, the solution may contain water, preservatives and soluble S-sulfoπated keratin peptide from 0.001 to 75% by woighl.

In a variation to the above three embodiments, the keratin source may be pre-soaked before step (a). An aim of pre-soakiπg is to pre-wet the keratin source which allows better penetration of the digestion liquor used in step (a) oxidative sulfitolysis. Uso of this step also avoids the need to use fiber swelling agents. A process for pro-soaking the keratin souice established may comprise immersing the keratin source, for example wool, with water. Immersion may take place ovor approximately 1-10 hours before draining and commencing step (a) oxidative sulfitolysis.

As noted above for each embodiment, the resulting S-sulfonated keratin peptide product from step (d) or step (e) if present may be further processed to concentrate the S-sulfonated keratin peptides in the resulting product. Processing steps may comprise an ion exchange step and a concentration step to remove most residual heavy metals and increase the peptide concentration. Any suitable ion exchange step or concentration step known to those or ordinaiy skill in the art may be used. In one embodiment, the residual heavy moial concentration is less than 50ppm. Preferably, the concentration is less than 10ppm.

Ion exchange may be completed by passing the S-sulfonated keratin peptide product through an ion exchange separator. The separator may be a protonated ion exchange with ion exchange or chelating resin. More specifically, one method used may comprise passing the S-sulfonated keratin. peptide product through a resin macroporous polystyrene based chelating resin containing an iminodiacetic functional group. By undertaking this step, residual heavy metals such as copper are removed to a level acceptable for nutritional and cosmetic applications.

Concentration noted above may be completed by techniques such lyophilisatioπ, spray drying or drum drying to produce a solid S-sulfonatod keratin peptide product or alternatively evaporation which typically produces a concentrated solution.

In a turther variation to the second embodiment and the first embodiment where acidification is completed, the resulting solution containing precipitate tollowiπg acidification is also filtered and clarified before completing an ion exchange step noted above. Filtration may be via a 500 micron fabric niter although other techniques known in the art may also be used. A holding time period step may also be utilized post filtration to allow any residual precipitate Io settle. The resulting filtrate is then clarified. One clarification method may comprise passing the mixture through a clarifying centrifuge although other clarification techniques known in the an may also be used.

The final S-sulfonated keratin peptide product of the above embooiments may also be formulated dependent on their end uses. Ono end use may be in personal care formulations whereby preservatives comprising phcnoxyethaπol. sodium bonzoate, potassium sorbate may be added. pH adjusters may also be added along with water to dilute the S-sulfonated keratin peptide solution. Personal care formulations in which the resulting keratin protein may be used on account of the protein properties comprise any of the following: conditioning shampoo, body/tacial cleanser/ shampoo, hair conditioner, hair gel, hair mouse, hair setting lotion, hairspray, pre-perming solution, post-perming solution, moisturizing cream, shower gel, foaming bath gel, mascara, nail polish, liquid foundation, shaving cream, and lipstick. Other personal care foirnulations that assist in achieving the properties noted above aro also encompassed within the invention for example a detergent that protects skin from drying.

It may be appreciated from the above description that the processes described offer a number of advantages over the art. In more detail, advantages comprise:

(a) The process may be easily tailored to produce desired S-sulfonated keratin peptide amino acid profiles ranging from substantially the same to low sulfur or to high sulfur.

(b) Processing of the keratin material to a hydrolyzed form is completed in one mixing vessel rather than the need to separate the material into different fractions between siilfitolysis and hydrolyzing. This results in a more time efficient process and greater yield.

(c) There is no need to separately hydrolyze different fractions of S-sulfonated keratin material resulting from the sulfitolysis step. All material is hydrolyzed at once and if necessary later separated. This also results in a more time efficient process and greater yield.

(d) An alternative process required that the end keratin amino acid profile be manipulated by separating out the dilferent fractions IFP, HSP, HGTP etc and then combining the different fractions in amounts that give the desired profile By contrast, the processes described avoid the need to separate and later re-mix with process step variations used to achieve a similar result. This avoids extra handling and reduces piocossing time.

(e) The above process reversibly protects the cystine / cysteine residues as cysteine S-sulfoπa'e Maintenance of the cystine / cysteine functionality is vital for efficacy of the material in cosmetic and nutritional applications.

(f) The process avoids the need for sulfitolysis at elevated temperatures (60°C or higher). Elevated temperatures are expected to result in significant conversion of cystine / cysteine to cysteic acid therefore removing desired functionality from the product. (g) The process removes heavy metals such as copper to levels suitable for cosmetic or ingestible applications and in a way that minimizes resource_%use, and in particular avoids the need for washing with significant amounts of water.

(h) The process eliminates the need to utilize certain compounds essential in the art such as hydrochloric acid. Working Examples

S Example 1 - Manufacturing a Hvdrolvzed S-Sulfonate.d Keratin Peptide

This Example describes one process to produce an S-sulfonated keratin peptide product. The process utilizes a first wool-soaking step whereby approximately 180 kg of chopped wool is added to a digestion tank and then 1500-2000L of water is added and mixed with the wool. Then mixture is then left to soak for approximately 2 ^"3A hours. As described above the aim is to pro-soak the wool which the inventors have0 found may aid penetration of the digestion liquor into the wool. Use of swelling agents may also be avoided by this step. Once soaking is complete, the water is drained from the digestion tank leaving pre-wet wool.

The second step in the Example process is mixing of reagent In this Example approximately 35kg of 0.91g/cc ammonium hydroxide is mixed with approximately 32 kg of copper sulfate pentahydrate in the absence of water. This mixture is then combined in a tank with 100-200L ot wateι. Additional water is then added to a5 final volume of approximately 110OL. Approximately 68 kg of 2M sulfuric acid is then added to the tank to lower the pH to approximately 8.6. The mixture tormed is a pale blue cupric ammonium complex. Separately, 82kg of anhydrous sodium sulfite is dissolved in 300L of warm water.

The third step in the Example process is an oxidative sulfilolysis digestion. The cupric ammonium complex mixture is transferred to the pre-wet wool. The sodium sulfite solution mixed above is then added. The0 resulting pH once all reagents are added Is approximately 9.5. After the addition of all the digestion chemicals, the digestion liquor temperature is raised to 25°C over approximately 10 minutes. During this time the digestion liquor is circulated through a distribution/sparge ring whilst being continuously mixed with a dual impeller pitched blade turbine mixer, Oxygen control is by natural air enlrainment using a digestion vessel with a diameterheight ratio of 1:1. At the end of the sulfltolysis digestion time period (approximately 2 Vi hours), approximately 63% of5 the tank volume of liquor is drained off tho wool. The inventors found that this equated to a removal of approximately 46% removal of copper from the remaining digested mixture.

The fourth step in tho Example piocess is a hold step. An equivalent amount of cold water is added back Io the mixture and tho mixture is then agitated conlinuously for 20 hours at 25°C. The length ot tho hold period allows the sulfonated peptides (in particular HSP and IFP tractions) in the sulfonated liquor to migrate into solution for hydrolysis. Compressed air is added at a pressure of 4 bar and at a rate of 60 Umin to end the sulfitolysis by oxidizing the remaining sulfites to sulfates.

The fifth step is enzyme hydrolysis. During this stop, the temperature of the sulfonated liquor is raised from 25"C to approximately 40"C to improve tho hydrolysis conditions without reducing the sulfonated species.

The temperature rise takes place over approximately 2 Vi hours Enzidase® PTX6L bacterial alkaline piotease5 enzyme is then added to the sulfonated liquor feedstock at a rale of approximately 15Og equating to approximately 600.000 DU/kg of protein and wool solids. 1 M Sodium hydroxide is also continuously added with the pH maintained at approximately 9.2. In this Example, the hydrolysis time period is approximately 20 hours.

The sixth step is acidification. The hydrolyzed protein is acidified over an approximate 2 1/2 hour time period with 2M sulfuric acid to lower tho pH to approximately 3.5. In this Example, potassium sorbate is added at a rate equivalent to a level of approximately 0.1% of lhe liquor volume to the mixture once the pH is reduced to approximately 5. The sorbate acts as a preservative. Precipitation of unhydrolyzed high molecular weight protein fractions results

The seventh step is filtration and settling. The dilute hydrolyzed protein mix is filtered using 500 micron in-line fabric filter to separate the precipitated higher molecular weight protein solids from the acid soluble peptide fraction containing (he non acid precipitatable high sulfur peptides. The filtrate is allowed to stand in this

Example for approximately 46 hours to allow further separation of the intermediate filament peptides which concentrates in turn the high sulfur peptide content in the supernatant above.

The eighth step is clarification. The dilute peptide supernatant, enriched with high sulfur peptides, is racked off abovo the settled solids and passed through a clarifier centrifuge The ninth step is ion exchange. The low pH dilute peptide liquid is pumped through a protonated ion exchange, typically at a rate of 3 bed volumes/ hour (BV/hr) where residual heavy metals, in particular copper, are deposited In this example, the Ion exchange resin is macroporous polystyrene based chelating resin containing an iminodiacetic functional group.

The tenth step is a concentration step, in this Example completed by evaporation in a flash vacuum evaporator

The eleventh step is a formulation step In this Example cosmetic and food grade preservatives are added

The resulting product from the abovo process is an S-sulfonated keratin peptide solution with a high sulfur amino acid content and in particular, a cysteine content of approximately 13%. The Example shows that the process may be completed as a One pot' process with no need for distinct breaks in processing to separate different Fractions

Example 2 - Manufacturing a Hydrolyzed S-Sulfonated Keratin Peptide

This Example describes un alternative process to produce an S-sulfonated keratin peptide product. The process utilizes a first wool-soaking step whereby approximately 135 kg of whole fiber wool is added to a digestion tank and then 1500-2000L of water is added and mixed with the wool The mixture is then IeIt to soak for approximately 2 \U hours As described above the aim is to pre-soak the wool which the inventors have found may aid penetration of the digestion liquor into the wool Use of swelling agents may also be avoided by tnis step Once soaking is complete, thu water is drained fiom the digestion lank leaving pro-wet woo! The socond step in tho Example process is mixing of reagent. In this Example, approximately 26kg of 0.91 g/cc ammonium hydroxide is mixed with approximately 24 kg of copper sulfate pentahydrate in the absence of water. This mixture is then combined in a tank with 100-200L of water. Additional water is then added to a final volume of approximately 110OL. Approximately 66 kg of 2M sulfuric acid is then added to the tank to lower lhe pH to approximately 8.6. The mixture formed is a pale blue cupric ammonium complex. Separately, 61kg of anhydrous sodium sulfite is dissolved In 300L of warm water.

The third step in the Example process is an oxidative sulfitolysis digestion. The cupric ammonium complex mixture is transferred to the pre-wet wool. The sodium sulfite solution mixed above is then added. The resulting pH once reagents are added is approximately 9.5. After the addition of all the digestion chemicals, the digestion liquor temperature is raised to 25'C. During this time the digestion liquor is circulated through a distribution/sparge ring whilst being continuously mixed with a dual impeller pitched blade turbine mixer. Oxygen control is by natural air entrainment using a digestion vessel with a diametcrhcight ratio of 1 :1. At the end of the sulfitolysis digestion time period (approximately 4 V* hours), approximately 74% of the tank volume of liquor is drained off the wool The fourth step in the Example process is a hold step. An equivalent amount of cold water is added back to the mixture and the mixture is then agitated continuously for approximately 16 V* hours at 25°C. The length of the hold period allows the sulfonated peptides (in particular HSP and IFP fractions) in the sulfonated liquor to migrate into solution for hydrolysis. Compressed air is added at a pressure of 4 bar and at a rate of approximately 60 L/min to end the sulfitolysis by oxidizing the remaining sulfites to sulfates. At this stage, the fibrous strands are chopped using a high shear pump.

The fifth stop is enzyme hydrolysis. During this step, the temperature of the sulfonated liquor is raised from 25"C to approximately 40°C to improve tho hydrolysis conditions without reducing the sulfonated species. The tempetature rise takes place over approximately 8 V5 hours. Enzidase® PTX6L bacterial alkaline protease enzyme is then added to the sulfonated liquor feedstock at a rate of approximately 1 15g equating to approximately 613.000 DU/kg of protein and wool solids. 1 M Sodium hydroxide is also continuously added with the pH maintained at approximately 9.2. In this Example, the hydrolysis time period is approximately 20 hours.

The sixth step is acidification. The hydrolyzed protein is acidified over an approximate 1 Vi hour time period with 2M sulfuiic acid to lower the pH to approximately 3.5. No preservative is added. Precipitation of nnhydrolyzed high molecular weight protein fractions results. The seventh step is filtration and settling. The dilute hydrolyzod protein mix is filtered using 500 micron in-line fabric filter to separate the precipitated higher molecular weight protein solids from the acid solubte peptide fraction containing the non acid precipitatable high sulfur peptides. The filtrate is allowed to stand in this Example (or approximately 46 hours to allow further sopaiation of the intermediate filament peptides v/hich in turn concentrates the high sulfur peptide content in the supernatant above. The eighth step is clarification. The dilute peptide supernatant, enriched with high sulfur peptides, is racked off above the settled solids and passed through a clarifϊer centrifuge.

The ninth step is ion exchange. The low pH dilute peptide liquid is pumped through a protonated ion exchange, typically at a rate of 2.5 bed volumes / hour (BV/hr), where residual heavy metals, in particular copper, are deposited. In this example, the ion exchange rcsiπ is macroporous polystyrene based chelating resin containing an iminodiacetic functional group.

The tenth step is a concentration stop, in this Example completed by evaporation in a flash vacuum evaporator. In this Example, the dilute peptide is pre-heated to approximately 75°C prior to the vacuum chamber.

The eleventh step is a formulation step. In this Example cosmetic and food grade preservatives are added depending on the end requirement

The resulting product from the above process is an S-sulfonated keratin peptide solution with a high sulfur amino acid content and in particular, a cysteine content of approximately 14%.

The Example shows that the process may be completed as a 'one pot" process with no need distinct breaks in processing to separate different fractions.

Example 3 - Low Sulfur Amino Acid Profile Manipulation

In this Example, a variation to the process is described whereby the process may be manipulated in order to produce keratin peptides with a low sulfur amino acid profile.

The process is completed as in Example I or Example 2 with the variation that in the oxidative sulfitolysis digestion step, the step is varied by increasing the digestion time; increasing the concentration of cupπ^'c ammonium complex and/or sodium sulfite solution, or by increasing both the time and concentration.

The extended time period may be greater than approximately 5 hours.

The increased concentration may be of cupric ammonium complex particularly, but also sulfite greatei than that described in Examples 1 and 2. The resulting product is a low sulfur amino acid peptide solution with cysteine content less than or equal to 8%

Example 4 - Low Sulfur Amino Acid Profile Manipulation

In this Example an alternative is provided to Example 3 Io produce keratin peptides with a low sulfur amino acid profile.

The process is completed as in Example 1 or Example 2 with the variation that the fourth 'hold' stop is increased in duration. As noted above, the length of the hold period allows the sulfonated HSP and IFP to migrate into solution for hydrolysis. Times greater than 20 houis are expected to cause a lowering in the sulfur amino acid profile. The resulting product is a low sυllur amino acid peptide r.olutiun with a cysteine content of less than or equal to 8%.

Example 5 - Low Sulfur Amino Acid Profile Manipulation

In lhis Example an alternative is provided to Example 3 and 4 to produce keratin peptides with a low sulfur amino acid profile.

The process is completed as in Example 1 or Example 2 wilh the variation that the fifth enzyme hydrolysis step is increased in duration. Additional time for the enzyme to act creates smaller peptide fragments from the IFP fraction resulting in a lower sulfur amino acid profile. Times greater than 20 hours are expected to cause a lowering in the sulfur amino acid profile. The resulting product is a low sulfur amino acid peptide solution with a cyslcine content of less than or equal to 8%.

Example 6 - Low Sulfur Amino Acid Profile Manipulation

In this Example an alternative is provided to Examples 3 to 5 to produce keratin peptides with a low sulfur amino acid profile.

The process is completed as in Example 1 or Example 2 with the variation that the sixth stop of acidification is omitted. Omitting this step results in a greater concentration of IFP and other lower sulfur fractions being present in the final solution.

The resulting product is a low sulfur amino acid peptide solution with a cysteine content of less than or equal to 8%.

Example 7 - Low Sulfur Amino Acid Profile Manipulation

In this Example an alternative is^'provided to produce keratin peptides with a low sulfur amino acid profile. The process is completed as in Example 1 or Example 2 using two or more combinations of the variations described in Examples 3 to 6.

By way of illustration, the low sullui amino acid profile may be produced by varying step three and step

• four as described in Examples 3 and 4. Alternatively, steps lour and five can be manipulated as described in

Examples 4 and 5. Alternately, step five can be manipulated as in Example 5 and step six omitted as in Example 6. Further combinations of two or more of the manipulations of Examples 3 to 6 may also be completed.

The resulting product is a low sulfur amino acid peptide solution with a cysteine content ot less than or equal to 8%. It should be appreciated from above Examples 3 to 7 is that the process may be varied in a variety of ways to tailor the final peptide amino acid profile to reduce the amino acid profile and therefore the resulting peptide properties. Example 8 - High Sulfur Amino Acid Profile Manipulation

In this Example a variation to the process is described whereby the process may be manipulated in order to produce a keratin peptide solution with a high sulfur amino acid profile. The process is completed as in Example 1 or Example 2 with the variation that in the third oxidative sulfitolysis step the step is varied by reducing the digestion time; reducing the concentration of cupric ammonium mixture and/or sodium sulfite solution; or by reducing both the time and concentration.

The reduced time period may be less than approximately 2 hours.

The reduced concentration may be amounts loss than the amount of cupric ammonium complex in particular, and sulfite used in Examples 1 and 2.

The resulting product is a high sulfur amino acid peptide solution with a cysteine content greater than 8%.

Example 9 - High Sullur Amino Acid Profile Manipulation In this Example an alternative is provided to Example 8 to produce keratin peptides with a high sulfur amino acid profile.

The process is completed as in Example 1 or Example 2 with the variation (hat the fourth hoid' step is reduced in duration. As noted above, the length of the hold period allows the sulfonated HSP and IFP to migrate into solution for hydrolysis. Times less than 16 hours are expected Io cause a lowering in the sulfur amino acid profile.

The resulting product is a low sulfur amino acid peptide solution with a cysteine content greater than 8%.

Example 10 - High Sulfur Amino Acid Profile Manipulation In this Example an alternative is provided to Examples 3 and 4 to produce keratin peptides with a high sulfur amino acid prolile

The process is completed as in Example 1 or Example 2 with the variation that the fifth enzyme hydrolysis step is reduced in duration. Reduced time for the enzyme to act limits the amount of time in which the enzyme can act to create smaller IFP peptide fragments resulting in a higher overall sulfur amino acid profile. Times less than 20 hours are expected to cause an increase in the sulfur amino acid profile.

The resulting product is a high sulfur amino acid peptide solution with a cysteine greater than 8%,

Example 1 1 - High SuHur Ammo Acid Prolile Manipulation

In this Example an alternative is provided to produce keratin peptides with a high sulfur amino acid profile. The process is completed es in Example 1 or Example 2 using two or more combinations of the variations described in Examples 8 to 10.

By way of illustration, the low sulfur amino acid profile may be produced by varying step three and step four as described in Examples 8 and 9. Alternatively, steps four and five can be manipulated as described in Examples 9 and 10. Further combinations of two or more of the manipulations of Examples 8 to 10 may also be completed.

The resulting product is a high sulfur amino acid peptide solution with a cysteine content greater than 8%. It should be appreciated from the above Examples 8 to 11 that the process may be tailored in a variety of ways to increase the final peptide sulfur amino acid profile and therefore the resulting peptide properties.

Example 12 - Personal Care Products and Formulations Containing the Solubilized Keratin Peptide

Examples are now provided of various personal care products using the keratin peptides produced by the process. It should be appreciated lhat due to the multiple beneficial properties, the keratin peptides are well suited to use in personal care products. For example, the proteins have the ability to bind to the skin and trap moisture in the skin therefore moisturizing the skin. Varying the sulfur amino acid content also allows tailoring of the peptides for specific personal care uses. The examples below are provided byway of illustration only and should not be seen as limiiing.

In each formulation 'keratin peptide' is described at an indicative level. Keratin peptide refers to keratin peptides that have been solubihzed, S-sulfonated and hydrolyzed using methods comprising those described above. Unless otherwise stated, it is convenient to provide the keratin peptides in the form of a dilute aqueous solution and include tho appropriate amount of this solution in the formulaϋoπ to achieve the keratin peptide level indicated. Percentages are expressed as w/v

Conditioning shampoo

Sodium lauryl sulphate 28% 25.0%

Sodium laureth-2-sulphate 70% 4 0

Cocamide DEA 70% 3 5

Cocamidopropyl betainυ (30%) 3.0

Keratin peplide 0.5

Sodium chloride q.s.

Cilric ncid q.s.

Fragrance q.s.

Preservativo q.s.

Water q.s. to 100 Procedure: Combine 35.Og water, sodium laureth sulphate and sodium lauryl sulphate. Heat to 65^*C until dissolved Add cocamide DEA and allow to cool Mix betame with water and add to phase A Add keratin peptide adjust the pH to 6 5 with citric acid Add preservative and fragrance as required, adjust to desired thickness with sodium chloride and add remaining water

Hair gel

Carbomer (Carbopol Ultre7 10) 0 5%

Oisodium EDTA 005

Glycerin 4 0

Tnelhdiiolamine (20%) 3 0

Keratin peptide 045

Preservative q s

Fragrance q s

Water q s to 100

Procedure Heat 60 Og of water to 70"C and add to carbopol, EDTA and glycerol Mix vigorously Cool Add triethanolamme lo adjust pH to β 3 Add keratin peptide Combine preservative and remaining wdter and add Mix thoroughly and add fragrance as desired

Clear Body/Foααl Cleanser and Shampoo AAmmmmoonniiuumm llaauuryl sulphato 28% 25 0%

Disodium laureth sulfosυccinate 20 0

Cocamidopropyl betame 8 0

Keratin peptide 0 5

Sodium chloride q s

Frayrancβ (parfυm) q s

Preservative q s

Water (aqua) q s to 100

Hair Conditioner

Cctπmoniuπi chloride 50%

Stearyl alcohol 4 5

Keratin peptide 025

Fragrance q s

Preservative q s

Water q s to 100 Hair Mousse

Keratin peptide 0 25%

Hydrogenated tallow l^ήmonw^' m chloride 0.20

Noπoxynol-10 0.35

Alcohol 10.0

Butanβ-48 10.0

Water q.s. to 100

Setting lotion Carbomer (Carbopol Ultrez 10) 2.0%

Mineral oil (light) 0.20

Keratin peptide 0.25

Alcohol 37.5

Fragrance q.s. Water q.s. to 100

Hairspray

VA/CrotonatesΛ/iπyl Neodeconoatυ Copolymer 1.607

(Resyn 28-2930)

Aminomethyl proμanol 0.15

PEG-75 lanolin 0.20

Keratin peptide 0.25

Alcohol 65.05

Butane 30 28.0

Pre-perming solution

TEA lauryl sulphale 30.0%

Cocamidopropyl dimethylamine oxide 10.0

Cocamide DEA 7.5

Cocamidopropyl botaino 200

Cocamide MEA 3.0

Keratin peptido 0.5

Fragrance q.S.

Preservative q.s

Waler q.s. Post-porming solution

Keratin peptide 0.5%

Cocamidopropyl dimethylamine oxido 10,0

PPG-5-ceteth-1 O-phosphate 0 5

Glycerin 3.0

Hydroxypropyl methylcellυlose 1.5

Fragrance q.s.

Preservative q.s.

Water q.s. to 100

Moisturising cream

Cetearyl alcohol and ceteareth-20 5.0%

Cetearyl Alcohol 2 0

Mineral oil (light) 5.0

Keratin peptide 0.5

Preservative 0.3

Fragrance q.s,

Water q.s. to 100

Hand and Body Lotion

Polyglyceryl-3 πmthylglucoso distearαte 4 0%

Stearyl/behenyl beeswaxatc 3.0

Ocryldodecaπo! 4 0

Avocado oil 6.0

Mineral oil 3 0

Jojoba oil 2.0

Keratin peptide 0.5

Ceramide III 0.2

Propylene glycol 3.0

Preservative q.s.

Fragrance (Parfum) q.s.

Water (aqua) q.s. to 100 Anti-Wrinkle Treatment Cream Sodium behenoyl lactylate 20%

Cetearyl alcohol 30

Glyceryl slearate 26 lsopropyl palmitate 60

Sunflower seed oil 60

Keratin peptide 05

Glycerine 30

Magnesium ascorbyl phosphate (and) lecithin 60

(Rovisome-C, R.I T.A)

Preservatrve qs

Water qs to 100

Facial Moisture Cream

Myπstyl lactate 30%

Lanelh-25 (and) coteth-25 (and) oleth-25 (and) 10

Steaιeth-25 (Solulan 25, Amerchol)

Mineral oil (70 vise ) 165

Petrolatum 30

Tocotricnol 10

Carbomer 934 075

Keratin peptide 05

Triethanolamine (10% aq ) 75

Preservative qs

Fragrance qs

Water q s to 100

Moisturising Body Lotion

Methyl glucose dioluate 20%

Methyl glucose sesquisteaiatθ 15

Methyl gluceth-20 distcaroto 15

Cetearyl alcohol (and) celcareth 20 15 lsopropyl palmitate 30

Ceramidc 3, hoxyldocanol 20

Methyl gluceth- 10 30

Keratin peptide 0.5 Carbomer 1342 0.2

Triethanolarniπe 0.2

Fragrance q.s.

Preservative q.s.

Water q.s. to 100

Catfonic Emollient Lotion lsostearamidopropyl laurylacetodimonium 5.0% chloride

Laclamide MEA 3 0 lsostearyl neopentøπoate 15.0

Myristyl myristate 1.0

Cetyl alcohol 4.0

Glyceryl isostearate 3.5

Keratin peptide 0.5

Preservative q.s.

Water q.s. to 100

Men's lαciαl Conditioner

Carbomer (Ultrez 10 Carbopol) 0.4%

Propylene glycol 1.0

PPG-5-bυteth 0.5

Beta glucan 2.0

PEG-60 hydrogenated castor oil 0.5

Triethanolamine (99%) 0.4

Keratin peptide 0.5

SD-39 C alcohol (Quantum) 5.0

Fragrance q.s.

Preservative q.s.

Water q.s. Io 100

Moisturising AHor Shavo Treυtment Ceteareth-12 land) cetcarclh-20 (and) cβlβaryl 6.0% alcohol (and) cetyl palmitate (and) glyceryl stearale (Emulflarie SE. Hcπkel) Cetearyl alcohol 1.0

Dicaprylyl ether 8.0

Octyldodecanol 4.0

Glycerin 3.0 Carbomer (Ultrez 10 Carbopol) 0.3

Keratin peptide 0.5

Bisabolol 0.2

Ethyl alcohol 3.0

Water (and) sodium hyaluronate, (and) wheat 4.0 {Triiicum vulgare) germ extract (and) saccharomyces

(nnd) cerevisiae exiract (Eashavβ, Pentapharm)

Triethanolamine q.s.

Fragrance q.s.

Preservative q.s.

Water q.s. to 100

Antioxidant cream

Glycerin (99.7%) 3.0%

Xanthan gum 0.15

Disodium EDTA 0.05

Hydrogenated polyisobutenn 1.0 lsopropyl palmitato 5.0

Petrolatum 0.75

Dimethicono 0.75

Cyclopontasiloxanβ 3 0

Steareth-2 1 0

PEG-100 stearate 1.9

Cetyl alcohol 2.0

Ethylhexyl palmitate 3.0

Polyacrylamide (and) C13-14 isoparaffiπ (and) 2.0 laurelh-7 (sepigel 305. Soppic)

Keratin peptide 0.5

Glycerin (and) water (and) WWs vinifcra (grapo) 0.5 seed extract (Collaborative)

Fragrance q.s. rreservauve qs

Water qs to 100

Liquid detergent

Sodium laureth sulphate 500%

Cocamide DEA 30

Keratin peptide 025

Sodium chloride qs

Preservative) qs

Citric acid qs

Water qs to 100

Shower Ge/

Sodium laureth sulphate 350%

Sodium lauroyl sarcosinate 50

Cocoamidoμtopyl betaine 100

Cocoamidopropyl hydioxyl sultdinυ 50

Glycerine 20

Keratin peptide 015

Tetrasodium CDTA 025

Citric acid qs

Fragrance qs

Preservative qs

Water q s to 100

Foβming bath gel

TEA lauryl sulphate 400%

Lauroyl diethanolamide 100

Linoleic diethanolamide 70

PEG 75 lanolin oil 50

Keratin peptide 025

Tetrasodium EDTA 05

Fragrance qs

Preservative q b

Dyes qs Water q.s. to 100

NaII Polish ■ First coat Keratin peptide 10.0% Sodium hydroxide (4%) 10.0 Keratin fraction (SHSP or SPEP) q.s. Sodium laυryl sulphate q.s. Dye or Pigment q.s. Water q s. to 100

Wa// Glosser

Keratin peptide 10.0%

Keratin fraction (SHSP) q.s.

Sodium hydroxide (4%) 10.0

Sodium lauryl sulphate q.s.

Water q.s. to 100

Mascara

PEG-8 3.0%

Xanthan gum 0.50

Tetrahydroxypropyl ethylenediamine 1.3

Carnauba wax 8.0

Beeswax 4.0

Isoeicosaπe 4.0

Polyisobutene 4 0

Stearic acid 5.0

Glyceryl stearate 1 0

Keratin peptide 0.25

Pigments 10.0

Polyurethane-1 8.0

VPΛ/A Copolymer 2.0

Preservative q.s.

Fragrance q.s.

Water q s. to 100 Liquid Foundation

Pϋlysoibale 80 01%

Potassium hydroxide 098

Keratin peptide 025

Titanium dioxidoΛalc, 80% 01

Talc 376

Yellow iron oxide/talc, 80% 08

Red iron oxide/talc, 80% 038

Black iron oxide/talc, 80% 006

Propylene glycol 60

Magnesium aluminum silicate 10

Cellulose gum 012 dι-PPG-3 myπstyl ethπr adipato 120

Cetearyl alcohol (and) coteth-20 phosphate (and) 30 dicctyl phosphate (Crudafob CS 20 Acid)

Steareth-10 20

Cetyl alcohol 062

Steareth-2 05

Preservative qs

Water qs to 100

Shaving Cream

Sodium cocosulfato 50%

Keratin peptide 025

Glyceπn 70

Oisodium lauryl sulfosuccinato 500

Disodium EDTΛ qs

Sodium chloridp qb

Citric cicid qs

Fragrance qs

Preservative qb

Water q s to 100

Lψstick Octyldodecoπol 22 Olo Oleyl alcohol 8.0

Keratin peptide 0.16 ^•

C3O-45 alkyl molhicoπe 20.0

Lanolin oil 14.0 Petrolatum 5.0

Bentone 36 (Rheox) 0.6

Tonox 20 (Eastman) 0.1

Pigment/castor oil 10.0

Preservative q.s. Cyclornethicoπe q.s. to 100

Sulfite Hair Stralghtener

Carbomer (Carbopol 940) 1.5%

Ammonium bisulphate 9.0

Diothylene urea 10.0

Cetearth 20 2.0

Keratin peptide 0.5

Fragrance q.s.

Ammonium hydroxide 28% q.s. to pH 7.2

Water q.s. Io 100

Post straightening ncut 'ralizing sotutk

Sodium bicarbonate 2.35%

Sodium carbonate 2.94

EDTA 0.15

Cetearth 20 0.2

Keratin peptide 0.5

Fragrance q.s.

Water q.s. to 100

Pre-relaxer Conditioner

Cationic polyamine 2.0% linidazolidinyl urea 0.25

Keratin peptide 0.5 Fragrance q.s.

Pieservative q.s.

Water q.s. to 100

Alkali Metal Hydroxide Straightcner (Lye)

Bentoπile 1.0%

Sodium Lauryl Sulphate 1.5

PEG-75 lanolin 1.5

Petrolatum 12.0

Cetearyl alcohol 12.0

Sodium hydroxide 3.1

Keratin pep 0.5

Fragrance q.s.

Water q.s. to 100

Post Relaxing Shampoo

Sodium lauryl sulphate 10.0%

Cocamide DEA 3,0

EDTA 0 2

Kαratin peptide 0.5

Citric acid q.s. to pH 5 0

Fragrance q.s.

Preservative q.s.

Water q.s. to 100

Heir tonic/cuticle cover

Glyceriπo 5 5%

EDTA 0.07

Carbomer (Cnrboμol Ultrez 10) 0.33

Triethanolamine (20%) 1.0

Koratin peptide 0.5

Ethanol 10.0

Preservative q.s.

Water q.s. to 100 Leave in hair conditioner

Cetyl alcohol 50%

Glyceryl slearale 30

Petrolatum 07 lsopropyl myristale 15

Polysorbate 60 10

Dirnethiconol & cyclomethicoπe 40

Glycerine 70

EDTA 01

D-pantheπol 02

Keratin peptide 05

Cyclomethicone 40

Fragrance qs

Preservative qs

Water qs to 100

Post Hαir-dyβing Conditioner

Quaternιum-40 20%

Keratin peptide 05

Amphoteπc-2 40

Hydroxyethyl cellulose 20

Phosphoric acid q s to pH 45

Fragrance qs

Water q s to 100

Temporary Hair Coloring Styling Gel

Dimethicone copolyol 15%

PPG-10 methyl glucose ether 10 Polyvinylpyrrolidone 25

Tπisopropaπolamlno 11

Carbomer {Carbopol 940) 06

Lπιιreth'23 10

Phenoxyelhanol 02 Keratin peptide 05 EDTA 0.01

D&C orange 4 0.12

Ext D&C Violet 2 0.02

FD&C yellow 6 002 Ethanol 5 0

Fragrance q s.

Water q.s. to 100

Aspects of tho present invention have been described by way of example only and it should be appreciated that modifications and additions may bu made thereto without departing from the scope thereof as defined in the appended claims.

Claims

What We Claim Is:

1. A process for manufacturing S-sulfonated keratin peptides wherein the process comprises the steps of:

(a) reacting a Keratin source via oxidative sulfitolysis to produce solids comprising S-sulfonated keratin fractions in digestion liquor;

(b) completing a partial to complete separation of the solids from the digestion liquor;

(c) mixing the separated solids with water and holding for a predetermined time to allow the S- sulfonated keratin fractions to migrate into the water and form a S-sultonated keratin fraction mixture;

(d) reacting the solids and S-sulfonated keratin fraction mixture via enzyme hydrolysis to produce S- sulfonatcd keratin peptides.

2. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides produced are less than 5OkDa in size.

3. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides produced are less than 10 kDa in size.

4. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides produced are soluble.

5. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides produced are reversibly S-sulfoπated and the functionality of the S-sulfoπated keratin peptides is retained.

6. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides produced have the same amino acid profile as the starting keratin source.

7. The process as claimed in claim 1 further comprising the step of manipulating the amino acid profile of the S-sulfonated keratin peptides such that the S-sulfonated keratin peptides have a different amino acid profile to the starting keratin source.

8. The process as claimed in claim 7 wheroin the step of manipulating the amino acid profile of the S- sulfoπated keratin peptides comprises forming S-sulfonoted keratin peptides having a high sulfur amino acid profile where the cysteine content of the S-sulfonated keratin peptides is gieater than 8 mol %.

9. The process as claimed in claim 8 wherein the cysteine content is between 10 mol% and 20 mol%.

10. The process as claimed in claim 7 wherein the step ol manipulating the amino acid profile of the S- sulfonated keratin peptides comprises forming S-sulfonated keratin peptides having a low sulfur amino acid profile where the cysteine content of the S-sulfonatoO keratin peptides is less than or equal to 8 mol%.

11. The process as claimed in claim 10 whoroin tha cysteine content is between 4 mol% and 8 mol%.

12. The process as claimed in claim 1 wherein the keratin source material is processed in the same vessel during steps (a) to (d).

13. The process as claimed in claim 1 wherein reacting a keratin source via oxidative sulfilolysis in step (a) comprises mixing tho keratin source with a cupiic ammonium complex, water, sulfuric acid and a sulfite source.

14. The process as claimed in claim 1 wherein the keratin source comprises whole fiber or chopped fiber.

15. The process as claimed in claim 1 wherein mixing thυ separated solids with water and holding for a predetermined time in step (c) comprises adding back Io tho solids an amount ol water to the solids as digestion liquor separated in step (b).

16. The process as claimed in claim 1 whoroin reacting the solids and solution via enzyme hydrolysis in step (d) comprises using at least one type of protease enzyme.

17 The process as claimed in claim 1 further comprising steps (c) and (0 after step (d) of:

(o) acidifying the S-sul(onated keratin peptides Io produce a supernatant comprising S-sulfonated keratin peptides having a high sulfur amino acid profile and a precipitate comprising partially or completely unhydrolyzed S-sulfonated keratin fractions: and.

(f) separating the precipitate fiom the supernatant.

18 The process as claimed in claim 17 further comprising the steps of reducing the pH of the supernatant to between approximately 3 and 4 sintl then holding the supernatant at this pH for between approximately 1 and 3 hours.

19. The process as claimed in claim 17 whurcin a presei vative is added to the supernatant duiing step

20. The process as claimed in claim 19 wherein the preservative comprises potassium sorbate

21. The process as claimed in claim 1 wherein the keratin source is pre-soaked before step (a).

22. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides from step (d) are further processed via an ion exchange step to remove heavy metals, including but not limited to Hg₁ Pb. Cu, Zn, Cd, from the S-sιιllonated keratin peptides

23. The process as claimed in claim 17 wherein the supernatant from step (0 is further processed via an ion exchange step to remove hoαvy metals from the supernatant

24. The process as claimed in claim 1 wherein the S-sulfonated keratin peptides from step (d) arc further processed to concentrate the S-sulfonated keratin peptides.