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Thermal and enzymatic recovering of proteins from untanned leather waste
Waste Management 21 (2001) 79±84
www.elsevier.nl/locate/wasman
Thermal and enzymatic recovering of proteins from untanned leather waste ZÏeljko Bajza a,*, Valerije VrcÏek b a
Faculty of Chemical Engineering and Technology, University of Zagreb, 10000 Zagreb, MarulicÂev trg 19, Croatia b Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Ante KovacÏicÂa 1, Croatia Accepted 8 May 2000
Abstract The laboratory trials of a process to treat untanned leather waste to isolate valuable protein products are presented. In this comparative study, both thermal and enzymatic treatments of leather waste were performed. The enzymatic method utilizes commercially available alkaline protease at moderate temperatures and for short periods of time. The concentration of the enzyme was 500 units per gram of leather waste which makes the method cost-eective. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis of untanned leather waste is determined. Chemical and physical properties of protein powder products from untanned leather waste were evaluated by spectrophotometric and chromatographic methods and by use of electron microscope. The results of microbiological assays con®rm that these products agree to food safety standards. This relatively simple treatment of untanned leather waste may provide a practical and economical solution to the disposal of potentially dangerous waste. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Leather processing is characterised by a large amount of liquid and solid waste. Leather mass losses in leather processing make up to 50%. The best way for their removal is to recover soluble proteins that may have commercial use. A number of authors have reported about chemical and enzymatic treatment of leather waste [1±6]. Leather waste can be processed into various useful products: glue, gelatin, arti®cial ®brous leathers, and collagen for medical use. Edible sausage casings, cosmetic microcrystalic collagen, collagen hydrolysate, fodder and foods are also being produced [6]. Hide, calculated according to the dry matter, usually contains 50 to 68% proteins, 0.6 to 9% fat, 15 to 50% ash and less than 5% water [7]. Treatment of the oal from ¯eshing machines has traditionally been limited to the chemical process of rendering. This is time consuming and necessitates the use of high amounts of energy. Exposure of the fat to high temperatures and strongly acidic or basic conditions will obviously yield products which will not give the most * Corresponding author. Tel.: +385-1-466-7526; fax; +385-1-4667526. E-mail address: [email protected] (Z. Bajza).
economical return. An alternative method is enzymatic processing, which is becoming increasingly more economical with the advent of biotechnology. These treatments can be carried out at low temperature, over short periods of time, yielding a protein product which can be used as a fertiliser or be disposed of by the sanitation departments. Fat products obtained by these enzymatic treatments have a low free fatty acid content and thereby have potential value to the chemical industry [8]. The enzyme collagen processing [8±13] and enzyme kinetics for these methodologies have been reported in the literature [10,14,15]. In this paper we present a comparative study on the thermal and enzymatic treatment of untanned leather waste. 2. Experimental 2.1. Materials Leather waste was obtained from commercial tanneries. In this study, we used lime treated leather (processing with 1.2% sodium sulphide, 1.5% sodium hydrosulphide and 3% of calcium hydroxide). Leather cuttings and ¯eshings were from bovine hide (the weight category of 35 kg).
0956-053X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0956-053X(00)00039-8
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Z. Bajza, V. VrcÏek / Waste Management 21 (2001) 79±84
Enzyme treatments were performed with alkaline proteases. A commercial preparation of alkaline proteases, Protoderm 100T (purchased from Pliva company), produced from submerged cultivation of the corresponding type of Bacillus genus was used. It contained 537 000 u/ml. The protease enzyme has optimal activity at pH 10±11 at 50±60 C. 2.2. Procedure Schematic presentations of the thermal and enzymatic methods are shown in Fig. 1. 2.2.1. Thermal treatment Ten kg (21.8 lb) of leather waste (sample I) was washed with running water inside a barrel for a period of 30 min (1). The deliming process was performed with 200% water and with the addition of 2% hydrochloric acid within the period of 2 h at 35 C. After repeated washing, the leather waste was placed in a cooking reactor (A) (Fig. 1). The cooking was performed by indirect vapour
warming within 2 h. Three layers were obtained: 24.8% of gelatin sediment (3), 57.5% of gelatin solution (2) and 8.4% of fat (Fig. 1). The gelatin sediment (3) was hashed up by the grinding machine and then dried on rollers (E) heated from inside (175 C) by the vapour under pressure of 6±8 bar. The powderlike leather ¯uor (8) was yielded. The gelatin solution (2) was transferred to a separator (B). The fat (4) solidi®es itself at the surface due to the cooling and can easily be removed in this separator. The water soluble hydrolysate (5) was obtained and concentrated in vacuum vapouriser (D). The concentrated gelatin solution (7) was dried either on rollers (E) or in the dispersion drying system (F) at 200 C which resulted in a gelatin powder product (9). 2.2.2. Enzymatic treatment One kg (2.2 lb) of leather waste (sample II) was prepared (see above) for the enzymatic treatment in the reactor (C). The enzymatic hydrolysis was performed with alkaline protease (Fig. 1). Enzymatic treatments were carried out at pH values of 9 to 10 at 56 C and a duration period of 120 min. Concentrations of enzymes were prepared in the following order: 500, 2500, 5000 and 15,000 units per gram of leather waste. Water-soluble hydrolysate (6), obtained from the enzymatic treatment and concentrated at the vacuum vaporiser (D), was afterwards dried in the dispersion drying system (F) to the enzymically hydrolyzed protein powder product (10). 2.3. Analyses Total protein was determined via total nitrogen. The obtained nitrogen concentration value (g/l) is multiplied by 6.25 to give the total protein concentration. Determination was performed by a colorimetric method with the Braun and Luebbe's automatic analyser (Traacs 800). Amino acid analyses were carried out qualitatively using paper chromatography and thin layer chromatography was used for quantitative analysis. Moisture content were determined by heating the samples at 50 C for 19 h. in a gravity oven. UV spectra were obtained by spectrophotometer Hewlett Packard 8452A, and cromatography experiments were carried out on PYE UNICAM 2201 Combicoldrac II system. Micrography was performed with Cambridge Stereoscan 600 raster electronic microscope (REM-SEM). Samples were plated with 10 to 20 nm thick golden layers using the Edwards-S-150-Sputer-Coater machine.
Fig. 1. Schematic presentation of the thermal (sample I) and enzymatic treatment (sample II) of the leather waste: (A) cooking reactor, (B) separator, (C) enzymatic reactor, (D) vacuum vaporiser (E) drying rollers machine, (F) dispersion drying system, (1) leather waste, (2) gelatin solution, (3) gelatin sediment, (4) fat, (5) water soluble hydrolysate, (6) water soluble hydrolysate, (7) concentrated gelatin solution, (8) powderlike leather ¯uor, (9) gelatin powder products, (10) enzymically hydrolyzed protein powder products.
3. Results and discussion Before processing the leather contained 76% of water and its pH was between 9 and 10. Protein analysis results showed that the content of the dry matter in powderlike leather ¯our (8) was 92.7% (29.2% in gelatin sediment
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(3) before drying), and in gelatin powder product (9) there was 93.0% of dry matter [4.2% in gelatin solution (2) before drying]. Fig. 2 shows UV spectra of powderlike leather ¯our (8), and the Fig. 3 shows its gel chromatogram. Photomicrograph of the leather after liming, and before dissolving, taken with the electron microscope, is presented in Fig. 4. Electron microscope photomicrographs of the gelatine powder product dried by the dispersion system (magni®cation=x1000) is shown in Fig. 5. Fig. 6
81
shows gelatin powder product dried on the rollers (magni®cation=1000), and Fig. 7 shows a photomicrograph of the powderlike leather ¯our dried on the rollers (magni®cation=2000). Fig. 8 shows the eect of the alkaline protease concentration on the solubility kinetics of the untanned
Fig. 5. Electron microscope micrograph of the gelatin, after the dispersion drying (magni®cation=1000). Fig. 2. UV spectra of the powderlike leather ¯uor: (a) before passing through the column; (b) 9th fraction after passing through the column; (c) 6th fraction after passing through the column.
Fig. 3. Gel chromatogram of the powderlike leather ¯uor: (a) using the absorbance of 254 nm, with the aborbance of 289 nm.
Fig. 4. Electron microscope micrograph of the leather (magni®cation= 500) after liming.
Fig. 6. Electron microscope micrograph of the gelatin, after the drying with the use of rollers (magni®cation=1000).
Fig. 7. Electron microscope micrograph of powderlike leather ¯uor, after the drying with the use of rollers (magni®cation=2000).
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leather, and Fig. 9 shows the relative change of the solubility during a time of 120 min (enzyme concentration of 10 u/ml). Dependence of 1/v on 1/S for the untanned leather solubilization process using an alkaline protease is shown in Fig. 10. According to the Lineweaver±Burke scheme good linearity was obtained (the correlation coef®cient is 0.99). Under conditions employed a value of constant has been calculated: Km=98.8 g/l and Vmax=1.355 g/l min. Km is the Michaelis-Menten constant for binding of enzyme with the substrate (hide) and Vmax is the maximal velocity of the enzyme reaction. Results of the microbiological examination of the hide and gelatin products, before and after drying, are shown in Table 1. Results of the amino acid composition of the hydrolysate obtained by the waste untanned leather enzyme hydrolysis are shown in Table 2. The enzyme concentration was 500 and 15,000 u/g and the samples were taken after 120 minutes of breakdown. Results of protein analysis show that the material is rich in protein. Collagen accounts for most of the protein. According to the literature, in hide there is 60±80% of proteins accounted to the dry weight. Our result of 79.4% of proteins is, therefore, in accord with reference data.
Fig. 8. In¯uence of alkaline protease concentration on solubility kinetics of untanned leather waste.
Fig. 9. Time dependence of the initial alkaline protease solubility rate.
Two dierent drying methods were used (Fig. 1): drying rollers (E) and dispersion drying system (F). Somewhat better results were obtained with the dispersion drying system. UV spectra (Fig. 2) of the powderlike leather ¯our are characterised by a broad shoulders between 260 and 280 nm (samples b and c). This absorbance pattern is most probably due to aromatic amino acid residues (tyrosine, phenylalanine, etc.) from leather proteins. Two dierent signals (Fig. 3) having intensities of 1.57 and 0.7 (sample a) and 2.25 and 0.95 (sample b) correspond to the fractions 6 and 9 at the gel chromatogram of powderlike leather ¯our. Nearly whole protein quantity eluted between the third and 15th fraction. These results also show that leather ¯our, obtained by the applied method, is rich in proteins. Electron microscope photomicrograph (Fig. 4) of the leather after liming and before dissolving, (magni®cation= 500) shows free, partially divided, ®bres of 5±9 mm thickness. Microscopic examination (Fig. 5) showed that regular particles are being formed in case of the dispersion drying, basically round shaped, on whose surfaces small recesses appear during the drying process. Smaller particles of 1010ÿ6 m in size are 25 times more abundant than particles of 4010ÿ6 m (Fig. 5). A transparent ®lm (Fig. 6) without specially formed particles is obtained by drying gelatin powder product with the use of the rollers. In the case of the powderlike leather ¯our dried with the use of the rollers, irregular shaped leaves and laminae are being produced. Fragments' sizes are within the range of 50 to 7010ÿ6 m (Fig. 7). It was determined that the concentration of alkaline protease (Figs. 8±10) in¯uenced the solubilization kinetics in a manner that the rate (curve slope) was higher at the beginning of the process and decreased during the process. An increase of the enzyme concentration from 500 to 15,000 u/g increases the solubility rate and the release of soluble proteins. That is in accord with the Michaelis±Menten concept of enzyme reaction kinetics which is described by equation:
Fig. 10. Dependance of 1/v on 1/S for the solubilization process of untanned leather with alkaline protease (Lineweaver±Burke diagram).
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Table 1 Microbiological examinations Sample amount (g/ml)
Microorganism
Gelatin sediment (3)
Leather ¯uor product (8)
Gelatin solution (2)
Gelatin powder product (9)
1 25 0.1 0.1 0.1 0.1 0 0 0 1 1 1
Microorganism count Salmonella Staphilococcus Sulpho-reducing Clostridium Proteus Escherichia coli Coliphorm bacteria b-hemolitic streptococcus Lipolytic bacteria Aerobic sporogenous bacteria Mould Yeast
792,000 0 0 Positive Positive Positive 0 0 0 0 0 0
100 0 0 0 0 0 0 0 0 0 100 0
92800 0 0 0 0 Positive 0 0 0 0 0 0
3400 0 0 0 0 0 0 0 0 0 0 0
Table 2 Concentration of amino acids in liquid phase, released from untanned leather after treatment with alkaline protease for 120 min
Glycine Proline Alanine Arginine Leucine Lysine Glutamine acid Asparagine acid Phenylalanine Methionine Histidine Serine Tyrosine a b
Sample Aa (mg/ml)
Sample Bb (mg/ml)
0.330 0.027 0.057 0.193 0.065 0.119 0.077 0.155 0.188 0.114 0.432 0.092 0.541
Trace Trace Trace Trace 0.033 Trace Trace Trace 0.089 Trace Trace Trace 0.183
Sample A, enzyme concentration 15,000 u/g. Sample B, enzyme concentration 500 u/g.
It was established with the use of thin layer paper chromatography (Table 2) that solid leather collagen is predominantly composed of 14 amino acids from which glycine makes one third, proline and hydroxyproline make the other third and all other amino acids make up the rest. The enzyme used most easily released those amino acids which in their composition have benzene ring (thyrosine and phenylalanine) or some other aromatic ring (hystidine). The type of amino acid being released later depends on its size and the bound functional clusters (arginine, asparagine acid, lysine, methionine, serine, glutamine acid). The amino acids which are the most abundant in collagen, glycine and proline, are being released at the end of the process. The quantities of the released amino acids depend on the size and structure of particular amino acid. Obtained amino acid solutions can be dried to the powder form which can be used in various industrial applications. 4. Conclusion
v vmax
c S c S KM
where v is the reaction rate, Vmax is the maximal velocity of the enzyme reaction, c(S) is a substrate concentration and Km is Michaelis±Menten constant. Microbial concentration in product samples is measured by direct count methods. Microbiological examinations (Table 1) showed that the micro organisms count in gelatin sediment (3) decreased from 792,000 per g to only 100 after drying process, and in gelatin solution (2) from 92,800 to 3400. Salmonella, Staphylococcus, Sulphoreducing Clostridium, Proteus and Coliforms bacteria were not registered at all. The results reveal that the leather ¯our and gelatin products are of the highest quality, i.e. the contamination level is very low. The products obtained by the applied method agree to food safety standards and can be used as animal feed additives.
A protocol was established to determine how eective the thermal and enzymatic treatments of untanned leather waste were in recovering the maximum amount of hydrolyzed protein with a minimum amount of residue. The methods that were developed have shown that the untanned leather waste can be successfully processed to the powderlike leather ¯our (8), gelatin products (9) and enzymically hydrolyzed protein powder products (10). The products of the satisfactory protein concentration were obtained. In this comparative study, we found the enzymatic treatment as a method of choice. It has also been demonstrated that it was not necessary to boil the leather waste before enzyme treatment. The method that was developed has shown that leather waste can be subjected to moderate pretreatment temperatures and that the enzyme can be added at these temperatures, thus eliminating the need to cool reaction mixture, which is, in the case of thermal treatment, a waste of energy.
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It was established that the enzyme concentration in¯uences the solubility kinetics of untanned leather waste in a way which is in accord with the Michaelis± Menten concept of enzyme reaction kinetics. We reported here the results of our attempts to achieve higher solubility and at the same time use of lower amounts of alkaline protease enzyme (500 units per g of leather waste), thus making the treatment more cost-eective. The procedure is simple, can be carried out without harmful odours, and the waste water contains no dangerous chemicals. With this process, therefore, not only the ecological problem is being solved, but also the valuable and useful products are being produced.
[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
References [1] Eisenbeiz F, Dumont E, Henke H. Angew Makromolekulare Chemie 1978;71:67.
[14] [15]
Urbanik MA. J Soc Leather Technol Chem 1971;55:320. Pechenard M. FP 1977;2:314±670. Heidemann E. J Soc Leather Technol Chem 1982;66:21. Bronowski K, Sagata J, Studniarski K. Leder 1979;30:8. Bajza Z. Master's thesis, Univesity of Zagreb, Zagreb, 1985. Jones MW, Whitmore RA. J Amer Leather Chem Ass 1975;70:91. Taylor MM, Diefendor EJ, Foglia TA, Bailey DG, Feairheller SH. J Amer Leather Chem Ass 1989;84:71. Mark HF, Bikales NM. Overberger, Ch. G. Menges, JG. Encyclopedia of Polymer Science and Engineering, vol. 3. New York: John Wiley and Sons, 1985. Michaelis L, Menten ML. Biochem Z 1913;49:333. Schurr JM, Mc Laren AD. Enzymologia 1965;29:315. Anon. Mextase, proteolytic enzyme product as additive in washing compounds. Delft: Royal Netherlands Fer. Ind. Ltd, 1967. Taylor MM, Diefendor EJ, Brown EM, Marmer WN. J Amer Leather Chem Ass 1992;87:380. Markovic I, Maric V, Johanides V. Biotechnology and Bioengineering 1988;32:18. Markovic I. Dissertation, University of Zagreb, Zagreb, 1981.
www.elsevier.nl/locate/wasman
Thermal and enzymatic recovering of proteins from untanned leather waste ZÏeljko Bajza a,*, Valerije VrcÏek b a
Faculty of Chemical Engineering and Technology, University of Zagreb, 10000 Zagreb, MarulicÂev trg 19, Croatia b Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Ante KovacÏicÂa 1, Croatia Accepted 8 May 2000
Abstract The laboratory trials of a process to treat untanned leather waste to isolate valuable protein products are presented. In this comparative study, both thermal and enzymatic treatments of leather waste were performed. The enzymatic method utilizes commercially available alkaline protease at moderate temperatures and for short periods of time. The concentration of the enzyme was 500 units per gram of leather waste which makes the method cost-eective. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis of untanned leather waste is determined. Chemical and physical properties of protein powder products from untanned leather waste were evaluated by spectrophotometric and chromatographic methods and by use of electron microscope. The results of microbiological assays con®rm that these products agree to food safety standards. This relatively simple treatment of untanned leather waste may provide a practical and economical solution to the disposal of potentially dangerous waste. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Leather processing is characterised by a large amount of liquid and solid waste. Leather mass losses in leather processing make up to 50%. The best way for their removal is to recover soluble proteins that may have commercial use. A number of authors have reported about chemical and enzymatic treatment of leather waste [1±6]. Leather waste can be processed into various useful products: glue, gelatin, arti®cial ®brous leathers, and collagen for medical use. Edible sausage casings, cosmetic microcrystalic collagen, collagen hydrolysate, fodder and foods are also being produced [6]. Hide, calculated according to the dry matter, usually contains 50 to 68% proteins, 0.6 to 9% fat, 15 to 50% ash and less than 5% water [7]. Treatment of the oal from ¯eshing machines has traditionally been limited to the chemical process of rendering. This is time consuming and necessitates the use of high amounts of energy. Exposure of the fat to high temperatures and strongly acidic or basic conditions will obviously yield products which will not give the most * Corresponding author. Tel.: +385-1-466-7526; fax; +385-1-4667526. E-mail address: [email protected] (Z. Bajza).
economical return. An alternative method is enzymatic processing, which is becoming increasingly more economical with the advent of biotechnology. These treatments can be carried out at low temperature, over short periods of time, yielding a protein product which can be used as a fertiliser or be disposed of by the sanitation departments. Fat products obtained by these enzymatic treatments have a low free fatty acid content and thereby have potential value to the chemical industry [8]. The enzyme collagen processing [8±13] and enzyme kinetics for these methodologies have been reported in the literature [10,14,15]. In this paper we present a comparative study on the thermal and enzymatic treatment of untanned leather waste. 2. Experimental 2.1. Materials Leather waste was obtained from commercial tanneries. In this study, we used lime treated leather (processing with 1.2% sodium sulphide, 1.5% sodium hydrosulphide and 3% of calcium hydroxide). Leather cuttings and ¯eshings were from bovine hide (the weight category of 35 kg).
0956-053X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0956-053X(00)00039-8
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Z. Bajza, V. VrcÏek / Waste Management 21 (2001) 79±84
Enzyme treatments were performed with alkaline proteases. A commercial preparation of alkaline proteases, Protoderm 100T (purchased from Pliva company), produced from submerged cultivation of the corresponding type of Bacillus genus was used. It contained 537 000 u/ml. The protease enzyme has optimal activity at pH 10±11 at 50±60 C. 2.2. Procedure Schematic presentations of the thermal and enzymatic methods are shown in Fig. 1. 2.2.1. Thermal treatment Ten kg (21.8 lb) of leather waste (sample I) was washed with running water inside a barrel for a period of 30 min (1). The deliming process was performed with 200% water and with the addition of 2% hydrochloric acid within the period of 2 h at 35 C. After repeated washing, the leather waste was placed in a cooking reactor (A) (Fig. 1). The cooking was performed by indirect vapour
warming within 2 h. Three layers were obtained: 24.8% of gelatin sediment (3), 57.5% of gelatin solution (2) and 8.4% of fat (Fig. 1). The gelatin sediment (3) was hashed up by the grinding machine and then dried on rollers (E) heated from inside (175 C) by the vapour under pressure of 6±8 bar. The powderlike leather ¯uor (8) was yielded. The gelatin solution (2) was transferred to a separator (B). The fat (4) solidi®es itself at the surface due to the cooling and can easily be removed in this separator. The water soluble hydrolysate (5) was obtained and concentrated in vacuum vapouriser (D). The concentrated gelatin solution (7) was dried either on rollers (E) or in the dispersion drying system (F) at 200 C which resulted in a gelatin powder product (9). 2.2.2. Enzymatic treatment One kg (2.2 lb) of leather waste (sample II) was prepared (see above) for the enzymatic treatment in the reactor (C). The enzymatic hydrolysis was performed with alkaline protease (Fig. 1). Enzymatic treatments were carried out at pH values of 9 to 10 at 56 C and a duration period of 120 min. Concentrations of enzymes were prepared in the following order: 500, 2500, 5000 and 15,000 units per gram of leather waste. Water-soluble hydrolysate (6), obtained from the enzymatic treatment and concentrated at the vacuum vaporiser (D), was afterwards dried in the dispersion drying system (F) to the enzymically hydrolyzed protein powder product (10). 2.3. Analyses Total protein was determined via total nitrogen. The obtained nitrogen concentration value (g/l) is multiplied by 6.25 to give the total protein concentration. Determination was performed by a colorimetric method with the Braun and Luebbe's automatic analyser (Traacs 800). Amino acid analyses were carried out qualitatively using paper chromatography and thin layer chromatography was used for quantitative analysis. Moisture content were determined by heating the samples at 50 C for 19 h. in a gravity oven. UV spectra were obtained by spectrophotometer Hewlett Packard 8452A, and cromatography experiments were carried out on PYE UNICAM 2201 Combicoldrac II system. Micrography was performed with Cambridge Stereoscan 600 raster electronic microscope (REM-SEM). Samples were plated with 10 to 20 nm thick golden layers using the Edwards-S-150-Sputer-Coater machine.
Fig. 1. Schematic presentation of the thermal (sample I) and enzymatic treatment (sample II) of the leather waste: (A) cooking reactor, (B) separator, (C) enzymatic reactor, (D) vacuum vaporiser (E) drying rollers machine, (F) dispersion drying system, (1) leather waste, (2) gelatin solution, (3) gelatin sediment, (4) fat, (5) water soluble hydrolysate, (6) water soluble hydrolysate, (7) concentrated gelatin solution, (8) powderlike leather ¯uor, (9) gelatin powder products, (10) enzymically hydrolyzed protein powder products.
3. Results and discussion Before processing the leather contained 76% of water and its pH was between 9 and 10. Protein analysis results showed that the content of the dry matter in powderlike leather ¯our (8) was 92.7% (29.2% in gelatin sediment
Z. Bajza, V. VrcÏek / Waste Management 21 (2001) 79±84
(3) before drying), and in gelatin powder product (9) there was 93.0% of dry matter [4.2% in gelatin solution (2) before drying]. Fig. 2 shows UV spectra of powderlike leather ¯our (8), and the Fig. 3 shows its gel chromatogram. Photomicrograph of the leather after liming, and before dissolving, taken with the electron microscope, is presented in Fig. 4. Electron microscope photomicrographs of the gelatine powder product dried by the dispersion system (magni®cation=x1000) is shown in Fig. 5. Fig. 6
81
shows gelatin powder product dried on the rollers (magni®cation=1000), and Fig. 7 shows a photomicrograph of the powderlike leather ¯our dried on the rollers (magni®cation=2000). Fig. 8 shows the eect of the alkaline protease concentration on the solubility kinetics of the untanned
Fig. 5. Electron microscope micrograph of the gelatin, after the dispersion drying (magni®cation=1000). Fig. 2. UV spectra of the powderlike leather ¯uor: (a) before passing through the column; (b) 9th fraction after passing through the column; (c) 6th fraction after passing through the column.
Fig. 3. Gel chromatogram of the powderlike leather ¯uor: (a) using the absorbance of 254 nm, with the aborbance of 289 nm.
Fig. 4. Electron microscope micrograph of the leather (magni®cation= 500) after liming.
Fig. 6. Electron microscope micrograph of the gelatin, after the drying with the use of rollers (magni®cation=1000).
Fig. 7. Electron microscope micrograph of powderlike leather ¯uor, after the drying with the use of rollers (magni®cation=2000).
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Z. Bajza, V. VrcÏek / Waste Management 21 (2001) 79±84
leather, and Fig. 9 shows the relative change of the solubility during a time of 120 min (enzyme concentration of 10 u/ml). Dependence of 1/v on 1/S for the untanned leather solubilization process using an alkaline protease is shown in Fig. 10. According to the Lineweaver±Burke scheme good linearity was obtained (the correlation coef®cient is 0.99). Under conditions employed a value of constant has been calculated: Km=98.8 g/l and Vmax=1.355 g/l min. Km is the Michaelis-Menten constant for binding of enzyme with the substrate (hide) and Vmax is the maximal velocity of the enzyme reaction. Results of the microbiological examination of the hide and gelatin products, before and after drying, are shown in Table 1. Results of the amino acid composition of the hydrolysate obtained by the waste untanned leather enzyme hydrolysis are shown in Table 2. The enzyme concentration was 500 and 15,000 u/g and the samples were taken after 120 minutes of breakdown. Results of protein analysis show that the material is rich in protein. Collagen accounts for most of the protein. According to the literature, in hide there is 60±80% of proteins accounted to the dry weight. Our result of 79.4% of proteins is, therefore, in accord with reference data.
Fig. 8. In¯uence of alkaline protease concentration on solubility kinetics of untanned leather waste.
Fig. 9. Time dependence of the initial alkaline protease solubility rate.
Two dierent drying methods were used (Fig. 1): drying rollers (E) and dispersion drying system (F). Somewhat better results were obtained with the dispersion drying system. UV spectra (Fig. 2) of the powderlike leather ¯our are characterised by a broad shoulders between 260 and 280 nm (samples b and c). This absorbance pattern is most probably due to aromatic amino acid residues (tyrosine, phenylalanine, etc.) from leather proteins. Two dierent signals (Fig. 3) having intensities of 1.57 and 0.7 (sample a) and 2.25 and 0.95 (sample b) correspond to the fractions 6 and 9 at the gel chromatogram of powderlike leather ¯our. Nearly whole protein quantity eluted between the third and 15th fraction. These results also show that leather ¯our, obtained by the applied method, is rich in proteins. Electron microscope photomicrograph (Fig. 4) of the leather after liming and before dissolving, (magni®cation= 500) shows free, partially divided, ®bres of 5±9 mm thickness. Microscopic examination (Fig. 5) showed that regular particles are being formed in case of the dispersion drying, basically round shaped, on whose surfaces small recesses appear during the drying process. Smaller particles of 1010ÿ6 m in size are 25 times more abundant than particles of 4010ÿ6 m (Fig. 5). A transparent ®lm (Fig. 6) without specially formed particles is obtained by drying gelatin powder product with the use of the rollers. In the case of the powderlike leather ¯our dried with the use of the rollers, irregular shaped leaves and laminae are being produced. Fragments' sizes are within the range of 50 to 7010ÿ6 m (Fig. 7). It was determined that the concentration of alkaline protease (Figs. 8±10) in¯uenced the solubilization kinetics in a manner that the rate (curve slope) was higher at the beginning of the process and decreased during the process. An increase of the enzyme concentration from 500 to 15,000 u/g increases the solubility rate and the release of soluble proteins. That is in accord with the Michaelis±Menten concept of enzyme reaction kinetics which is described by equation:
Fig. 10. Dependance of 1/v on 1/S for the solubilization process of untanned leather with alkaline protease (Lineweaver±Burke diagram).
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83
Table 1 Microbiological examinations Sample amount (g/ml)
Microorganism
Gelatin sediment (3)
Leather ¯uor product (8)
Gelatin solution (2)
Gelatin powder product (9)
1 25 0.1 0.1 0.1 0.1 0 0 0 1 1 1
Microorganism count Salmonella Staphilococcus Sulpho-reducing Clostridium Proteus Escherichia coli Coliphorm bacteria b-hemolitic streptococcus Lipolytic bacteria Aerobic sporogenous bacteria Mould Yeast
792,000 0 0 Positive Positive Positive 0 0 0 0 0 0
100 0 0 0 0 0 0 0 0 0 100 0
92800 0 0 0 0 Positive 0 0 0 0 0 0
3400 0 0 0 0 0 0 0 0 0 0 0
Table 2 Concentration of amino acids in liquid phase, released from untanned leather after treatment with alkaline protease for 120 min
Glycine Proline Alanine Arginine Leucine Lysine Glutamine acid Asparagine acid Phenylalanine Methionine Histidine Serine Tyrosine a b
Sample Aa (mg/ml)
Sample Bb (mg/ml)
0.330 0.027 0.057 0.193 0.065 0.119 0.077 0.155 0.188 0.114 0.432 0.092 0.541
Trace Trace Trace Trace 0.033 Trace Trace Trace 0.089 Trace Trace Trace 0.183
Sample A, enzyme concentration 15,000 u/g. Sample B, enzyme concentration 500 u/g.
It was established with the use of thin layer paper chromatography (Table 2) that solid leather collagen is predominantly composed of 14 amino acids from which glycine makes one third, proline and hydroxyproline make the other third and all other amino acids make up the rest. The enzyme used most easily released those amino acids which in their composition have benzene ring (thyrosine and phenylalanine) or some other aromatic ring (hystidine). The type of amino acid being released later depends on its size and the bound functional clusters (arginine, asparagine acid, lysine, methionine, serine, glutamine acid). The amino acids which are the most abundant in collagen, glycine and proline, are being released at the end of the process. The quantities of the released amino acids depend on the size and structure of particular amino acid. Obtained amino acid solutions can be dried to the powder form which can be used in various industrial applications. 4. Conclusion
v vmax
c S c S KM
where v is the reaction rate, Vmax is the maximal velocity of the enzyme reaction, c(S) is a substrate concentration and Km is Michaelis±Menten constant. Microbial concentration in product samples is measured by direct count methods. Microbiological examinations (Table 1) showed that the micro organisms count in gelatin sediment (3) decreased from 792,000 per g to only 100 after drying process, and in gelatin solution (2) from 92,800 to 3400. Salmonella, Staphylococcus, Sulphoreducing Clostridium, Proteus and Coliforms bacteria were not registered at all. The results reveal that the leather ¯our and gelatin products are of the highest quality, i.e. the contamination level is very low. The products obtained by the applied method agree to food safety standards and can be used as animal feed additives.
A protocol was established to determine how eective the thermal and enzymatic treatments of untanned leather waste were in recovering the maximum amount of hydrolyzed protein with a minimum amount of residue. The methods that were developed have shown that the untanned leather waste can be successfully processed to the powderlike leather ¯our (8), gelatin products (9) and enzymically hydrolyzed protein powder products (10). The products of the satisfactory protein concentration were obtained. In this comparative study, we found the enzymatic treatment as a method of choice. It has also been demonstrated that it was not necessary to boil the leather waste before enzyme treatment. The method that was developed has shown that leather waste can be subjected to moderate pretreatment temperatures and that the enzyme can be added at these temperatures, thus eliminating the need to cool reaction mixture, which is, in the case of thermal treatment, a waste of energy.
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Z. Bajza, V. VrcÏek / Waste Management 21 (2001) 79±84
It was established that the enzyme concentration in¯uences the solubility kinetics of untanned leather waste in a way which is in accord with the Michaelis± Menten concept of enzyme reaction kinetics. We reported here the results of our attempts to achieve higher solubility and at the same time use of lower amounts of alkaline protease enzyme (500 units per g of leather waste), thus making the treatment more cost-eective. The procedure is simple, can be carried out without harmful odours, and the waste water contains no dangerous chemicals. With this process, therefore, not only the ecological problem is being solved, but also the valuable and useful products are being produced.
[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
References [1] Eisenbeiz F, Dumont E, Henke H. Angew Makromolekulare Chemie 1978;71:67.
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