Journal of the Society of Leather Technologists and Chemists, Vol. 90 p.29

IntroductionDevelopment of leather manufacture, chiefly in Europe,

is limited to a considerable extent by the large volume ofprotein waste produced, which is estimated, by particularauthors, at 40-60% of the mass of starting raw material.The problem has admittedly received attention since theend of the 1930s but, solid protein waste, however, hasbeen mostly disposed of until now to landfill despite thematerial in question being a potential secondary industrialraw material.

When considering processing solid leather waste, thestarting operation is partial hydrolysis making possiblesubsequent chemical modification of the hydrolysate in anaqueous medium, as well as quite simple separation and re-cleaning of polypeptides formed from substancespotentially contaminating the collagen waste from variousstages of leather manufacture. The preferred procedure ispartial enzymatic hydrolysis, which is the least demandingon energy, capable of being applied to leather waste bothtanned, greased or otherwise treated as well as non-tannedcollagen waste including that from the manufacture ofmeat product casings and also collagen waste fromabbatoirs. Hydrolysates of non-tanned collagen waste canbe used to advantage without further modification ashumectants in cosmetic preparations for skin and haircare.1-4 As far as concerns the processing of solid tannedwaste from leather production, enzymatic hydrolysis isinteresting for its potential of relatively easily separatingCr3+ tanning compounds which may be ecologicallycontroversial.

Hydrolysates, obtained as quite dilute aqueoussolutions, can be stored only with difficulty. Evaporatingexcess quantities of water (after separation of tanningchrome salts or other substances contaminatinghydrolysate) is quite an expensive matter and complicatesto a certain extent the application of hydrolysates inindustrial practice.

In the widely discussed application of proteinhydrolysates as fertilisers in agricultural technology, this is

exactly the reason why the hydrolysates obtained areincapable of competing in price with current types ofagricultural fertilisers. In addition, their employment as acomponent in livestock feed comes up against low nutritivevalue of collagen proteins (polypeptides) and against thesuspicion that feed mixes prepared from them may play nominor role in spreading the BSE syndrome.

Glues

The mean molecular mass of hydrolysates obtainedthrough enzymatic procedures usually ranges within limitsof 15-50kDa.5 These are capable, in a similar fashion togelatine or glues, of producing thermo-reversible gelsalthough, of course, at considerably higher concentrationsof dry matter. The rigidity of such gels (similarly to glues)depends on their mean molecular mass (weighted meanMw) and dry matter concentration. At dry matterconcentrations of 20-35 % (w/w), gels of enzymatichydrolysates usually exhibit rigidities of 150-350g-1

(°Bloom).6 Their quality, assessed in a standard manner, isthus not too high and makes it impossible to consider theirapplication as adhesives for functional bonds with theexception, maybe, for sticking labels (e.g., on glass bottlesand such) that should be removable with water. Theeconomic advantage of such applications, when comparedwith lower quality glues or even starch adhesives, is hardlyappreciable.

In aqueous solutions, collagen hydrolysates behave like‘substances with positive sorption’, but do not displaystronger surface activity if not further processed. This is thebasis for contemplating their use in the form of additivesfor retarding concrete setting in building. Nevertheless, thenecessity to concentrate hydrolysate solutions (for storage)is a factor limiting the economy of these applications evenin such considerations.

The environmental necessity to process leather wasteand the benefits of processing it through enzymatichydrolysis are not, then, sufficiently supported by theabove mentioned applications as having any economic

MODIFICATION OF CHROME-TANNED LEATHER WASTEHYDROLYSATE WITH EPICHLORHYDRINF. LANGMAIER, P. MOKREJS, R. KARNAS, M. MLÁDEK and K. KOLOMAZNÍKFakulty of Technology, Tomas Bata University, Nám. TGM 275, CZ 276 72, Zlín, Czech Republic.

Summary Wider use of chrome-tanned leather waste hydrolysate as a secondary industrial raw material is impeded by theunclear economic effect of most proposed practical applications. More attention should deservedly go tohydrolysate utilised as a biodegradable packing material (packing for agricultural chemicals including herbicides,insecticides, pesticides, fertilizers etc.) The appropriate cross-link density enables us to control the watersolubility of hydrolysate, its biodegradation and the rate at which active substances are released from suchpacking. The properties may significantly influence the economic effect of such an application. Increasing thecross-link density of hydrolysate by reacting it with dialdehydes easily leads to the formation of thermo-irreversible gels which are difficult to process. The main attention, therefore, is aimed at cross-linking hydrolysatewith epichlorhydrin.

In an aqueous environment, epichlorhydrin reacts with the primary amino groups of hydrolysate as amonofunctional agent through its chlorine atom. At temperatures around 60°C an equilibrium is attained in about60 min. characterised by approx. 80% of the primary amino groups present having reacted. Reaction of theoxirane ring of epichlorhydrin proceeds at considerably higher temperatures (~200°C). Cross-linking ofhydrolysate with epichlorhydrin is thus a two-stage reaction, which may be regarded as an advantage for someapplications.

29

˘

30

advantage. Achieving an adequate economic effect is morelikely to result from utilising specific hydrolysateproperties, possibly emphasised through appropriatechemical modification of their functional groups, than by ahigh demand for non-modified hydrolysates.

Surfactants

One of the more realistic possibilities consists in usingleather waste hydrolysates for manufacturing surfactants of‘Lamepon’ type - through acylation of primary aminogroups of hydrolysates with acyl chlorides of fatty acids(anionic surfactant types), or through their quaternation,which is best with benzyl chloride (various types ofamphoteric surfactants).7 The relatively high meanmolecular mass of enzymatic hydrolysates results inreduced solubility of surfactant derivatives made from themin water, which leads to a lower production yield.Acceptable yields can be attained if the mean molecularmass of enzymatic hydrolysates is reduced to belowapprox. 1.0kDa. That is more readily achieved through acidhydrolysis (e.g. in HCl) than through alkaline hydrolysis orthrough prolonged enzymatic hydrolysis.8 Subsequentacylation or quaternation has to be performed in an alkalinemedium (pH>10.5 ),which somewhat complicates thisindustrial application.

Adhesives

On closer study of the properties of hydrolysate, it wasfound that its addition to aminoplast type adhesives, atapprox. 5% (w/w) level, may considerably limit emissionsof formaldehyde gas from their cured adhesive films. Theeffect is more pronounced if addition of hydrolysate isaccompanied with adapted, optimised conditions for curingadhesive bonds.9-11 Results evaluating formaldehydeemissions from cured films by the official bottlemethod12-13 confirmed that added hydrolysate may reduceformaldehyde emissions from free adhesive films by asmuch as 70%.

Packaging materials

A topic much discussed lately, concerns problems ofbiodegradable polymers based on renewable raw materialsources, due to their employment in packing technology.Besides renewability of raw material sources, the propertythey are appreciated for is degradation rate, on terminationof package life. That, (degradation rate) when comparedwith hitherto most widespread synthetic polymers based onnon-renewable crude oil, is several times greater and posesvirtually no environmental hazards. Among biodegradablematerials, proteins of both animal and vegetable originoccupy a prominent position. It was proved that treatinghydrolysates of chrome-tanned leather waste withglutaraldehyde allowed the production of biodegradablematerials14 applicable to packing of chemicals in farmingtechnology (herbicides, insecticides, fertilisers, etc.). Inaddition, the degree of hydrolysate cross-linking withglutaraldehyde allows efficient control of the rate at whichactive substances are released from such packing intosurroundings, which is a very interesting effect from thepractical point of view.

Similar results may be probably also attained by reactinghydrolysates with other cross-linking agents. One of them,easily and economically available, is epichlorhydrin, whichwas more applied in the past to grafting cellulose materials(e.g., for immobilising various enzymes, etc). It is knownthat under favourable conditions epichlorhydrin can

activate nucleophile groups of a protein matrix (mainly-NH2 or -OH groups) with formation of epoxidecompounds which subsequently react with remainingnucleophile groups of polypeptides. Reactivity of thefunctional groups of peptides considered goes down in thesequence -SH > -NH2 > -OH > -COOH.

Mention may be even sporadically found ofepichlorhydrin reacting with guanidine or imidazolegroups.15 It is assumed that cross-linking of proteins withepichlorhydrin has the character of a two-stage process thatmay bring certain advantages. These problems are dealtwith in this work.

Experimental procedures

Starting materials:Collagen hydrolysate (H) in powder form was prepared

by enzymatic hydrolysis of chrome-tanned leather waste bya procedure according to reference 16 and drying inpulverising drier. Its characteristics are shown in Table I.

Epichlorhydrin (EPICH), 99% from Sigma-Aldrich,Cz,Sigma-Aldrich Cat. 2005-2006, E 1055-500ml, CAS No.106-89-8, boiling point 115-117°C Sodium hydroxide,analytical quality, from Lachema Co., CZ

Working procedureThe stock solution of hydrolysate of chrome-tanned

leather waste was prepared by dissolving powderedhydrolysate in 0.01N NaOH to make a solution of 5.0%(w/w) concentration, whose pH level was maintained at11.0. To 50ml stock solution in a reaction flask underreflux, heated over a water bath to 60°C, epichlorhydrinwas added with good stirring over 15 min, in a quantitycorresponding to mass fractions from 0.05 to 0.376. Thereaction was performed at this temperature for 60 minmaintaining good stirring.

After cooling the reaction mixture to room temperature,an aliquot proportion of the mixture was employed forspectrophotometric determination (by reaction withninhydrin) of primary amino group content.17 The resultsobtained are graphically represented in Fig. 1, and confirmthat under these conditions a certain equilibrium state isestablished in the reaction mixture.

In the first series of experiments, an excess ofepichlorhydrin was provided over the primary aminogroups of hydrolysate (see data in Table II). No gelformation occurred, and as that is normally a side-effect ofhydrolysate cross-linking, we conclude that, epichlorhydrinunder the given conditions does not behave like abifunctional, cross-linking, agent.

Lower reactivity of the oxirane cycle of epichlorhydrin(as compared with the chlorine atom) to -SH, -NH2 and

TABLE ICharacteristics of the collagen hydrolysate used in the work

Dry substance % 92.99Amide nitrogen in dry substance % 14.85Ash in dry substance % 4.94Cr content in dry substance ppm 28.15Ca content in dry substance ppm 2746.62Mg content in dry substance ppm 4798.00Primary amino groups in dry substance mmol-NH2.g-1 0.216Average molecular weight (numerical mean MN), kDa 17.75

31

even -OH groups is a problem often discussed.18 For thesake of completeness in the experiments we performed, thetime dependence of unreacted chlorine atoms and oxiranerings of epichlorhydrin (EPICH) was investigatedemploying an FTIR technique using a commercial IRspectrophotometer Genesis FTIR (ATI Matsson, U.S.A.).

The Cl-C bond is usually assigned absorption peaks inthe frequency range of 686-648cm-1 or 731-723cm-1, whichare quite serrated. Their exact position depends on thestructure of remaining carbon-chain, and from theviewpoint of analytical determination, they are notregarded as being too reliable. Nevertheless, the timecourse of relative absorbance in both the regions mentioned(648-686cm-1 and 723-731cm-1) is shown in Fig. 2 anddemonstrates in convincing manner the decrease in Cl-groups of the epichlorhydrin in reaction mixture.

To the contrary, determination of the content of oxiranerings in various reaction mixtures is often based on IR

absorption in the 901-917cm-1 range, possibly in the lessspecific 840-854 cm-1 range.19 More specific absorption inthe range 901-917cm-1 was employed in FTIRmeasurements, and the values thus determined of startingand/or final contents of the oxirane ring of liquid reactionmixtures are included in Table II, together with contents ofunreacted and reacted amino groups.

Relatively lower reactivity of oxirane groups whencross-linking collagen hydrolysates may be compensated(similarly to epoxide resins) by an elevated reactiontemperature. Based on analogous cross-linking of epoxideresins, we may assume that such a reaction will take placeat a temperature above 120-150°C. Therefore, this stage ofthe reaction was studied with solid films prepared fromliquid reaction mixtures through vacuum drying at apressure of 1.0mm Hg and at 60°C. The actual cross-linking reaction was observed with these films employingdifferential scanning calorimetry (DSC) over thetemperature interval 25-250 or 300°C (DSC 2010, TAInstruments, Del., U.S.A.). Exothermal DSC peaks relatingto a condensation reaction of oxirane ring with primaryamino groups of hydrolysate were detected in thetemperature range 190-230°C. A typical record of suchmeasurements is shown for illustration in Figure 3.

In the experiments of series I, as follows from data ofTable II, considerable proportions of primary amino groupsof hydrolysate react with epichlorhydrin in the liquidphase. Following condensation of the oxirane ringin thesolid phase may be then unfavourably affected by the ratioof epichlorhydrin to primary amino groups in the dried filmof reaction mixture. Thus, we introduced, in addition,experimental series II (performed in liquid phase throughsame procedure) immediately before preparing the solidfilm, hydrolysate content in reaction mixture was increasedby 30% ( related to starting content of H in reactionmixture), which corresponds to increasing the content ofprimary amino groups in the dried film of reaction mixtureby 0.167mmol. Experiments of series II are also includedin Table II.

The presence of free (unreacted) EPICH in films usedfor DSC measurements is unlikely due to the samplepreparation (EPICH: b.p.760 ≈ 117.9°C, b.p.400 ≈98°C, b.p.200

≈62.0°C and b.p.100 ≈62.0°C).20 The area of exothermalDSC peaks detected may be thus regarded as proportionate

Figure 1. Reacted primary amino groups of hydrosylate (-NH2) relativeto mass fraction of epichlorhydrin in initial reaction mixture.

Rel

ativ

eab

sorb

ance

Reaction time, HFigure 2. Time dependence of IR absorption peaks in region 648-686cm-1

and 713-731cm-1 characteristic of -Cl group in liquid reaction mixturewhen modifying the hydrosylate with epichlorhydrin in alkalineenvironment.

Figure 3. Typical DSC results for dried film of reaction mixture ofhydrosylate with epichlorhydrin for the thermal interval of 25°C-300°Cat dT/dt = 5°C/min.

EPICH weight fraction in reaction mixture XEPICH = 0.234EPICH weight fraction in reaction mixture XEPICH = 0.355EPICH weight fraction in reaction mixture XEPICH = 0.376

Hea

tflo

w(W

/g)

Temperature (°C)

32

to the enthalpy of the cross-linking reaction of oxirane ringof EPICH, with the remaining -NH2 groups of H for,according to finds by various authors,21,22 its value lies in aquite narrow interval of 102.5 ± 2.5kJ/mol. Results of DSCmeasurements are summarised for ready survey in TableIII.

DiscussionSynthetic polymers, nowadays so widespread in

packaging technology, are a source of certain problemsbecause of their limited recycling possibilities. Aconsiderable proportion of packages, on finishing their life,are disposed of by incineration or to landfill. Disposal ofpolymeric packages by incineration has to be performed athigh temperatures to prevent formation of degradationproducts possessing unfavourable (often carcinogenic)properties. This has a negative effect on costs of theirdisposal. The long degradation time of synthetic polymersin landfills, currently ranging up to hundreds of years,exerts pressure for permanent expansion of landfillsnecessitating subsequent recultivation. Biodegradablepolymers, for which the time required for microbialdegradation in landfill falls in a range of weeks or, not morethan months, thus represent a very attractive alternative inthis field. In addition, these polymers come from renewableraw material sources (mostly animal or vegetable proteins),and this may also be a very significant circumstance for thefuture.

Hydrolysates of chrome-tanned leather waste, withoutcertain chemical modification, are not directly exploitablein this field, above all due to their ready water solubility.However, increasing their cross-link density (essentiallyanalogous to tanning) enables us to arrive at materials wellsuited for application in packing technology, and,moreover, regulation of cross-link density allows to achievetime-controlled release of active substances from thesepackings.

Agents applicable to modifying hydrolysate of chrome-tanned leather waste are substances of bifunctionalcharacter, capable of entering into cross-linking reactionswith its functional groups. Choice is limited by arequirement to perform the cross-linking reaction in anaqueous or, at most, an aqueous-alcoholic environment(solubility of hydrolysate). In the not very wideconcentration range of both reactants, cross-linkingreactions usually lead to formation of initially thermo-reversible, subsequently thermo-irreversible gels swellingin water.

The ability of swelling in water gradually deteriorateswith increasing cross-link density, and the gel acquires thecharacter of thermosetting (reactoplastic), poorly soluble,non-fusible material whose processing by standardprocedures is somewhat more complicated. At the sametime, its biodegradability also deteriorates.

An alternative solution is cross-linking of hydrolysatewith bifunctional cross-linking agents possessing markedlydifferent reactivities of their functional groups as, forexample, epichlorhydrin. In an an aqueous-alkalineenvironment such a reaction does not lead to gel formation(phenomenon accompanying cross-linking of collagenhydrolysates) despite the fact that a decrease in the primaryamino groups of the hydrolysate demonstrates clearlyenough the reaction of both substances. A state ofequilibrium is established in the reaction mixture,characterised by 70-85% of initially present primary aminogroups of hydrolysate having reacted (see Figure 1).

The detected decrease in -Cl groups of epichlorhydrin inthe reaction mixture corresponds to a decrease in primaryamino groups, while the found decrease in oxirane rings ofepichlorhydrin merely ranges within limits of experimentalerror. In an aqueous-alkaline environment, epichlorhydrinthen binds to hydrolysate at one point and actualhydrolysate cross-linking does not occur in this stage of thereaction. The mutual ratio of reacted EPICH and -NH2

TABLE IICharacteristics of chrome-tanned leather waste hydrolysate modified with epichlorhydrin

XEpich Primary amino groups Epichlorhydrin Ratio of EPO:NH2 groups

ReactedReactedUnreacted mmol

mmol %

Starting

mmol Mmol %

Startingmmol

Reactedmmol

Series I.0.053 0.2293 0.3097 57.40 0.5841 0.3033 51.93 1.08 0.98

0.074 0.1916 0.3474 64.45 0.8276 0.3236 39.10 1.53 0.93

0.094 0.1428 0.3952 73.32 1.0710 0.3394 31.69 1.99 0.86

0.131 0.1034 0.4269 79.20 1.5578 0.3643 23.38 2.89 0.85

0.171 0.0746 0.4644 86.16 2.1420 0.3863 18.03 3.97 0.83

0.203 0.0846 0.4544 84.30 2.6289 0.4015 15.32 4.88 0.88

Series II.

0.053 0.3564 0.1820 33.77 0.5841 0.3042 52.08 1.08 1.67

0.094 0.3545 0.1845 34.23 1.0710 0.3399 31.74 1.99 1.84

0.171 0.2608 0.2782 51.61 2.1420 0.3864 18.04 3.97 1.39

0.234 0.2889 0.2502 53.50 3.1644 0.4155 13.13 5.87 1.66

0.286 0.1379 0.4011 74.41 4.1380 0.4234 10.23 7.68 1.06

0.335 0.1051 0.4339 80.50 5.2091 0.4419 8.48 29.66 1.02

0.376 0.1041 0.4339 80.50 6.2310 0.4569 7.33 11.56 1.05

%

33

groups of H (see data in Table II) ranges within the limits1:0.83-1.84, so that amino groups of hydrolysate obviouslybond to epichlorhydrin as a virtual monomer unit. Rathersimilar conclusions were arrived at by authors studying theimmobilisation of some biologically active proteins.18

Utilisation of epichlorhydrin in this reaction largelydepends on its mass fraction in the initial reaction mixture.With a low mass fraction, the percentage proportion ofreacted epichlorhydrin fluctuates around 50%, thisproportion gradually decreases with increasing massfraction of EPICH in the starting reaction mixture until,with a mass fraction of epichlorhydrin exceeding approx.0.29, the percentage of hydrolysate-bonded epichlorhydrinis less than 10 % (related to its initial quantity in reactionmixture). Quite a large quantity of unreacted EPICH thusremains in the reaction mixture and has to be removed priorto its further processing. These facts are shown by data inTable II.

According to results of DSC measurements, the reactionof the oxirane ring takes place (again very probably with-NH2 groups of H) only at temperatures of 180-200°C (seeTable III). Cross-linking of H accordingly requiresapplication of the two-stage technique well-known in thefield of epoxide or other types of thermosetting polymers.

The area of the exothermal peak on DSC curves, whichcorresponds to the reaction of oxirane ring with functionalgroups of hydrolysate, characterises its energetic contentand is proportionate to the enthalpy of the cross-linkingreaction (unreacted EPICH was removed by drying thereaction mixture). According to findings by someauthors,21,22 molar enthalpy of the reaction of oxirane ringwith amino groups corresponds to practically constant level102.15 ± 2.5kJ/mol, so that the energetic content of theDSC peak enables us to estimate the density of cross-linksformed in films under observation. Such data, together withthermal coordinates of corresponding exothermal DSCpeaks and contents of oxirane rings of a given film, aresummarised in Table III.

The data presented make it obvious that cross-linkdensity is affected by the ratio of EPICH to primary aminogroups in the film. When amino groups dominate (approx.up to 2.1-2.2 in the ratio of EPICH : NH2- groups), thecontent of cross-links in film decreases with an increase inthis ratio, while with further increase of the ratio to over 2.2produces a slight increase in cross-link density of films.Such a dependency is shown in Figure 4.

This find is interesting in view of potentially regulatingthe cross-link density in the end-product, which affects notonly processing properties but also solubility in an aqueous

environment and hence, as a consequence, also itsbiodegradation rate. This circumstance is particularlyinteresting from a practical viewpoint as it offers potentialfor time-controlled releasing of active substances from

XEPICH Ratio EPICH:NH2

in film

ThermaL coordinates and enthalpy ofExothermal DSC peak

Tinit. oC Tmax oC TfinaloC Enthalpy

Q (J/g)

mmol/g ofcross-linksin film

Experiments of series I.0.053 1.323 203.0 215.0 233.8 0.31 0.00330.074 1.689 195.4 209.2 233.0 1.06 0.0100.094 2.377 190.7 201.5 232.3 0.82 0.0080.131 3.523 190.5 202.9 232.9 1.11 0.0110.171 5.178 190.8 203.1 232.0 1.84 0.0180.203 4.746 193.8 204.6 230.0 1.92 0.019Experiments of series II0.053 0.588 203.1 212.0 237.2 19.20 0.1880.094 0.659 201.5 211.2 237.1 16.78 0.1640.171 0.916 185.0 202.4 236.0 14.38 0.1410.234 0.924 181.5 200.4 232.5 13.95 0.1360.286 1.416 187.2 205.6 227.8 8.40 0.0820.355 1.661 183.8 201.5 230.0 5.60 0.0550.376 1.742 181.5 201.50 230.8 4.80 0.047

Note:*) Mass fraction of epichlorhydrin (EPICH) in liquid reaction mixture with hydrolysate**) Calculated from mean value of enthalpy of condensation reaction of epoxy groups withamino groups 102.15 kJ/mol – see / 20,21 /

TABLE IIICharacteristics of DSC exothermal peaks of dry films of reaction mixtures from TABLE 1

Figure 4. Influence of ratio of epichlorhydrin (oxirane groups) toprimary amino groups in epichlorhydrin cross-linkedhydrosylate films.

34

such packages,23 and that may be utilised in a number ofmodern applications for chemicals in farming technology,whether those in question are fertilisers, herbicides,pesticides, insecticides, etc., where the non-foodstuffcharacter of initial protein material (hydrolysate of chrome-tanned leather waste with very low content of Crcompounds - see data in Table I) has no essentialsignificance for packing such materials, while theeconomic aspects are undoubtedly more interesting.

Modification of hydrolysates with epichlorhydrin toproduce biodegradable packing materials is to be carriedout as a two-step process: in alkaline solution (pH≈11.0)epichlorhydrin is bonded to hydrolysate -NH2 groups withits chlorine group representing the reaction centre. At thisstage the hydrolysate solution (preferably with 10-15% ofdry matter content) may be used directly. The reactionproduct remains water-soluble and the obvious ‘casting’method may be used for film preparation. Curing of thefilms proceeds in the second stage by heating the films to210°C for 5 minutes.

The biodegradation rate of the films depends on cross-link density. The experiments in this direction are not yetfully finished, but preliminary results lead to conclusion,that with cross-links density of 0.1-0.15mmol/g thebiodegradation time does not exceed 1-2 months. As theexperiments were carried out at laboratory scale only, theeconomy of the whole process remain to be investigated.

Acknowledgement

The work presented was executed within the frameworkof grant VZ MSM7088352102. The authors wish toextend their thanks to the Ministry of Education of theCzech Republic for financial support to this work.

(Received July 2005, revised August 2005)

References 1. Hood, L. L., Collagen in Sausage Casings - in Pearson A. M., Dutson

T. R. and Bailey A. J. (Eds.), Advances in Meat Research, Vol. 4.,VanNorstrand, Reinhold, N.Y., (1987), pp.109-129.

2. Langmaier, F., Mladek, M., Kolomaznik, K. et al., CollagenousHydrolysates from Untraditional Sources of Proteins., Int. J.Cosmetic Sci., 2001, 23, 193.

3. Langmaier, F., Mladek, M., Kolomaznik, K. et al., Isolation of Elastinand Collagen from Long Cattle Tendons as Raw Material for theCosmetic Industry., Int. J. Cosmetic Sci., 2002, 24, 273-279 .

4. Osborn, W. N., Collagen - in Gennadios, A. (Ed.), Protein BasedFilms and Coatings, CRC-Press, Boca Raton/Fl, USA, (2002),pp.445-465.

5. Langmaier, F., Kolomaznik, K., Sukop, S. et al., Products ofEnzymatic Decomposition of Chrome-Tanned Leather Waste., J. Soc.Leather Technol. Chem., 1999, 83, 187-195 .

6. Langmaier, F., Stibora, M., Mladek, M. et al., Gel-Sol Transitions ofChrome-Tanned Leather Waste Hydrolysate, J. Soc. Leather Technol.Chem., 2001, 85, 100-105 .

7. Langmaier F., Mladek M., Kolomaznik K. et al., Hydrolysates ofChromed Waste as Raw Material for the Production of Surfactants.,Tenside, Surfactants, Detergents, 2002, 39 (No1) 47-51.

8. Langmaier F., Mladek M., Kolomaznik K. et al., Degradation ofChromed Leather Waste Hydrolysates for the Production ofSurfactants., Tenside, Surfactants, Detergents, 2002, 39, (No 2) 31-39.

9. Langmaier, F, Sivarova, J., Mladek, M. et al., Curing Adhesives ofUrea-Formaldehyde Type with Collagen Hydrolysates of Chrome-Tanned Leather Waste., J. Thermal Anal. Calor., 2004, 75. 205-219.

10. Langmaier, F., Sivarova, J., Mladek, M. et al., Curing of Urea-Formaldehyde Adhesives with Collagen Type Hydrolysates UnderAcid Condition., J. Thermal Anal. Calor., 2004, 75, 1015-1023.

11. Langmaier, F., Kolomaznik, K., Mladek, M. et al., Curing Urea-Formaldehyde Adhesives with Hydrolysates of Chrome-TannedWaste from Leather Production., Int. J. Adhesion Adhesives, 2005, 25,101-108.

12. Langmaier, F., Dubec, R., Mladek, M. et al., Influence of CuringConditions for Urea-Formaldehyde Adhesive Films of FormaldehydeEmissions., Holz als Roh- u. Werkstoff (European J. WoodWoodproducts) - in press

13. CEN 717-3 (1996): Wood based Panels, Determination ofFormaldehyde Release by ‘Bottle’ Method.

14. Langmaier, F., Kolomaznik, K. and Mladek, M., Modifying Productsof Enzymatic Breakdown of Chrome-Tanned Leather Waste withGlutaraldehyde., J. Soc. Leather Technol. Chem, 2003,. 87, 55-61.

15. Bäumert, H. G. Fassold, H., Cross-linking Techniques., MethodsEnzymol., 1989, 172, 584-595.

16. Kolomaznik K., Mladek M., Langmaier F. et al., Experience inIndustrial Practice of Enzymatic Dechromation of Chrome Shavings.,J. Amer. Leather Chem. Ass., 2000, 94, 55- 63.

17. Davidek, J., Hrdlicka J., Kavanek M. et al., Laboratorní príruckaanalysy potravin, SNTL Pratur 1977, pp.200-208.

18. Wong, S. S. (Ed.) Chemistry of Protein Conjugation and Cross-linking., CRC Press Inc., Boca Raton, Fl., USA (1993), pp.308-310.

19. Martin, D. M., Ormaetxea, M., Harismendy I. et al., Cure Chemo-Rheology of Mixtures Based on Epoxy Resins and Ester Cyanates.,Europ. Polymer J. 1999, 35, 57-68.

20. O’Neil M. J., Smith A., Heckelman E. et al., The Merck Index, 13thEd., Merck & Co Inc., Whitehouse Station N.Y, USA 2001, p.3641.

21. Jenninger, W., Schawe J. E. K. and Alig I., Calorimetric Studies ofIsothermal Curing of Phase Separating Epoxy Networks., Polymer,2000, 41, 1577-1588.

22. Bakker C J., St John N. A. and Geiorge G. A., SimultaneousDifferential Scanning Calorimetry and Near Infra Red Analysis of theCuring of Tetraglycidyldiaminodiphenyl Methane withDiaminodiphenyl Sulphone., Polymer, 1003, 34, 716-725.

23. Bowman, B. J. and Ofiner, C. M., Hard Gelatin Capsules in:Gennadios, A.(Ed.) Protein Based Films and Coatings, CRC Press,Boca Raton Fl, USA, (2002) pp.381-387.

˘ ˘