ph.d thesis - improvement of conventional leather making processes
TRANSCRIPT
IMPROVEMENT OF CONVENTIONAL LEATHER MAKING PROCESSES TO
REDUCE THE ENVIRONMENTAL IMPACT
Doctoral Thesis directed by
Dr. José Costa López Dr. Jaume Cot Cosp
Eduard Hernàndez Balada
Barcelona, December 2008
Programa de doctorat d’Enginyeria del Medi Ambient i del Producte
Bienni 2006-2008
El Dr. JOSÉ COSTA LÓPEZ, Catedràtic del Departament d’Enginyeria Química de la
Universitat de Barcelona,
CERTIFICA QUE:
El treball d’investigació titulat “IMPROVEMENT OF CONVENTIONAL
LEATHER MAKING PROCESSES TO REDUCE THE ENVIRONMENTAL
IMPACT” constitueix la memòria que presenta l’Enginyer Químic EDUARD
HERNÀNDEZ BALADA per a aspirar al grau de doctor per la Universitat de
Barcelona. Aquesta tesi doctoral ha estat realitzada dins del programa de doctorat
“Enginyeria del Medi Ambient i del Producte”, bienni 2006-2008, en el Departament
d’Enginyeria Química de la Universitat de Barcelona.
I per què així consti als efectes oportuns, signa el present certificat a Barcelona, a 8 de
Desembre de 2008.
Dr. José Costa López
Codirector de la tesi doctoral
El Dr. JAUME COT COSP, professor de recerca del Departament d’Ecotecnologies
de l’Institut d’Investigacions Químiques i Ambientals de Barcelona (IIQAB),
pertanyent al Consell Superior d’Investigacions Científiques (CSIC)
CERTIFICA QUE:
El treball d’investigació titulat “IMPROVEMENT OF CONVENTIONAL
LEATHER MAKING PROCESSES TO REDUCE THE ENVIRONMENTAL
IMPACT” constitueix la memòria que presenta l’Enginyer Químic EDUARD
HERNÀNDEZ BALADA per a aspirar al grau de doctor per la Universitat de
Barcelona. Aquesta tesi doctoral ha estat realitzada dins del programa de doctorat
“Enginyeria del Medi Ambient i del Producte”, bienni 2006-2008, en el Departament
d’Enginyeria Química de la Universitat de Barcelona.
I per què així consti als efectes oportuns, signa el present certificat a Barcelona, a 8 de
Desembre de 2008.
Dr. Jaume Cot Cosp
Codirector de la tesi doctoral
Luck is what happens when preparation meets opportunity
Seneca
Life is what happens to you while you are busy making other plans
John Lennon
Acknowledgments
ACKNOWLEDGMENTS
Hi ha moltes persones a les quals dec un enorme reconeixement en la realització de la
present tesi doctoral. El suport incondicional i a distància de la meva família ha estat
sens dubte cabdal. Voldria destacar particularment als meus pares, avis, germà i tiets,
que em van fer sentir tan aprop quan ens separava tot un oceà. A tota la resta de
família, els dec un sentit agraïment per tots els àpats de rebuda i comiat cada cop que
tornava a Catalunya.
Al Professor Jaume Cot (o Jaume a seques, com em va fer acostumar a dir-li) li dono
les gràcies per obrir-me les portes del Consell Superior d’Investigacions Científiques
(CSIC) i haver fet possible la meva estada al Departament d’Agricultura dels Estats
Units (USDA). La seva bondat, humiltat, sentit de l’humor i capacitat científica són un
exemple a seguir.
No em voldria oblidar dels companys del CSIC que em van donar suport i ànims quan
vaig decidir fer les Amèriques. Especial menció mereixen el Drs. Albert Manich, Agustí
Marsal, Fernando Fernández i Merche Catalina. A tota la resta d’amics i companys
del CSIC, que són massa nombrosos per enumerar-los tots, moltes gràcies per les
enriquidores estones passades junts.
També voldria agraïr al codirector de tesi Dr. Costa, així com al Prof. Mans i Dra.
González, el suport rebut durant els darrers anys i la generositat per ajudar-me a fer
tràmits a distància.
I would like to acknowledge a lot of amazing people I met during my stay at the United
States of America. I must start thanking my three overseas “aunts”: Maryann Taylor,
Ellie Brown and Lorelie Bumanlag. Their kindness, generosity and wisdom were
absolutely priceless. With them, I felt home.
Special mention goes to my first office mate at the USDA, Brian Coll, who took the
patience to teach me proper English and “show me around”. I would also like to thank
Joe Lee, not only for sharing his endless knowledge in leather, but for his true
friendship during all this time. To all of you, I will truly miss you.
Acnowledgements
I would also like to extend a BIG thank-you to the Fats, Oils and Animal Coproducts
Research Unit staff, and to Dr. William Marmer in particular. Thanks for believing in
me and giving me the opportunity to stay at the USDA two more years beyond the initial
appointment.
Visit of my parents to the USDA. Top – With Ms. Maryann Taylor Bottom – With Dr. Ellie Brown
Table of contents
Table of Contents SUMMARY .................................................................................................................. i 1. HIDES AND SKINS ................................................................................................ 1
1.1 History of leather processing ...................................................................... 2
1.2 Economics of hides and skins in the U.S. market ...................................... 3
1.3 Structure of hides and skins ........................................................................ 5
1.4 Conversion of hides and skins into leather ................................................. 9
2. PRESERVATION OF RAW HIDES AND SKINS ............................................... 14
2.1 Causes and signs of decay of hides and skins .......................................... 15
2.2 Preservation of raw hides and skins with common salt ............................ 18
2.2.1 Salt pack curing ......................................................................... 18
2.2.2 Brine raceways .......................................................................... 18
2.2.2.1 Brine curing in the industry ......................................... 20
2.2.2.2 Advantages and disadvantages of brine curing ........... 21
2.4 Alternatives to brine curing ...................................................................... 23
2.4.1 Alternative methods ................................................................... 24
2.4.2 Alternative chemicals ................................................................ 26
3. FILLERS IN THE LEATHER INDUSTRY .......................................................... 28
3.1 Looseness and veininess .......................................................................... 29
3.1.1 Veins ......................................................................................... 29
3.1.2 Grain break ................................................................................ 30
3.2 The upgrading of veiny or coarse break leather ……............................... 31
3.3 Whey and whey products ......................................................................... 32
3.3.1 Description and characterization ............................................... 32
3.3.2 Uses of whey ............................................................................. 35
3.4 Gelatin ...................................................................................................... 36
3.4.1 Description and characterization ............................................... 37
3.4.2 Uses of gelatin ........................................................................... 39
3.4.3 Gelatin in the leather industry ................................................... 39
3.5 Enzymes .................................................................................................... 40
Table of contents
3.5.1 Enzymes in the leather industry ................................................ 42
3.5.2 Microbial transglutaminase ……….......................................... 42
3.5.3 Reactivity of TGase with gelatin and whey proteins ................ 45
4. MATHEMATICAL MODEL OF RAW HIDE CURING WITH BRINE ............. 48
4.1 Letter of acceptance .................................................................................. 50
4.2 Abstract ..................................................................................................... 51
4.3 Resum ....................................................................................................... 52
4.4 Introduction .............................................................................................. 53
4.5 Theory ....................................................................................................... 53
4.6 Experimental ............................................................................................. 59
4.6.1 Materials .................................................................................... 59
4.6.2 Methods ..................................................................................... 59
4.6.3 Analyses ..................................................................................... 59
4.6.3.1 Chloride concentration determination ........................ 59
4.6.3.2 Fluorescence imaging ................................................. 59
4.7 Results and discussion .............................................................................. 60
4.7.1 Epifluorescence microscopy ...................................................... 60
4.7.2 Determination of diffusion coefficients …................................. 61
4.7.3 Determination of optimum brine curing conditions ….............. 63
4.8 Conclusions .............................................................................................. 64
4.9 Definition of terms ................................................................................... 65
4.10 References .............................................................................................. 66
4.11 Acknowledgments .................................................................................. 67
5. EVALUATION OF DEGREASERS AS BRINE CURING ADDITIVES ............... 68
5.1 Letter of acceptance ..................................................................................... 70
5.2 Abstract ........................................................................................................ 71
5.3 Resum .......................................................................................................... 72
5.4 Introduction ................................................................................................. 73
5.5 Experimental ................................................................................................ 74
5.5.1 Materials ....................................................................................... 74
5.5.2 Methods ........................................................................................ 74
5.5.2.1 Stratigraphic study ......................................................... 74
Table of contents
5.5.2.2 Degreaser study .......................................................... 75
5.5.3 Analyses ..................................................................................... 75
5.5.3.1 Determination of moisture and ash content ................ 75
5.5.3.2 Determination of fat content ....................................... 75
5.5.3.3 Determination of thermal stability .............................. 76
5.5.3.4 Back-scattered/Low Vacuum Scanning Electron
Microscopy (SEM-BSE) ......................................................... 76
5.5.3.5 Statistical analysis ....................................................... 77
5.6 Results and Discussion ............................................................................. 77
5.6.1 Stratigraphic study ..................................................................... 77
5.6.2 Degreasing study ....................................................................... 81
5.7 Conclusions .............................................................................................. 85
5.8 References ................................................................................................ 86
5.9 Acknowledgments .................................................................................... 87
6. PROPERTIES OF BIOPOLYMERS PRODUCED BY TRANSGLUTAMINASE
TREATMENT OF WHEY PROTEIN ISOLATE AND GELATIN ......................... 88
6.1 Letter of acceptance .................................................................................. 90
6.2 Abstract ..................................................................................................... 91
6.3 Resum …................................................................................................... 91
6.4 Introduction .............................................................................................. 92
6.5 Experimental ............................................................................................. 93
6.5.1 Materials .................................................................................... 93
6.5.2 Sample preparation .................................................................... 94
6.5.3 Analyses ..................................................................................... 95
6.5.3.1 Gel strength ................................................................. 95
6.5.3.2 Viscosity ..................................................................... 95
6.5.3.3 Rheology ..................................................................... 95
6.5.3.4 SDS-PAGE ................................................................. 96
6.5.3.5 Statistical modeling .................................................... 96
6.6 Results and discussion .............................................................................. 96
6.6.1 Gel strength ................................................................................ 96
6.6.2 Viscosity .................................................................................... 98
6.6.3 Rheological properties ............................................................. 100
Table of contents
6.6.4 SDS-PAGE .............................................................................. 101
6.7 Conclusions ............................................................................................ 103
6.8 References .............................................................................................. 104
6.9 Acknowledgments .................................................................................. 107
7. WHEY PROTEIN ISOLATE: A POTENTIAL FILLER FOR THE LEATHER
INDUSTRY .............................................................................................................. 108
7.1 Letter of acceptance ................................................................................ 110
7.2 Abstract ................................................................................................... 111
7.3 Resum ..................................................................................................... 112
7.4 Introduction ............................................................................................ 113
7.5 Experimental ........................................................................................... 114
7.5.1 Materials .................................................................................. 114
7.5.2 Methods ................................................................................... 114
7.5.2.1 Preparation of WPI-Gelatin blends ........................... 114
7.5.2.2 Application of WPI-Gelatin blends to wet blue leather
................................................................................................ 115
7.5.2.3 Retan/Color/Fatliquor (RCF) .................................... 116
7.5.2.4 Drying ....................................................................... 116
7.5.3 Analyses ................................................................................... 116
7.5.3.1 Mechanical properties ............................................... 116
7.5.3.2 Subjective evaluation ................................................ 116
7.5.3.3 Protein concentration determination ......................... 117
7.6 Results and discussion ............................................................................ 117
7.6.1 Shoe upper wet blue ................................................................ 117
7.6.2 Upholstery wet blue ................................................................. 121
7.6.3 Mechanical properties .............................................................. 123
7.7 Conclusions ............................................................................................ 126
7.8 References .............................................................................................. 127
7.9 Acknowledgments .................................................................................. 128
8. GENERAL CONCLUSIONS AND RECEOMMENDATION ........................... 129
8.1 Preservation of raw hides and skins with brine. Conclusions ................. 130
8.2 Preservation of raw hides and skins with brine. Recommendations ....... 131
Table of contents
8.3 Obtaining and characterization of potential fillers for leather.
Conclusions ................................................................................................... 132
8.4 Obtaining and characterization of potential fillers for leather.
Recommendations ......................................................................................... 133
9. REFERENCES ..................................................................................................... 135
10. NOTATION ........................................................................................................ 144
11. GLOSSARY OF TERMS ................................................................................... 148
12. RESUM EN CATALÀ ....................................................................................... 157
List of figures
List of Figures
FIGURES OF CHAPTER 1
Figure 1.1 – Leather articles …………………………………………....................... 3
Figure 1.2 – Typical distribution of a cow’s weight after the slaughtering .................. 5
Figure 1.3 – Composition of hide ................................................................................. 5
Figure 1.4 – Structure of collagen ................................................................................ 6
Figure 1.5 – Schematic cross-section of a bovine hide ................................................ 7
Figure 1.6 – Structural comparison of hides and skins ................................................. 8
Figure 1.7 – Unit processes and operations in leather processing ................................ 9
Figure 1.8 – Pile of wet blue leather ........................................................................... 11
FIGURES OF CHAPTER 2
Figure 2.1 – (a) Scratches on the grain of leather; (b) Leather damaged by bites of
insects ......................................................................................................................... 15
Figure 2.2 – Pinholes (open grain) in sheepskin ........................................................ 17
Figure 2.3 – (a) Process flow drawing of a typical brine curing system; (b) Raceway
brine cure tank ............................................................................................................ 19
Figure 2.4 – Red heat damage on salted skins ............................................................ 23
FIGURES OF CHAPTER 3
Figure 3.1 – Veininess in calfskin ......................................................................................... 29
Figure 3.2 – Scanning electron microscope images of blue stock without and with
visible veins ................................................................................................................ 30
Figure 3.3 – Samples of shoe upper leather showing coarse break and fine break .... 30
Figure 3.4 – Processing of whey protein isolate ......................................................... 32
Figure 3.5 – Snack fortified with whey protein isolate .............................................. 36
Figure 3.6 – Transformation of collagen into gelatin in the course of hydrolysis ..... 36
Figure 3.7 – Conversion of chrome shavings into gelatin .......................................... 40
Figure 3.8 – Role of an enzyme in a given reaction ................................................... 40
List of figures
Figure 3.9 – Lock and key analogy for enzymes and substrate .................................. 41
Figure 3.10 – Temperature and pH activity profiles of microbial transglutaminase .. 43
Figure 3.11 – Cross-linking of gelatin chains upon reaction with microbial
transglutaminase ......................................................................................................... 45
Figure 3.12 – Effect of mTGase concentration, incubation time, pH and incubation
temperature on breaking strength of a 10% (w/w) type A gelatin .............................. 46
Figure 3.13 – Reduction of a disulfide bond by two thiol-disulfide exchange reactions
involving DTT ............................................................................................................ 47
FIGURES OF CHAPTER 4
Figure 4.1 – Mathematical model of the curing process of a raw hide ..................... 54
Figure 4.2 – Dimensionless sodium chloride concentration field within the hide
during the curing process ............................................................................................ 56
Figure 4.3 – Dimensional sodium chloride concentration field within the hide during
the curing process for various soaking numbers ........................................................ 57
Figure 4.4 – Epifluorescent microscopic images of a cross section of a hide at
different stages of curing ............................................................................................ 60
Figure 4.5 – Determination of transport parameter � from experimental data. The
graph corresponds to c0p = 30% (w/v) and Na = 3 ..................................................... 62
FIGURES OF CHAPTER 5
Figure 5.1 – Stratigraphic distribution of water, ash, and hide salt saturation in a hide
treated for various intervals of time with a 500% float of an initial 95 °SAL brine .. 79
Figure 5.2 – Denaturation temperature (TD) of the grain, middle, and flesh layers
of a hide treated for various intervals of time with a 500% float of an initial 95 °SAL
brine ............................................................................................................................ 80
Figure 5.3 – Composite images of hide samples, collected at different curing times
by low vacuum, mixed signal SEM imaging .............................................................. 81
List of figures
FIGURES OF CHAPTER 6
Figure 6.1 – Gel strengths of gels formed from (a) bovine type B gelatin, 1 to 10%
(w/w), and (b) WPI-gelatin blends, 10% WPI with 0 to 3% added gelatin ............... 98 Figure 6.2 – Viscosities of solutions of (a) gelatin, (b) WPI and (c) WPI-gelatin
blends ....................................................................................................................... 100
Figure 6.3 – Time sweep analysis of 10% (w/w) WPI and WPI-gelatin blends ...... 101
Figure 6.4 – SDS-PAGE gels of WPI, gelatin, and WPI-gelatin blends .................. 102
FIGURES OF CHAPTER 7
Figure 7.1 – Flow diagram for retan, color and fatliquor formulation of upholstery
and shoe upper wet blue ........................................................................................... 116
Figure 7.2 – mTGase and protein uptake profiles by shoe upper wet blue pretreated
with a solution containing 0, 2.5 or 5% mTGase and treated with a solution of
5% WPI + 0.5% gelatin ............................................................................................ 118
Figure 7.3 – mTGase and protein uptake profiles by upholstery wet blue pretreated
with a solution containing 0 or 2.5% mTGase and treated with a solution of
2.5% WPI + 0.25% gelatin ……………………………………………………....... 122
Figure 7.4 – Subjective properties of upholstery crust leather ................................. 123
Figure 7.5 – Effect of the various treatments of leather with WPI and mTGase on
the tensile strength of upholstery and shoe upper crust leather ................................ 124
Figure 7.6 – Effect of the various treatments of leather with WPI and mTGase on
the Young’s modulus of upholstery and shoe upper crust leather ............................ 125
Figure 7.7 – Effect of the various treatments of leather with WPI and mTGase on
the tear strength of upholstery and shoe upper crust leather .................................... 125
FIGURES OF CHAPTER 12
Figure 12.1 – Model matemàtic del procés de curat d’una pell crua ........................ 160
Figure 12.2 – Mostres de cuir amb un toc de flor gruixut o fi .................................. 163
Figure 12.3 – Reacció de crosslinking entre molècules proteiques amb l’enzim
mTGase ..................................................................................................................... 165
List of figures
Figure 12.4 – Força de gel, viscositat, i mòdul elàstic (G’) d’una barreja proteica
composta per 10% (w/w) WPI i diferents quantitats de gelatina (de 0.5 a 3%),
en un ambient reductor ............................................................................................. 166
Figura 12.5 – Imatges de microsopia epifluorescent del wet blue després d’haver
Estat tractat amb una solució etiquetada de WPI i gelatina ...................................... 167
List of tables
List of Tables
TABLES OF CHAPTER 1
Table 1.1 – U.S. hides and skins production ........................................................................... 4
Table 1.2 – U.S. hide and skin exports by country .................................................................. 4
Table 1.3 – Typical reagents needed and wastes produced during the manufacturing
of leather .................................................................................................................................. 13
TABLES OF CHAPTER 2 Table 2.1 – Typical range of emission factors for conventional leather processing .................. 22
TABLES OF CHAPTER 3
Table 3.1 – Composition of different whey products .............................................................. 33
Table 3.2 – Summary of properties of the basic constituent proteins in whey ....................... 34
Table 3.3 – Composition of whey protein isolate .................................................................... 35
Table 3.4 – Physicochemical properties of types A and B of gelatin ...................................... 37
Table 3.5 – Dependence of molecular weight on the bloom strength of type B gelatin ......... 38
Table 3.6 – List of proteinaceous substrates reported to be reactive towards TGase ............. 44
TABLES OF CHAPTER 4
Table 4.1 – Transport coefficient � for various conditions of initial brine concentration (c0p)
and soaking Number (Na) ...................................................................................................... 62 TABLES OF CHAPTER 5 Table 5.1 – Effect of commercial degreasers on brine curing [0.5% w/w] ............................. 82
Table 5.2 – Effect of degreaser 1 on brine curing ................................................................... 83
Table 5.3 – Effect of sophorolipid on brine curing ................................................................. 84
TABLES OF CHAPTER 7 Table 7.1 – Uptake rate coefficient k for various treatments ................................................ 118
Table 7.2 – Subjective evaluation of shoe upper crust leather .............................................. 120
Summary
i
Summary The research reported in the present PhD dissertation was developed in its totality at the
United States Department of Agriculture (USDA), Eastern Regional Research Center
(ERRC) located in Wyndmoor, Pennsylvania.
During my stay at the ERRC from July 2005 to October 2008, I was assigned to two
CRIS (Current Research Information System) projects. The main goals of each project
are shown below. Further information about them can be found at the ERRC website
(http://cris.csrees.usda.gov/).
1. New and efficient processes for making quality leather
Project number: 1935-41440-013-00D
Lead Scientists: Dr. William N. Marmer and Dr. Cheng-Kung Liu
Objectives: Develop new technology for preparing hides for tanning. Establish drying
and finishing processes and develop in-line nondestructive tests for improving the
quality and durability of leather. Additional funding was obtained to expand the scope
of hide preparation research by investigating ways to impart efficiency to short-term
hide preservation (brine-curing).
2. Sustainable technologies for processing of hides, leather, wool and associated
byproducts
Project number: 1935-41440-014-00D
Lead Scientist: Dr. Eleanor M. Brown
Objectives: 1. Functional modification, leather and leather byproducts. Develop a
foundation for the use of new chemical and biochemical technologies (a) in the
production of high quality chrome-free leathers; (b) in expanding the range of high
Summary
ii
value biomaterial applications for solubilized proteins from leather byproducts. 2.
Functional modification, wool: modify wool to impart functionality for improved
performance and expanded uses of domestic wool.
Therefore, my assignments were divided into two different areas.
1. New and efficient processes for making quality leather
The preservation of raw hides and skins with a highly concentrated sodium chloride
solution (brine) is the most traditional and cost effective method. Nevertheless, it is a
lengthy process and detrimentally impacts the environment due to the disposal of salt
during the hide's conversion into leather. A mathematical model that described the
diffusion of sodium chloride in the hide during the curing process was developed in
order to search for the optimum brine curing conditions such as brine concentration and
float percentage. The diffusion of salt into the hide was characterized by the transport
coefficient �, which was found to be in the order of 10-5 s-1. From the model it was
found that the use of an initially saturated brine (35.9 g NaCl/100 ml water) as well as a
minimum float of 500% yielded an optimal diffusion rate. These findings corroborated
the generally accepted rule which states that about five kg of brine per one kg of hide is
required for a proper curing. The model also revealed that hides that were cured with
diluted brines (e.g. 20 or 25 g NaCl/100 ml water) would not receive a proper cure
regardless of the float percentage used. Importantly, the model put in evidence that the
results obtained were highly dependent on the hide salt saturation level required to reach
a proper cure, which was targeted at 85%. Finally, the proposed mathematical model
may be used to optimize the curing process under any given conditions and thus
rationalize the amount of salt and time employed to properly preserve raw hides and
skins.
Another goal of the project was to find ways to accelerate the uptake of sodium chloride
by the hide during the cure. By accomplishing this, the turn-around times in raceways
would be reduced and thus additional curing capacity created. By means of a
stratigraphic analysis of the hide at varying curing time intervals, it was found that salt
entered the hide mainly from the flesh side whereas water was withdrawn from both
sides of the hide, with the epidermis acting as a semipermeable membrane. These
findings were supported by epifluorescence microscopy and scanning electron
Summary
iii
microscopy in back-scattered electron mode. Bearing in mind that the adipose tissue
that adheres to the flesh side of the hide is believed to slow down the penetration of salt,
three commercial degreasers as well as a glycolipid-based surfactant (sophorolipid, SL)
were tested as brine curing additives. One of the three commercial degreasing agents
was proved to significantly enhance the uptake of salt by the hide during the cure,
simultaneously as it decreased the fat content. Trials with the SL also turned out
successfully; when the SL was used above its solubility limit, the degreasing ability was
comparable to that of commercial degreasers. The usage of SL in the leather
manufacturing has not yet been attempted on a commercial scale, but the promising
results here presented as well as their antimicrobial properties make them a very
promising candidate.
2. Sustainable technologies for processing of hides, leather, wool and associated
byproducts
The usage of leather byproducts in later stages of tanning is established practice,
although not well publicized in the literature. One of the products tanners frequently
use as a post tanning agent is called filler. Fillers minimize the veiny, loose and pipey
areas of the hide to obtain a uniform leather product. Back in the 1970’s, extracts of
vegetable tannins were used for that purpose. Recently, leather industry suppliers offer
filling agents such as polymers, resins and proteins. One of the proteins more
commonly used in U.S. tanneries was casein. Current casein prices in the American
market ($5.8-5.9/lb) encouraged the search for cheaper sources of protein.
Whey protein isolate (WPI), a byproduct of the cheese industry ($0.9-1.2/lb), and
gelatin, a byproduct of the leather industry ($2.4/lb), were selected for that purpose.
Biopolymers formed by the enzymatic crosslinking of dissimilar proteins have the
potential for generating novel products. Thus, small amount of gelatin was added to the
less expensive WPI and reacted with the enzyme microbial transglutaminase (mTGase)
under reducing conditions. The improvement in physical properties over either protein
component, given the same treatment, suggested the possibility of greater utilization and
new products from these coproducts.
Next, a biofiller composed of WPI and small amounts of gelatin was evaluated as a
filling agent for shoe upper and upholstery wet blue. Also, the effect of pretreating the
Summary
iv
wet blue with mTGase was examined. The outcome of the process was assessed by
subjective properties of the crust leather such as grain break, fullness, handle or color.
The general appearance of both shoe upper and upholstery leather was markedly
improved upon treatment with the biofiller, yielding fuller crust leather with a tighter
grain break and enhanced color. Importantly, the proteins contained in the filler were
not considerably removed by further processing. Furthermore, although treated samples
were a little stiffer and presented slight lower tear strength than the untreated samples,
the various treatments did not negatively affect the mechanical properties of the crust
leather. The pretreatment of the wet blue leather with mTGase affected the kinetics of
protein uptake and also contributed in the further improvement of the grain break in
samples with very bad original break. Noteworthy, it was proved that a 200% float
satisfactorily enabled the proteins to be taken up by the wet blue. If this technology is
to be transferred to the industry, use of a shorter float could be feasible due to a stronger
mechanical action. Further research that explores the possibility of using even cheaper
sources of protein as a raw material for bio-based leather products is an interesting
option currently being examined at the ERRC laboratories.
CHAPTER 1
Hides and skins
Chapter 1
2
1. HIDES AND SKINS
1.1 History of leather processing
From the earliest civilizations right to the present time, leather has been considered
nature’s product with inherent beauty and universal appeal. Prestige, durability,
physical properties and eye-appeal are some of the many natural qualities of this
product.
The process of leather making predates recorded history. The earliest record of the use
of leather dates from the Paleolithic period, when primitive man removed the hides and
skins from wild animals after having hunted them for food. They used those hides and
skins to make coats and footwear. Primitive man learned that skins rotted away after a
relatively short time. Throughout the following centuries, they discovered that if the
skins were stretched out and allowed to dry in the sun, they became stiff and hard but
also lasted much longer (Covington, 1997). Much more time had to pass until it was
eventually discovered that the bark of certain trees contained tannin or tannic acid
which could be used to convert raw skins into what we recognize today as leather.
Although it is hard to substantiate chronologically the exact time this tanning method
materialized, many claim that it had to be around 5,000 BC (Thomson, 1981).
Later on, wall paintings and artifacts in Egyptian tombs indicated that leather was used
for military equipment, sandals, clothes, gloves, buckets, bottles, etc. Also the Romans
used leather on a wide scale for footwear, clothes, and military equipment including
shields, saddles and harnesses. As a matter of fact, the manufacture of leather was
introduced to Britain by the Roman invaders.
Through the centuries, leather manufacture expanded steadily and by medieval times
most towns had a tannery located on the stream or river, which they used as a source of
water for processing and as a source of power for their water wheel driven machines.
During the middle age, leather was used for all kind or purposes: footwear, clothes,
bags, cases, upholstery of chairs and couches, book binding and military uses.
With the discovery and introduction of basic chemicals such as lime and sulfuric acid,
leather production slowly became a chemically based series of processes. Until the later
part of the 19th century, there were relatively few changes in the methods used to
Hides and skins
3
produce leather. However, the industrial revolution did not bypass tanning. A wider
range of dyestuffs, synthetic tanning agents and oils were introduced during that time.
Together with precision machinery, these changes and continued innovations to the
present day have combined to make tanning into a viable, modern manufacturing
industry.
Figure 1.1 – Leather articles. From left to right, a woman’s bag, leather seats in automobile and leather jacket.
1.2 Economics of hides and skins in the U.S. market
The hides and meatpacking industries may be regarded as a bridge between production
of the hide as a byproduct of the food industry and its manufacture into leather goods,
for which it provides a basic raw material.
Hides need some form of preservation after being removed from the animal. Once this
is accomplished, they can be shipped great distances (e.g. overseas) or stored until used.
Thus, the preserved hide becomes an article of international commerce. The supply of
hides is determined by the amount and type of meat in people’s diet. For instance, the
United States is a hide exporting nation since its appetite for beef exceeds the capacity
for tanning of the industry. On the contrary, Japan has a strong demand for leather
goods but a limited hide supply, which turns it into a hide importing nation
(Thorstensen, 1993).
Table 1.1 shows the estimate values of total slaughtered cattle in the Unites States over
the last years. On average, about 60% of the hides produced in the U.S. from 2001 to
2005 were exported, in their majority to Asia (Table 1.2). Latest figures available from
the United States Hide Skin & Leather Association (USHSLA) stated that an 80% of the
hides produced in the U.S. in 2008 were exported, half of which were shipped to China
Chapter 1
4
and Hong Kong (Reddington, 2008). This trend concludes that the United States’
exporting character is currently growing even further.
Table 1.1 – U.S. hides and skins production (1,000 hides)
Year Total slaughter Exports Imports Net exports
2001 35,530 23,471 1,721 21,750
2002 35,734 20,783 1,299 19,484
2003 35,647 19,330 1,153 18,177
2004 32,880 18,704 1,316 17,388
2005 32,535 19,200 1,355 17,845
Source: U.S. Leather Industry Statistics (2006 edition)
Table 1.2 – U.S. hide and skin exports by country (1,000 hides)
Destination 2005 2003 2001
China 8,191 5,434 5,417
Korea 4,089 4,860 7,602
Taiwan 1,718 1,941 2,751
Hong Kong 1,331 2,534 1,382
Mexico 1,287 1,348 1,647
Thailand 651 819 888
Italy 594 826 920
Japan 333 475 1,343
Vietnam 165 15 1
Brazil 141 160 76
India 107 11 41
Source: U.S. Leather Industry Statistics (2006 edition)
At the present time it is not uncommon for cattle hides to be produced in the United
States, converted into leather in China and shipped back to the U.S. to make garments.
As a matter of fact, hide sales to China are soaring. By the end of April 2006, U.S.
exports sales to China amounted to 47% of all exports worldwide which compares with
36% a year earlier (Leather International, 2006).
Hides and skins
5
1.3 Structure of hides and skins
Raw hides and skins are byproducts of the meat industry, and in turn are the raw
material of the leather industry. Figure 1.2 depicts that only about one third of the
animal live weight is manufactured as edible meat. Hide, hair, bones and organs
account for an approximate 23% of the animal’s weight.
Figure 1.2 – Typical distribution of a cow’s weight after the slaughtering.
Hides and skins are removed from the carcass of the animal during the slaughtering
process. By convention, the tanner employs the word hide to refer to the skin covering
large animals, such as cows, steers, horses, buffalos, etc., and the word skin is mostly
used to refer to smaller animals such as calves, sheep, goats, pigs, etc. The term hide is
never applied to the small animals.
The approximate composition of a freshly flayed hide is as follows (Figure 1.3).
Figure 1.3 – Composition of hide (Sharphouse, 1971).
Chapter 1
6
Collagen, with a content of 29%, is the main structural protein in the hide. Up to twenty
eight different types of collagen are described in literature. Among them, fibril-forming
collagens types I, II, III, V and XI are the majority components of skin, cartilage and
bone (van der Rest and Garrone, 1991). Collagen molecules consist of three
polypeptide chains, each coiled in a left-handed helix. The three chains are thrown into
a right-handed triple superhelix stabilized by periodic hydrogen bonds (Rich and Crick,
1955; Ramachandran and Kartha, 1955). The triple helices, also known as
tropocollagen, associate laterally and longitudinally to form microfibrils. These, in turn,
form fibrils, aggregates of which constitute various forms of connective tissue (Figure
1.4).
Figure 1.4 – Structure of collagen.
Other structural proteins present in the hide are elastin (0.3%) and keratin (2%). The
former helps skin to return to its original position when it is poked or pinched, whereas
the latter is the constituent protein of the hair. The figure for keratin varies widely
depending on the amount of hair present. Among the non-structural proteins, albumens
and globulins account for 1% and mucins and mucoids 0.7%.
An interesting approach of seeing the structure of a hide is to examine a cross section
(Figure 1.5).
Hides and skins
7
Figure 1.5 – Schematic cross-section of a bovine hide
(Sharphouse, 1971).
Starting from the hair side the following elements are found:
• Hair. It is composed by an actively growing root zone embedded in the skin and
a visible dead hair shaft above the skin. Hair is primarily keratin, a sulfur-
bearing protein. The structure of a hair follicle is quite complex and up to five
concentric layers can be differentiated (Wagner and Bailey, 1999).
• Epidermis. It is the outermost layer of skin. It consists of a variety of cell layers
produced in the geminative basal cell region located at the base of the dermis. It
is hard, quite inert chemically, and constantly in the state of flaking.
• Sweat glands. They release sweat and undesirable body wastes through the
pores of the skin.
• Sebaceous glands. They release oil into the hair and onto the surface of the skin
in order to maintain a proper body temperature in warm-blooded animals.
• Corium. It consists of a network of collagen fibers intimately woven. In the
upper part of the corium, the fibers are very thin and tightly woven and towards
the center they are coarser and stronger. The orientation of the fibers and
Chapter 1
8
whether they are loosely or tightly-woven will determine some characteristics of
the resultant leather.
• Flesh. It is composed of varying amounts of fatty adipose tissue, blood vessels,
nerves and voluntary muscle.
Hides and skins differ in their structure, depending upon the habits of life of the animal,
season of the year, age, sex and breeding. Figure 1.6 depicts the different structure of a
goatskin, sheepskin and cattle hide. The amount of hair and fat present as well as the
tightness of the structure are three important parameters used to differentiate them. For
instance, goatskin presents less hair and fat and a more firm structure than sheepskin
and cattle hide. Cattle hide presents fat in both sides of the hide and has a tighter
structure than that of sheepskin but more open than goatskin.
Figure 1.6 – Structural comparison of hides and skin (Thorstensen, 1993).
Hides and skins
9
1.4 Conversion of hides and skins into leather
The conversion of a raw hide or skin into leather requires numerous processing steps,
which may be grouped in the following five categories (Figure 1.7).
Figure 1.7 – Unit processes and operations in leather processing (Saravanabhavan et al., 2003).
1. Hide preservation. Treatment given to raw hides or skins just removed from
the carcass of the animal to minimize putrefaction and bacterial action, but
enabling the skins to be rehydrated conveniently in preparation for tanning.
Sometimes the preservation of hides is carried out after cutting away the
subcutaneous tissues or flesh, which accounts for about 20% of the weight of the
hide. This process is known as fleshing.
2. Beamhouse (Pretanning). It refers to the processes in the tannery between the
removal of the skins or hides from storage and their preparation for tanning.
Despite this definition, hide preservation is typically not considered a
beamhouse process. Beamhouse operations are of tremendous importance in the
ultimate quality of the leather. The following operations are considered
beamhouse:
a. Trimming. Cut away useless or unwanted material from the edges of raw
hides or skins to give them a better shape.
Preserved Hides and Skins
Pelt Wet Blue Leather
Crust Leather
Finished Leather
Finishing Operations Preservation Pretanning Tanning
Post tanning Operations
Trimming Soaking
Unhairing Liming
Deliming Bating
Scudding Pickling
Wringing Splitting
Retanning Dying
Fatliquoring Setting Drying
Conditioning Staking Toggling Buffing
Spraying Plating
Unit Operations
Outcome
Stages
Chapter 1
10
b. Soaking. Treat hides or skins with water, sometimes with the addition of
a disinfectant, to cleanse them, remove salt and other soluble matter, and
to rehydrate and soften them.
c. Unhairing. Removal of hair or wool from hides or skins. Various
chemicals may be used for this purpose: lime-sulfide, an oxidizing agent
in acid solution, or an enzyme preparation.
d. Liming. Treatment of hides or skins with lime intended to loosen hair,
fat, flesh, etc. Sometimes it is done simultaneously to the unhairing.
e. Deliming. Removal of alkali and pH adjustment for bating.
f. Bating. Enzymatic action for the removal of unwanted inter-fibrillary
proteins, as well as any remaining hair roots, epidermal structure and
fatty cells.
g. Scudding. Working over the grain surface of limed, or bated, pelt with a
blunt-bladed tool, by hand or machine, to eliminate hair fragments,
pigment granules, lime soaps and other impurities.
h. Pickling. Treatment of pelts with an acid liquor, such as a solution of
sulfuric acid and sodium chloride, to preserve them or to prepare them
for tanning, especially chrome tanning.
3. Tanning. Tanning is defined as a process by which putrescible biological
material is converted into a stable material which is resistant to microbial attack
and has enhanced resistance to wet and dry heat (Gustavson, 1956). Chrome
tanning is the most frequently used method to tan hides, mainly due to the short
time it takes (4 to 6 hours) and because it produces leather that combines both
the best chemical and physical properties sought after in the majority of leather
uses (Leather Facts, 1973). A hide or a skin that has been subjected to the usual
beamhouse processes, chrome-tanned and left wet receives the name of wet blue
(Figure 1.8). Wet blue may be stored or exported in this state.
Hides and skins
11
Figure 1.8 – Pile of wet blue leather.
Alternatives to chrome tanning include vegetable tanning, carried out by means
of tanning agents contained in the barks, woods, fruits, leaves, etc., of plants.
Although they have not been implemented in the industry, natural products like
genipin (Ding et al., 2007) or salts of metals such as aluminum (Brown and
Dudley, 2005) or titanium (Peng et al., 2007) were reported to be successfully
tan the skins and hides.
4. Post tanning operations. The order of processes varies considerably for
different leathers. The control and choice of these operations will determine the
characteristics of the leather made.
a. Wringing. Removal of excess moisture from the stock for a proper
splitting.
b. Splitting. Adjustment of the thickness of the hide to the specified
requirements. The cut off layer, named split, is still a valuable raw
material for making into sueded type of leathers. Some hides may need
further thickness adjustment in a process called shaving.
c. Retanning, Coloring and Fatliquoring (RCF). These three operations
have vastly different purposes, but they are considered as a unit because
one follows the other without interruption. To retan is to subject an
already tanned leather to a further tanning treatment to modify its
properties. Coloring, also called dyeing, gives the required color to the
Chapter 1
12
leather. The aim of fatliquoring is to soften the leather by the use of oils
that lubricate the fibers.
d. Setting out. Multiple purpose operation, which smoothes and stretches
the leather while squeezing out excess moisture from it.
e. Drying. Removal of all but equilibrium water from the stock. Moisture
content after this step should be around 10-12%.
5. Finishing operations. The act of making completely tanned leather more
attractive, serviceable and durable. It can include one or more of the following
processes:
a. Conditioning. Application of a fine mist of water to raise the water
content to about 25%.
b. Staking. Mechanical operation that softens and flexes the leather.
c. Toggling. The straining and fixing of leather onto frames with toggles.
The purpose is to dry leather keeping it under tension.
d. Buffing. Abrade or grind a leather surface, especially the grain surface,
by a moving band of abrasive paper or cloth.
e. Spraying. Apply a liquid in the form of very fine droplets designed to
enhance the appearance and/or give the grain or flesh surface special
properties.
f. Plating. Mechanical finishing process used to subject the surface finish
of leather to a high pressure from a heated, polished plate or cylinder to
obtain desired smoothness, flow-out, gloss and film formation.
Hides and skins
13
Table 1.3 summarizes the reagents typically used in the tannery for the conversion of
hides and skins into leather, as well as the different wastes generated at each one of the
processing stages.
Table 1.3 – Typical reagents needed and wastes produced during the manufacturing of leather
(Rao et al., 2003)
Reagents Liquid Waste Solid Waste Air pollutants
Beamhouse operations
Water Lime
Sulfide Enzymes
Salt & Acid
Salt Protein Lime
Dusted salt Trimming Fleshing
Hair
Sulfide
Tanning
Water Chrome
Vegetable tannins
Salt Chrome Tannins
Splits Shavings
Vegetable bark
Post tanning operations
Water Chrome Syntans
Dyes Fatliquors
Dyes Greases Syntans Chrome
Finishing
Binders Pigments
Organic solvents Formaldehyde
Lacquers
Tanned wastes Buffing dust
Organic solvents Buffing dust
Formaldehyde
CHAPTER 2
Preservation of raw hides and skins
Chapter 2
15
2. PRESERVATION OF RAW HIDES AND SKINS
2.1 Causes and signs of decay of hides and skins
The purpose of preserving hides and skins is to temporarily prevent deterioration from
the time they are removed from the animal until they are processed into a product that is
no longer susceptible to putrefaction or rotting. A hide that has not been properly cured
will yield a finished leather of poor quality. Therefore, this very first step plays an
essential role in the quality of the finished product.
Hide damage can be classified into two categories: physical damage and putrefaction.
• Physical damage. Occurs before the slaughter of the animal. It becomes an
important issue because these defects will have an adverse effect on the finished
leather even if the hides receive a proper cure. It includes tears, scratches, cuts,
hook marks, contamination with dirt (manure), insect attack, etc (Figure 2.1).
Figure 2.1 – (a) Scratches on the grain of leather; (b) Leather damaged by bites of insects (Tancous et al., 1959).
• Putrefaction. The main object of preservation is to fight the damage caused by
bacteria and the proteolytic enzymes produced by them. Bacteria are one-celled
microorganisms that multiply very rapidly when they feel comfortable in the
surrounding environment. These conditions are summarized in the following
list:
a) Food. Bacteria secrete a digestive juice that contains enzymes. The
function of these enzymes is to nourish bacteria by breaking down
molecules of the substrate (hide). Three different kinds of bacteria are
a b
Preservation of raw hides and skins
16
involved in the degradation of skin proteins: anaerobic, aerobic and
facultative (which can grow with or without oxygen). Anaerobic
bacteria deteriorates the proteins into the stage of amino acids and
therefore it is one of the most dangerous. A species named Clostridium
histolyticum was the first to be reported to produce the enzyme
responsible for collagen degradation, collagenase (Mandl et al., 1958).
b) Water. Bacteria need water to live, grow, multiply and produce
enzymes. Nevertheless, there are some bacteria that have become
accustomed to dry conditions that form dormant spores which are
capable of reproducing when moisture is available again, sometimes
even after years. The critical moisture, below which level the skin is not
conducive for bacterial attack was found to be at around 50% (Stuart and
Frey, 1938).
c) pH. Bacteria require a near neutral or slightly alkaline environment to
survive. Generally, microorganisms can not survive in an environment
with a pH very much lower than 5 or higher than 8.
d) Temperature. Bacteria are more likely to reproduce in a warm
surrounding, leading to poor cure efficiency and loss of hide substance.
Literature reported that optimum temperature for bacteria was between
15 to 37 °C and that a better preservation was attained if curing was
carried out at a temperature between 10 to 18 °C (McLaughlin and
Rockwell, 1922).
e) Pre-curing period. This term defines the time comprised between the
slaughtering of the animal and the commencement of the curing process.
It was reported that a five hours period led to degenerative changes in the
cells lying around the sweat glands, and that another 6 hours led to
structural damage of the skin and the breaking down of the polypeptide
chains into dipeptides (Chattopadhyay, 1998).
Chapter 2
17
f) Presence of certain chemicals. Microorganisms can be killed by a
variety of chemical substances. These chemical poisons may be used as
short term preserving agents or along with salt in order to obtain hides
that will remain preserved in a long term period. A bacteriostat is a
chemical agent that stops or inhibits the activity of bacteria at whichever
state they are in. A bactericide is a chemical agent that kills bacteria
outright. They used to be based on mercury compounds or chlorinated
phenols, but they were ruled out with the enforcement of more strict
environmental laws.
Typically, preserved hides may be graded according to the following classification:
• Very good. No sign of putrefaction, raw skin smell, fresh appearance.
• Good. No sign of putrefaction, smells good, not so fresh as raw skin, appearance
good but not so fresh.
• Fair. Signs of putrefaction present, slight putrid smell, dull appearance.
• Poor. Several bacterial damage, strong putrid smell, slimy appearance.
Hair slippage, bad odor, the presence of mold and grain damage (e.g. discoloration) are
amongst the most common signs of a hide decay. Hair slippage has been used as an
indicator by packers and tanners that hides or skins have not been preserved to the
fullest degree. It is a sign that either autolytic or bacterial degeneration has occurred to
the extent that the hair and epidermis is loosened and can be easily removed. This
phenomenon may be accompanied by degradation of the hair follicle leaving a hole in
the grain (Didato et al., 1999) (Figure 2.2).
Figure 2.2 – Pinholes (open grain) in sheepskin.
Preservation of raw hides and skins
18
2.2 Preservation of raw hides and skins with common salt
Preservation of hides with sodium chloride (NaCl) is the most frequent curing method
used currently. The hygroscopic nature of sodium chloride reduces the water content of
the hide as well as lowers the water activity of the remaining moisture (Bailey, 2003).
Sodium chloride takes the water away from the bacteria that inhabit the surface of the
hide. Yet an exception to this statement are the halophilic bacteria, which grow in a
concentrated salty environment (see Section 2.3.).
There are two distinct processes that use sodium chloride as a preserving agent: salt
pack curing and brine curing.
2.2.1 Salt pack curing
Also known as green salting, is a more antique method of salt preservation. It is not
common in the United States or Europe but it is extensively applied in warm countries
like India. It consists of sprinkling solid salt onto the flesh surface of the hide, usually
about one kilogram of salt per kilogram of hide. As the solid salt slowly diffuses into
the hide, the water inside migrates out. Successive layers of salt and hides are added to
the pack until a height of four to five feet. Because of the slow diffusion of salt into the
hide, a considerable time (approximately 30 days) is needed to insure a satisfactory and
uniform curing.
2.2.2 Brine raceways
This method is extensively used in American and European hide processing facilities. It
consists of a huge vat containing a very concentrated or saturated solution of sodium
chloride (brine) where hides are suspended for a minimum of 18 h. The brine is kept
close to saturation by circulating it through a salt-box or Lixator, or by having excess
salt in the brining vat. Brine curers also add bactericide in the brine to keep low the
presence of bacteria both in the hide and in the vat.
Chapter 2
19
Figure 2.3 – (a) process flow drawing of a typical brine curing system (Thorstensen, 1993) (b) raceway brine cure tank, provided with two small paddles to keep the solution agitated (courtesy of Diamond Crystal Salt Co.).
Tanners and meatpackers monitor the concentration of brine in the raceway with an
instrument called salometer. This hydrometer is a long, narrow, graduated stem
attached to a weighted bulb. It is more buoyant the higher the salinity of the liquid and
therefore sinks less deeply into the liquid. The salometer scale ranges from 0 °SAL
(pure water) to 100 °SAL (saturated brine).
There are other hydrometers that can be used for the same purpose, which measure the
specific gravity or the sodium chloride percentage by weight. The specific gravity is the
relation between the weight of a certain volume of any substance and the weight of the
same volume of water. This value rises from 1.000 (pure water) to 1.198 (saturated
brine at 25 °C).
At 25 °C, 100 ml of saturated brine hold 31.7 g of salt, or likewise 100 g of saturated
brine holds 26.5 g of sodium chloride. Saturation levels can also be referred as g of salt
per 100 ml of water. In this case, 35.9 g of salt saturates 100 ml of pure water, at 25 °C.
The following equation relates brine concentration (g NaCl/100 g brine) and salometer
degrees (°SAL), at 15 °C.
[ ] 0004.0789.3)( −⋅=° NaClSALSalometer
a
b
Preservation of raw hides and skins
20
It is important to note than the solubility of sodium chloride is slightly dependent on
temperature, with values ranging from 35.7 to 39.8 g/100 g water, at 0°C and 100 °C
respectively.
Despite the low knowledge of technology involved, there are a few aspects that need to
be taken into consideration when curing hides and skins in a raceway.
• Brine needs to be kept always in fast motion. Otherwise the hides settle to the
bottom and will not receive a proper cure.
• Brine needs to be changed regularly due to the accumulation of dirt and manure
in the bottom of the vat. The removal of dirt, clay, manure and urine during
brining results in hides cleaner than those obtained in salt pack curing. If no
action is taken, the actual float decreases and the hides are more likely to receive
a poor cure. Float (or float percentage) stands for the ratio between the volume
of brine and the volume of hides.
• A large float and a high concentration of brine are essential to attain a proper
cure of the hides. A generally accepted rule stipulates a minimum 500% float
(about 5 m3 of brine per m3 of hide) in order to reach a good cure.
2.2.2.1 Brine curing in the industry
Brine curing is performed in conjunction with one of two hide processing methods:
green fleshing or cured fleshing.
1. Green fleshing + Brine curing. It consists of fleshing the hides first and then
brine cure. This is the option of choice for packers that flesh in operations
adjacent to their kill floors. There are three main reasons that justify this modus
operandi:
• Green fleshing has allows the removed flesh to be processed in a
rendering plant, since it is not contaminated with salt, thus preserving
some value in the flesh. In fact, production of biodiesel from solid
tannery waste is being currently considered (Kolomaznik, 2008).
Chapter 2
21
• With the adipose tissue mostly removed, salt penetrates more quickly
and to a greater extent into the hide. This fact also leads to an additional
weight increase in the hides fleshed before cure (Meat Industry, 1979).
• Hides fleshed before the cure show significantly lower bacterial levels
than hides that have not been prefleshed.
2. Brine curing + cured fleshing. In this case, the hide is cured first and then
fleshed. Packers who do not have adjacent hide processing facilities or hide
processors that are located at some distance from their clients, usually choose
this method. Similarly, this method presents two advantages.
• The 18 h curing time in the raceway gives some time to soften and
remove the feedlot mud that adheres to the hair of cattle (especially from
January through April).
• From an operational point of view, the hide has to be handled twice: put
it in the raceway, cured and fleshed. Conversely, green fleshing requires
the hide to be washed in a fresh water tank first, fleshed, returned to the
brine raceway, cured and then removed. Therefore, the hide needs to be
handled three times and more wash tank/raceway capacity is needed.
2.2.2.2 Advantages and disadvantages of brine curing
Brine curing of hides and skins presents the following advantages and disadvantages.
The most relevant advantages are the following:
• It is relatively inexpensive in comparison to the other preservation methods or
curing materials (current commodity prices for sodium chloride are around
$0.11/kg).
• A raceway can process a high volume of hides and skins (various thousands per
day).
• There is not high tech knowledge involved.
• It involves the usage of safe chemicals.
The most important disadvantages of the brine-curing method are detailed next.
Preservation of raw hides and skins
22
• Water pollution. Production of leather entails a high consumption of water. In
fact, the conversion of 1 ton of raw hides into leather requires between 20 and
40 m3 of water (Ramasami and Prasad, 1991), which will be almost fully
discharged upon completion of the process.
Soaking of brine cured hides releases about 40-50% of the total dissolved solids
(TDS) of the whole leather making process (Table 2.1). Technology to treat a
stream heavily contaminated with TDS and chlorides (e.g. membrane processes)
is not cost effective. In addition, the presence of common salt in irrigation water
can be extremely adverse, leading to an increase in soil salinity and reduction of
crop yields (Daniels, 1998).
Table 2.1 – Typical range of emission factors for conventional leather processing
Parameters Soaking
Volume of effluent 6 - 9
Biological oxygen demand (BOD) 6 - 24
Chemical oxygen demand (COD) 18 - 60
Total solids 200 - 500
Dissolved solids 190 - 400
Suspended solids 15 - 60
Chloride as Cl- 90 - 250
Total chromium as Cr -
Note: all values expressed in kg/ton of hide or skin processed. They were obtained from the formula (concentration x volume of effluent)/ton of leather processed (Ramasami et al., 1998).
• The large amount of salt required. Most industrial curing operations try to
maximize salt concentration in the brine to 97 °SAL (25.6 g NaCl/100 g brine).
• Despite the historically low cost of common salt, economics of brine curing of
hides and skins has been affected recently by increasing commodity prices for
sodium chloride (a 10-15% increase over the past few years) (Godsalve, 2007).
• The enormous amount of hides being cured at once makes it difficult to ensure
that they remain in the vat a minimum of 18 hours. Brining in a raceway is a
batch process and hides are not tagged. It is probable that hides are removed
Chapter 2
23
from the vat in a different order that they were put in. These hides will be
undercured and are likely to present deterioration in long term storage.
• Overloading the vat with hides causes the float to fall below the recommended
500%. With a lower float, the concentration of salt in the brine decreases
rapidly, which also leads to a slower rate of diffusion of salt into the hide.
Under this scenario, it is likely that a hide removed from the raceway after 18
hours may not be fully cured.
• Red heat damage. Hides that have received a proper cure are more susceptible
to be attacked by bacteria that grow in a concentrated salt environment.
Figure 2.4 – Red heat damage on salted skins (Source: BLC Leather Technology).
Archaea, an extremely halophilic bacteria belonging to the family
Halobacteriaceae, was demonstrated to cause significant damage on brine cured
hides (Bailey and Birbir, 1993). A concentration of at least 1.5 to 2 M NaCl was
needed for growth and optimally most species required 2 – 4 M NaCl (Grant et
al., 2001). A study showed that 98% of brine cured American hides were
contaminated with those microorganisms (Bailey and Birbir, 1993).
Furthermore, a total of 332 extremely halophilic Archaeal strains were isolated
and 94% of these strains were protease positive (Bailey and Birbir, 1993).
2.4 Alternatives to brine curing
Issues associated to the preservation of raw hides and skins with common salt
encouraged the search for alternatives. Over the last 30 years, different substances were
assayed as preserving agents, in either salt-less or less salt methods. Unfortunately for
Preservation of raw hides and skins
24
the majority of cases, they were not applied in the industry due to the high cost
involved. Also, new proposed techniques that efficiently preserved the hides were
studied, but again the high capital investment ruled them out. A quick summary of
these new methods and substances is listed next.
2.4.1 Alternative methods
• Drying. It is the earliest method of preservation of raw hides and skins. Even
though this method is still used by primitive men to cover huts or making tents,
it presents a few disadvantages:
a) If the drying is too slow, which is likely to happen in a cold and wet
climate, putrefaction may occur before the moisture is low enough to
prevent the bacterial attack.
b) If the drying is too fast and the temperature too high, it is likely that
some areas of the hide start to become gluey, thus preventing inner layers
from drying.
c) Hides are thicker than skins and therefore more difficult to dry out.
d) Increase of slipperiness of the pelt during fleshing.
e) The dried hides may be subject to insect attack, which is as bad as the
damage caused by bacteria.
• Cooling and chilling. Some countries use cooling systems to preserve skins.
The preservation time depends on the temperature, going from 3 weeks to 2 days
for temperatures of 0 and 15 °C, respectively. There are three methods to cool
raw hides and skins (Kanagaraj and Chandra Babu, 2002).
Chapter 2
25
a) Cooled air treatment. Just flayed hides are cooled at 3 to 5 °C for about
one hour, which preserves them in a sufficient extent to confront the
transport from the slaughterhouse to the tannery without decaying
(approximately 12 hours).
b) Addition of ice. The addition of ice cubes or flakes in a container that
encloses hides cause a drop of about 20 °C and then can be stored for 24
hours without further treatment. This technique was improved by the use
of a preservative solution to produce ice.
c) Use of dry ice. The use of dry ice cools hides to temperatures of -35 °C
and effectively preserves them for a minimum of 48 hours. The use of
carbon dioxide, however, entails risk of suffocation.
• Freezing. It is a medium term preservation technique that consists of storing
hides at temperatures around -10 to -20 °C. Freezing is rarely used nowadays
due to the high investment required and to the micro-distortion within the fiber
structure caused by the formation of ice crystals.
• Freeze drying. In this case, water is removed by means of applying low
pressure at low temperatures, which gives a flexible and porous cured hide. This
preservation technique cuts back money on shipping and makes the soaking
process easier. However, the operational costs are too high for common use.
• Gamma irradiation. The irradiation of hides with gamma rays along with the
application of a bactericide was reported as a successful salt-less preservation
technique. However, the high cost capital investment required and the difficulty
to control the minimum amount of radiation needed to attain a proper cure
impeded its commercial adoption (Bailey, 1999).
• Electron beam irradiation. The application of electron-beam-irradiation to
cattle hides, also known as EvergreenTM technique, yields a preservation level
Preservation of raw hides and skins
26
comparable to that of brine. This high tech method, however, presented the
same disadvantages as the irradiation with gamma rays (Bailey et al., 2001).
2.4.2 Alternative chemicals
• Formaldehyde (H2CO). The use of low amounts of this aldehyde was proved to
efficiently preserve the hide but its usage was ruled out due to its toxicity
(Sharphouse and Kinweri, 1978).
• Soda ash (Na2CO3). Even though soda ash is not a true preservative, its high
alkalinity created an environment where bacteria and proteolytic enzyme activity
was inhibited. This method was only analyzed as a short term preservation
technique (storage time up to 8 days) (Rao and Henrickson, 1983).
• Potassium chloride (KCl). It could be effectively used as a curing agent.
Furthermore, this substance presented a few advantages over the traditional
brine curing:
a) Production of less excess salt.
b) Possible effective use of the water stream as a plant fertilizer.
c) More rapid kinetics on uptake of salt.
d) Elimination of halophilic bacteria.
However, preservation with potash did not take off due to the more expensive
cost of potash over brine. In addition, the low solubility of potassium chloride at
low temperatures made this technique unviable in cold climate tanneries (Bailey
and Gosselin, 1996).
• Boric acid (H3BO3). This substance, used either in a salt-less or less-salt
method, effectively cured skins for storage times up to two weeks. The main
Chapter 2
27
advantage is the reduction by more than 80% of the TDS and chlorides in the
effluents. However, the cost for that new susbtance was about 15-20% higher
than for the traditional brine curing (Kanagaraj et al., 2005).
• Silica gel. This powerful dehydrating agent was proved to be effective in short
and long term preservation of raw hides and skins (Kanagaraj et al., 2001;
Munz, 2007). The advantages of this method are various:
a) Reduction of TDS and chlorides content in soaking liquors.
Furthermore, these liquors can replace pure water for irrigation
purposes.
b) Stronger dewatering of the hide which does not affect the subsequent
rehydration in the soaking step.
c) No impact on the quality of the final product.
However, the production cost of silicate was double that of common salt, and
therefore this method has not been extensively used yet.
• Azardirachta Indica. This herbal preservation agent, commonly named Neem
tree, was demonstrated to be an efficient agent in the curing of goatskins. By
using it instead of sodium chloride, both TDS and solid waste discharge were
reduced, and the quality of the finished crust leather was not adversely affected
(Preethi et al., 2006).
CHAPTER 3
Fillers in the leather industry
Chapter 3
29
3. FILLERS IN THE LEATHER INDUSTRY
3.1 Looseness and veininess
The presence of veins and loose or pipey areas in finished leather figure amongst the
most important concerns that tanners are facing in today’s leather processing. Thus, the
upgrading of leather that presents these defects is one of the most value adding
opportunities for a tanner.
3.1.1 Veins
Although the term veiny is widely accepted among tanners, the term “prominent blood
vessels”, which includes veins, arteries and their branches is more appropriate. A vein
can be compared to a tube which is either empty or full of blood and which is located in
the deeper or very superficial layers of the dermis. Veininess was firstly believed to be
caused by an improper bleeding of the animal after death, followed by coagulation of
animal blood in the blood vessels of the skin (Orthmann and Higby, 1929). Later on,
many more causes were believed to exert an influence on the appearance of veins: the
age, diet and breed of the animal, the climate, the period of slaughter, the preservation
method, the manufacturing process, etc. Belly and neck areas of the hide are most
likely to suffer from veininess.
Veins are very easily recognizable; they are very apparent in stock that is inspected
visually (Figure 3.1).
Figure 3.1 – Veininess in calfskin.
A significant difference in the structure of veiny and non-veiny leather can also be
observed when looking them under a scanning electron microscopy (Figure 3.2).
Fillers in the leather industry
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Figure 3.2 – Scanning electron microscope images of blue stock (a) without and (b) with visible veins. The flesh side of the hide lies on the bottom of the picture, and the grain side on the top.
3.1.2 Grain break
The grain break, also known as break of leather, is characterized by the wrinkles formed
on the surface of leather when it is bent grain inward. A fine break, characterized by the
presence of many fine wrinkles per linear inch, is more pleasing to the eye and thus
more desirable than a coarse break (Figure 3.3). Because no objective standard has
been established to evaluate the break of hides, it remains a matter of subjective
personal inspection.
Figure 3.3 – Samples of shoe upper leather showing (a) coarse break, and (b) fine break.
The break is a naturally occurring characteristic of the skin or hide, although it can be
influenced by processing. Typically, the butt has a finer break than the shoulder and
belly areas.
Frequently, grain break, pipiness and looseness are used interchangeably. These three
conditions have in common that the grain separates to some degree from the corium.
a b
a b
Chapter 3
31
The grain break, pipiness and looseness can be caused by inherent condition of the hide
or conditions associated to tannery processing.
3.2 The upgrading of veiny or coarse break leather
One common way to address this issue is by introducing a substance into the voids that
exist between the fibers of the leather. This substance is called filler or filling agent.
Filler’s objective is to give more body and substance to the leather by reducing
looseness and diminishing the appearance of veininess.
The nature of fillers has changed over time. At first, tanners used extracts of vegetable
tanning agents, barium compounds, glucose, flour and gum as fillers (Harris, 1974).
More recently, filling agents were made from conventional petroleum feedstocks, which
are becoming increasingly expensive. Therefore, the utilization of products from
renewable resources, and particularly those from waste proteins, became highly
interesting. Gelatin chemically crosslinked with glutaraldehyde was demonstrated to be
an effective filling agent, entering the loose areas of the hide and remaining attached to
the leather upon further processing (Chen et al., 2001). However, the potential toxicity
of glutaraldehyde advised against this method.
Our laboratory at the Eastern Regional Research Center (ERRC) has been working over
the last several years in the modification of various waste proteins with the enzyme
microbial transglutaminase (mTGase), with the goal of obtaining a product that could be
used as a filler. Unlike glutaraldehyde, mTGase is a nontoxic and food grade protein
crosslinker (see section 3.5.2). Gelatin, either commercial or experimental obtained
from tannery waste, whey, whey protein isolate (WPI) and sodium caseinate were
demonstrated to be reactive substrates towards the enzyme mTGase, and their reacted
products could be effectively used as potential fillers for leather (Taylor et al., 2001;
2002; 2003; 2004; 2005; 2006a; 2006b; 2007). These proteins, used alone or in
combination, were effectively bound to the leather and not significantly removed upon
further processing. In addition, the mechanical properties of the finished leather were
not adversely affected by the filling treatment (Taylor et al., 2008). Current prices of
sodium caseinate ($5.8/lb) and gelatin ($2.6/lb) emphasized the need for further
research into cheaper sources of protein to generate fillers for leather, like the less
expensive whey ($0.31/lb) or WPI ($1.05/lb) (USDA Agricultural Marketing Service).
Fillers in the leather industry
32
Other technologies to upgrade the quality of finished leather have also been
investigated. A recent publication reported the use of polymeric micro-spheres that
penetrate selectively into the loose areas of the leather to subsequently expand by the
application of saturated steam (Tegtmeyer et al., 2007).
Next, a brief description of the products used in the preparation of new fillers for leather
(whey protein isolate, gelatin and microbial transglutaminase) is given (sections 3.3 to
3.5).
3.3 Whey and whey products
3.3.1 Description and characterization
Whey is a liquid that separates from clotted milk during manufacture of cheese and after
coagulation of the caseins at pH 4.6 and 20 °C (Eigel et al., 1984).
The majority component in whey is lactose (70-72%), followed by minerals (12-15%)
and whey proteins (8-10%). The whey protein fraction can be selectively or totally
removed from raw whey and concentrated by using various membrane processes (e.g.
diafiltration, electrodialysis, nanofiltration) or ion exchange columns (Figure 3.4).
Membrane processing of whey is more cost effective than ion exchange technology, but
it also yields a whey concentrate with a higher content of fat and less heat stability than
that obtained using the ion exchange columns.
Figure 3.4 – Processing of whey protein isolate (U.S. Dairy Export Council).
Chapter 3
33
Depending on the removal of non-protein constituents, whey products may be classified
as whey protein concentrate (WPC) or whey protein isolate (WPI). The composition of
these products is shown in Table 3.1.
Table 3.1 – Composition of different whey products (Jelen, 2002)
Product type Total protein (%) Lactose (%) Minerals (%)
Whey powder 12.5 73.5 8.5
Whey protein concentrate (WPC) 65.0-80.0 4.0-21.0 3.0-5.0
Whey protein isolate (WPI) 88.0-92.0 <1 2.0-3.0
Next, a brief description of the main constituent proteins in whey and whey products is
given.
Beta-lactoglobulin (�-Lg) is the most abundant of the whey proteins. It comprises 10%
of the total milk protein or about 58% of the whey protein. It has about 15, 43 and 47%
�-helix, �-sheet, and unordered structure, respectively (Kinsella, 1984).
The structure of �-Lg is pH and temperature dependant. Above its isoelectric point (pH
= 5.2), the protein exists as a dimer with a molecular weight of 36.7 kDa. However,
above pH 7.5 and below pH 3.5, the dimer dissociates to its monomeric form. Between
pH 3.5 and 5.2 the dimer polymerizes to a 147 kDa octomer (Cayot and Lorient, 1997).
The conformation of �-Lg can also be modified by temperatures above 65 °C, at which
the protein undergoes denaturation accompanied by conformational transitions that
expose highly reactive �-NH2 groups (Kinsella, 1984).
Alpha-lactalbumin (�-La) is the second most abundant protein in whey. It comprises
2% of the total milk protein which is about 13% of the total whey protein. A single
polypeptide chain contains 123 amino acid residues, which include eight cysteines
covalently linked by four disulfide bonds. The protein is arranged in a spherical and
highly compact structure that undergoes thermal denaturation at approximately 62 to 65
°C, depending on the pH (Rüegg et al., 1977; de Wit and Swinkels, 1980). Due to an
unusually high apparent heat resistance, �-La is extensively renatured upon cooling.
Fillers in the leather industry
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Bovine serum albumin (BSA) carries insoluble fatty acids in the blood circulatory
system, which in turn further stabilizes the protein molecule against heat denaturation
(de Wit, 1998).
Immunoglobulins (Ig) refer to four classes of glycoproteins ranging in size from 15 to
1,000 kDa: IgG1 and IgG2, IgA, IgM and IgE. These proteins consist of two 20 kDa
polypeptide chains and two 50 to 70 kDa polypeptide chains which are crosslinked by
disulfide bonds.
Lactoferrin (LF) is a 78 kDa glycoprotein consisting of a single polypeptide chain
linked to two glycans by N-glycosidic linkages. Its average concentration in cow’s milk
is 10 mg/ml but LF is found in higher concentration in whey protein products. LF is not
only a source of amino acids but also a regulatory factor with broad biological roles.
Table 3.2 summarizes the most relevant physicochemical properties of the proteins
contained in whey.
Table 3.2 – Summary of properties of the basic constituent proteins in whey (Eigel et al., 1984)
Property �-Lg �-La BSA Ig
Isoelectric point 5.2 4.2-4.5 4.7-4.9 5.5-8.3
Concentration in whey, g/l 2-4 0.6-1.7 0.4 0.4-1.0
Concentration in whey, % (w/w) 56-60 18-24 6-12 6-12
Molecular weight, Da 18,400 14,000 66,000 �146,000
Total amino acid residues/mol 162 123 582 NA
Cysteine residues/mol 5 8 35 NA
Disulfide residues/mol 2 4 17 NA
Sulfhydryl residues/mol 1 0 1 NA
Lysine residues/mol 15 12 59 NA
Glutamic acid residues/mol 16 8 59 NA
Chapter 3
35
The composition of WPI is shown in Table 3.3.
Table 3.3 – Composition of whey protein isolate (Morr and Foegeding, 1990)
Parameter Range Mean ± S.D.
Moisture 2.40-5.57 3.75 ± 1.34
Protein 88.6-92.7 91.0 ± 1.73
�-Lg 67.6-74.8 70.2 ± 3.3
�-La 8.3-17.5 14.3 ± 4.3
BSA 7.2-10.9 8.6 ± 1.6
Ig 5.9-7.5 6.9 ± 0.7
Lactose 0.42-0.46 0.44 ± 0.02
Total lipids 0.39-0.67 0.57 ± 0.13
Ash 1.37-2.15 1.82 ± 0.33
n=2.
3.3.2 Uses of whey
The United States is the world’s largest whey exporter; latest available figures from the
U.S. Dairy Export Council (USDEC, 2006) show an increase in exportation by 104%
between 2001 and 2006. Whey used to be considered a bothersome byproduct of the
cheese manufacture because of the high value of biological oxygen demand (40,000
mg/kg). Therefore, it constituted a major ecological issue if disposed off as a waste
material. However, the image of whey is changing rapidly and becoming a highly
priced resource which may be used within the same dairy installation or destined
somewhere else for other applications (e.g. low-cost additive to animal feed).
Due to its balanced amino acid profile and nutritious value, the main use of whey and
whey products fall in the food industry. In fact, whey is one of the preferred sources of
protein for bodybuilders. Whey can also be used to fortify a wide variety of food in
order to enhance its nutritional quality and functional attributes. Researchers at the
ERRC developed texturized whey proteins that can be used to increase up to 35% the
protein content in foods such as breakfast cereals, corn puffs or cheese curls (Onwulata
et al., 2001). (Figure 3.5).
Fillers in the leather industry
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Figure 3.5 – Snack fortified with whey protein isolate.
Also, various non-food uses for whey have been explored, such as incorporation into
foamed insulation materials for the construction industry or use as a binder for iron ore
pellets (Chambers and Ferretti, 1979). Currently, the conversion of whey into valuable
materials such as biosorbents, biopolymers, fat substitute or bacteriocins is being
thoroughly examined.
3.4 Gelatin
Gelatin is obtained by the partial hydrolysis of collagen, which is the main protein in
skin, bones, hides and white connective tissues of the animal body. Gelatin does not
exist in nature and is classified as a derived protein because it is obtained from collagen
by a controlled partial hydrolysis. This hydrolytic process may be carried out with acid,
alkali or enzymes and in all cases entails the partial separation and rupture of the three
polypeptide chains of the triple-helix of the parent collagen (Figure 3.6). The degree of
hydrolysis attained leads to gelatin of various molecular weights, which may range from
20 to 250 kDa, with an average molecular weight between 50 and 70 kDa.
Figure 3.6 – Transformation of collagen into gelatin in the course of hydrolysis. Triple helix collagen molecules break off into random coiled structured gelatin.
Hydrolysis
Chapter 3
37
Gelatin is classified as type A or B. The former is produced by acid processing of
collagenous raw materials, typically pigskin. The latter is produced by means of an
alkaline or lime process, and cattle hides or bones are the most common starting
material.
3.4.1 Description and characterization
The physical and chemical properties of gelatin depend on factors such as the source of
collagen, the method of manufacture, the extraction conditions, the pH, and the
chemical nature of additives or impurities. Table 3.4 summarizes the most relevant
properties for these two types of gelatin.
Table 3.4 – Physicochemical properties of types A and B of gelatin (Glicksman, 1969)
Property Type A Gelatin Type B Gelatin
Moisture (%) 8-12 8-12
pH 3.8-5.5 5.0-7.5
Isoelectric point 7.0-9.0 4.7-5.1
Bloom strength (g) 50-300 50-275
Viscosity (mps) 20-70 20-75
Ash (%) 0.3 0.5-2.0
Cystine (%) 0.1 None-trace
Lysine (%) 11.3-11.7 11.1-11.4
Glutamic acid (%) 4.1-5.2 4.5-4.6
The capability to form heat-reversible gels is one of the most useful and unique
properties of gelatin solutions. The gelation process is believed to proceed through
three stages: first, the individual molecular chains are rearranged in a helical structure.
Next, crystallites are formed by the union of two or three ordered segments. Finally, the
formed structure is stabilized by lateral interchain hydrogen bonding within the helical
regions (Von Hippel, 1967).
The bloom strength gives an indication of the strength of a gel formed from a gelatin
solution of known concentration. The strength of the gel is function of the
concentration, the intrinsic strength of the gelatin sample, the pH, the temperature, and
Fillers in the leather industry
38
the presence of additives. Also, the bloom strength is proportional to the average
molecular weight (Table 3.5).
Table 3.5 – Dependence of molecular weight on the bloom strength of type B gelatin
Bloom strength Average molecular weight (kDa)
50 – 125 (Low Bloom) 20 – 25
175 – 225 (Medium Bloom) 40 – 50
225 – 325 (High Bloom) 50 – 100
Source: Sigma Gelatin data sheet (1996).
Aside from gel strength, there are other relevant physical and chemical properties to
characterize gelatins.
• Viscosity. Gelatin exhibits Newtonian behavior above 40 °C, and non-
Newtonian at temperatures between 30 and 40 °C. Viscosity of aqueous
solution of gelatin increases with gelatin concentration and decreasing
temperatures. Viscosity is also affected by the same factors as those mentioned
for gel strength.
• Solubility. Gelatin is soluble in water and in most highly polar organic solvents,
and practically insoluble in less polar solvents. The ratio of exothermic solvent
absorption is characteristic of each particular gelatin.
• Amphoteric character. The hydrolysis of collagen creates terminal amino and
carboxyl groups that make gelatin positively charged in strong acidic solutions
and negatively charged in a strong alkaline environment. This important
characteristic allows gelatin to be reactive with a wide range of substances.
• Swelling. This property is important in the solvation capacity of gelatin. The
rate of swelling is affected by factors such as pH, temperature, time and the
presence of electrolytes. Swelling is an essential characteristic to consider if
gelatin is intended to be used in photographic film processing or as
pharmaceutical capsules coating.
• Stability. Even though dry gelatin has a shelf life of many years if stored at
room temperature, it decomposes above 100 °C. In addition, aqueous solutions
of gelatin are highly susceptible to bacterial activity and breakdown by
proteolytic enzymes.
Chapter 3
39
3.4.2 Uses of gelatin
Gelatin has a wide variety of applications, mainly in the food, pharmaceutical and
photographic industries. Food applications are centered on edible products such as
candy, marshmallow, pies, ice cream, wafers, etc. The addition of small amounts of
gelatin during the manufacture of these products contributes in preventing the
crystallization of sugar or ice, inhibiting water separation, increasing the viscosity or
stabilizing the foam. Most edible gelatin is type A, but type B is also used.
Type A and B gelatins are also formulated in the manufacture of soft and hard capsules
for the pharmaceutical company. In addition, the use of gelatin as coating agents for
tablets is becoming more predominant due to the extra protection that provides against
medication adulteration.
Gelatin also has a long tradition as a binder in light sensitive products, particularly in
the manufacture of photographic film. Over the last 40 years, the photographic industry
has progressively switched from hide-derived gelatin to that obtained from bones.
3.4.3 Gelatin in the leather industry
Interestingly, gelatin can also be isolated from chrome shavings. Shavings are small
pieces of leather shaved off when the thickness of wet or dry tanned leather is rendered
uniform by a bladed cylinder. Therefore, this treatment can add value by using a
cleaner production pathway to a valuable product extracted from what was previously
hazardous waste.
The conversion of this waste material into gelatin can be done with enzymes (Cabeza et
al., 1997) or by means of classic hydrolysis (Catalina et al., 2007) (Figure 3.7). Gelatin
obtained through this technology was reported to have a potential application in
microencapsulation (Cabeza et al., 1999) and finishing agents for leather (Catalina et
al., 2007).
Fillers in the leather industry
40
Figure 3.7 – Conversion of chrome shavings into gelatin.
3.5 Enzymes
Enzymes are proteins capable of catalyzing (i.e. accelerating) a reaction in which
various substrates are converted to products through the formation of an intermediate
enzyme-substrate complex. The objective is accomplished by lowering the energy
required to reach the substrate transition state (Figure 3.8).
Figure 3.8 – Role of an enzyme in a given reaction.
Chapter 3
41
As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do
they alter the equilibrium of these reactions. However, enzymes do differ from most
other catalysts by being much more specific. The specific action of an enzyme with a
single substrate can be explained using a lock and key analogy. In this analogy, the
lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate)
fits into the key hole (active site) of the lock (enzyme). Smaller keys, larger keys, or
incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do
not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock
(Figure 3.9).
Figure 3.9 – Lock and key analogy for enzymes and substrate, first postulated in 1894 by Emil Fischer.
The activity of an enzyme depends on various factors: temperature, pH, concentration of
substrates and reactants, diffusion of the site of activity, and presence of certain
substances such as ions, inhibitors (molecules that decrease enzyme activity) or
activators (molecules that enhance enzyme activity).
It is important to point out the difference between enzyme activity and enzyme
concentration. The former may be measured as units of change in a specified substrate
over a period of time. The latter is defined as the quantity of enzyme present in the
sample. Therefore, doubling the concentration of enzyme does not entail doubling its
activity in any case.
Fillers in the leather industry
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3.5.1 Enzymes in the leather industry
Enzymes have been around in the leather industry for a long time and are used at
different stages of the beamhouse. Proteolytic enzymes are used in soaking, liming and
bating, with the mission of opening up the fibers, increasing area yield, improving the
cleanliness of hides and mitigate the problem of fine or short hair (Alexander, 1988).
Alkaline serine protease isolated from Aspergillus tamari was recently reported to be an
effective dehairing agent which eliminates the nuisance of sulfide in tannery effluent
disposal (Anandan et al., 2007). However, one has to bear in mind that the use of
excessive amounts of enzymes may lead to undesirible effects such as loose leather and
abraded grain.
Enzymes have been applied with success in gelatin isolation from chrome shavings also.
Pepsin and trypsin were reported to be the most promising enzymes on the isolation of
high quality products from tannery wastes (Cabeza et al., 1997). However, cost was a
major deterrent to using research grade enzymes. In 2000, Taylor et al. found a
commercial trypsin preparation that proved to be not only efficient in solubilizing the
shavings but also cost effective.
3.5.2 Microbial transglutaminase
Protein-glutamine: amine �-glutamyl-transferase (EC 2.3.2.13), commonly known as
transglutaminase (TGase) (Webb, 1992), is an enzyme capable of forming inter- or
intra-molecular crosslinks in many proteins. The enzyme catalyzes an acyl transfer
reaction between the �-carboxamide group of peptite-bound glutamine residues as acyl
donors and primary amines as acceptors. When the �-amino group of peptide-bound
lysine acts as acyl acceptor, an �-(�-glutamyl) lysine crosslink is formed.
In the absence of amine substrates, TGase can catalyze the deamidation of glutamine
residues during which water molecules are the acyl acceptors.
Chapter 3
43
Transglutaminase can also catalyze an acyl transfer reaction between the �-carboxamide
group of peptite-bound glutamine residues and various primary amines.
Transglutaminase’s activity depends strongly on pH and temperature, regardless of the
substrate. Optimum conditions of pH and temperature are 7 and 50 °C, respectively.
Figure 3.10 – (a) Temperature and (b) pH activity profiles of microbial transglutaminase (Source: Ajinomoto, Inc.).
Although the earliest papers that reported on TGase were released in the late 1950’s, it
was not until the 1980’s that research on this enzyme and particularly its reactivity
towards food proteins arose. By that time, TGase was isolated from mammalian
systems such as pig liver or plasma coagulation and its activity was found to be
dependent on the availability of calcium ions (Folk, 1980). The unfeasibility to produce
large amounts of TGase triggered off the research to find a more cost-effective way to
produce the enzyme. In 1989, Ando et al. were the first investigators to isolate TGase
from a microorganism, the Streptoverticillium mobaraense. The advantages of this new
microbial transglutaminase (mTGase) over its predecessor were tremendous:
inexpensive, readily available, broad specificity and an activity independent of calcium
ion concentration. Ajinomoto Co., Inc. and Amano Pharmaceutical Co. Ltd. jointly
granted a patent shortly
a b
Fillers in the leather industry
44
after that discovery and began to produce the enzyme in an industrial scale. Later on,
the enzyme was also identified in Physarum polycephalum (Klein et al., 1992), fish
(Yasueda et al., 1994) and plants (Pallavicini et al., 1992).
After the enzyme became readily available and economical, the industry started to show
interest on the ability of the enzyme to modify proteins functionality. In the 1990’s, the
literature exploded with scientific papers reporting the effects of mTGase on food
proteins.
Table 3.6 lists examples of various food proteins that were successfully treated with
TGase or mTGase and the effect exerted on their functional properties.
Table 3.6 – List of proteinaceous substrates reported to be reactive towards TGase
Substrate Effect of transglutaminase Reference
Gelatin, caseinate, soy protein isolate, egg yolk • Increase in breaking strength of gels Sakamoto et al.,
1994
Caseinate • Increase of gel forming ability,
modification of breaking strength, strain and cohesiveness of the gels formed.
Nonaka et al., 1992
Whey protein + Soybean 11S globulin
• Modification of heat stability, emulsifying properties and foaming capacity and stability of formed biopolymers.
• Obtaining of films with better mechanical properties and more resistance to solubilization.
Yildirim et al., 1996, 1997, 1998
Soy protein
• Increase in solubility, decrease in surface hydrophobicity, improvement of emulsifying and foaming properties, reduction of bitterness.
Babiker et al., 1996a
Gluten • Improvement of solubility, foaming,
emulsifying and surface functional properties.
Babiker et al., 1996b
�-lactoglobulin • Enhancement of heat stability, increase of viscosity and gel forming ability
Tanimoto and Kinsella, 1988
Chapter 3
45
3.5.3. Reactivity of TGase with gelatin and whey proteins
The ultimate goal of enzymatic modification of proteins is to improve their
functionality. Protein functional properties can be defined as “physico-chemical
properties that influence the structure, appearance, texture, viscosity, mouthfeel, or
flavor retention of the product” (Morr and Ha, 1993).
Gelatin is a reactive substrate towards the enzymatic action of mTGase, meaning that
the reaction will take place under the right conditions of pH, temperature and substrate
and enzyme concentration (Figure 3.11).
Figure 3.11 – Cross-linking of gelatin chains upon reaction with mTGase.
Sakamoto et al. (1994) studied the optimum reaction conditions of mTGase with gelatin
and found that an incubation period of 4 hours with 30 units mTGase/g gelatin, at pH 6
and 50 °C yielded gels with higher breaking strengths (Figure 3.12).
Fillers in the leather industry
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Figure 3.12 – Effect of (a) mTGase concentration, (b) incubation time, (c) pH and (d) incubation temperature on breaking strength of a 10% (w/w) type A gelatin (Sakamoto et al., 1994).
All kind of gelatins are susceptible to undergo enzymatic modification: pigskin gelatin,
both types A and B (McDermott et al., 2004; de Carvalho and Grosso, 2004), and fish
gelatin, from skin and bones of cod, haddock, and pollock (Yi et al., 2006) or the skin
of bigeye snapper and brownstripe red snapper (Jongjareonrak et al., 2006).
There are proteins that need to be treated prior to undergo the enzymatic reaction with
mTGase. This is the case of the two most abundant proteins in WPI, �-Lg and �-La.
As opposed to gelatin, the globular structure of these two proteins causes the reactive
groups to be buried and therefore inaccessible to the action of the enzyme. A less
globular structure of these proteins can be achieved by undergoing a denaturation
process, which enhances their reactivity towards the enzyme (Færgemand et al., 1997).
Denaturation of whey proteins can be achieved using different methods. The use of a
reductant or the application of heat are among the two most common. DTT is generally
(a) (b)
(c) (d)
Chapter 3
47
the reductant of choice for cleaving disulfide bonds given that it is needed in much
lower concentration than other reductants (e.g., glutathione, cysteine) to complete the
reaction (Grazú et al., 2003). DTT breaks down the disulfide bond by two sequential
thiol-disulfide exchange reactions, resulting in DTT becoming a six-member ring
structure. (Figure 3.13).
Figure 3.13 – Reduction of a disulfide bond by two thiol-disulfide exchange reactions involving DTT.
Whey proteins can also be denatured by the action of heat. Upon heating, whey
proteins undergo a conformational transition that unfolds their structures making the
reactive groups available. As an example, sulfhydryl groups react with the disulfide
bonds in a thiol-disulfide exchange reaction. This reaction, along with interactions of
other nature (e.g. electrostatic, hydrophobic) are responsible for the aggregation of the
thermally denatured whey proteins, which may result in the formation of gels.
Even though the apparent order of denaturation of individual whey proteins is Ig > BSA
> �-Lg > �-La, their rates of denaturation are strongly influenced by factors such as pH,
ionic composition, or total solids concentration.
CHAPTER 4
Mathematical model of raw hide curing with brine
Chapter 4
49
4. MATHEMATICAL MODEL OF RAW HIDE CURING WITH BRINE
Authors: Eduard Hernàndez Balada1,2, William N. Marmer1, Karel Kolomazník3, Peter
H. Cooke1 and Robert L. Dudley1.
1U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA
2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain
3Tomas Bata University, Mostni 5139, 760 01 Zlín, Czech Republic
Manuscript published at the Journal of the American Leather Chemists´Association
(JALCA), Vol. 103 (5), 167-204, 2008.
Mathematical model of raw hide curing with brine
50
4.1 Letter of acceptance
THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS
ASSOCIATION c/o The American Leather Chemists Association, 1314 50th Street, Suite 103,
Lubbock, Texas 79412 Mobile phone: (616) 540-2469 E-mail: [email protected] November 19, 2007 Dr. William N. Marmer USDA, Agricultural Research Service Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor, PA 19038 Dear Dr. Marmer, This will acknowledge receipt and acceptance for publication of the manuscript entitled “Mathematical Model of Raw Hide Curing with Brine”. It is tentatively scheduled for publication in the May 2008 issue of the Journal. The manuscript now lists you as the corresponding author. As instructed in your cover letter of November 14, 2007, I will change the manuscript to footnote Dr. Hernàndez as the corresponding author. Could you please sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution. Sincerely yours, Robert F. White Journal Editor cc: Dr. Eduard Hernàndez Balada
Chapter 4
51
4.2 Abstract
The most common method of preserving raw hides is brine curing with sodium
chloride. However, this process has three important disadvantages: first, the length of
time that it takes, which is a minimum of 18 hours; second, the insufficient degree of
curing reached in some hides due to an overload and possibly the low efficiency of the
brine raceway; and finally, the environmental impact associated with the discharge of
large quantities of electrolytes in the soaking step. Our long term goal is to address all
three issues. Initially, we have carried out a study of the salt uptake and its diffusion
mechanism in order to attempt a reduction in the curing time. A continuous reaction
mathematical model of a closed one dimensional system that describes the diffusion of
sodium chloride in the hide during the curing process was chosen in the search for the
optimum brine curing conditions such as the optimum brine concentration and percent
float. The effect of these two parameters on the values of transport coefficient � was
reported. Brine diffusion into the hide was tracked by measurement of the chloride
concentration of the residual brine solution. In addition, a piece of hide was cured with
a fluorescently labeled brine solution and analyzed by means of epifluorescent
microscopy for direct visualization of the sodium location within the hide.
Mathematical model of raw hide curing with brine
52
4.3 Resum
El mètode més comú per a preservar pells crues és el curat amb salmorra. Tot i així,
aquest procés presenta tres desavantatges: en primer lloc, el temps que requereix, un
mínim de 18 hores; en segon lloc, l’insuficient nivell de curat que s’assoleix en algunes
pells a causa de la sobrecàrrega i baixa eficàcia del tanc de curat; i finalment, l’impacte
mediambiental associat a la descàrrega d’elevades quantitats d’electròlits en l’operació
de remullat. El nostre objectiu a llarg terme és resoldre els esmentats problemes.
Inicialment, estudiem l’absorció de sal i el seu mecanisme de difusió per tal de reduir el
temps de curat. Un model matemàtic de reacció contínua d’un sistema unidimensional
tancat fou seleccionat per descriure la difusió del clorur de sodi a la pell durant el procés
de curat per tal de trobar les condicions òptimes de curat, tals com la concentració
òptima de salmorra i el percentatge òptim de bany. L’efecte d’aquests dos paràmetres
en els valors del coeficient de transport � són presentats. La difusió de sal a la pell va
ser monitoritzada mitjançant la determinació de concentració de clorurs en la solució
residual de salmorra. A més, una mostra de pell fou curada amb una solució de
salmorra etiquetada amb una substància fluorescent i analitzada mitjançant microscopia
epifluorescent per a la visualització directa del sodi en la pell.
Chapter 4
53
4.4 Introduction
Raw hides and skins are ~ 60-70% water and ~ 25-30% protein. In this form the hide is
susceptible to bacterial activity within hours after being removed from the carcass. The
autolytic degradation of skins/hides is assumed to be due to a combined action of tissue
enzymes and bacteria, the latter requiring moisture to be viable.1 Curing is the process
that provides an environment in which bacteria cannot survive. Several curing agents
have been reported in the literature, e.g., potassium chloride,2 silica gel,3,4 boric acid,5
and herbal-based products.6 Common salt, in spite of its inherent impact on the
environment and the large amount required, is the most popular and inexpensive
material used to preserve hides and skins. A suitable improved method would yield
savings in salt, shipping and effluent treatment costs as well as a diminished
environmental impact. Mathematical modeling has been reported to be a powerful tool
in the optimization of processes such as soaking of salted cattle hides.7,8 Curing has
been modeled in substrates such as cheese9,10 and meat11, but has not been studied for
the particular case of raw hides.
The aim of this work is to develop and verify a mathematical model that describes the
diffusion of sodium chloride in the hide during the curing process. Upon its
verification, the model is applied in the search of the optimum values of process
variables such as brine concentration, float percentage and curing time.
4.5 Theory
We propose a continuous reaction model to describe the diffusion of sodium chloride
from the bath containing brine solution to the surface of the solid phase (hide). The
model assumes that salt will further diffuse into the hide’s inner volume where it will
form a non-stationary concentration field (Figure 4.1). It also assumes that diffusion
takes place only into the flesh side and that hide parameters such as thickness, surface
and properties of both hair and flesh sides will remain constant throughout the whole
process.
Mathematical model of raw hide curing with brine
54
Figure 4.1 – Mathematical model of the curing process of a raw hide.
Curing can be considered as a counter diffusion in which sodium chloride soaks into the
skin as water simultaneously washes out. Equation (1) describes a non-stationary one
dimensional concentration field inside the inner volume of the solid phase, defined by
Fick’s second law. Boundary condition (1a) assumes that sodium chloride diffuses into
the hide from the flesh side only. Terms are defined at the end of the paper.
),(),(2
2
τττ
xxc
Dxc
∂∂⋅=
∂∂
bx <<0 0>τ (1)
0),0( =∂∂ τxc
(1a)
Equation (2) corresponds to a mass balance of a closed system in which salt flow at the
hide surface is equal to accumulation speed of sodium chloride in the bath. Equations
(2a) and (2b) are the initial boundary conditions (� = 0). They assume a null initial
content of sodium chloride in the fresh hide and a constant initial value of brine
concentration in the bath respectively.
)(),( 00 τ
ττ
∂∂⋅=
∂∂⋅⋅− c
Vbxc
DS (2)
0)0,( =xc (2a)
pcc 00 )0( = (2b)
Chapter 4
55
Equation (3) is valid under an ideal mass transfer from the bulk solution to the surface
of the solid phase.
)(),( 0 τετ cbc ⋅= (3)
The introduction of dimensionless parameters (equations 4a to 4e) has been
demonstrated to be a useful tool in the model development.
pc
cC
0⋅=
ε (4a)
pc
cC
0
00 = (4b)
bx
X = (4c)
2bD
Fo
τ⋅= (4d)
VV
Na 0= (4e)
The dimensionless time, also called Fourier’s number [F0], assesses the proximity of the
process to the equilibrium, i.e. equilibrium is reached when F0 � �. The dimensionless
soaking number [Na] expresses the ratio between the volumes of liquid and solid
phases. The replacement of the dimensionless parameters into the previous model leads
to a new dimensionless model (Eq. (5a) to (5f)).
),(),( 02
2
00
FXX
CFX
FC
∂∂=
∂∂
10 << X 00 >F (5a)
)(),1( 000 FCFC = (5b)
)(),1( 00
00 F
FCNa
FXC
∂∂
⋅−=∂∂
ε (5c)
0),0( 0 =∂∂
FXC
(5d)
0)0,( =XC (5e)
Mathematical model of raw hide curing with brine
56
1)0(0 =C (5f)
the analytical solution of which can be obtained by means of Laplace’s transformation:
( ) �∞
=
⋅−
⋅⋅−⋅
−⋅
⋅⋅⋅+
+=
1 )sin()sin(
)cos(
)cos(2,
20
nnn
n
nn
gFn
o
gNagg
gg
egXNa
NaNa
FXCn
εεε (6)
Where gn are the roots of the transcendent equation (7).
ε
nn
gNagtg
⋅−=)( (gn > 0) (7)
The three dimensional concentration field graphic corresponding to Eq. (6) is shown in
Figure 4.2.
Figure 4.2 – Dimensionless sodium chloride concentration field within the hide during the curing process.
Chapter 4
57
Replacing Eq. (7) into Eq. (6) and rearranging terms (for X = 1), we obtain an equation
that illustrates the variation of brine concentration with time.
( ) �∞
=
⋅−
⋅++
⋅++
=1
220
20
2n n
gF
o gNaNa
eNa
NaNa
FCn
εεε
(8)
In addition, integration of Eq. (6) leads to Eq. (9) which calculates the optimal time in
order to reach a certain content of sodium chloride in the skin, C (integral average
concentration).
�∞
=
⋅−
⋅+⋅+⋅−
+=
1222
2
20
2)(n n
gF
o gNaNae
NaNa
NaFC
n
εεε (9)
Figure 4.3 shows the curves of the integral average concentration for various values of
soaking number.
Figure 4.3 – Dimensional sodium chloride concentration field within the hide during the curing process for various soaking numbers.
Mathematical model of raw hide curing with brine
58
Determination of Diffusion Coefficients
The value of effective diffusion coefficient of sodium chloride in the hide can be
evaluated from experimental data. Crank12 suggested an equation for the diffusion
coefficients study at short times:
τλπ
ττ ⋅⋅+⋅=∞−
−=
NaNa
cccc
CP
P 12)()(
)(00
000 (10)
From the mass balance
VcVcVc p ⋅⋅+⋅=⋅ ∞∞ 00000 ε (11)
We get
ε+⋅=∞ NaNac
c P00 (12)
Transport parameter � is defined as a ratio of the effective diffusion coefficient D’ to
pore half length (a) square.
22
'aD
bD ==λ (13)
when the factor for the tortuosity of the pores:
ba=ξ (14)
� is an important value from an engineering point of view since it includes two
phenomena not considered in the presented model, which are the transport of water
from the hide to the bath and the interaction between sodium chloride and water counter
flows during the curing.
Chapter 4
59
4.6 Experimental
4.6.1 Materials
Fresh cow hides were purchased from a local abattoir. They were soaked for 2 h (with
surfactant) and then fleshed. Approximately 6 × 10 in (15 × 25 cm) pieces were cut and
stored at -20°C. They were thawed at 4°C just before use. Food grade sodium chloride
of purity minimum 99.82% was obtained from US Salt Corporation (Watkins Glen,
NY). All other chemicals were reagent grade and used as received.
4.6.2 Methods
Thawed hide was cut into square pieces of approximately 4 × 4 in (10 × 10 cm) with an
average weight of ~ 100 g. They were transferred to a Dose drum (Model PFI 300-34,
Dose Maschinenbau GmbH, Lichtenau, Germany), and tumbled at 6 rpm with brine
solution for varying time intervals after which they were pulled out of the drum, hand-
squeezed to wipe excess water, sealed in plastic bags and placed in the refrigerator. A
fraction of the residual brine solution was also collected at different time intervals. Two
sets of experiments were carried out: a constant 300% (v/v) float (volume of brine
solution/volume of hide) at different initial salt concentration levels (20%, 25%, 30%
and 35% (w/v), which correspond to 64, 80, 96 and 100 °SAL, respectively) and a
constant 30% (w/v) initial salt concentration (weight of NaCl/volume of solution) at
different float percentages (300%, 500%, 750% and 1000% v/v). Density of hide was
assumed to be ~ 1g/cm3.
4.6.3 Analyses
4.6.3.1 Chloride concentration determination
Chloride concentration was determined by classical Mohr titration.13 Residual brine
samples were diluted (1:100 v/v) in nano pure water prior to titration. All samples were
run in triplicate.
4.6.3.2 Fluorescence imaging
CoroNa ™ Green Sodium Indicator fluorescent dye (Invitrogen, Carlsbad, CA) was
used as a probe of sodium ions diffusing into raw hide from brine solution. A piece of
raw hide of approximately 1 × 1 in (2.5 × 2.5 cm) was immersed in a beaker containing
500% v/v float of a 30% w/v NaCl solution and 5µM of the fluorescent dye and gently
Mathematical model of raw hide curing with brine
60
agitated. A 2-3 mm wide slice of the sample was excised manually with a stainless steel
razor blade (cutting from flesh surface toward the grain) at regular intervals of
incubation time, then mounted onto Petri dishes for imaging, using a Leica MZ FLIII
stereomicroscope (Leica Microsystems, Bannockburn, IL, USA) equipped for
epifluorescence and with a model DC200 color charge couple device camera system at
2.5x magnification. Samples were irradiated with blue (470/40 nm) and UV (360/40
nm) light, and images of the fluorescence were acquired at 0.1 (blue) and 0.44 (UV)
seconds of exposure time. Control samples, immersed in 500% v/v nano-pure water
and 5µM dye, were examined under the same conditions of concentration and time to
assess the penetration of the fluorescent dye in the absence of salt, and a blank sample
was also examined to evaluate possible autofluorescence of the untreated raw hide.
4.7 Results and discussion
4.7.1 Epifluorescence microscopy
The diffusion of labeled sodium ions into the cross section of hide samples was
followed by means of epi-fluorescent microscopy. In Figure 4.4, increased fluorescence
indicating diffusion of salt started on the flesh side and gradually moved toward the hair
side.
Figure 4.4 – Epifluorescent microscopic images of a cross section of a hide at different stages of curing. The hide was cured with 30% (w/v) labeled sodium chloride solution.
15min 30min 1h 2h 3h 4h 5h 28h 48h
Flesh
Hair
15min 30min 1h 2h 3h 4h 5h 28h 48h
Flesh
Hair
Flesh
Hair
Chapter 4
61
The lack of fluorescence development at the hair side validated the mathematical
assumption described by Eq. (1a); this can be attributed to the existence of a thin
protective barrier of sebaceous oil.14 The series of images demonstrate the advance of
fluorescence due to sodium ions into the cross sections of hide as well as increases in
fluorescence intensity throughout curing time. Signal saturation in the area near the
flesh side, denoted by a very bright fluorescence, was observed after 5 h of curing. The
apparent retrograde movement of the labeled sodium between 2 h and 5 h may be due to
the shrinking of the hide caused by dehydration. Surprisingly, sodium ions did not
seem to reach the hair side even after 48 h of curing. This could be due to many factors.
In order to determine if the penetrability of the dye is a technical factor, a sample of
hide incubated in an aqueous solution of dye for 24 hours was examined under the
microscope and then transferred to a beaker with concentrated brine solution for 24
more hours. The dye did not fluoresce in the uncured sample but showed a high
fluorescence after being cured for 24 hours (graphs not shown). However, fluorescence
was absent or undetectable in the upper part of the corium, leading to the possibility that
the dye may not penetrate into the tightly-woven and dense structure of the corium,
possibly due to its size (MW = 586 Da). The use of scanning electron microscopy with
energy dispersive X-Ray spectroscopy (SEM-EDS) and elemental mapping to measure
the amount and location of salt in a brine-cured hide is an alternative method to
fluorescence imaging and this approach is planned.
4.7.2 Determination of diffusion coefficients
The diffusion of salt in the hide was evaluated by means of the transport coefficient �,
which can be calculated from the slope of the straight line that results from plotting
C0(t) versus square root of time (Eq. 10). Taking into account early published results12
and the accuracy of our measurements, the linear dependence holds approximately as
far as to the value of C0(t) = 0.6. Figure 4.5 depicts this correlation for the particular
case of c0p = 30% (w/v) and Na = 3.
Mathematical model of raw hide curing with brine
62
Figure 4.5 – Determination of transport parameter � from experimental data. The graph corresponds to c0p = 30% (w/v) and Na = 3.
As seen in Table 4.1, all � values, except from that of c0p=35% (w/v), are on the order of
10-5 s-1. These results are of the same order of magnitude as those reported in the
mathematical model of soaking15, which suggests that the diffusion of salt does not
significantly differ between curing and soaking. A numerical value of � for c0p=35%
(w/v) may not be reliable, because the brine was initially supersaturated and the model
was developed for homogeneous solutions solely. Note than a saturated brine solution
holds 31.7g of salt in 100 ml of solution, (c0n) at 25°C.16
TABLE 4.1 Transport coefficient � for various conditions of initial brine concentration (c0p) and
soaking number (Na) Na = 3 C0p = 30% (w/v)
C0p (% w/v) �·105 (s-1) R2 C0p (% w/v) �·105 (s-1) R2
20 4.2 0.835 3 5.3 0.949
25 3.8 0.921 5 9.0 0.887
30 5.3 0.949 7.5 4.1 0.905
35 10.7 0.776a 10 8.6 0.876 aR2 < critical value for � = 0.05.
A comparison of the individual values of � is not simple, since they may be affected by
some of the following factors: 1. The thickness of the hide, which may vary throughout
the process and exerts a strong effect on the value of �, as seen in Eq. (13). 2. The pore
length, which varies among the hides and is hardly measurable. 3. The dry matter
content of the hide, the variation of which may extensively modify �. In fact, � may
drop up to two orders of magnitude between a wet and a dry hide.15,17 4. The
Chapter 4
63
temperature, which affects the diffusion rate and was sometimes difficult to keep at
25°C during the process. 5. The influence of the error in the measurement, i.e. the
difficulty to measure a small chloride concentration diminution with the Mohr method
despite the very low coefficient of variation (CV) found for this technique (< 1%).
Even so, one can draw the conclusion on the effect that both c0p and Na exert on the
values of �. Increasing values of c0p yielded larger values of � as a consequence of an
increasing gradient concentration between the solid phase and the solution, which is in
accordance with Fick’s second law of diffusion. This fact corroborates a general
practice applied in curing raceways, where solid salt is periodically added to the brine
solution to keep it close to saturation (� 97 °SAL). The float percentage also exerts a
remarkable effect on the values of transport parameter �. Larger floats yielded faster
diffusion of salt into the hide, even though that effect became less significant for Na >
5. That experimental observation corroborates the common practice in tanneries, which
operate at Na ~ 4 even though the generally accepted rule requires a Na � 5 in order for
hides to receive a proper cure.18 In addition, a large float will help maintain an almost
constant salt concentration. The outstandingly low value of � obtained for Na = 7.5
may be due to factors inherent to the hide, e.g., poor fleshing, which slows down salt
penetration, agglutination of the fibers and content of dry matter.
4.7.3 Determination of optimum brine curing conditions
An 85% salt saturation of the water remaining in the hide was established as a minimum
standard in order to attain a proper degree of cure.19 One can calculate the theoretical
minimum soaking number needed to attain this saturation percentage in the equilibrium,
that is, at infinite time, and without further additions of salt into the solution. From Eq.
(8), if � � then Fo � �; thus the second summand can be neglected, giving:
Na
Nacc
Cop +
==ε
00 (15)
Replacing 0cc ⋅= ε and rearranging for Na,
Mathematical model of raw hide curing with brine
64
cc
cNa
p −⋅⋅=
0εε
(16)
Using ncc 085.0 ⋅⋅= ε , a porosity of � = 0.5 and c0p = 30 % (w/v), and assuming that all
pores are filled up with brine solution, a soaking number of 4.4 is needed to reach 85%
saturation. Solving Eq. (16) for c0p = 20 and 25% (w/v), negative values of Na are
obtained, indicating the unfeasibility to attain 85% saturation. On the other hand, the
minimum soaking number would drop to 2.8 if the cure was started out with a saturated
brine solution (31.7 % (w/v), or 100 °SAL). Notice that these values depend on the
porosity of the hide, which varies from one to another and within itself.20 Therefore,
slightly different values of minimum Na’s would be obtained using another value of
porosity.
Working out the value of c0p from Eq. (16) and using Na = 3, we received a minimum
initial brine concentration of 30.8% (w/v) in order to achieve the target saturation level.
By means of Eq. (9), the plot of which is depicted in Figure 3, one is able to calculate
the curing time needed to reach an 85% salt saturation in the hide. As just mentioned
above, this level cannot be achieved for c0p = 20, 25 and 30% (w/v) and Na = 3.
However, a time of 4.2 h is obtained for a 35% (w/v) supersaturated brine and Na = 3.
The 85% saturation is also achieved in 4.9, 7.7 and 3.4 h if the hide is cured with a brine
of c0p = 30% (w/v) and Na = 5, 7.5 and 10, respectively. These values are substantially
lower than the 18 hours that usually are required for a full cure in a normal float (Na~5).
The calculations of those times contain the parameter �, and therefore are affected by
the same factors mentioned in the previous section. In spite of this, it is interesting to
note the decrease of curing time with increasing float percentages, except from the
erratic value obtained for Na = 7.5.
4.8 Conclusions
Over 20 millions brine-cured hides were exported by the U.S. in 2006 (U.S. Leather
Industry Statistics, 2007). Increasing commodity prices for sodium chloride over the
past few years together with issues associated with water pollution set the alarm off in
the leather and meatpacking industries. The purpose of research reported in this article
Chapter 4
65
was to optimize the brine curing of hides and skins under specific process conditions by
means of mathematical modeling. The diffusion of salt into the hide was characterized
by the transport coefficient �, which was found to be in the order of 10-5 s-1. The usage
of saturated brine as well as large floats (>500%) yielded higher values of �, therefore
higher diffusion rates. From the model it was also possible to determine the minimum
float and initial brine concentration needed to attain an 85% salt saturation in the hide.
This saturation level was not achieved employing brines of initial concentration of 20
and 25% (w/v) independently of the float percentage used. For 30 and 35% brines, a
minimum float of ~ 440% and ~280% was found, respectively. Using a 30% (w/v)
brine, the targeted 85% saturation is attained in shorter times as the % float increases,
and one may expect the same trend for any other initial brine concentration. The
established 85% salt saturation in the hide obviously plays a critical role in the search
for optimum conditions of curing, and the need to attain this saturation level for a
proper cure will be discussed in our next contribution.
4.9 Definition of terms
a: pore half length of the skin [m]
b: thickness of cured hide [m]
c: concentration of sodium chloride in the hide moisture, at a distance x from the
boundary ( > 0) [mol m-3]
c0: concentration of sodium chloride in the bath ( > 0) [mol m-3]
c0n: concentration of saturated sodium chloride solution at 25°C [mol m-3]
c0p: initial concentration of sodium chloride in the bath ( = 0) [mol m-3]
c0�: equilibrium concentration of sodium chloride in the bath [mol m-3]
C : dimensionless concentration integral average [1]
C, C0: dimensionless concentrations [1]
D: diffusion coefficient of sodium chloride in the hide [m2 s-1]
D’: effective diffusion coefficient of sodium chloride in the hide [m2 s-1]
F0: Fourier number/dimensionless time [1]
Na: soaking number [1]
S: outer surface of the solid phase (skin) [m2]
V: volume of skin [m3]
V0: volume of brine solution [m3]
Mathematical model of raw hide curing with brine
66
X: dimensionless distance [1]
Greek symbols
�: porosity of solid state [1]
�: transport coefficient [s-1]
: time (s) 4.10 References
1. Rao, B.R., and Henrickson, L.; Short-term preservation of cattlehide. JALCA 78, 48-
53, 1983.
2. Bailey, D.G., and Gosselin, J.A.; The preservation of animal hides and skins with
potassium chloride. JALCA 91, 317-333, 1996.
3. Kanagaraj, J., Chandra Babu, N.K., Sadulla, S., Suseela Rajkumar, G., Visalakshi
V., and Chandra Kumar, N.; Cleaner techniques for the preservation of raw goat
skins. J. Clean Prod. 9, 261-268, 2001.
4. Munz, K.H.; Silicates for raw hide curing. JALCA 102, 16-21, 2007.
5. Kanagaraj, J., John Sundar, V., Muralidharan, C., and Sadulla, S.; Alternatives to
sodium chloride in prevention of skin protein degradation–a case study. J. Clean
Prod. 13, 825-831, 2005.
6. Preethi, V., Rathinasamy, V., Kannan, N., Babu, C., and Sehgal, P.K.; Azardirachta
Indica: a green material for curing of hides and skins in leather processing. JALCA
101, 266-273, 2006.
7. O’Brien, D.J.; A mathematical model for unsteady state salt diffusion from brine-
cured cattlehides. JALCA 78, 286-299, 1983.
8. Kolomazník, K., Janacova, D., Vasek, V., and Blaha, A.; Chemical engineering and
automatics control in leather technology. Advanced Technologies: Research-
Development-Application (Lalic, B. ed.) Verlag Robert Mayer – Scholz, Germany,
pp. 475-516, 2006.
9. Guinee, T.P., and Fox, P.F.; Sodium chloride and moisture changes in Romano-type
cheese during salting. J. Dairy Res. 50, 511-518, 1983.
10. Turhan, M., and Kaletunç, G.; Modeling of salt diffusion in white cheese during
long term brining. J Food Sci. 57, 1082-1085, 1992.
Chapter 4
67
11. Bertram, H.C., Holdsworth, S.J., Whittaker, A.K., and Andersen, H.J.; Salt diffusion
and distribution in meat studied by 23Na nuclear magnetic resonance imaging and
relaxometry. J. Agric. Food Chem. 53, 7814-7818, 2005.
12. Crank, J.; The mathematics of diffusion, 2nd ed. Clarendon Press, Oxford, London.
1975.
13. Quantitative Analysis, 4th ed. (Pierce, Haenisch and Sawyer eds.) John Wiley &
Sons Inc., New York, 1958.
14. Sharphouse, J.C.; Types of hides and skins and principal uses. In Leather
Technician’s Handbook (Leather Producer’s Association eds.) Northampton, UK,
pp. 20-36, 1971.
15. Blaha, A., and Kolomazník, K.; Mathematical model of soaking. Part I. J. Soc.
Leather Tech. Chem. 73, 136-140, 1988.
16. Kallenberger, W.E.; Heat, humidity and cure quality. JALCA 82, 365-371, 1987.
17. Blaha, A., Kolomazník, K., and Dederle, T.; Mathematical model of the soaking
process. Part II. J. Soc. Leather Tech. Chem. 73, 172-174, 1989.
18. Bailey, D.G.; The preservation of hides and skins. JALCA 98, 308-319, 2003.
19. Trade Practices for Proper Packer Cattlehide Delivery, 3rd ed., Leather Industries of
America and U.S. Hide, Skin & Leather Association, pp. 12-19, 1993.
20. Allsop, T.F., and Passman, A.; Porosity measurement as a means of determining the
degree of processing of lamb pelts. J. Soc. Leather Tech. Chem. 87, 49-54, 2003.
4.11 Acknowledgments
The authors would like to thank Eleanor Brown, Laurelie Bumanlag, Gary Di Maio,
Rafael García, Michael Kurantz, Joseph Lee, John Phillips, Maryann Taylor and
Michaela Uhlíová for their technical support and assistance.
CHAPTER 5
Evaluation of degreasers as brine curing additives
Chapter 5
69
5. EVALUATION OF DEGREASERS AS BRINE CURING ADDITIVES Authors: Eduard Hernàndez Balada1,2, William N. Marmer1, Peter H. Cooke1 and John
G. Phillips1.
1U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA
2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain
Manuscript accepted for publication at the Journal of the American Leather
Chemists´Association (JALCA). Tentatively scheduled for publication in the May 2009
issue.
Evaluation of degreasers as brine curing additives
70
5.1 Letter of acceptance
THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS ASSOCIATION
c/o The American Leather Chemists Association, 1314 50th Street, Suite 103, Lubbock, Texas 79412
Mobile phone: (616) 540-2469 E-mail: [email protected] November 12, 2008 Dr. William N. Marmer c/o Eastern Regional Research Center, USDA 600 East Mermaid Lane Wyndmoor, PA 19038-8598 Dear Dr. Marmer, Your manuscript "Evaluation of Degreasers as Brine Cure Additives" has been accepted for publication in JALCA. It is tentatively scheduled for the May 2009 issue. Please have the appropriate research leader sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution to JALCA. Sincerely yours, Robert F. White Journal Editor
Chapter 5
71
5.2 Abstract
The length of time needed for brine curing of raw hides and skins, a minimum of 18 h,
is a time consuming process. In this paper we initially report the results of an
investigation of the stratigraphic distribution of sodium chloride and water in fleshed
hides cured for varying intervals of time. We demonstrated that salt entered the hide
mainly from the flesh side. Water, on the other hand, was withdrawn from both sides of
the hide; the epidermis acted as a semipermeable membrane. Three commercial
degreasers as well as a glycolipid surfactant (sophorolipid) were tested as brine curing
additives and their efficiency evaluated according to the moisture, salt, salt saturation
and fat content levels in the brine cured hide. One of the commercial degreasers, when
used at 0.5% w/w, significantly removed fat from the hide as well as enhanced the
uptake of salt. The sophorolipid also was an effective degreasing agent, decreasing the
fat content of the brine-cured hide and, if used in excess, significantly increasing the
uptake of salt. The data presented here confirmed that the usage of an appropriate
degreasing agent in the brine is a suitable option for reducing the turn-around times in
raceways and thus creating additional curing capacity.
Evaluation of degreasers as brine curing additives
72
5.3 Resum
El període de temps que comporta la preservació de pells crues amb salmorra, que és
d’un mínim de 18 hores, és un important inconvenient del procés. En el present article
mostrem els resultats d’un estudi estratigràfic que descriu la distribució del clorur de
sodi i aigua en pells descarnades curades durant distints intervals de temps. Demostrem
que la sal penetra la pell majoritàriament pel costat de la carn. L’aigua, pel seu cantó,
migra cap a fora per les dues cares de la pell, amb l’epidermis actuant com una
membrana semipermeable. Tres desengreixants disponibles comercialment i un
tensioactiu glucolipídic (soforolípid) varen ser provats com a additius en la preservació
de pells amb salmorra, i la seva eficàcia fou mesurada d’acord amb els nivells d’aigua,
sal, saturació de sal i greix de la pell curada. Un dels desengreixants, emprat al 0.5%
w/w, rebaixà de forma significativa el contingut de greix a la pell, augmentant també
l’absorció de sal. El soforolípid demostrà també ser un agent desengreixant efectiu,
disminuint el contingut de greix de la pell curada, i en cas de ser emprat en excés,
augmentant també significativament l’absorció de sal. Les dades presentades en aquest
article confirmen que l’ús d’un apropiat agent desengreixador amb la salmorra és una
opció viable per tal de reduir el temps de processat de pells i per tant crear una major
capacitat de producció.
Chapter 5
73
5.4 Introduction
Curing of raw hides and skins with common salt (sodium chloride) is the most popular
preservation method used currently. Sprinkling of solid salt on the flesh surface of the
hide, known as green-salting, is extensively used in warm countries like India.
Conversely, most American and European hide processing facilities typically treat their
hides in raceways filled with a highly concentrated solution of sodium chloride (brine).
The economics of brine curing of hides and skins have been affected recently by
increasing commodity prices for sodium chloride.1 Furthermore, the large amount of
salt needed creates environmental issues when it is soaked out, contributing to 40% of
the total dissolved solids generated in the entire process of leather manufacturing.2
In our previous paper3 we developed a mathematical model that described the diffusion
of sodium chloride into the hide during the curing process. We concluded that the
usage of saturated brine as well as a minimum float of 500% yielded an optimal
diffusion rate. We also proved by means of epifluorescence microscopy that salt
diffused into the hide mainly from the flesh side, which presents a less compact
structure and greater porosity than the grain and upper corium. However, the fatty or
adipose tissue present on the flesh side was reported to retard the diffusion of salt into
the hide.4 Thus, a poor fleshing of the hide prior to the cure may further aggravate this
phenomenon.
The use of degreasing agents in the leather manufacturing process is quite widespread
and they are mainly utilized during soaking, liming and deliming to decrease the fat
content of the wet blue below the norm value.5 However, there is no literature
regarding the usage of these degreasers in the cure. If the use of a degreasing agent
along with the brine could accelerate the penetration of salt into the hide, then turn-
around times in the raceways would be reduced and thus additional curing capacity
would be created.
Sophorolipds (SL) are glycolipid surfactants produced in large quantities by the yeast
Candida bombicola. Among their desirable properties, they are biodegradable, non-
ecotoxic, and non-foaming.6 Sophorolipids are currently used in the cosmetic industry
and as an active ingredient of dishwashing detergents.7 The potential for their use in the
leather industry as a degreasing agent is therefore worthy of consideration.
Evaluation of degreasers as brine curing additives
74
In the present paper we first carried out a stratigraphic study to monitor the distribution
of sodium chloride and water in a hide throughout the curing process. The effect of the
cure on the collagen denaturation temperature was also examined. Furthermore, we
used scanning electron microscopy in backscattering electron mode to establish changes
in the distribution of sodium chloride within the hide. Next, we evaluated the effect that
commercial degreasers exerted on the penetration of salt into the hide, as well as their
fat removal efficiency. Finally, the possibility of using SL as brine curing enhancers
was investigated.
5.5 Experimental
5.5.1 Materials
Two fresh cow hides were purchased from a local abattoir. They were soaked in 200%
water with 0.15% Boron TS (Rohm & Haas Co., Spring House, PA) and 0.10% Proxel
(Chemtan Co. Inc., Exeter, NH) for 2 h and then fleshed. Approximately 6 × 10 in (15
× 25 cm) pieces were cut and stored at -20 °C. They were thawed at 4 °C just before
use. Food grade sodium chloride of minimum purity 99.82% (US Salt Corporation,
Watkins Glen, NY) was used in the brine preparation. Three commercial degreasing
agents marketed to the leather industry and identified as degreasers 1, 2 and 3, as well as
the experimental sophorolipid (SL) developed in our research facility, were used
without further purification. All other chemicals were reagent grade and used as
received.
5.5.2 Methods
5.5.2.1 Stratigraphic study
Thawed hide was cut into square pieces of approximately 4 × 4 in (10 × 10 cm) with an
average weight of approximately 100 g. They were transferred to a Dose drum (PFI
300-34; Dose 131 Maschinenbau GmbH, Lichtenau, Germany) and tumbled at 6 rpm in
a 500% float of 95 °SAL brine solution (25.1 g NaCl/100 g brine) for varying time
intervals, after which they were removed from the drum, hand-squeezed to wipe off
excess water, split into three layers (flesh, middle and grain), sealed in plastic bags and
stored at 4 °C until analysis.
Chapter 5
75
5.5.2.2 Degreaser study
Pieces of hides of approximately 100 g were tumbled at 6 rpm in a 500% float of
initially saturated brine (100 °SAL, 26.4 g NaCl/100 g brine) for 16 h. To the test
samples, the required volume of degreasers 1, 2 or 3 was added to the brine held in the
drum and tumbled for five minutes to ensure a uniform distribution of the product
within the brine. Due to its low solubility, the SL solution was prepared in the
laboratory by adding the required amount of SL powder to the saturated brine solution
and stirring for at least 24 h, after which the solution was dumped into the drum. An SL
solution that had been filtered through a sintered glass funnel immediately after 24 h of
stirring was also tested. Control samples to which no degreaser was added were also
run for all experiments. The concentration of degreasers or SL was always expressed
with respect to the combined weight of hide and brine. No further salt or brine was
added to the drums during the experiments. Brine density was determined by
gravimetric analysis. Hide density was assumed to be 1g/cm3.
5.5.3 Analyses
5.5.3.1 Determination of moisture and ash content
An oval arch punch (C.S. Osborne & Co., Harrison, NJ), 7/8 inches (2.2 cm) in
diameter, was used to sample the pieces of hide. Samples were clipped to minimize
variability due to long hair. Next, they were weighed on an analytical balance into dry
tared crucibles and dried in a vacuum oven at 80 °C for 18 h. After cooling in a
desiccator they were weighed and percentage moisture was calculated. Dry samples
were placed in a muffle furnace and ashed at 600 °C for 2 h. After cooling in a
dessicator, they were weighed and percentage ash was calculated. All samples were run
at least in triplicate.
5.5.3.2 Determination of fat content
In order to accurately compare results among different samples, fat content8 is
expressed in terms of moisture and ash free basis (MAFB).9 All samples were run in
triplicate.
Evaluation of degreasers as brine curing additives
76
5.5.3.3 Determination of thermal stability
Differential Scanning Calorimetry (DSC) analysis was performed on a Multi-Cell
Differential Scanning Calorimeter CSC-4100 (Calorimetry Sciences Corporation,
Lindon, UT). An aliquot of raw or cured hide of approximately 100 mg was weighed
into a stainless steel pan and placed in the calorimeter. Another pan containing
approximately the same weight of a 5% NaCl solution was used as a reference. When
raw hide samples were analyzed, nanopure water was used as a reference. Both sample
and reference capsules were weighed before and after the DSC run in order to determine
any possible weight loss by leakage. Samples were heated from 10 ºC to 120 ºC at
1.5 ºC/min. The peak maximum temperature was taken as the hide’s denaturation
temperature (TD). All samples were run in duplicate.
5.5.3.4 Back-scattered/Low Vacuum Scanning Electron Microscopy (SEM-BSE)
Backscattered electrons consist of high-energy electrons originating in the electron
beam that are reflected or backscattered out of the specimen interaction volume. The
brightness of the SEM-BSE image tends to increase with the atomic number. Two
major advantages over conventional SEM in the secondary electron image mode are the
high-quality atomic number contrast images produced and the simplicity of the
technique, which does not require a conductive coating.10
A piece of raw hide of approximately 1 × 1 in (2.5 × 2.5 cm) was immersed in a beaker
containing 500% v/v float of a saturated brine solution and gently agitated for varying
intervals of incubation time. A 2-3 mm wide slice of the sample was excised manually
with a stainless steel razor blade (cutting from flesh surface toward the grain), mounted
on aluminum stubs, and then irradiated at an accelerating voltage of 25 kV in low
vacuum mode (spot size 3.0, pressure 0.98 Torr) using a Quanta 200 FEG
environmental scanning electron microscope (FEI Company, Hillsboro, OR). Samples
were imaged as montages at a magnification of 50×. Individual montages of images at
each curing time were processed and analyzed using Fovea 3.0 plug-ins (Reindeer
Graphics, Asheville, NC) for PhotoShop, v. 7 (Adobe Systems, Inc., San Jose, CA).
The montages were spatially filtered with a Gaussian blur (3 pixels) to reduce local
noise and a line profile was drawn in each image extending from a point at the grain
surface to a point at the flesh surface. The line profiles were then plotted as gray level
Chapter 5
77
versus distance (data not shown), and the ranges of gray levels for different curing times
were plotted.
5.5.3.5 Statistical Analysis
Data were analyzed by analysis of variance followed by a Dunnett’s test to compare
each treatment (degreasing agent) with the control. Further comparisons were made
using orthogonal polynomial contrasts to test the linear and quadratic trends due to
degreaser 1 concentration. An orthogonal contrast was also used in the sophorolipid
study to determine the significance of the filtering effect.
5.6 Results and discussion
5.6.1 Stratigraphic study
The layer-wise distribution of water and salt (ash) in a hide cured for various intervals
of time was monitored (Figures 5.1a and 5.1b). Typically, the flesh layer included the
flesh itself and the attached adipose tissue; the middle layer represented the majority of
the corium or true skin; and the grain layer included the junction of grain and corium
and the epidermis. The initial moisture content was highest in the region of the
epidermis and decreased toward the center of the corium. Whereas the diminishing
moisture content of the flesh layer leveled off at 54% after 2 h of cure, the moisture
content of the middle and grain layers continued to diminish over a 24-h period to less
than 45% (Figure 5.1a). The salt content of a raw hide was found to be less than a
0.5%. As curing progressed, salt content increased in all three strata: the flesh layer
contained the highest amount of salt at all curing times (Figure 5.1b). Noteworthy, the
salt content of the flesh layer after 30 min and 1 h of cure was 6.7 and 7.1%,
respectively, but only 1.6 and 1.9%, respectively, in the grain layer.
These figures demonstrated that salt entered the hide mainly from the flesh side, which
is in agreement with the findings of McLaughlin and Theis.11 It was also confirmed that
the epidermis acted as a semipermeable membrane, which allowed the withdrawal of
water by osmosis but did not allow salt to penetrate through it.12 Thus, whereas water
was extracted from the hide by both sides, only the flesh side was susceptible to
extensive absorption of salt, which then diffused into the hide and towards the grain.
Evaluation of degreasers as brine curing additives
78
The porous nature of the hide may have also contributed to the diffusion of salt through
the hide by capillarity.
By means of the equation 100359.01
%%
% ��
���
�⋅��
���
�=WaterSalt
Saturation , the hide salt
saturation levels at various curing times were calculated (Figure 5.1c). O’Flaherty13
claimed that a 70% salt saturation of the water remaining in the hide with a maximum
of 50% moisture was needed for a proper cure. Later on, a new minimum standard of
85% salt saturation was established by the U.S. Hide, Skin & Leather Association.14
The salt saturation of the flesh layer was the highest of all three layers at all curing
times. The reason was found in the rapid dehydration experienced in the early stages of
the cure along with the almost exclusive penetration of salt from the flesh side of the
hide. The differences of hide salt saturation levels among the three layers become less
pronounced after 16 h of cure. Although we did not sample the hides beyond the 24 h
curing time frame, one could assume that the hide and surrounding brine reached
equilibrium after about one day of brining. In fact, it was previously reported that a
hide continuously brined for 48 h took up moisture as well as salt;4,15 brining a hide
beyond the equilibrium did not necessarily entail a more proper cure.
Chapter 5
79
Figure 5.1 – Stratigraphic distribution of (a) water (b) ash and (c) hide salt saturation in a hide treated for various intervals of time with a 500% float of an initial 95 °SAL. Each point is the mean of three values.
The denaturation or melting temperature (TD) of the grain, middle and flesh layers of a
hide cured for various time intervals was monitored (Figure 5.2). The hide’s collagen,
upon heating, undergoes a transition from the triple helix to a randomly coiled form in
which the three chains are separated.16 The flesh layer was the first to reach a constant
TD of 78 °C after only 2 h of cure. On the other hand, about 8 h of cure were needed to
level off the temperature curve of the middle and grain layers (TD = 82 °C). The
increase of the thermal stability of the hide upon brining could be ascribed to two
factors. The first and most important factor is the dehydration of the hide. Kopp et al.17
developed a mathematical function that showed an exponential decrease of TD with
increasing moisture content. The second factor is protein salting out and aggregation.
The brine, despite the disruption of some hydrogen bonds due to the withdrawal of
water, induces this phenomenon, which was reported to further stabilize the collagen
molecules, thus increasing the values of TD.18.
35 40 45 50 55 60 65 70 75
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
5
10
15
20
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
20
40
60
80
100
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
35 40 45 50 55 60 65 70 75
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
35 40 45 50 55 60 65 70 75
0 4 8 12 16 20 24
Flesh layer Middle layer
35 40 45 50 55 60 65 70 75
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
5
10
15
20
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
5
10
15
20
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
20
40
60
80
100
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
0
20
40
60
80
100
0 4 8 12 16 20 24
Flesh layer Middle layer Grain layer
Time (h)
1a
1c
1b
Evaluation of degreasers as brine curing additives
80
60
65
70
75
80
85
0 4 8 12 16 20 24
Flesh layerMiddle layerGrain layer
Time (h)
Figure 5.2 – Denaturation temperature (TD) of the grain, middle, and flesh layers of a hide treated for various intervals of time with a 500% float of an initial 95 °SAL brine. Each point is the mean of two values. Cross-sectioned samples of hides, cured up to 24 h duration, were examined in a
scanning electron microscope using a backscatter detector at low vacuum. Although the
backscatter signal is not specific for sodium chloride, results of backscattered intensity
variations were expected to reveal general trends related to diffusion of the dissolved
salt that could be further explored and mapped by energy dispersive x-ray
microanalysis.
The range of intensity in backscattered images was adjusted to maximize the dynamic
range of the detector by adjusting contrast and brightness values with the waveform
monitor using the 24-h cured sample. Then images of all the cured samples were
collected under the same conditions. Line profiles drawn on images from the grain side
to flesh side of an uncured hide (0 h) had a narrow range of low brightness values (69-
77) relative to the values set for the 24-h sample. At 0 h, higher values were located in
the lower half or flesh-side of the hide. Similar line profiles drawn on backscatter
images of cured hides indicated that the range of intensities increased monotonically
with longer curing times, with brightness values extending from a low around 20 to a
high above 200 (16 h), and the higher values were located within the flesh side of the
cross-section, with a boundary slowly moving toward the grain side of the cross-section
as the hours of curing increased (Figure 5.3).
Chapter 5
81
Figure 5.3 – Composite images of hide samples, collected at different curing times by low vacuum, mixed signal SEM imaging. Image heights were adjusted to an average value in order to facilitate comparisons. At 0 hours (control), the superficial brightness was low and variable in the lower, flesh side and uniformly brighter in the upper, grain side; the range of gray levels was narrow, between 69 and 77. At 1-24 hours of curing, the average values of brightness were increased almost monotonically, mainly from the flesh side, but not uniformly; some bright and dark patches were present through 1, 2, 4, 8 and 16 hours. At 24 hours, the brightness was the most uniform, suggesting that the concentrations of Na and Cl were evenly distributed in the hide.
5.6.2 Degreasing study
The effect of three commercial degreasers on the content of water, salt and extractable
fat of the hide after 16 h of brine curing was evaluated (Table 5.1). A degreaser
concentration of 0.5% (w/w) with respect to the combined weight of hide and brine was
Evaluation of degreasers as brine curing additives
82
used. Samples that had been brine-cured in the presence of degreaser 1 showed a
significantly higher salt content than the control, to which no degreaser had been added.
Furthermore, degreaser 1 was shown to be the most efficient as far as its fat-removal
capacity, significantly decreasing the extractable fat content with respect to the control.
Samples that had been treated with degreasers 2 and 3, despite tending to increase salt
content and decrease extractable fat, were not statistically more efficient than the
control. The composition of degreasers 2 and 3 was likely the cause of a lower
efficiency than degreaser 1. Although the specific compositions of these commercial
products are not disclosed, they are typically composed of a blend of nonionic
surfactants, modifiers and adjuvants. The hydrophilic-lipophilic balance (HLB) is a
widely used scale in the technology of surfactants; HLB values run from 0 to 20 and
indicate whether a surfactant has greater wetting (low end of the scale) or emulsifying
properties (high end of the scale). It is likely that degreaser 1 had a slightly higher HLB
than the other two products and hence a higher tendency toward forming oil-in-water
emulsions. As a matter of fact, the emulsification of fat in water was reported to be a
crucial step for the efficiency of the degreasing process.19
TABLE 5.1
Effect of commercial degreasers on brine curing [0.5% w/w]
Treatment Moisture
(%)
Salt
(%)
Hide salt
saturation (%)
Fat
(%, MAFB)
Control 46.5 ± 1.5 11.8 ± 0.9 70.9 ± 7.0 11.8 ± 2.6
Degreaser 1 47.9 ± 3.0 12.9 ± 0.9* 75.2 ± 1.1 7.3 ± 0.6*
Degreaser 2 46.5 ± 1.0 12.1 ± 0.8 72.1 ± 4.1 8.8 ± 3.6
Degreaser 3 46.7 ± 1.7 12.2 ± 0.4 73.1 ± 4.1 8.8 ± 2.2
Average of 8 values Values with * are significantly different from the control (Dunnett’s test, p<0.05)
Next, degreaser 1 was selected to study the effect of its concentration on the content of
water, salt and extractable fat of a hide after 16 h of curing with brine (Table 5.2). All
three tested concentrations of degreaser (0.25, 0.5 and 1% w/w) significantly defatted
the hide. Furthermore, a concentration of 0.5% also effected significantly higher salt
and hide salt saturation values than did the control. The statistical analysis showed
Chapter 5
83
evidence of a significant quadratic trend with increasing concentration of degreaser 1,
with a maximum occurring at a critical concentration between 0.5% and 1%. The usage
of a concentration larger than this critical point neither further increased the uptake of
salt nor additionally decreased the fat content. That could be ascribed to the fact that
the concentration of surfactants in degreaser 1 was well above the critical micelle
concentration.
TABLE 5.2
Effect of degreaser 1 on brine curing
Treatment Moisture
(%)
Salt
(%)
Hide salt
saturation (%)
Fat
(%, MAFB)
Control 53.4 ± 2.0 15.2 ± 0.6 79.5 ± 1.3 16.8 ± 2.2
Degreaser 1 [0.25%] 50.4 ± 2.0 13.8 ± 1.1 76.3 ± 3.5 7.4 ± 2.4*
Degreaser 1 [0.5%] 57.2 ± 4.2 17.1 ± 1.7* 83.5 ± 2.7* 3.8 ± 0.9*
Degreaser 1 [1%] 56.4 ± 2.9 16.5 ± 0.7 81.7 ± 1.9 9.7 ± 2.9*
Average of 6 values Values with * are significantly different from the control (Dunnett’s test, p<0.05)
The feasibility of using a sophorolipid (SL; a fermentation product, under investigation
at this location) as a degreasing agent for raw hides was examined. Due to the low
solubility of SL in the brine, the effect of filtering the SL solution prior to its use was
also examined. By doing this, we ensured that the brine solution contained the
maximum amount of soluble SL. Hide samples cured with the unfiltered brine-SL
solution displayed significantly higher values of salt and salt saturation than the control
(Table 5.3). The extractable fat was also significantly lower than the control. When a
filtered brine-SL was used, the values for moisture, salt, and extractable fat content were
significantly different from those of the control. Hence, SLs were demonstrated to have
a degreasing activity on the hide. The percentage of fat removed from the hide was 40.5
and 27.5% for the unfiltered and filtered SL-brine solution, respectively. These figures
are similar to those obtained for the commercial degreasers (from 25.4% for degreasers
2 and 3 to an average of 53.4% for degreaser 1). The undissolved SL likely provided a
reservoir of active surfactant as the initial concentration of solubilized SL suffered from
dilution or inactivation.
Evaluation of degreasers as brine curing additives
84
TABLE 5.3
Effect of sophorolipid on brine curing
Treatment Moisture
(%)
Salt
(%)
Hide salt
saturation (%)
Fat
(%, MAFB)
Control 45.0 ± 1.4 11.8 ± 0.6 72.8 ± 1.5 15.3 ± 0.5
SL Non-filtered 46.6 ± 0.8 12.5 ± 0.3* 74.9 ± 0.8* 9.1 ± 2.3*
SL filtered 49.7 ± 1.6* 13.0 ± 0.5* 73.0 ± 1.1 11.1 ± 1.9*
Average of 5 values Values with * are significantly different from the control (Dunnett’s test, p<0.05)
From Tables 5.1, 5.2 and 5.3 it can be seen that test samples with a significantly higher
amount of salt than the controls also exhibited a significantly lower fat content.
However, this statement was not verified in the other direction. It was also observed
that a significant effect of the degreaser on the content of salt and fat did not necessarily
entail a significant increase in the salt saturation levels. That could be attributed to the
wetting properties of the degreasers, which did not significantly alter the amount of
remaining moisture in the hide after the 16 h curing time frame (Tables 5.1 and 5.2).
It is important to bear in mind that the results presented in this study are based on the
analysis run on different areas of two cow hides. Variance in the results can be
attributed to some extent to the variability always found in biological material. In
addition, although the hides were washed, fleshed, cut up and stored in the freezer on
the same day that the animal was slaughtered, some damage to the hide due to a delayed
cure might have taken place. A delayed cure of only a few hours was reported to slow
down the diffusion of salt during the early stages of the cure.11 Furthermore, and for the
particular case of fat content, some variability could be attributed to the horizontal
distribution of grease, which is more abundant in the looser and more flexible areas
(e.g., belly;20). Despite all these facts, the coefficients of variation (CV) obtained for
the determination of moisture, salt and salt saturation were 3.97, 5.76 and 3.54%,
respectively, which indicate good precision.21 In the particular case of the
determination of extractable fat, a CV of 21.3% was relatively precise, considering the
low analytical values of that parameter.
Chapter 5
85
5.7 Conclusions
The purpose of research reported in this article was to evaluate the possibility of using
degreasers as brine curing additives, with the aim of enhancing the uptake of salt by the
hide. In the stratigraphic study carried out in order to get a better understanding of the
curing process, we found that the epidermis acted as a semipermeable membrane,
enabling the diffusion of water from the hide and into the surrounding float as well as
hindering the diffusion of salt into the hide, which occurred from the flesh side
overwhelmingly. These findings were corroborated by the pictures taken with a
scanning electron microscope run in the back-scattered electron mode. Furthermore, the
thermal stability of the hide increased upon curing, mainly due to the simultaneous
dehydration and salting out processes of the constituent collagen. If 0.5% (w/w) of a
commercial degreaser, made of a blend of nonionic surfactants, had been added to the
brine, the fat content of that hide was significantly decreased and the uptake of salt was
also significantly enhanced. Since another two commercial degreasers exhibited less
stimulation of salt uptake, we concluded that the composition of the degreaser was a
critical parameter for this specific purpose. The sophorolipid tested showed remarkable
degreasing properties and enhanced the uptake of salt by the hide if it was used above
the solubility limit. These facts along with its low-foam properties and low cost (from
$1 to 3/kg;22) make it an attractive choice of surfactant. One may hypothesize that the
addition of small amounts of a proteolytic enzyme along with a degreasing agent would
enhance the fat removal action, since it would facilitate the breakdown of the
proteinaceous membrane of the fat-containing sac.23 Nevertheless, the detrimental
effect that this treatment could have on the grain is an important drawback.
In this paper, we gave an overview of the brine curing process of raw hides and also
suggested the use of an additive (e.g., commercial degreasers, sophorolipid) to enhance
the uptake of salt and remove a major amount of the fat. It is important to note that
actual raceways or vats are operated continuously. Thus, it will be essential to find a
way to remove the fat that builds up in the vat.
Evaluation of degreasers as brine curing additives
86
5.8 References
1. Personal communication, Ed Godsalve, WE CO., 1991, Inc., 2006.
2. Rajamani, S. Cleaner tanning technologies in the beam house operation. UNIDO
report, pp. 9-18, 1998.
3. Hernàndez Balada, E.; Marmer, W.N.; Kolomazník, K.; Cooke, P.H.; Dudley, R.L.
Mathematical model of raw hide curing with brine. JALCA 103, 128-134, 2008.
4. Stuart, L.S.; Frey, R.W. Effect of adipose tissue fat on the green-salting of heavy
hides. JALCA 35, 414-418, 1940.
5. Stockman, G.B.; Rangarajan, R.; Didato, D.T. The replacement of
nonylphenolethoxylates (NPEs) as degreasing agents in wet blue manufacture.
World Leather, October 2005.
6. Garcia, R.A.; Solaiman, D.K.Y. Government scientists find new uses for rendered
products. Render, June 2008.
7. Solaiman, D.K.Y. Applications of microbial biosurfactants. Inform 16, 408-410,
2005.
8. Taylor, M.M.; Diefendorf, E.J.; Phillips, J.G.; Feairheller, S.H.; Bailey, D.G. Wet
process technology I. Determination of precision for various analytical procedures.
JALCA 81, 4-18, 1986.
9. Donmez, K.; Kallenberger, W.E. Total crude fat determination in hides. JALCA 89,
369-390, 1994.
10. Robinson, B.R.; Nickel, E.H. A useful new technique for mineralogy: the
backscattered-electron/low vacuum mode of SEM operation. Am. Mineral. 64,
1322-1328, 1979.
11. McLaughlin, G.D.; Theis, E.R. Science of hide curing. JALCA 17, 376-399, 1922.
12. Tancous, J.J. A study of a drawn condition, caused through osmosis, encountered in
some brine-cured hides. JALCA 58, 143-154, 1963.
13. O’Flaherty, F. Curing study part 1: Moisture to ash relationship. Leather and Shoes
138, 31-34, 1959.
14. Trade Practices for Proper Packer Cattlehide Delivery, 3rd ed., Leather Industries of
America and U.S. Hide, Skin & Leather Association, pp. 12-19, 1993.
15. Strandine, E.J.; DeBeukelaer, F.L.; Werner, G.A. Stratigraphic distribution of
sodium chloride and water in fresh and brined steer hide. JALCA 46, 19-34, 1951.
16. Miles, C.A.; Bailey, A.J. Thermal denaturation of collagen revisited. Proc. Indian
Acad. Sci. (Chem. Sci.) 111, 71-80, 1999.
Chapter 5
87
17. Kopp, J.; Bonnet, M.; Renou, J.P. Effect of collagen crosslinking on collagen-water
interactions (a DSC investigation). Matrix 9, 443-450, 1989.
18. Komsa-Penkova, R.; Koynova, R.; Kostov, G.; Tenchov, B.G. Thermal stability of
calf skin collagen type I in salt solutions. Biochim. Biophys. Acta Protein Struct.
Mol. Enzymol. 1297, 171-181, 1996.
19. Thanikaivelan, P.; Rao, J.R.; Nair, B.U.; Ramasami, T. Progress and recent trends
in biotechnological methods for leather processing. Trends Biotechnol. 22, 181-
188, 2004.
20. Balderston, L. The distribution of grease in leather. JALCA 17, 405-407, 1922.
21. Taylor, M.M.; Diefendorf, E.J.; Artymyshyn, B.; Hannigan, M.V.; Phillips, J.G.;
Feairheller, S.H.; Bailey, D.G. The chemical and physical analysis of contemporary
thru-blue tanning technology, 1984. Animal Biomaterials Laboratory, ERRC,
Philadelphia, PA 19038. Available from senior author at
22. Sun, X.X.; Choi, J.K.; Kim, E.K. A preliminary study on the mechanism of harmful
algal bloom mitigation by use of sophorolipid treatment. J. Exp. Mar. Biol. Ecol.
304, 35-49, 2004.
23. Langridge, D.A.; Long, A.J.; Addy, V.L. The impact of sebaceous grease on the
leather industry. JALCA 101, 45-50, 2006.
5.9 Acknowledgments
The authors appreciate the assistance of the following: Dr. Richard Ashby, Guoping
Bao, Dr. Eleanor Brown, Nicholas Latona, Joe Lee, Dr. Justin Martin, Paul Pierlott, Dr.
Daniel Solaiman, Maryann Taylor and Amanda Tiscavitch.
CHAPTER 6
Properties of biopolymers produced by transglutaminase treatment of whey protein isolate and gelatin
Chapter 6
89
6.PROPERTIES OF BIOPOLYMERS PRODUCED BY TRANSGLUTAMINASE
TREATMENT OF WHEY PROTEIN ISOLATE AND GELATIN
Authors: Eduard Hernàndez Balada1,2, Maryann M. Taylor1, John G. Phillips1, William
N. Marmer1 and Eleanor M. Brown1.
1U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 E Mermaid Lane, Wyndmoor, PA 19038, USA 2Department of Chemical Engineering, University of Barcelona, c/ Martí i Franquès 1,
08028 Barcelona, Spain
Manuscript accepted for publication at the Bioresource Technology journal. Tentatively
scheduled for publication around March 2009.
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
90
6.1 Letter of acceptance
Chapter 6
91
6.2 Abstract
Byproduct utilization is an important consideration in the development of sustainable
processes. Whey protein isolate (WPI), a byproduct of the cheese industry, and gelatin,
a byproduct of the leather industry, were reacted individually and in blends with
microbial transglutaminase (mTGase) at pH 7.5 and 45 °C. When a WPI (10% w/w)
solution was treated with mTGase (10 U/g) under reducing conditions, the viscosity
increased four-fold and the storage modulus (G') from 0 to 300 Pa over 20 hours.
Similar treatment of dilute gelatin solutions (0.5 to 3%) had little effect. Addition of
gelatin to 10% WPI caused a synergistic increase in both viscosity and G', with the
formation of gels at concentrations greater than 1.5% added gelatin. These results
suggest that new biopolymers, with improved functionality, could be developed by
mTGase treatment of protein blends containing small amounts of gelatin with the less
expensive whey protein.
6.3 Resum
La utilització de subproductes és important en el desenvolupament de processos
sostenibles. El concentrat de sèrum de proteïna (WPI, per les seves sigles en anglès), un
subproducte de la indústria del formatge, i la gelatina, un subproducte de la indústria
pelletera, van ser reaccionats per separat i conjuntament amb la transglutaminassa
microbiana (mTGase) a pH 7.5 y 45 ºC. Quan una solució (10% w/w) de WPI era
tractada amb mTGase (10 U/g) sota condicions reductores, la viscositat es multiplicava
per quatre i el mòdul elàstic (G’) passava de 0 a 300 Pa en 20 hores. Un tractament
similar de solucions diluïdes de gelatina (0.5 a 3%) va sorgir poc efecte. L’addició de
gelatina a un 10% de WPI va provocar un efecte sinèrgic en ambdós viscositat i G’, amb
la formació de gels a concentracions de gelatina superiors a 1.5%. Aquests resultats
suggereixen que nous biopolímers, amb funcionalitat millorada, poden ser
desenvolupats mitjançant el tractament de mescles de proteïnes amb mTGase, contenint
petites quantitats de gelatina juntament al més econòmic sèrum de proteïna.
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
92
6.4 Introduction
Whey and gelatin are both proteinaceous agricultural byproducts. Whey is an abundant,
inexpensive and readily available byproduct of the cheese industry. The United States
is the world’s largest whey exporter; latest available figures from the U.S. Dairy Export
Council (USDEC, 2006) show an increase in exportation by 104% between 2001 and
2006. Despite the exports, a considerable amount of whey is wasted through disposal,
and would be available for potential new uses. These uses could be for food or nonfood
products.
Animal hides, skins and bones, major byproducts of the meat industry, are rich sources
of collagen, the structural protein of connective tissue. Gelatin, produced by partial
hydrolysis of collagen, is a polydisperse mixture of varying sized collagen fragments.
Gelatins are classified by type, edibility, and gel strength. Type A gelatin is obtained by
acid treatment usually of pigskins, while type B is obtained by alkaline treatment of
cattle hides or bones (Rose, 1992). An early step in leather manufacturing is the liming
of the hide to assist in hair removal and opening up of the collagen fiber bundle for
access by tanning chemicals. The hide may then be split; the upper layer would be
tanned to make leather, and the lower layer would provide a rich source for the
extraction of type B gelatin. Gelatin extracted from limed hides is edible, and is used in
sausage casings, as an ingredient in a variety of food products, as well as in
pharmaceuticals. Inedible gelatin may be surplus lower grade material from production
of edible gelatins or may be from an inedible source such as leather waste (Taylor et al.,
1994). The highest value market for inedible gelatin traditionally was the photographic
industry, where the demand for gelatin has declined markedly in recent years. The
Bloom value, a measure of gel strength of a 6.67% (w/w) gelatin, is often used to
predict the behavior of a gelatin in a particular application. Gelatin suppliers typically
give a nominal Bloom value for the gelatins they market, in this case 75 g. High Bloom
(greater than 225 g) gelatins are taken from the highest molecular weight fraction of
crude gelatin and are relatively uniform. High and low molecular weight gelatin
fractions are blended to produce low Bloom (less than 125 g) gelatins, and variation
between lots may be expected.
Transglutaminase (TGase, EC 2.3.2.13) is a calcium-dependent enzyme that catalyzes
an acyl transfer reaction between the γ-carboxyamide of a protein or peptide bound
Chapter 6
93
glutamine and a primary amine (Folk and Chung, 1973). An ε( γ-glutamyl)lysine bond
is formed when the primary amine is the ε-amino group of a lysine residue. In 1989,
Ando et al. used Streptoverticillium mobaraense to produce a calcium-independent
form of the enzyme, namely microbial transglutaminase, mTGase.
A variety of individual food proteins, including gelatin, casein and whey proteins have
been polymerized by the formation of mTGase-mediated intermolecular crosslinks
(Sakamoto et al., 1994; Rodríguez-Nogales, 2005). Biopolymers formed by the
enzymatic crosslinking of dissimilar proteins have the potential for generating novel
products (Motoki and Nio, 1983; Yildirim and Hettiarachchy, 1997; Oh et al., 2004).
The most abundant proteins in whey, β-lactoglobulin and α-lactalbumin, are globular
and contain two and four disulfide bonds, respectively (Farrell et al., 2004). The
reduction of disulfide bonds in globular proteins, prior to mTGase treatment, has been
shown (Færgemand et al., 1997) to increase access to primary amino groups.
Dithiothreitol (DTT) is generally the reductant of choice for cleaving disulfide bonds,
given that it is needed in much lower concentration than other reductants to complete
the reaction (Grazú et al., 2003).
In this study, biopolymers produced by treating gelatin or whey protein isolate (WPI)
and blends of WPI and gelatin with mTGase under reducing or nonreducing conditions
are characterized. In blends, WPI, the less expensive of the protein sources, is the
primary component and the focus is on the effect of including small amounts of gelatin
with the WPI in these biopolymers.
6.5 Experimental
6.5.1 Materials
mTGase, Activa TG-TI (Ajinomoto USA Inc., Paramus, NJ) with an active range of pH
4.0 to 9.0 at 0 to 70 °C was used without further purification. The mTGase activity, in
the presence of the maltodextrose carrier, under the assay conditions of Folk and Chung
(1985) was approximately 100 U/g. Type B gelatin, alkaline extracted from bovine
skin, was obtained from Sigma (St. Louis, MO), and characterized in this laboratory as
115 g Bloom. WPI, Alacen 895, containing 93.2% protein (manufacturer’s data), was
generously supplied by NZMP (formerly New Zealand Milk Products; Lemoyne, PA).
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
94
DTT was obtained from Calbiochem (San Diego, CA). All other chemicals were
reagent grade and used as received.
6.5.2 Sample preparation
Solutions of gelatin or WPI ranging from 1% to 10% (w/w) were prepared by
suspending protein powder in the required weight of deionized water. WPI-gelatin
solutions (10% WPI with 0.5 to 3% gelatin) were prepared by suspending the required
amounts of WPI and gelatin powders in deionized water and stirring for about 30 min to
ensure a uniform suspension. To test the effect of a reducing environment, a 10% DTT
(w/v) solution was prepared and the volume necessary to give a concentration of 10 mg
DTT per g protein was included in the preparation of selected samples. Samples were
allowed to swell at room temperature for four hours, and then adjusted to pH 7.5 with 1
N NaOH or 1 N HCl. Samples were then heated at 38 ºC for one hour, cooled to room
temperature and stored overnight at 4 ºC.
Typical conditions for mTGase-mediated crosslinking of gelatin are pH 6.5 and 50 °C
for 4 h (Taylor et al., 2001). For whey proteins, the optimum conditions are pH 7.5 and
40 °C for 8 h (Truong et al., 2004). To provide a basis for comparison, all reactions in
this study were performed at pH 7.5 and 45 °C for 5 h, considering that WPI was the
major protein component in the mixtures.
For crosslinking experiments, protein samples were prepared in 10 ml less than the
required volume. The appropriate concentration of mTGase was then prepared in 10 ml
of water and this solution added with stirring to gelatin, WPI, or WPI-gelatin solutions
to give the desired final protein concentration. Samples were readjusted to pH 7.5 and
incubated for 5 h at 45 ºC in a shaker bath under mild agitation. Reaction products were
then heated at 90 ºC for 10 min to deactivate the mTGase. Control samples, without
mTGase, were subjected to the same thermal treatment.
Gelatin and WPI solutions (1 to 10%) were treated with mTGase at 10 U/g of protein.
The effect of enzyme concentration (0 to 10 U/g of protein) was also investigated for
gelatin and WPI solutions at 10%. Finally, the effect of gelatin (0.5 to 3%) was tested
by adding gelatin to a 10% WPI sample containing mTGase at either 0 or 2 U/g of total
protein.
Chapter 6
95
6.5.3 Analyses
6.5.3.1 Gel strength
The gelatin used for this study was characterized by the standard Bloom test (AOAC
Method 948.21). The strength of a gel formed in a standard 59 mm Bloom jar from a
6.67% (w/w) solution of gelatin at pH 6.5 was measured on a TA.XT2 Texture
Analyzer (Stable Micro Systems, Surrey, UK). Gel strengths of experimental samples
were determined by the modified small sample Bloom test (Wainewright, 1977).
Samples were transferred to 39 mm weighing bottles, to a height of 40 mm, cooled to
room temperature and then chilled for 17 h at 10 ºC in a constant temperature bath.
Each sample was then placed under a 0.5 in. diameter analytical probe, which then was
driven into the sample to a depth of 4 mm at 1mm/s. The measured force, using the
correction factor (1.389) previously determined for small samples (Taylor et al., 1994),
was expressed in grams. After the determination of gel strength, samples were melted
at 60 °C for viscosity measurements.
6.5.3.2 Viscosity
Viscosity was determined using a Model LV 2000 Rotary Viscometer (Cannon, State
College, PA) equipped with a low Centipoise Adapter and a jacketed sample chamber
connected to a refrigerated bath circulator, Model RTE-8 (Neslab, Portsmouth, NH).
An 18 ml aliquot of each sample was added to the sample chamber and equilibrated for
at least 10 min. Viscosity was measured at a spindle speed of 60 rpm, corresponding to
a shear rate of 73.42 s-1. After the viscometer had been stabilized for one minute,
readings were taken at 60 °C for gelatin as recommended by Rose (1992) and at 25 ºC
for WPI and blended biopolymer solutions.
6.5.3.3 Rheology
Dynamic oscillatory rheometric measurements were carried out in a controlled stress
AR-2000 Rheometer (TA Instruments, New Castle, DE) at room temperature (21 ± 2
ºC) after enzyme deactivation and equilibration of samples for one hour. The sample
was placed between parallel 25 mm diameter plates and the gap between them was set
to 2.5 mm. Excess sample was trimmed off and a thin layer of mineral oil applied to the
exposed free edges of the sample to prevent moisture loss. Time sweep measurements
were used to study the evolution of the storage modulus G' as a function of time. A
strain sweep test was performed at a constant frequency of 0.1 Hz to find an oscillation
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
96
stress value that was within the linear viscoelastic range. This value, 0.5 Pa, was then
used to perform time sweep measurements at a constant 0.1 Hz frequency. Storage
modulus G' was recorded and analyzed over 20 h, at a rate of two points per hour.
6.5.3.4 SDS-PAGE
Inter-protein crosslinking was evaluated by polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS-PAGE) (Laemmli, 1970) using precast gradient gels (4 to
15%). Gels were calibrated using the broad range (BRM) SDS calibration standard
(Bio-Rad, Hercules, CA) that contains a mixture of nine proteins ranging in size from
6.5 to 200 kDa. Samples (approximately 0.5 mg) of lyophilized protein dissolved in
sample buffer (10 mM Tris-HCl at pH 8.0 containing 1 mM EDTA, 25 mg/ml SDS, 50
µl/ml β-mercaptoethanol and 0.1 µl/ml bromphenol blue) were heated at 40 ºC for 4 h.
Separation was achieved using a Phast System (Pharmacia Biotech Inc., Piscataway,
NJ). Gels were stained with Coomassie Blue.
6.5.3.5 Statistical modeling
Because x,y plots of the data points always showed curvature, second order models in
%gelatin or %WPI were generated by regression analysis of viscosity and gel strength
data for products from the treatment of combinations of gelatin, WPI, and blends with
mTGase and DTT. These models are of the form: Response = a + b*Gelatin +
c*Gelatin*Gelatin, where a, b, and c are regression coefficients. Analysis of covariance
(ancova) was performed on the models to determine whether the coefficients were
significantly different between treatments. Comparisons between treatments of the
slopes and second order coefficients were performed using orthogonal contrasts (Littell
et al., 2002). All tests of significance were performed at the P<0.05 level.
6.6 Results and discussion
6.6.1 Gel strength
The effects on gel strength of increasing concentrations of gelatin (1 to 10%) in the
presence or absence of mTGase and DTT were investigated. The second order model
showed that gel strengths of all samples significantly increased (P<0.05) with increasing
gelatin concentration and were greatest at each concentration for samples containing
gelatin alone (Figure 6.1a). Because type I collagen, the primary source of gelatin, has
no disulfide bonds, the addition of DTT to gelatin was not expected to have a noticeable
Chapter 6
97
effect on gel strength, and at concentrations below 6% gelatin, the addition of DTT did
not impact the gel strength. At higher concentrations of gelatin (6 to 10%), however,
gel strengths were significantly (P<0.05) reduced by up to 15%. One possible
explanation is the presence of type III collagen, which is generally co-located with type
I collagen in connective tissue (van der Rest et al., 1990). The individual chains of type
III collagen are disulfide-linked (Boudko and Engel, 2004), and separation of these
chains may have interfered with the partial renaturation that normally accompanies
gelation. Inclusion of mTGase in dilute (1 to 5%) gelatin solutions, either with or
without DTT, resulted in gel strengths significantly (P<0.05) lower than for gelatin
alone (Figure 6.1a). The formation of intramolecular crosslinks, which would inhibit
the attainment of collagen-like structure, is favored over intermolecular ones in dilute
solutions (Sakamoto et al., 1994). At higher (6 to 10%) gelatin concentrations where
mTGase-mediated intermolecular crosslinking is more favorable, the resulting gel
strengths were about 90% of those for gelatin alone.
When the effect of mTGase (1 to 10 U/g gelatin) was determined, no trend could be
observed in the gel strength of a 10% gelatin (data not shown). The range of values was
between 188 g and 225 g, with an average gel strength of 207 ± 12 g, significantly
(P<0.05) lower than the 254 ± 1 g determined for 10% gelatin without enzyme. In the
presence of the reducing agent, DTT, the activity of mTGase was enhanced, as reported
by Kolodziejska et al. (2006), and gel strengths (263 ± 14 g) were comparable to those
for gelatin alone.
The formation of mTGase-mediated WPI gels has been previously reported at WPI
concentrations greater than 10% (Færgemand et al., 1997). Under the conditions of
these experiments, no gel formation was observed for WPI alone at concentrations up to
10%, with or without mTGase, under reducing or nonreducing conditions. Gels did
form when small amounts of gelatin (0.5 to 3%) were included in 10% WPI solutions
(Figure 6.1b). Gelatin alone at these concentrations forms only very weak gels, and so
long as the reaction conditions were not reducing, the gel strength was not significantly
altered whether the gelatin was alone or in a 10% WPI solution, with or without
mTGase at 2 U/g WPI-gelatin. Under reducing conditions, a dramatic increase in gel
strength was seen for mTGase-treated WPI-gelatin blends, the significance of which
was confirmed by the second order statistical model (P<0.05). Because reducing
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
98
conditions and gelatin were essential for enhancing the gel strength, it is likely that
crosslinking was between whey proteins and gelatin chains. The reduction of disulfide
bonds in the globular whey proteins by the action of DTT would expose reactive groups
to the action of the mTGase. In the absence of DTT, the WPI proteins were not
sufficiently unfolded to serve as effective substrates for mTGase.
Figure 6.1 – Gel strengths of gels formed from (a) bovine type B gelatin, 1 to 10% (w/w), and (b) WPI-gelatin blends, 10% WPI with 0 to 3% added gelatin. Gelatin gels were prepared in the presence or absence of mTGase (10 U/g protein) and DTT (10 mg/g protein), WPI-gelatin gels in the presence or absence of mTGase (2 U/g protein) and DTT (10 mg/g protein). Each data point represents a separate measurement. Abbreviations are: G - gelatin, W - WPI, R - reducing conditions (DTT), E - enzyme (mTGase).
6.6.2 Viscosity
The viscosities at 60 ºC, of gelatin solutions (1 to 10%), with and without DTT and
mTGase, were determined (Figure 6.2a). Neither DTT nor mTGase alone had a
significant effect on gelatin viscosity at this temperature. Similar to the case for gel
strength, the effect of mTGase under reducing conditions was not significant at gelatin
concentrations in the 1 to 6% range. Above 7% gelatin, the effect was a significantly
dramatic increase (P<0.001) in viscosity to values greater than 1800 mPa·s, beyond the
limit of the viscometer. These results are consistent with the formation of
intramolecular crosslinks at low gelatin concentrations and both intra- and
intermolecular crosslinks at higher concentrations (Clark and Courts, 1977; Yi et al.,
2006).
The viscosities at 25 °C of solutions of WPI (1 to 10%) with and without DTT and
mTGase were determined (Figure 6.2b). At WPI concentrations less than 7%, neither
Chapter 6
99
DTT nor mTGase, alone or together, had any significant effect on the viscosity of the
solution.
Between 7% and 10% WPI, mTGase alone did not affect the viscosity (data not shown),
and by inference had little ability to crosslink the proteins. In this concentration range,
the viscosity was decreased slightly when disulfide bonds were reduced and the protein
conformation was less well defined. A highly significant effect on the viscosity
(P<0.001) was observed as a result of the treatment that combined the action of DTT
and mTGase on a 10% WPI solution; a four-fold increase in viscosity was attained.
Viscosities measured at 25 °C for blends of gelatin (0.5 to 3%) with 10% WPI were
always higher than those for gelatin alone (Figure 6.2c). When mTGase was included
in blends of 10% WPI with gelatin, the increase in observed viscosity was not
significant. When both DTT and mTGase were included in the blend, their effect on
viscosity became more significant (P<0.001). In fact, viscosities for blends of 10%
WPI with more than 1.5% gelatin could not be measured because the solution formed a
gel that did not melt below 60 °C.
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
100
6.6.3 Rheological properties
The storage modulus (G') was determined for 10% WPI, blends of 10% WPI with 3%
gelatin, and samples treated with mTGase under reducing or nonreducing conditions
(Figure 6.3). A 10% WPI solution, which remained liquid at 20 °C, exhibited a constant
value less than 1 for G'. In contrast, for a 10% WPI solution treated with mTGase under
reducing conditions, G' remained nearly constant and low for the first 10 h and then
increased to 300 Pa between 10 and 20 h, suggesting the formation of a stable and
permanent gel structure (Comfort and Howell, 2002). Because G' can be related to the
amount of crosslinking, the similar and flat response of the WPI sample in a reducing
environment and the WPI sample with mTGase under nonreducing conditions suggest
that no significant crosslinking occurred under these conditions. WPI-gelatin blends
under reducing conditions without mTGase or with mTGase in a nonreducing
environment showed a small increase in G' between 10 and 20 hours, possibly due to
noncovalent interactions between gelatin and whey protein molecules. Chen and
Dickinson (1999) suggested that electrostatic interactions, hydrogen bonding or
Figure 6.2 – Viscosities of solutions of (a) gelatin, (b) WPI and (c) WPI-gelatin blends prepared as described for Fig. 6.1. Viscosities were measured at 60 °C for gelatin solutions and at 25 °C for WPI or WPI-gelatin blends. Abbreviations are as reported for Figure 6.1.
Chapter 6
101
hydrophobic interactions that have a physical nature and are temperature-dependent
might contribute to the appearance of weak gel-like behavior. A similar study of 3%
gelatin samples with or without mTGase (2 U/g gelatin) produced a constant G' value
near zero (data not shown). Blends of 10% WPI with 3% gelatin after incubation with
mTGase (2 U/g protein) under reducing conditions showed an exponential increase of
G' from approximately 70 Pa to approximately 5,000 Pa within the first ten hours. This
remarkable gain suggests the existence of an initial gelled network that became more
reticulated and stable with time (Eissa et al., 2004). Similar enhancements in storage
modulus were reported for blends of gelatin with small amounts of chitosan, a larger
polymer, and mTGase (Chen et al., 2003).
Figure 6.3 – Time sweep analysis of 10% (w/w) WPI and WPI-gelatin blends. Blends contain 10% (w/w) WPI and 3% (w/w) gelatin, mTGase was 10 U/g protein for WPI and 2 U/g protein for WPI-gelatin blends, DTT was 10 mg/g protein. Abbreviations are as designated for Figure 6.1.
6.6.4 SDS PAGE
Gelatin, WPI, and blended biopolymers were analyzed by SDS PAGE. Ten percent
gelatin (Figure 6.4, lane 2) reflects a polydisperse mixture of different sized collagen
fragments with some aggregated material in the stacking gel at the top, and faint bands
visible through the general smear in the separating gel. Although more distinct bands
can be seen in SDS PAGE patterns for gelatins isolated from a specific source (Taylor
et al., 1994) or high Bloom (250 g) commercial gelatins (Tosh et al., 2003), the pattern
seen here is typical of low Bloom (115g) commercial gelatins (Taylor et al., 2004),
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
102
which are sold on the basis of gel strength, and are blends of gelatins from different
preparations. The pattern for 10% gelatin treated with mTGase under reducing
conditions (Figure 6.4, lane 3) shows only a faint smear, representing small collagen
fragments that most likely lack the lysine or glutamine sidechains necessary for
mTGase-mediated crosslinking. Although samples prepared for lanes 2 and 3 were of
the same weight, the faintness of the pattern in lane 3 and the lack of material in the
stacking gel and the upper range of the separating gel suggests the formation of
aggregates so large they could not penetrate the stacking gel (Taylor et al., 2004). A
similar observation (Sharma et al., 2002) was interpreted to suggest that the protein had
become so highly polymerized that it could not penetrate the stacking gel; they also
demonstrated that although the highly polymerized products did not appear on the gels,
their presence could be confirmed by chromatography.
The SDS PAGE patterns for 10% WPI alone (Figure 6.4, lane 4) or after incubation
with mTGase 5 U /g under nonreducing conditions (Figure 6.4, lane 5) show the major
whey proteins, β-lactoglobulin (MW approximately 18 kDa) and α-lactalbumin (MW
approximately 14 kDa) as well as faint bands for bovine serum albumin (MW
approximately 66 kDa) and lactoferrin (MW approximately 86 kDa). Incubation of
10% WPI with mTGase 5 U /g under reducing conditions (Figure 6.4, lane 6) resulted in
bands, above both the separating gel and the stacking gel, showing formation of high
molecular weight polymers in addition to the typical WPI bands. An earlier study
(Færgemand et al., 1997) produced similar results.
1 2 3 4 5 6 7 8 9
Figure 6.4 – SDS-PAGE gels: lane 1, molecular weight markers, ranging in size from 200 kDa at the top to 6.5 kDa at the bottom; lane 2, gelatin 10% (w/w); lane 3, 10% gelatin after treatment with mTGase (10 U/g) under reducing conditions; lane 4, WPI 10% (w/w); lane 5, 10% WPI after treatment with mTGase (5 U/g); lane 6, 10% WPI after treatment with mTGase (5 U/g) under reducing conditions; lane 7, WPI 10% (w/w) with gelatin 1.5% (w/w); lane 8, WPI 10% (w/w) with gelatin 1.5% after treatment with mTGase (2 U/g); and lane 9 WPI 10% (w/w) with gelatin 1.5% after treatment with mTGase (2 U/g) under reducing conditions. This is a composite figure produced from two gels that were run simultaneously.
Chapter 6
103
The addition of 1.5% gelatin to 10% WPI had little effect on the gel pattern (Figure 6.4,
lane 7). In an earlier study, when small amounts of WPI were added to 10% gelatin
samples (Taylor et al., 2006), the whey component was clearly visible in the gel
patterns, and its participation in the formation of crosslinked biopolymers could easily
be monitored. Here, the lack of defined molecular weight bands in patterns for gelatin
and the low concentration of gelatin in the blends contribute to its invisibility.
Incubation of the WPI-gelatin blend with mTGase under nonreducing conditions
resulted in the appearance of a high molecular weight band, possibly representing
crosslinked gelatin fragments, but had little effect on the WPI pattern (Figure 6.4, lane
8). The gel pattern (Figure 6.4, lane 9) for a similar sample incubated under reducing
conditions shows a decrease in intensity of the WPI bands, an increase in high
molecular weight aggregates and a decrease in total protein. The pattern is consistent
with the formation of WPI-gelatin biopolymers in addition to aggregates of the
individual biopolymers. Although it is clear from the gel strengths and rheological data
that the inclusion of 1.5% gelatin in 10% WPI induces gel formation and enhances
physical properties, the biopolymers formed range in size from those formed by WPI
when treated with mTGase under reducing conditions to the much larger aggregates that
do not penetrate the gel.
6.7 Conclusions
Both gelatin (ICIS, 2006) and whey (FASS, 2006) are relatively low value byproducts
of the American food industry. While the actual prices vary over time the ratio is
relatively constant at approximately 5:1 (gelatin:whey). The addition of minor amounts
of relatively low quality gelatin to whey protein improves the strength and stability of
gels formed by the action of mTGase in a reducing environment. As a byproduct of the
meat industry, and a breakdown product of collagen, gelatin comes in a range of
qualities. The higher quality gelatins find a variety of uses in food, pharmaceutical and
industrial products and are heavily traded. Whey is a lower value byproduct that can be
recovered from the waste stream of the cheese industry. The use of mTGase to catalyze
the formation of crosslinks in and between protein molecules is well established, as is
the efficacy of its usefulness with either gelatin or WPI. When a small amount of
gelatin was added to WPI, before mTGase treatment under reducing conditions, a
dramatic rise in viscosity, higher gel strengths, and the appearance of high molecular
weight bands due to inter-protein crosslinking in SDS-PAGE gel patterns than for either
Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin
104
gelatin or WPI treated separately were observed. These results suggest that the reducing
environment partially unfolds the whey proteins, increasing access to glutamine and
lysine side chains, and that the gelatin chains crosslink the whey proteins to form a
network. The improvement in physical properties over either protein component, given
the same treatment, suggests the possibility of greater utilization and new products from
these byproducts.
6.8 References
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Motoki, M., 1989. Purification and characteristics of a novel transglutaminase
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Boudko, S.P., Engel, J., 2004. Structure formation in the C terminus of type III collagen
guides disulfide cross-linking. J. Mol. Biol. 335, 1289-1297.
Chen, J., Dickinson, E., 1999. Interfacial ageing effect on the rheology of a heat-set
protein emulsion gel. Food Hydrocoll. 13, 363-369.
Chen, T., Embree, H.D., Brown, E.M., Taylor, M.M., Payne, F.P., 2003. Enzyme-
catalyzed gel formation of gelatin and chitosan: Potential for in situ applications.
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Clark, R.C., Courts, A., 1977. The chemical reactivity of gelatin. In: Ward, A.G.,
Courts, A. (Eds.), The Science and Technology of Gelatin, Academic Press,
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Comfort, S., Howell, N.K., 2002. Gelation properties of soya and whey protein isolate
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Eissa, A.S., Bisram, S., Khan, S.A., 2004. Polymerization and gelation of whey protein
isolates at low pH using transglutaminase enzyme. J. Agric. Food Chem. 52,
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Færgemand, M., Otte, J., Qvist, K.B., 1997. Enzymatic cross-linking of whey proteins
by Ca2+-independent microbial transglutaminase from Streptomyces lydicus.
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Chapter 6
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Farrell, H.M., Jr, Jiménez-Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K.,
Hicks, C.L., Hollar, C.M., Ng-Kwai-Hang, K.F., Swaisgood, H.E., 2004. Nomenclature
of the proteins of cows' milk--sixth revision. J. Dairy Sci. 87, 1641-1674.
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Prices-F-J.html
Kolodziejska, I., Piotrowska, B., Bulge, M., Tylingo R, 2006. Effect of transglutaminase
and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide on the solubility of fish
gelatin-chitosan films. Carbohydr. Polym.. 65, 404-409.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227, 680-685.
Littell, R.C., Stroup, W.W., Freund, R.J. 2002. SAS for Linear Models, Fourth Edition.
Cary, NC: SAS Institute Inc.
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Oh, J.H., Wang, B., Field, P.D., Aglan, H.A., 2004. Characteristics of edible films made
from dairy proteins and zein hydrolysate cross-linked with transglutaminase. Int.
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ntID=1450
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catalyzed cross-linking of goat milk proteins. Process Biochem. 41, 430-437.
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Inedible Meat By-Products. Advances in Meat Research, Elsevier Applied
Science, New York, pp. 217-263.
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Sakamoto, H., Kumazawa, Y., Motoki, M., 1994. Strength of protein gels prepared with
microbial transglutaminase as related to reaction conditions. J. Food Sci. 59,
866-871.
Sharma, R., Zakora, M., Qvist, K.B., 2002. Susceptibility of an industrial α-lactalbumin
concentrate to cross-linking by microbial transglutaminase. Int. Dairy J. 12,
1005-1012.
Taylor, M.M., Diefendorf, E.J., Marmer, W.N., Brown, E.M., 1994. Effect of various
alkalinity-inducing agents on chemical and physical properties of protein
products isolated from chromium-containing leather waste. J. Amer. Leather
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Taylor, M.M., Cabeza, L.F., Marmer, W.N., Brown, E.M., 2001. Enzymatic
modification of hydrolysis products from collagen using a microbial
transglutaminase. I. Physical properties. J. Amer. Leather Chem. Assoc. 96, 319-
332.
Taylor, M.M., Marmer, W.N., Brown, E.M., 2004. Molecular weight distribution and
functional properties of enzymatically modified commercial and experimental
gelatins. J. Amer. Leather Chem. Assoc. 99, 129-141.
Taylor, M.M., Marmer, W.N., Brown, E.M., 2006. Preparation and characterization of
biopolymers derived from enzymatically modified gelatin and whey. J. Amer.
Leather Chem. Assoc. 101, 235-248.
Tosh, S.M., Marangoni, A.G., Hallett, F.R., Britt, I.J., 2003. Aging dynamics in gelatin
gel microstructure. Food Hydrocoll. 17, 503-513.
Truong, V.D., Clare, D.A., Catignani, G.L., Swaisgood, H.E., 2004. Cross-linking and
rheological changes of whey proteins treated with microbial transglutaminase.
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Yi, J.B., Kim, Y.T., Bae, H.J., Whiteside, W.S., Park, H.J., 2006. Influence of
transglutaminase-induced cross-linking on properties of fish gelatin films. J.
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Yildirim, M., Hettiarachchy, N.S., 1997. Biopolymers produced by cross-linking
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62, 270-274.
6.9 Acknowledgments
The authors would like to thank Lorelie Bumanlag, Paul Pierlott, Michael Tunick and
Carlos Carvalho for their technical support in this work.
CHAPTER 7
Whey protein isolate: a potential filler for the leather industry
Chapter 7
109
7. WHEY PROTEIN ISOLATE: A POTENTIAL FILLER FOR THE LEATHER
INDUSTRY
Authors: Eduard Hernàndez Balada1,2, Maryann M. Taylor1, Eleanor M. Brown1,
Cheng-Kung Liu1 and Jaume Cot3.
1U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA 2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,
08028 Barcelona, Spain 3 Consejo Superior de Investigaciones Científicas (CSIC), Research and Development
Center, Ecotechnologies Department, Jordi Girona 18-26, 08034 Barcelona, Spain
Manuscript accepted for publication at the Journal of the American Leather
Chemists´Association. Tentatively scheduled for publication in the March 2009 issue.
Whey protein isolate: a potential filler for the leather industry
110
7.1 Letter of acceptance
THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS ASSOCIATION
c/o The American Leather Chemists Association, 1314 50th Street, Suite 103, Lubbock, Texas 79412
Mobile phone: (616) 540-2469 E-mail: [email protected] September 11, 2008 Ms. Maryann M. Taylor USDA, Agricultural Research Service Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor, PA 19038 Dear Ms. Taylor, This will acknowledge receipt and acceptance for publication of the manuscript entitled “Whey Protein Isolate: A Potential Filler for the Leather Industry”. It is tentatively scheduled for publication in the March 2009 issue of the Journal. Could you please sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution. Sincerely yours, Robert F. White Journal Editor cc: Dr. Eduard Hernandez Balada
Chapter 7
111
7.2 Abstract
The upgrading of leather that presents loose areas and poor grain break is one of the
most value adding opportunities for a tanner. Typically, petroleum-based products are
used to improve the final appearance and feel of crust leather. In this study, we
demonstrate that blends composed of whey protein isolate (WPI), a byproduct of the
cheese industry, and small amounts of gelatin, a byproduct of the leather industry, could
be effectively used as filling agents for both shoe upper and upholstery leather. Wet
blue leather from three different areas in the hide (butt, belly and neck) was treated with
the WPI-gelatin blend, retanned, colored and fatliquored, and their subjective and
mechanical properties evaluated. The effect of pretreatment of the wet blue samples
with various concentrations of the enzyme microbial transglutaminase (mTGase) was
also examined. It was found that the rate of uptake of the WPI-gelatin blend by
upholstery wet blue increased four-fold when it was pretreated with a 2.5% mTGase
solution. Conversely, this rate was decreased when shoe upper was pretreated with
increasing amounts of mTGase. The subjective properties (e.g. handle, fullness, color
and grain break) of both shoe upper and upholstery leather that were treated with the
WPI-gelatin blend were significantly improved over the controls. Importantly, the grain
break of the belly area of samples that were pretreated with enzyme (both upholstery
and shoe upper) was remarkably improved. Hence, fillers mainly composed by the less
expensive WPI were demonstrated to be effective filling agents for both upholstery and
shoe upper leather.
Whey protein isolate: a potential filler for the leather industry
112
7.3 Resum
La millora de cuirs que presenten àrees buides i poca fermesa de flor és un dels majors
desafiaments que té l’adobador per afegir-los-hi valor. Típicament, matèries primeres
derivades del petroli són emprades en la millora de l’aspecte final i tacte del cuir. En el
present estudi, demostrem que barreges formades per sèrum concentrat de proteïna
(WPI, per les seves sigles en anglès), un subproducte de la indústria del formatge, junt
amb petites quantitats de gelatina, un subproducte de la indústria pelletera, es poden
emprar de forma efectiva com agents de rebliment en cuirs per tapisseria i calçat.
Diferents zones de pells adobades amb crom (flancs, coll i part posterior de l’animal)
van ser tractades amb una barreja de WPI-gelatina, tornades a adobar, colorejades i
engreixades, i les mostres avaluades respecte a les propietats subjectives i mecàniques.
L’efecte del pretractament de cuir amb vàries concentracions de l’enzim
transglutaminassa microbiana (mTGase) fou també examinat. Es va trobar que la
velocitat d’absorció de la mescla de WPI-gelatina per part del cuir per tapisseria
s’incrementava per quatre si era pretractat amb una solució de 2.5% mTGase. En canvi,
aquesta velocitat disminuïa quan el cuir per calçat era pretractat amb quantitats creixents
de mTGase. Les propietats subjectives (tacte, cos, color i fermesa de flor) de les pells
per tapisseria i calçat que van ser tractades amb la mescla WPI-gelatina van millorar
sensiblement en comparació amb els controls. En particular, la fermesa de flor de les
mostres dels flancs que van ser pretractades amb l’enzim (tant en cuirs per tapisseria
com per calçat) va ser millorada notablement.
Chapter 7
113
7.4 Introduction
The presence of loose areas with poor grain break in finished leather is one of many
concerns that tanners are facing in today’s leather processing. This problem becomes
particularly significant in the neck and belly areas of the hide with the belly area
exhibiting a looser break.1 Fillers are materials used to fill the interstices of the leather
and make the looseness less pronounced which in turn should improve cutting yields.
Over the last several years, this laboratory has explored alternatives to petroleum-based
fillers. Chen et al.2 demonstrated that collagen hydrolysate crosslinked with
glutaraldehyde could be suitable for filling low quality leather. More recent research has
focused on the use of gelatin polymerized by the action of microbial transglutaminase
(mTGase) (EC 2.3.2.13), an enzyme capable of forming crosslinks in a wide variety of
proteins. Both commercial and experimental alkali-extracted gelatins were effectively
crosslinked with mTGase, yielding products with improved functional properties.3-6
This enzyme also proved to be effective in the crosslinking of gelatin with sodium
caseinate, a dairy industry byproduct.7 Enzymatically modified gelatin and casein were
successfully applied as fillers in wet blue leather. It was later found that these modified
proteins were not removed during the washing process.8 Nevertheless, the relatively
elevated cost of gelatin and casein encouraged the search for a cheaper source of
renewable proteins. Whey and whey protein isolate (WPI), byproducts of the cheese
manufacturing industry, fulfill that condition and were also effectively reacted with
mTGase yielding viable products for use as filling agents.9,10
We recently demonstrated that the addition of small amounts of gelatin to whey protein
isolate (WPI) in the presence of mTGase and the reducing agent dithiothreitol (DTT)
yielded novel products with improved physical properties (e.g., viscosity, gel strength,
degree of polymerization) over either protein component.11 The main goal of that study
was to obtain biopolymers with unique properties at low cost, hence using WPI as the
majority component of the WPI-gelatin blend.
In the present study, we examine the suitability of biopolymers produced by combining
WPI with small amounts of commercial low Bloom gelatin as a filling agent for shoe
upper or upholstery leather. The effectiveness of the various treatments was assessed by
Whey protein isolate: a potential filler for the leather industry
114
measuring the physical, mechanical and subjective properties of crust leather from three
different areas of the hide (butt, belly and neck).
7.5 Experimental
7.5.1 Materials
Microbial transglutaminase, Activa TG-TI (approximately 100 units/g), a commercial
mTGase formulation containing 99% maltodextrose as a carrier, with an active range of
pH 4.0 to 9.0 at 0 to 70°C, was obtained from Ajinomoto USA Inc. (Paramus, NJ) and
used without further purification. Type B gelatin, alkaline extracted from bovine skin,
and characterized in this laboratory as 115 g Bloom, was obtained from Sigma (St.
Louis, MO). WPI, Alacen 895, containing 93.2% protein (manufacturer’s data), was
generously supplied by NZMP (formerly New Zealand Milk Products; Lemoyne, PA).
Dithiothreitol (DTT) was obtained from Calbiochem (San Diego, CA). A Bicinchoninic
Acid (BCA) Kit for protein determination was purchased from Sigma (St. Louis, MO).
Trutan PA-65 and Trutan PRP-77 were obtained from the former Pilar River Plate Corp.
(Newark, NJ); Havana Dye (Derma Havana R Powder) was obtained from Clariant
Corporation (Charlotte, NC).; Altasol-CAM, Altasol 310-L and Eureka 400R were
obtained from Atlas Refinery, Inc. (Newark, NJ). Basyntan NNOL and Basyntan DLE
were obtained from BASF Corporation (Charlotte, NC). Upholstery and shoe upper wet
blue leather was obtained from commercial tanneries. All other chemicals were reagent
grade and used as received.
7.5.2 Methods
7.5.2.1 Preparation of WPI-Gelatin blends
One day prior to the treatment, the required amounts of WPI and gelatin powders were
suspended in water (200% float), mixed well and allowed to sit at room temperature for
at least two h. The amount of protein powder was calculated on the basis of the weight
of wet blue. Next, a 10% DTT (w/v) solution was prepared and the volume necessary
to give a concentration of 10 mg DTT per g of WPI was added. The pH was then
adjusted to 7.5 with 1 N NaOH or 1 N HCl and heated at 38 ºC for one h, cooled to
room temperature and stored overnight at 4 ºC. It is worth noting that all the
proteinaceous blends discussed in the present paper were prepared without adding
Chapter 7
115
mTGase to the mixture. By doing this, the WPI-gelatin blend can be stored for an
extended period of time without danger of it becoming a permanent gel.
7.5.2.2 Application of WPI-Gelatin blends to wet blue leather
Wet blue samples from the butt, belly, and neck were tumbled with water in a Dose
drum (Model PFI 300-34, Dose Maschinenbau GmbH, Lichtenau, Germany) for 30 min
at 50°C, drained and refloated (200% float, 50°C) in 4% sodium bicarbonate solution,
based on the wet blue weight. Upon pH stabilization, the float was drained, mTGase
solution (2.5% or 5% mTGase) with a 200% float was added and samples were
drummed for one h at room temperature (22 ± 4°C). Note that the mTGase
concentrations stated throughout the manuscript are mTGase + carrier concentration.
The float was drained and a WPI-gelatin solution was added (200% float). Control
pieces to which no enzyme or protein mixture was added were also run with a 200%
float of water. The samples were tumbled for one h at RT and then for five h at 45°C.
The floats were then drained and the samples washed twice for 10 min at 50°C (200%
float), drained, patted dried and stored at 4°C. Previous work carried out in our
laboratory typically employed a 400% float in all the above mentioned processing
steps.8-10,12 By cutting back the float to a half we reduce the consumption of water and
also increase the concentration of protein in the solution, which ultimately leads to a
higher concentration gradient between the solution and the leather.
Aliquots of 3 ml were extracted from the drum after varying time intervals throughout
the treatment of the samples with the mTGase or the WPI-gelatin solutions. Aliquots of
water after each wash were also collected to estimate the amount of protein that was
removed from the wet blue. One drop of 5% sodium azide solution was added to each
aliquot and they were stored at 4°C until needed to run the protein determination assay.
The following day, both treated and untreated samples were weighed and the amount of
reagents needed for the Retan-Color-Fatliquor (RCF) calculated.
Whey protein isolate: a potential filler for the leather industry
116
7.5.2.3 Retan/Color/Fatliquor (RCF)
Control and test samples were retanned, colored and fatliquored in separate drums.
Shoe upper and upholstery samples followed slightly different procedures (Figure 7.1a
and 7.1b, respectively).
Retan/Color
• 150% float @ 30°C
• Add 2% Trutan PA-65 (20 min @ 30°C)
• Add 4% PRP-77 (30 min @ 30°C)
• Add 4% 400R (5 min @ 60°C)
• Add 6% Havana Dye (60 min @ 60°C)
• Add 1% formic acid and tumble until dye is exhausted, @ 60°C
• Batch wash x 3 (200% float @ 60°C, 10 min)
Fatliquor
• Refloat in 150% float @ 60°C
• Add 10% Altasol CAM and 2% Eureka 400R (60 min @ 60°C)
• Add 1.5% formic acid (target pH 3.0-3.5)
• Drain and wash for 5 min
Toggle dry, mill for 24 h, store for 48 h @ constant temp. & RH
Mechanical Properties & Subjective Evaluation
Retan/Color
• 150% float @ 30°C
• Add 2% Trutan PA-65 (20 min @ 30°C)
• Add 4% PRP-77 (30 min @ 30°C)
• Add 4% 400R (5 min @ 60°C)
• Add 6% Havana Dye (60 min @ 60°C)
• Add 1% formic acid and tumble until dye is exhausted, @ 60°C
• Batch wash x 3 (200% float @ 60°C, 10 min)
Fatliquor
• Refloat in 150% float @ 60°C
• Add 10% Altasol CAM and 2% Eureka 400R (60 min @ 60°C)
• Add 1.5% formic acid (target pH 3.0-3.5)
• Drain and wash for 5 min
Toggle dry, mill for 24 h, store for 48 h @ constant temp. & RH
Mechanical Properties & Subjective Evaluation
Retan/Color
• 75% float @ 43°C
• Add 10% DLE Syntan, 2% Basyntan NNOL (5 min @ 43°C)
• Add 1% Havana dye in 25% float @ 60°C (45 min @ 60°C)
• Add 0.5% formic acid and tumble until dye is exhausted, @ 60°C
• Batch wash x 3 (100% float @ 55°C, 10 min)
Fatliquor
• Refloat in 75% float @ 55°C
• Add 5% Altasol CAM, 1% Eureka 400R
• Run 30 min @ 55°C
• Add Altasol 310L in 10% water @ 55°C
• Run 10 min @ 55°C
Haul, horse overnight, set out, toggle dry, store for 48 h @ constant temp. & RH
Mechanical Properties & Subjective Evaluation
Retan/Color
• 75% float @ 43°C
• Add 10% DLE Syntan, 2% Basyntan NNOL (5 min @ 43°C)
• Add 1% Havana dye in 25% float @ 60°C (45 min @ 60°C)
• Add 0.5% formic acid and tumble until dye is exhausted, @ 60°C
• Batch wash x 3 (100% float @ 55°C, 10 min)
Fatliquor
• Refloat in 75% float @ 55°C
• Add 5% Altasol CAM, 1% Eureka 400R
• Run 30 min @ 55°C
• Add Altasol 310L in 10% water @ 55°C
• Run 10 min @ 55°C
Haul, horse overnight, set out, toggle dry, store for 48 h @ constant temp. & RH
Mechanical Properties & Subjective Evaluation Figure 7.1 – Flow diagram for retan, color and fatliquor formulation of (a) upholstery and (b) shoe upper wet blue.
7.5.2.4 Drying
After RCF, samples were removed from the drum and allowed to dry. Shoe upper crust
leather was allowed to hang freely, whereas stretching (toggling) was applied to
upholstery crust leather. Once dry, the leathers were conditioned, put into plastic bags
for one day and then staked twice. Shoe upper crust samples were not milled;
upholstery crust samples were milled for about 24 h. All samples were then kept on a
shelf in the conditioning room at 20°C and 65% relative humidity for at least three days.
7.5.3 Analyses
7.5.3.1 Mechanical properties
Tensile strength, Young modulus and tear strength were determined as described in a
previous publication.12
7.5.3.2 Subjective evaluation
Experimental and control crust leathers were assessed for handle, fullness, grain
tightness (break), color and general appearance by hand and visual examination.
a b
Chapter 7
117
Handle is defined as the sensation or feeling of certain physical properties of leather,
such as flexibility and smoothness, which can be perceived by touch with fingers and
hands���Fullness refers to the way a loop of leather feels in the palm of the hand when
compressed. A full leather fills the palm while a flat leather has more of a cardboard
effect. Grain break is the pattern of tiny wrinkles formed when the leather is bent grain
inward. Leather was rated on a scale of 1 to 5 for each functional property by two
experienced tanners, where higher numbers indicate a better property.
7.5.3.3 Protein concentration determination
Protein concentrations in the float, at different stages of the treatment, were determined
using the bicinchoninic acid (BCA) assay13 according to the directions supplied with the
kit. Samples were centrifuged at 13,400 rpm for 30 min in a microcentrifuge
(Eppendorf MiniSpin plus, Westbury, NY). One ml of protein supernatant was
removed and typically a 1:25 (v/v) dilution was prepared in order to fall within the
linear concentration range for the assay (200 to 1,000 µg/ml protein). A 50 µl aliquot of
the diluted solution was mixed with 1.0 ml of BCA reagent and incubated at 37°C for
30 minutes. The absorbance of a sample solution at 562 nm minus a reagent blank was
compared with a standard curve using known concentrations of bovine serum albumin.
7.6 Results and discussion
7.6.1 Shoe upper wet blue
We first investigated the uptake of mTGase and WPI-gelatin blends by shoe upper wet
blue at both 2.5% and 5% mTGase concentration levels. Similar trends for the uptake
of mTGase were obtained for samples that were pretreated with 2.5 or 5% mTGase. In
both cases, the curve leveled off after only 20 minutes and the bath was not exhausted
after one hour of drumming (Figure 7.2a).
After draining the mTGase solution, a blend of 5% WPI and 0.5% gelatin, with respect
to weight of wet blue, was added and drummed one hour at RT followed by 5 h at 45°C.
A protein uptake of 98% was achieved with wet blue that was not treated with mTGase,
whereas wet blue pretreated with 2.5% and 5% mTGase reduced the percentage to 86%
and 83%, respectively (Figure 7.2b).
Whey protein isolate: a potential filler for the leather industry
118
0
10
20
30
40
50
60
0 10 20 30 40 50 60
5% mTGase2.5% mTGase
Time (min)
0
20
40
60
80
100
0 1 2 3 4 5 6
5% mTGase2.5% mTGase0% mTGase
Time (h) Figure 7.2 – (a) mTGase and (b) protein uptake profiles by shoe upper wet blue pretreated with a solution containing 0, 2.5 or 5% mTGase and treated with a solution of 5% WPI + 0.5% gelatin. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.
The absorption of the protein by the wet blue follows first order reaction kinetics. Hence,
[ ][ ] tAA ⋅−=��
�
����
�κ
0
ln
where [A] and [A]0 are the protein concentration remaining in the drum at time t and t =
0, respectively and k is the uptake rate coefficient. Table 7.1 shows k and the
correlation coefficient values for the uptake of WPI-gelatin by shoe upper wet blue and
shoe upper wet blue pretreated with 2.5% or 5% mTGase. The most rapid absorption,
reflected by the highest value of k, was obtained for the wet blue that was not pretreated
with enzyme (k = 0.608 h-1), followed by the one pretreated with 2.5% (k = 0.413 h-1)
and 5% mTGase (k = 0.323 h-1), respectively.
TABLE 7.1
Uptake rate coefficient k for various treatments
Treatment Wet blue % mTGasea % WPIa Gelatina k (h-1) R2
A Shoe upper 0 5 0.5 0.608 0.994
B Shoe upper 2.5 5 0.5 0.413 0.851
C Shoe upper 5 5 0.5 0.323 0.950
D Upholstery 0 2.5 0.25 0.365 0.830
E Upholstery 2.5 2.5 0.25 1.377 0.956 aPercentages calculated with respect to weight of wet blue.
a b
Chapter 7
119
The wet blue was washed twice immediately after draining the proteinaceous solution.
No detectable level of protein was found in washes of wet blue pretreated with 5%
mTGase. At 2.5% and 0% mTGase there was a protein removal of approximately 6%
and 8%, respectively, and approximately 75% of that protein was washed out in the first
wash. Part of that washed out protein could be due to the unbound protein adhered to
the hide or to the insufficient draining of the drum before the addition of water.
All crust samples were evaluated with respect to handle, fullness, grain tightness (break)
and color. The samples were rated on a scale of 1 to 5, with 1 being the worst and 5
being the best. From these ratings, an overall rating in which the grain break was
weighted more than the other ratings was also presented. Table 7.2 reports the results
on the above mentioned subjective properties of shoe upper wet blue subjected to
treatments A, B or C. Values that were equal to or better than controls are underlined.
Whey protein isolate: a potential filler for the leather industry
120
TABLE 7.2
Subjective evaluationa,b,c
Treatment A Treatment B Treatment C
Hide
area Property Control Test Control Test Control Test
Handle 4 4 4 3 3.5 4.5
Fullness 4 5 4 5 4 4.5
Break 5 5 5 5 1.5 3.5
Color 3 5 3 5 4 4
Butt
Overall 4 5 4 5 3 4
Handle 3 4 3 2 2 4.5
Fullness 3 5 4 5 2 4.5
Break 4 5 5 5 1 4
Color 3 5 3 5 2.5 3.5
Belly
Overall 3 5 4 5 1.5 4.5
Handle 3 4 3 2 3.5 4.5
Fullness 4 5 4 5 4 4.5
Break 5 5 5 5 2.5 4
Color 2 5 2 5 3 3.5
Neck
Overall 3 4 1.5 4.5 2.5 4.5 aScale 1-5, 1=worst, 5=best bn=2 cA, B and C stand for treatments of shoe upper with a solution containing 5% WPI + 0.5% gelatin and pretreated with a solution of 0, 2.5 or 5% mTGase, respectively. All percentages were calculated with respect to the weight of wet blue and added in a 200% float
In all treatments, the test pieces were found to be equal to or superior to the control
pieces. Only the handle of the leather pretreated with 2.5% mTGase was rated slightly
lower than the control. Focusing on the grain break, it is important to note that the wet
blue samples used for the 0% and 2.5% mTGase batches had a good break before the
treatment while the 5% mTGase wet blue samples exhibited a poor break. The samples
that already exhibited a good break showed neither a significant improvement in break
Chapter 7
121
nor any detrimental effect. The 5% mTGase wet blue sample clearly showed significant
improvement in leather from all areas of the hide when comparing the break to the
control.
Next, we examined the effect of reducing the WPI to 2.5% and gelatin to 0.25% and
mTGase to 2.5%, for samples that exhibited a poor break. Approximately 80% of the
protein was taken up by the wet blue, with a rate coefficient rate of k = 0.262 h-1.
During the wash procedure, approximately 6% of the protein was removed, all of it in
the first wash. Although the break of the crust leather fared better than the control, the
improvement was not as dramatic as when a 5% mTGase treatment followed by 5%WPI
+ 0.5% gelatin was used (data not shown). These results suggest that 5% WPI + 0.5%
gelatin filled the leather better than 2.5% WPI + 0.5% gelatin, particularly in the belly
area.
7.6.2 Upholstery wet blue
The ability of WPI and gelatin to fill and improve upholstery wet blue was examined.
Given the smaller thickness of upholstery (1.0-1.2 mm) compared to shoe upper (2.0-
2.4 mm), a lower concentration of WPI and gelatin was selected to make up the
proteinaceous blend (2.5% WPI + 0.25% gelatin). The effect of an enzymatic
pretreatment of the samples with 2.5% mTGase prior to the addition of the
proteinaceous blend was also evaluated. About half the amount of the enzyme was
picked up by the wet blue within the first 30 minutes, and the curve leveled off
thereafter (Figure 7.3a). A complete uptake of protein was reached within the first 3 h
of tumbling for wet blue that was pretreated with 2.5% mTGase. Conversely, the
protein uptake trend for samples that were not pretreated with the enzymatic leveled off
at approximately 90% after 4 h of drumming (Figure 7.3b).
Whey protein isolate: a potential filler for the leather industry
122
0
10
20
30
40
50
60
0 10 20 30 40 50 60Time (min)
0
20
40
60
80
100
120
0 1 2 3 4 5 6
0% mTGase2.5% mTGase
Time (h) Figure 7.3 – (a) mTGase and (b) protein uptake profiles by upholstery wet blue pretreated with a solution containing 0 or 2.5% mTGase and treated with a solution of 2.5% WPI + 0.25%. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.
A remarkably faster uptake of protein was observed for samples that were pretreated
with mTGase, as can be seen from the uptake coefficient values (Table 7.1).
Approximately 5% of the protein was removed in the first wash regardless of the
enzymatic pretreatment. A non detectable amount of protein was removed during
subsequent washes.
The treatment of upholstery wet blue with 2.5% WPI + 0.25% gelatin considerably
improved the handle, fullness, and color of the resulting crust leather. Most
importantly, the break of the belly and butt areas was significantly improved when the
wet blue was pretreated with 2.5% mTGase prior to the addition of the proteinaceous
blend (Figure 7.4).
a b
Chapter 7
123
Figure 7.4 – Subjective properties of upholstery crust leather. D and E stand for treatments of upholstery with a solution containing 2.5% WPI + 0.25% gelatin and pretreated with a solution of 0 or 2.5 mTGase, respectively. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.
7.6.3 Mechanical properties
Leathers from three different areas of the hide, neck, belly and butt, were tested for
mechanical properties. This report will present the test results from belly area only, the
primary area of concern. The three areas demonstrated the same tendency towards the
change of two major variables: percent WPI and percent mTGase. A 3-D regression
plot of the resultant tensile strength as a function of percent WPI and percent mTGase
simultaneously for both upholstery and shoe upper leather samples was developed
(Figure 7.5). The tensile strength decreases slightly with increasing percent WPI for
both upholstery and shoe upper leather. However, for upholstery leather, the tensile
strength increases significantly with percent mTGase, whereas for shoe upper leather,
Butt
0
1
2
3
4
5
Control Treatment D Treatment E
Belly
0
1
2
3
4
5
Control Treatment D Treatment E
Neck
0
1
2
3
4
5
Control Treatment D Treatment E
Handle
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
Butt
0
1
2
3
4
5
Control Treatment D Treatment E
Belly
0
1
2
3
4
5
Control Treatment D Treatment E
Neck
0
1
2
3
4
5
Control Treatment D Treatment E
Handle
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
Handle
BreakFullness
ColorOverall
BreakFullness
ColorOverall
Whey protein isolate: a potential filler for the leather industry
124
the tensile strength shows little change with percent mTGase. It is worthy to note that
as demonstrated in Figure 7.5, shoe upper leather has greater tensile strength than
upholstery leather and this is ascribable to the fact that shoe upper leather is thicker and
has more fiber network to resist the fracture.
Figure 7.5 – Effect of the various treatments of leather with WPI and mTGase on the tensile strength of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.
Young’s modulus is a value indicating the stiffness of leather. Young’s modulus of
upholstery leather increases significantly with both percent WPI and percent mTGase
(Figure 7.6a). On the other hand, for shoe upper leather Young’s modulus also
increases significantly with percent mTGase, but changes very little with percent WPI
(Figure 7.6b). Looking closely at Figure 7.6, one can notice that the range of Young’s
modulus values are higher for shoe upper leather than upholstery leather. Besides the
fact that shoe leather is thicker, this variation could be due to the use of different types
of fatliquors.
a b
Chapter 7
125
Figure 7.6 – Effect of the various treatments of leather with WPI and mTGase on the Young’s modulus of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.
Tear strength decreases with both percent WPI and percent mTGase for upholstery
leather (Figure 7.7a). However, for shoe upper leather the tear strength responds quite
differently to the changes of percent mTGase and percent WPI. As demonstrated in
Figure 7.7b, tear strength of shoe upper leather decreases pronouncedly with percent
mTGase but is relatively unchanged with percent WPI.
Figure 7.7 – Effect of the various treatments of leather with WPI and mTGase on the tear strength of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.
a b
a b
Whey protein isolate: a potential filler for the leather industry
126
7.7 Conclusions
Current prices of sodium caseinate ($5.8/lb)14 and gelatin ($2.6/lb)15 emphasized the
need for research into cheaper sources of protein to generate fillers for leather. The less
expensive whey protein isolate ($1.05/lb)14 along with small amounts of gelatin were
successfully applied as a filling agent for upholstery and shoe upper leather. Subjective
properties such as fullness, handle and color of the resulting crust leather were
significantly improved. Furthermore, grain break for upholstery and shoe upper leather
fared markedly better when samples were pretreated with mTGase. The enzymatic
pretreatment of wet blue leather with mTGase also affected the protein uptake ratio.
The uptake coefficient for upholstery leather pretreated with 2.5% mTGase increased
four-fold over samples that were not enzyme pretreated. The trend was reversed for
shoe upper leather with a drop in the uptake coefficient value from 0.608 h-1 to 0.323 h-1
for samples that were not pretreated with mTGase or pretreated with 5% mTGase,
respectively. We further demonstrated that a 200% float satisfactorily enabled the
proteins to be taken up by the wet blue. If this technology is to be transferred to the
industry, use of a shorter float could be feasible due to a stronger mechanical action.
Importantly, the proteins are not considerably removed by washing, regardless of the
enzymatic pretreatment. Another advantage presented herein is that the proteinaceous
blend was added to the drum without any enzyme pretreatment, thus its preparation
becomes more convenient and feasible. Even though the various treatments did not
negatively affect the mechanical properties of the crust leather, filled samples were a
little stiffer and presented slight lower tear strength than the controls. Further research
that explores the possibility of using even cheaper sources of protein as a raw material
for bio-based leather products is an interesting option currently being examined in our
laboratory.
Chapter 7
127
7.8 References
1. Tancous, J. J., Roddy, W. T., and O’Flaherty, F.; Defects due to natural
characteristics of the skin or hide. In: Skin, Hide and Leather Defects (The Western
Hills Publishing Company), pp. 2-18, 1959.
2. Chen, W., Cooke, P. H., DiMaio, G. L., Taylor, M. M., and Brown, E. M.; Modified
collagen hydrolysate, potential for use as a filler for leather. JALCA 96, 262-267,
2001.
3. Taylor, M. M., Cabeza, L. F., Marmer, W. N., and Brown, E. M.; Enzymatic
modification of hydrolysis products from collagen using a microbial
transglutaminase. I. Physical properties. JALCA 96, 319-332, 2001.
4. Taylor, M. M., Liu, C. K., Latona, N. P., Marmer, W. N., and Brown, E. M.;
Enzymatic modification of hydrolysis products from collagen using a microbial
transglutaminase. II. Preparation of films. JALCA 97, 225-234, 2002.
5. Taylor, M. M., Liu, C. K., Marmer, W. N., and Brown, E. M.; Enzymatic
modification of hydrolysis products from collagen using a microbial
transglutaminase. III. Preparation of films with improved mechanical properties.
JALCA 98, 435-444, 2003.
6. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Molecular weight distribution
and functional properties of enzymatically modified commercial and experimental
gelatins. JALCA 99, 129-141, 2004.
7. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Characterization of biopolymers
prepared from gelatin and sodium caseinate for potential use in leather processing.
JALCA 100, 149-159, 2005.
8. Taylor, M. M., Bumanlag, L., Marmer, W. N., and Brown, E. M.; Use of
enzymatically modified gelatin and casein as fillers in leather processing. JALCA
101, 169-178, 2006.
9. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Preparation and characterization
of biopolymers derived from enzymatically modified gelatin and whey. JALCA 101,
235-248, 2006.
10. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Evaluation of polymers prepared
from gelatin and casein or whey as potential fillers. JALCA 102, 111-120, 2007.
11. Hernàndez Balada, E., Taylor, M. M., Phillips, J. G., Marmer, W. N., and Brown, E.
M.; Properties of biopolymers produced by transglutaminase treatment of whey
protein isolate and gelatin. Bioresour. Technol. In press.
Whey protein isolate: a potential filler for the leather industry
128
12. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Effect of fillers prepared from
enzymatically modified proteins on mechanical properties of leather. JALCA 103,
128-137, 2008.
13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,
Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C.;
Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85, 1985.
14. http://www.ams.usda.gov/AMSv1.0/.
15. http://www.icis.com/Articles/2006/08/28/2015785/Chemical-Prices-F-J.
7.9 Acknowledgments
The authors would like to thank Lorelie Bumanlag, Gary Di Maio, Rafael García,
Nicholas Latona, Renée Latona and Joe Lee for their technical support and assistance.
CHAPTER 8
General conclusions and recommendations
Chapter 8
130
8. GENERAL CONCLUSIONS AND RECOMMENDATIONS
The main conclusions extracted from this work are compiled and discussed in this
section. Given that the research reported in the thesis was divided into two different
projects, the conclusions are also presented in two sections.
8.1 Preservation of raw hides and skins with brine. Conclusions
� A continuous reaction mathematical model was verified to describe the diffusion
of sodium chloride in the hide during the curing process.
� It was demonstrated that the infusion of sodium chloride into the hide occurred
from the flesh side overwhelmingly. That was ascribed to the semipermeability
of the epidermis, which enabled the diffusion of water from the hide and into the
surrounding float but also hindered the diffusion of salt into the hide.
� The thermal stability of the hide increased upon curing, mainly due to the
simultaneous dehydration and salting out processes of the constituent collagen.
The denaturation temperature of the hide increased gradually from the flesh to
the grain during the cure, corroborating that the absorption of salt took place
mainly from the flesh side.
� The diffusion of sodium chloride into the hide was characterized by the transport
coefficient �, which was found to be in the order of 10-5 s-1. By comparing the
individual values of � obtained for various values of initial brine concentration
and float percentage.
� The concentration of brine was found to play a critical role on the effectiveness
of the curing process. A proper cure of the hides could not be achieved by using
diluted brines (20 and 25% w/v, or 64 and 80 ºSAL, respectively) regardless of
the float percentage used.
� If employing an initially saturated brine (100 ºSAL), the usage of a minimum
float of 280% was found to be necessary in order to properly cure hides. A float
of 440% was needed if a less concentrated brine of 96 ºSAL was selected.
� It was concluded that the usage of a saturated brine (35% w/v or 100 ºSAL) as
well as a minimum float of 500% yielded higher values of �, therefore higher
diffusion rates. These results corroborated a general practice applied in curing
raceways, where a 97 ºSAL is typically employed in a 500% float.
� It was proven that the amount of fat adhered to the flesh side retarded the
diffusion of salt into the hide. The addition of a commercial degreasing agent,
General conclusions and recommendations
131
made of a blend of nonionic surfactants, along with the brine significantly
decreased the fat content of the hide and significantly enhanced the uptake of
sodium chloride. In addition, the composition of the degreaser was found to be
a critical parameter for this specific purpose.
� A concentration of degreaser between 0.5 and 1% w/w (with respect to the
combined weight of hide and brine) was proven to be sufficient to significantly
defat the hide and facilitate the uptake of salt.
� A glycolipid surfactant produced by the yeast Candida bombicola, sophorolipid,
was effectively tested as brine curing enhancer agent. When used at a
concentration above its solubility limit of 0.5% w/w (with respect to the
combined weight of hide and brine), the sophorolipid showed remarkable
degreasing properties and enhanced the uptake of salt by the hide.
� By using an appropriate degreasing agent in the brine, turn-around times in
raceways could be reduced and thus additional curing capacity could be created.
8.2 Preservation of raw hides and skins with brine. Recommendations
For further investigation, the following recommendations are proposed.
� In the presented model, it was assumed that the thickness of the hide, b, was
constant throughout the curing. Nevertheless, it is well known that a hide
shrinks to a certain extent during the first stages of the cure. It would be
interesting to remake the mathematical model in which the thickness of the hide
would be a variable of the process instead of a parameter.
� More research needs to be done in order to establish a minimum salt saturation
level that ensures a proper preservation of the hides, currently set at 85%. A
comprehensive study that studies the relationship between the quality and
characteristics of the cured hide, the degree of saturation and the storage
conditions (e.g. temperature, time, humidity) needs to be carried out.
� More research needs to be done to increase the solubility of sophorolipids. By
accomplishing this, their field of applications would be greatly widened and
hide dealers would be more receptive towards its usage in an industrial scale.
� It is essential to find a way to remove the fat that builds up in the curing vats or
raceways, which are operated continuously.
� Hide dealers do not have a reliable method to assess the degree of curing of a
hide. Typically, they evaluate the completeness of curing by making a cut
Chapter 8
132
through the hide and looking for a uniform blue color on the cut surface that
indicates complete curing. Therefore, a non-destructive rapid test method for
cure validation is needed.
8.3 Obtaining and characterization of potential fillers for leather. Conclusions
� Both gelatin and whey protein isolate (WPI), which are relatively low value
byproducts of the American food industry, were proved to be reactive substrates
towards the enzyme microbial transglutaminase (mTGase).
� A reducing environment was needed in order to partially unfold the globular
whey proteins and thus expose the reactive glutamine and lysine groups to the
action of the mTGase.
� A dramatic rise in gel strength, viscosity and elastic modulus was observed
when a small amount of gelatin (ranging from 0.5 to 3% w/w) was added to 10
% w/w WPI and reacted with mTGase under reducing conditions. Also, the
appearance of high molecular weight bands due to inter-protein crosslinking in
SDS-PAGE gel patterns suggested that gelatin chains crosslinked the whey
proteins to form a network.
� The synergistic effect found between WPI and gelatin suggested that new
biopolymers, with improved functionality, could be developed by mTGase
treatment of protein blends containing small amounts of gelatin with the less
expensive WPI.
� Bioproducts composed of WPI along with small amounts of gelatin were
successfully applied as filling agents for upholstery and shoe upper leather.
� Wet blue leather treated with the WPI-gelatin blend, retanned, colored and
fatliquored, exhibited a significant improvement in fullness, handle and color,
with respect to the samples that were not treated.
� Grain break for upholstery and shoe upper leather fared markedly better when
wet blue samples were pretreated with mTGase, prior to the treatment with the
WPI-gelatin blend.
� The protein uptake rate coefficient was affected by whether the wet blue was
pretreated with mTGase or not. The coefficient for upholstery leather pretreated
with 2.5% mTGase increased four-fold over samples that were not enzyme
pretreated (from 0.365 h-1 to 1.377 h-1). A reverse trend was observed for shoe
upper leather, with a drop in the uptake coefficient value from 0.608 h-1 to 0.323
General conclusions and recommendations
133
h-1 for samples that were not pretreated with mTGase or pretreated with 5%
mTGase, respectively.
� The proteins were not largely removed by washing, regardless of the enzymatic
pretreatment.
� The mechanical properties of the filled crust leather were not adversely modified
with respect to the sample that did not undergo the treatment. However, treated
samples were a little stiffer and presented slight lower tear strength than
untreated samples.
8.4 Obtaining and characterization of potential fillers for leather.
Recommendations
� The enzyme microbial transglutaminase has the potential of polymerizing a wide
variety of proteins and the ability of crosslinking dissimilar proteins for
generating novel products. Therefore, the research for other sources of protein
and the study of their reactivity towards the mTGase is strongly encouraged. In
the case of the production of potential fillers for leather, it would be interesting
to look into the possibility of using keratin, the main protein in the cattle hair
and wool, as a cheap and readily available source of protein.
� The reaction between the proteinaceous substrate and a carbodiimide (e.g. 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) prior to the
enzymatic treatment with mTGase would be an interesting approach to increase
the reactivity of the proteins towards the enzyme.
� Scaling up of the filling process. Once the new biopolymer has been succesfully
tested as a filling agent for leather in the lab scale, it should be scaled up to a
pilot plant size, prior to eventually transferring the technology to the industry.
By scaling up the process, lesser concentrations of reactants and volume of float
would be needed due to a stronger mechanical action. A continuous monitoring
of the protein concentration in the drum should be carried out in order to assess
the efficiency of the process and whether the proteinaceous solution should be
dumped or reutilized for another batch.
� From an industrial point of view, it would be interesting to make the filler from
the tannery'ssolid waste (e.g. chrome shavings). By doing this, they would not
only adding value to a byproduct that it is usually landfilled, but they would also
Chapter 8
134
be increasing the value of low quality hides that will be treated with the product.
Also, they would be closing the loop within the industry itself.
CHAPTER 9
References
Chapter 9
136
9. REFERENCES
Alexander, K.T.W. (1988). Enzymes in the tannery – catalysts for progress? Journal of
the American Leather Chemists’ Association�83(9): 287-316�
Anandan, A., Marmer, W.N., Dudley, R.L. (2007). Isolation, characterization and
optimization of culture parameters for production of an alkaline protease isolated
from Aspergillus tamari. Journal of Industrial Microbiology and Biotechnology�
34(5): 339-347��
Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H.,
Motoki, M. (1989). Purification and characteristics of a novel transglutaminase
derived from microorganisms. Agricultural and Biological Chemistry 53(10): 2613-
2617.
Babiker, E. F. E., Khan, M.A.S., Matsudomi, N., Kato, A. (1996a). Polymerization of
soy protein digests by microbial transglutaminase for improvement of functional
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CHAPTER 10
Notation
Chapter 10
145
10. NOTATION
SYMBOL /
ACRONYM DESCRIPTION UNITS
a Pore half length of the skin (m)
[A] Protein concentration remaining in the drum at time t (g dm-3)
[A]0 Protein concentration remaining in the drum at time t = 0 (g dm-3)
Ancova Analysis of covariance (-)
b Thickness of cured hide (m)
BCA Bicinchoninic acid (-)
BSA Bovine serum albumin (-)
c Concentration of sodium chloride in the hide moisture, at a
distance x from the boundary ( > 0) (mol m-3)
c0 Concentration of sodium chloride in the bath ( > 0) (mol m-3)
c0n Concentration of saturated sodium chloride solution at 25 °C (mol m-3)
c0p Initial concentration of sodium chloride in the bath ( = 0) (mol m-3)
c0� Equilibrium concentration of sodium chloride in the bath (mol m-3)
C Dimensionless concentration integral average (-)
C, C0 Dimensionless concentrations (-)
CV Coefficient of variation (-)
D Diffusion coefficient of sodium chloride in the hide (m2 s-1)
D’ Effective diffusion coefficient of sodium chloride in the hide (m2 s-1)
DSC Differential scanning calorimetry (-)
DTT Dithiothreitol (-)
F0 Fourier number/dimensionless time (-)
G’ Elastic or storage modulus (Pa)
GLR Gray level range (-)
HLB Hydrophilic-lipophilic balance (-)
Ig Immunoglobulins (-)
k Protein uptake rate coefficient (h-1)
LF Lactoferrin (-)
MAFB Moisture and ash free basis (-)
mTGase Microbial transglutaminase (-)
MW Molecular weight (Da, kDa)
Notation
146
SYMBOL /
ACRONYM DESCRIPTION UNITS
Na Soaking number (-)
R2 Correlation coefficient (-)
RCF Retan-Color-Fatliquor (-)
RH Relative humidity (%)
RT Room temperature (ºC)
S Outer surface of the solid phase (skin) (m2)
SAL Brine saturation degree (ºSAL)
SD Standard deviation (-)
SDS-PAGE Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (-)
SEM Scanning electron microscopy (-)
SEM-BSE Back-scattered low vacuum scanning electron microscopy (-)
SL Sophorolipid (-)
t Time (min, h)
TD Hide’s denaturation temperature (ºC)
TDS Total dissolved solids (kg/ton hide)
TGase Transglutaminase (-)
USDEC United States Dairy Export Council (-)
USHSLA United States Hide Skin & Leather Association (-)
UV Ultraviolet (-)
V Volume of skin (m3)
V0 Volume of brine solution (m3)
WPC Whey Protein Concentrate (-)
WPI Whey Protein Isolate (-)
x Distance (m)
X Dimensionless distance (-)
Chapter 10
147
Greek symbols
SYMBOL /
ACRONYM DESCRIPTION UNITS
�-La Alpha-lactalbumin (-)
�-Lg Beta-lactoglobulin (-)
� Porosity of solid state (skin) (-)
� Transport coefficient (s-1)
Time (s)
ξ Tortuosity (-)
CHAPTER 11
Glossary of terms
Chapter 11
149
11. GLOSSARY OF TERMS
– A –
Adipose tissue. Form or connective tissue in whose cell fat is deposited and stored.
More frequently found in the flesh layer of hide or a skin.
– B –
Back (of animal). Main portion of hide, obtained by cutting off the two bellies. Note
that in North America a back is a half cattle hide (or side) after the removal of the head
and belly.
Bacteria. Bacteria are the smallest organisms that can complete their life cycle
independently. They lack a nucleus and membrane-bound organelles. They may be
autotrophic or heterotrophic and occur in a wide range of environments. They are
abundant on the remains of dead plants and animals and some cause disease in other
living organisms. Bacteria are also responsible for such process as fermentation and
decomposition.
Bacterial damage. Hides and skins damaged and rendered evil-smelling by bacterial
damage.
Bactericide. Agent or treatment that specifically kills bacteria.
Belly. The underside of a hide between the fore and the hind legs.
Break. Tiny wrinkles formed on the grain side of the leather when it is bent inward.
Brine. A strong solution of salt and water used for preserving raw stock in the
preparation of leather.
Butt. The part of the hide after the bellies and shoulders have been removed.
Glossary of terms
150
Byproduct. A secondary or incidental product deriving from a manufacturing process,
a chemical reaction or a biochemical pathway, and is not the primary product or service
being produced (e.g, gelatin, whey).
– C –
Casein. A nitrogenous substance prepared by precipitation of skim milk. Large
quantities of casein are used by the tanning industry in making leather finishes.
Cattle hide. The skin of a fully grown bovine animal.
Chrome leather. A bluish-green leather which has been chrome tanned. Also known
as wet blue.
Coarse grain. Grain surface that is somewhat rough, due to the nature of the pelt and
the method of treatment during tannage etc., and in which the hair or wool follicles are
large, forming a prominent pattern (see also loose and pipey grain).
Collagen. Protein contained in connective tissue, cartilage and bones, the chief protein
of raw hides and skins.
Crosslink. Chemical links between the molecular chains of polymers.
Crosslinking agent. Highly reactive products like polyisocyanates or polyfunctional
aziridine compounds to achieve film-forming properties of different finish formulations.
Crosslinking reaction. Process of joining free polymer chains with each other by side
linkages to form a two or three dimensional network.
Crust leather. Leather which, after tanning, has not been further processed.
Curing. The treatment of raw hides and skins after flaying to retard bacterial action and
putrefaction.
Chapter 11
151
– D –
Degrease. To remove grease by any method.
Delayed salting. Salting which has been delayed for so long a period after flaying that
damage may have been caused through putrefaction, etc.
– F –
Filler. Any substance which is capable of entering into the voids that exist between the
fibers of leather and remain there.
Filling. Introduction of conditioning substances into the leather to give weight and
body.
Fine grain. Leather whose grain is smooth and the hair follicles are minute.
Fleshing. Removal of any adipose tissue on the flesh side of the skins.
Float. Aqueous liquor in which a process such as curing, pickling or tanning is
performed.
Fresh hide. Undressed, uncured hides taken directly from an animal’s carcass.
Fullness. Property of a leather to have pleasing handle and not being flat and empty.
To achieve fullness or an improved, pleasing handle, filling agents are used in finish
formulations. They deposit additional material between the leather fibres and are used
especially for leathers which feel thin or empty.
– G –
Gelatin. An organic colloidal substance made from animal bones, skins or hide
fragments.
Glossary of terms
152
Grain. Indicates the outer or hair side of hide or skin in cases where it is split into two
or more thicknesses, or to unsplit skins which are finished on the grain side.
Grain layer. The top layer of the corium including the hair follicles.
Green fleshing. Fleshing in the raw state.
Green hides. Hides which have not been salted, dried, or otherwise cured for
preservation.
– H –
Hair side. The side of the skin or hide on which the hair grows; the grain side.
Hair slip. Slipping or loosening of the hair in hides or skins due to putrefaction.
Halotolerant bacteria. Class of bacteria that do not require salt (NaCl) for growth but
can tolerate salt and are capable of growth in 20% salt environment. Halotolerant
bacteria are frequently equated with halophiles, a group of archaeobacteria that require
salt for growth.
Handle. Sensation or feeling of certain physical properties of leather, such as flexibility
and smoothness, which can be perceived by touch with fingers and hands.
Hide. The outer covering of a mature, fully grown animal of the larger kind (e.g. cattle,
horses, buffaloes, camels, elephants).
– L –
Leather. The hide or skin of an animal which has been treated chemically so as to
make it a non-putrescible substance impervious to and insoluble in water.
Chapter 11
153
Loose and pipey grain. Grain layer which is loosely attached to the underlying main
corium layer and forms folds or wrinkles when the leather is bent grain inwards. Often
caused by staleness and can also be caused by mechanical processes.
– P –
Paddle. Semi-cylindrical vessel (of metallic, wood, concrete or plastic materials) fitted
with a revolving paddle wheel for keeping skins and liquors in motion. This type of
vessel could be used in different beamhouse processes such as soaking, liming, and
rinsing (see also vat).
Pelt. A hide or skin, usually when raw, with the hair or wool left on. Most frequently
used to designate the skins of fur bearing animals.
Pipey. Leather whose grain layer separates away from the corium creating a void
between them.
Preservative. An agent used to prevent decay, decomposition, putrefaction, etc.
Preserve. To treat something in such a way as to protect it against harmful influences.
Putrefaction. Hides and skins damaged and rendered evil-smelling by bacterial
damage.
– R–
Raceway brining. Raceway, such as a tank shaped like a racecourse, in which brine
solution and hides are moved around by a paddle, for brine curing of hides.
Raw stock. The hides and skins used for making leather, as referred to in a completely
untanned state.
Rawhide. The usual American name for cattle hide that has been dehaired and limed.
Glossary of terms
154
Red heat. Red colouration found on the flesh side of salted hides after storage. Caused
by salt tolerant (halophilic) bacteria that are aerobic so they stay on the surface of the
hide. Long term storage of hides with red heat can lead to pitting of the surface.
– S–
Salometer. An instrument for measuring the weight of a salt solution per unit volume
and thus giving a measure of its salt content. Saturation equals to 100°SAL.
Salting. Any treatment of hides and skins with a salt for preservation.
Salt diffusion. Penetration of salt into the fibrous tissue of the hide or skin.
Salt uptake. Amount of salt taken up, or absorbed, when hides or skins are treated with
salt.
Saturated brine. Saturated solution of sodium chloride; used for brining hides.
Shoe upper leather. A shoe leather used for the upper portions. Predominantly from
cattle hides and calfskins, although a high variety of skins are used.
Short term curing. Treatment of hides and skins by a method which will preserve
them for a few days only.
Shoulder. The part of the hide that is bounded by the face and cheeks on the top, the
butt on the bottom, and the two bellies on the side.
Skin. A general term of the outer covering of an animal. The raw skins of a mature
fully-grown animal of the smaller kinds (e.g. sheep, goat, pigs).
Sodium chloride. Colourless crystalline compound, NaCl, occurring naturally as halite
and in sea water; common salt. Sodium chloride is used in great quantities for the
conservation of raw hides and skins and in leather making (pickling).
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155
Surfactant. Substance introduced into a liquid to alter (usually to increase) its
spreading, wetting and similar properties (particularly properties depending on surface
tension); can cause foaming and hinder biological activity.
– T –
Tanner. People whose job is to convert animal hides or skins into leather by any
process.
Tannery. An establishment where hides or skins are converted into crust, wet tanned
or finished leather.
Tanning. The processing of perishable rawhides and skins by the use of tanning
materials, into the durable and permanent form of leather.
– U –
Unsaturated brine. Solution of water and salt (brine) that has, at 25 oC, a specific
gravity under 1,198 or contains less than 36,0 g of salt per 100,0 g of water.
Upholstery leather. A general term for leathers used for furniture, airplanes, buses and
automobiles. The staple raw material consists of spready cattle hides, split at least once
and in many cases two or three times. The top or grain cuts go into the higher grades
and the splits into the cheaper.
– V –
Vat. Water-tight vessel, of wood, brick, concrete, etc., usually above ground level,
for storing liquids, preparing solutions, giving liquid treatments, etc.
Vat curing. Method of curing in which hides are laid one by one in a pit and covered
by salt, the pit being finally filled with brine.
Veininess. A prominent vein pattern in hides which becomes visible in the finished
leather, often due to poor bleeding which encourages bacterial growth.
Glossary of terms
156
Veins. Tube through which blood circulates in an animal body. When visible in
leather the cause is usually poor bleeding or staleness.
Veiny. Leather in which the pattern of the blood vessels is visible, or unusually
prominent, on the grain or flesh side, usually through use of stale hides or skins.
CHAPTER 12
Resum en català
Chapter 12
158
12. RESUM EN CATALÀ La recerca presentada a la present dissertació doctoral va ser desenvolupada en la seva
totalitat al Departament d’Agricultura dels Estats Units (USDA), Eastern Regional
Research Service (ERRC) a Wyndmoor, Pennsilvània.
Durant la meva estada al ERRC des del Juliol de 2005 fins a l’Octubre de 2008, se’m
van assignar dos projectes CRIS (Current Research Information System). Els principals
objectius de cada projecte es detallen a sota. Més informació pot ser trobada a la pàgina
web de l’ERRC (http://cris.csrees.usda.gov/).
1. Nous i eficaços processos en la producció de cuir de qualitat
Projecte Nº: 1935-41440-013-00D
Científics en cap: Dr. William N. Marmer i Dr. Cheng-Kung Liu
Objectius: Desenvolupar noves tecnologies per la preparació de pells per l’adobatge.
Establir els processos de secat i acabament així com mètodes no destructius amb la
finalitat de millorar la qualitat i durabilitat de la pell. Preparació de la pell: fons
addicionals destinats a la recerca i optimització de la preservació de pells amb salmorra.
2. Tecnologies sostenibles pel processat de pells, cuirs, llana i subproductes
associats
Projecte Nº: 1935-41440-014-00D
Científics en cap: Dr. Eleanor M. Brown
Objectius: 1. Modificació funcional de pells i subproductes de la pell: desenvolupar una
fundació en l’ús de noves tecnologies químiques i bioquímiques (a) en la producció de
cuirs d’alta qualitat lliures de crom; (b) expandir les aplicacions de subproductes
proteics de la indústria pelletera en el camp dels biomaterials. 2. Modificació funcional
Resum en català
159
de la llana: modificar la llana per tal de millorar-ne la funcionalitat i expandir-ne el seu
camp d’aplicacions.
1. Nous i eficaços processos en la producció de cuir de qualitat
En un món globalitzat, és cada cop més comú trobar articles fabricats a l’estranger amb
matèries primeres provinents de tercers països. En el cas particular del mercat de les
pells i cuirs als Estats Units d’Amèrica, més de la meitat de les pells provinents dels
escorxadors americans són exportades a països com Xina, Corea, Taiwan o Mèxic.
Prèviament a la seva exportació, les pells crues han de ser sotmeses a un procés de
preservació o conservació.
L’objectiu de la preservació és prevenir de forma temporal el deteriorament de les pells
des del moment que són extretes de l’animal fins que són convertides en un producte
que ja no és putrescible (Bailey, 2003). La putrefacció de la pell és deguda als enzims
proteolítics produïts per bactèries, microorganismes unicel·lulars que es multipliquen
molt ràpidament quan les condicions són favorables. La presència de menjar (les
proteïnes de la pell) i aigua, junt amb les condicions adequades de pH (neutral) i
temperatura (entre 20 i 37 ºC), fa que la pell crua sigui un substrat idoni per al ràpid
creixement bacterià, i per tant per a la putrefacció de la pell. Una pell que no ha estat
preservada de forma apropiada tindrà com a conseqüència l’obtenció d’un producte
acabat (cuir) de baixa qualitat.
La preservació de pells crues amb clorur de sodi (NaCl) rep el nom de curat i és el
mètode més tradicional i econòmic. A Europa i als Estats Units, el curat es duu a terme
en uns tancs de gran capacitat omplerts amb una solució quasi saturada de clorur de sodi
(salmorra). Les pells es fan circular en els tancs durant un mínim de 18 hores, després
de les quals són retirades, escorregudes i apilonades. En el curat es dóna un doble
procés de difusió; per una banda, el clorur de sodi migra cap a l’interior de la pell,
mentre que per l’altra, part de l’aigua continguda inicialment a la pell es desorbeix i
passa a formar part de la solució de salmorra continguda en el tanc. Típicament, l’aigua
és el component majoritari de la pell crua (60-70%), i el contingut de sals minerals és
només d’un 0.5-1% (Sharphouse, 1971). Després del procés de curat, el percentatge
d’aigua disminueix fins a un 45-50%, i el de sals minerals incrementat fins a un 10-
Chapter 12
160
15%. Mitjançant aquest parell de valors, hom pot trobar el nivell de saturació de sal de
la pell.
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O’Flaherty va assegurar el 1953 que una pell estava adequadament preservada quan el
contingut d’aigua remanent a la pell després del curat era com a màxim del 50%, i amb
un grau mínim de saturació de sal del 70%. Més endavant, la organització americana
U.S. Hide, Skin & Leather Association va assegurar que el nivell de saturació mínim de
l’aigua remanent a la pell després del curat havia de ser igual o superior al 85%. Tot i
així, a data d’avui no s’ha publicat cap article que estudiï exhaustivament el grau de
saturació mínim que es necessita assolir per tal de garantir la correcta preservació de les
pells, ni l’efecte que les condicions d’emmagatzematge de les pells (temperatura, temps,
humitat...) hi exerceixen.
El procés de curat té dues variants. Una, anomenada green fleshing, en la qual les pells
son descarnades primer i curades posteriorment. El descarnat consisteix en eliminar
gran part del teixit adipós de la pell, la qual cosa provoca una absorció més ràpida de la
sal durant el curat. L’altra opció és curar les pells sense haver estat descarnades
prèviament, la qual cosa facilita l’eliminació del fang i fems adherits al pèl de l’animal,
i també un menor ús d’aigua en comparació al green fleshing.
La preservació de pells crues amb salmorra presenta una sèrie d’avantatges, la més
important de les quals és el relatiu baix cost de la matèria primera emprada (1 kg de
clorur de sodi val aproximadament 11 cèntims de dòlar). Tot i així, el preu del clorur de
sodi s’ha vist incrementat en un 10-15% durant els darrers anys (Ed Godsalve, 2007).
L’àcid bòric (Kanagaraj et al., 2005), el clorur de potassi (Bailey i Gosselin, 1996), el
formaldehid (Sharphouse i Kinweri, 1978) o el gel de sílica (Kanagaraj et al., 2001;
Munz, 2007) són exemples de substàncies que han estat assajades com a agents
conservants de pells crues, però el seu cost, sensiblement superior al del clorur de sodi,
ha provocat que no s’apliquessin a nivell industrial. A part de la vessant econòmica, el
curat de pells amb NaCl és una tecnologia segura, que no requereix amplis
coneixements ni una preparació complicada, i que permet processar varis milers de pells
Resum en català
161
diàries. Com qualsevol altre procés, el curat de pells amb clorur de sodi presenta alguns
inconvenients. Un d’ells és la pol·lució de l’aigua, ja que la sal absorbida durant el
procés de curat és en gran part abocada als rius en el procés de remullat de les pells.
L’elevada salinitat dels efluents pot acabar afectant la salinitat del sòl provocant una
reducció del rendiment de les collites (Daniels, 1998). Des del punt de vista
operacional, el fet que es tractin tantes pells al dia i que el procés sigui discontinu, pot
provocar que hi hagi pells que siguin retirades del tanc abans de les 18 hores establertes,
i per tant amb un grau de curat insuficient.
Tot i que el procés de curat ha estat modelitzat en substrats tals com el formatge
(Turhan i Kaletunç, 1992) o la carn (Bertram et al., 2005), mai no ho ha estat en el cas
de les pells crues. Per tal de buscar les condicions òptimes del procés de curat, tals com
la concentració de salmorra o el volum de bany, es va desenvolupar un model
matemàtic que descriu la difusió del clorur de sodi a la pell durant el procés de curat. El
model proposat s’anomena de reacció contínua i assumeix que el clorur de sodi forma
un gradient de concentració no estacionari dins la pell a mesura que s’hi va difonent
(Figura 12.1).
Figura 12.1 – Model matemàtic del procés de curat d’una pell crua (veure notació en el capítol 10).
La difusió de sal a la pell es va caracteritzar pel coeficient de transport �, el valor del
qual es va trobar de l’ordre de 10-5 s-1. Els resultats obtinguts eren del mateix ordre de
magnitud que els trobats en el model matemàtic del remullat (Blaha i Kolomazník,
1988), la qual cosa va suggerir que la difusió del clorur de sodi no diferia gaire entre les
operacions inverses de curat i remullat.
Chapter 12
162
Mitjançant el model es va esbrinar que els valors més alts de � s’obtenien al utilitzar una
salmorra inicialment saturada (35.9 g/100 ml aigua) així com un mínim de 500% de
volum de bany. Els resultats obtinguts corroboraren una norma generalment acceptada
que estipula que es necessiten un mínim de 5 kg de salmorra per cada kg de pell crua
per tal d’assolir un bon nivell de preservació (Bailey, 2003). El model també revelà que
quan s’empraven salmorres diluïdes de 20 ó 25 g NaCl/100 ml aigua, les pells no rebien
un bon nivell de curat, independentment del volum de bany aplicat o el temps. El
model també va evidenciar que els resultats obtinguts depenien altament del nivell de
saturació de sal que havia d’assolir la pell, que actualment està establert al 85%. Es pot
concloure que el model matemàtic presentat pot ser utilitzat per tal d’estimar la quantitat
de sal, volum de bany i temps que es necessiten per preservar eficaçment pells crues
sota unes condicions d’operació donades.
Un altre objectiu important del projecte d’investigació era trobar maneres d’accelerar
l’absorció de clorur de sodi a la pell crua durant el procés de curat. Assolint aquest
objectiu, el temps de processat en els banys es veuria reduït i per tant es crearia una
capacitat addicional de processat.
En primer lloc, es va dur a terme un estudi estratigràfic que permetés fer un seguiment
de les quantitats d’aigua i clorur de sodi a la pell durant el procés de curat. Es va
observar que la sal entrava a la pell majoritàriament per la cara de la carn, mentre que
l’aigua es desorbia per totes dues cares, amb l’epidermis actuant com una membrana
semipermeable. L’epidermis, en altres paraules, era permeable a l’aigua però no al
clorur de sodi. Aquestes observacions van ser corroborades pels resultats obtinguts amb
microscopia epifluorescent i microscopia d’escaneig electrònic.
Les variables susceptibles de ser modificades en el procés de curat són les següents:
concentració de salmorra, volum de bany, temps, acció mecànica, temperatura i
utilització d’additius. Donat que les tres primeres variables ja havien estat estudiades i
optimitzades en el model anteriorment presentat i que l’efecte de l’acció mecànica i
temperatura no eren de l’interès de la indústria, es va decidir optar per l’estudi
d’utilització d’additius en el procés de curat.
Resum en català
163
Tal i com s’ha apuntat prèviament, la presència de teixit adipós adherit al costat de la
carn de la pell es va demostrar que disminuïa la velocitat de penetració de la sal (Stuart i
Frey, 1940). Tenint en compte la relació directa entre la quantitat de greix a la pell i la
quantitat de sal absorbida, es va decidir emprar tres desengreixants comercials així com
un tensioactiu glucolipídic experimental (soforolípid, SL), desenvolupat als laboratoris
de l’ERRC, com a additius del procés de curat. Els desengreixadors comercials estaven
compostos per una barreja de tensioactius no iònics de la família dels etoxilats
d’alcohol. Els desengreixadors seleccionats eren comercialitzats per a la seva utilització
en la indústria d’adobatge de pells, tot i que eren concebuts per a altres operacions com
ara el remullat o encalcinat de les pells. Les propietats més destacables dels SL són la
seva biodegradabilitat, nul·la ecotoxicitat i baixa escumositat. Els SL estan actualment
emprant-se en la indústria cosmètica i també com a ingredient actiu dels sabons per a
rentavaixelles (Solaiman, 2005). A més, el cost d’obtenció d’aquests productes és
relativament baix, d’entre $1 i $3/kg (Sun et al., 2004). Un dels majors inconvenients
que presenten els SL és la seva limitada solubilitat, tot i que s’està duent a terme una
recerca extensiva per tal d’augmentar i millorar la funcionalitat i aplicabilitat d’aquests
productes.
Un dels tres desengreixadors comercials potenciava significativament l’absorció de sal,
al mateix temps que disminuïa la quantitat de greix a la pell. Els assajos amb el SL van
demostrar que si era emprat per sobre del límit de solubilitat, la capacitat
desengreixadora era comparable a la dels desengreixadors comercials. La implantació
dels SL en el els processos de producció de cuir no s’ha dut a la pràctica encara, però els
esperançadors resultats aquí presentats així com les seves propietats antimicrobials el
converteixen en un candidat molt prometedor. Malauradament, la seva limitada
solubilitat fa que més recerca sigui necessària per tal d’obtenir un producte que pugui
competir amb els que estan actualment al mercat.
Chapter 12
164
2. Tecnologies sostenibles pel processat de pells, cuirs, llana i subproductes
associats
La presència de venes i d’àrees buides i sense cos en el cuir acabat és un dels problemes
més importants que els adobadors han de fer front avui en dia. En cas que el cuir
presenti venes prominents o àrees buides, l’àrea aprofitable es pot veure notablement
reduïda, així com el benefici econòmic de l’adobador. Per tant, la millora del cuir que
presenta aquests defectes és una bona oportunitat per augmentar el valor del producte
final.
Una vena es pot comparar a un tub que tan pot estar ple com buit de sang i que està
localitzat a una zona profunda o superficial de la dermis. Tot i que al principi es creia
que estava causada per un dessagnament deficient de l’animal (Orthmann i Higby,
1929), posteriors teories aposten també per altres motius com l’edat, dieta o raça de
l’animal. Normalment, els flancs i coll de l’animal són les àrees més susceptibles de
patir aquest tipus de problema.
Un altre dels defectes més recurrents que apareix algunes vegades en el cuir acabat és la
mala qualitat de flor, caracteritzada per la formació d’arrugues gruixudes a la superfície
del cuir quan aquest es torça. Un bon toc de flor està caracteritzat per la formació d’un
gran nombre d’ arrugues fines, molt juntes les unes a les altres, i que resulten més
plaents a la vista (Figura 12.2). Un exemple gràfic d’aquesta característica seria el plec
que es forma a la part de davant de les sabates de pell al caminar. Típicament, l’àrea
dels flancs i el coll acostumen a tenir una qualitat de flor pitjor que d’altres àrees de
l’animal (esquena, part posterior de l’animal).
Figura 12.2 – Mostres de cuir amb un toc de flor (a) gruixut i (b) fi.
a b
Resum en català
165
Un mètode extensament aplicat per solventar o minimitzar els esmentats problemes és
mitjançant l’aplicació d’una substància anomenada filler o reblidor. L’objectiu dels
reblidors és donar més cos i substància al cuir tot reduint-ne la buidor i disminuint-ne la
prominència de venes. Per tal d’aconseguir aquest propòsit, els reblidors s’introdueixen
en els buits que existeixen entre les fibres del cuir.
La naturalesa dels reblidors ha canviat durant els darrers anys. Inicialment, s’empraven
extractes de tanins vegetals, compostos de bari o glucosa (Harris, 1974). Més
recentment, altres substàncies com polímers i resines han estat utilitzades com a
reblidors. L’augment de preu dels productes derivats del petroli juntament amb una
major conscienciació mediambiental dels adobadors va provocar que les teneries
busquessin altres fonts per a la fabricació de reblidors. Si fos possible emprar
subproductes d’altres processos o indústries, no només es solucionaria el problema dels
cuirs de baixa qualitat, sinó que també s’estaria donant valor afegit a aquests
subproductes.
Un dels primers intents en aquesta línia de recerca va ser emprant gelatina, un
subproducte de la indústria d’adobatge de pells (Chen et al., 2001). Es va demostrar
que la gelatina, prèviament reticulada amb glutaraldehid, podia ésser utilitzada de forma
efectiva com a reblidor de cuirs. La citotoxicitat del glutaraldehid i l’elevat cost de la
gelatina ($5.7/kg) va reconduir la recerca cap a la búsqueda d’altres fonts renovables de
proteïna i agents reticulants no tòxics. L’elevat preu de la caseïna ($12.8/kg), que havia
estat una de les proteïnes més àmpliament emprades en les teneries americanes,
desaconsellava seguir aquesta línia. Finalment, es va optar per una de les proteïnes de
rebuig més econòmiques en el mercat americà actual, com és el sèrum de proteïna i els
seus derivats, que al seu torn són subproductes de la indústria del formatge. El preu de
mercat d’aquests productes depèn en gran part de la concentració de proteïna, i van des
de $0.66/kg pel sèrum de proteïna (aproximadament un 15% en pes de proteïna) fins als
$2.30/kg pel concentrat de sèrum de proteïna, WPI per les seves sigles en anglès (al
voltant d’un 90% en pes de proteïna).
Als laboratoris de l’ERRC es va treballar durant els darrers anys en la modificació de
subproductes proteics amb transglutaminassa microbiana (mTGase), un enzim que
catalitza la reacció entre un grup �-amino d’un residu de lisina i un grup �-carboxiamida
Chapter 12
166
d’un residu de glutamina, obtenint un entrecreuament (crosslinking) covalent de les
proteïnes, que pot ser inter o intramolecular (Figura 12.3).
Figura 12.3 – Reacció de crosslinking entre molècules proteiques amb l’enzim mTGase
S’ha demostrat que l’actuació d’aquest enzim sobre proteïnes de naturalesa molt diversa
provoca l’obtenció d’estructures polimèriques amb propietats reològiques i funcionals
modificades, la qual cosa permet ampliar els seus camps d’aplicació. La gelatina, tant
la comercial com la obtinguda a partir de residus sòlids de les teneries, el sèrum de
proteïna i els seus concentrats, i la caseïna, així com barreges binàries d’aquestes
substàncies, van ser reaccionades de forma efectiva amb l’enzim mTGase, i els seus
productes de reacció van ser aplicats amb èxit com a reblidors (Taylor et al., 2004;
2005; 2006a; 2006b; 2007). A més, els reblidors, un cop adherits al cuir, no n’eren
despresos en etapes posteriors de processat. Finalment, les propietats mecàniques del
cuir acabat no van ser modificades sensiblement pels diferents reblidors estudiats
(Taylor et al., 2008).
En els casos estudiats, la solució proteica estava formada per un component majoritari
dels considerats cars (gelatina, caseïna) i un de minoritari més econòmic (sèrum de
proteïna, WPI). Per tal de continuar aquesta línia de recerca i aconseguir produir un
bioproducte més econòmic, es va estudiar l’efecte d’afegir una quantitat petita de
gelatina al més econòmic WPI, i reaccionar-los amb mTGase. Es va trobar que
mitjançant l’addició d’una petita quantitat de gelatina al WPI i posterior reacció amb
l’enzim, s’obtenien uns productes les propietats físiques dels quals (força de gel,
viscositat, mòdul elàstic) eren millorades respecte a si la gelatina o WPI eren
Resum en català
167
reaccionats amb l’enzim de forma individual (Figura 12.4). També es va concloure que
feia falta disposar d’un ambient reductor per tal que els grups reactius de les dues
majors proteïnes constituents del WPI, �-lactoglobulina i �-lactalbúmina, amagats a
causa de la geometria esfèrica de les molècules, fossin susceptibles de reaccionar amb
l’enzim mTGase.
Un cop caracteritzats els nous biopolímers es va procedir a estudiar-ne la seva
aplicabilitat com a reblidors de pells de baixa qualitat. En la present tesi, es va estudiar
l’eficàcia d’aquests productes tant en cuirs per tapisseria (d’un gruix que oscil·la entre
1.0 i 1.2 mm) com per calçat (gruix entre 2.0 i 2.4 mm), ambdues adobades amb sals de
crom (wet blue). Es van introduir dues importants diferències respecte als estudis
realitzats prèviament a l’ERRC, amb l’objectiu d’optimitzar el procés des del punt de
vista econòmic. Per una banda, no es va afegir mTGase en la preparació de les
solucions proteiques; així, es retallava el cost de matèries a emprar i també s’evitava
que el producte esdevingués massa viscós abans de ser utilitzat. Per l’altra banda, es va
decidir dur a terme el procés de rebliment amb un volum de bany del 200%, la meitat
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ul e
làstic G
' (Pa)
Test
Control
a a
b
c
Figura 12.4 – (a) Força de gel, (b) viscositat i (c) mòdul elàstic (G’) d’una barreja proteica composta per 10% (w/w) de WPI i diferents quantitats de gelatina, en un ambient reductor (1% DTT, respecte el pes de WPI). Control: 0 U mTGase/g proteïna; Test: 2 U mTGase/g proteïna. Nota: els valors de viscositat dels tests que contenien un 1.5% o més de gelatina excedien el límit del rang de mesura del viscosímetre (1,800 cP). El test del gràfic c correspon a una barreja de 10% (w/w) WPI + 3% (w/w) gelatina, amb 2 U mTGase/g proteïna, en un ambient reductor (1% DTT, respecte el pes de WPI).
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del que havia estat aplicat típicament. Finalment, també es va analitzar l’efecte que el
pretractament del wet blue amb diferents quantitats de mTGase exercia en la qualitat
final del cuir.
L’estudi va concloure que l’aparença general del cuir, tant el destinat a tapisseria com el
destinat a calçat, fou significativament millorada després del tractament amb l’agent de
rebliment, obtenint un cuir més ple, de color més intens i un toc de flor més plaent a la
vista i al tacte. Tot i que aquestes propietats són subjectives i van ser avaluades per dos
adobadors experts, la millor qualitat del cuir que havia estat tractat respecte el que no ho
havia estat era evident fins i tot per a persones aleatòries sense coneixements específics
en aquesta matèria.
El seguiment de l’absorció de proteïna durant l’operació de rebliment va concloure
tendències inverses pel casos del wet blue per tapisseria o per calçat. El coeficient
d’absorció del wet blue per tapisseria pretractat amb una solució de mTGase era quatre
vegades superior al del que no ho havia estat; en canvi, el coeficient d’absorció del wet
blue per calçat que no havia estat pretractat amb mTGase era quasi el doble del d’aquell
que sí que havia estat pretractat amb la solució de l’enzim. Independentment de
l’enzim, un cop les proteïnes eren absorbides pel wet blue, el percentatge desprès en les
posteriors etapes de rentat era poc significatiu (entre un 0 i un 6%).
L’absorció de proteïnes pel wet blue també es podia visualitzar mitjançant microscopia
epifluorescent (Figura 12.5). En aquest cas, les diferents proteïnes van ser complexades
amb uns colorants que fluorescien a una determinada longitud d’ona. Aquesta tècnica,
tot i ser només qualitativa, permetia localitzar les proteïnes de rebliment en el sí del wet
blue i també avaluar la seva presència després del procés de rentat.
Resum en català
169
Figura 12.5 – Imatges de microsopia epifluorescent del wet blue després d’haver estat tractat amb una solució etiquetada de WPI i gelatina. La fluorescència del complex WPI-colorant fluorescent s’aprecia amb un intens color vermellós (imatge superior) i la de la gelatina-colorant fluorescent adopta un color verd intens (imatge inferior). La flor del wet blue es troba a la part superior de les imatges.
Addicionalment, es va demostrar que un volum de bany del 200% era suficient per
aconseguir que les proteïnes fossin absorbides pel wet blue. És molt probable que si
aquesta tecnologia acaba essent implantada a la indústria, sigui factible dur a terme
aquesta operació en un volum de bany fins i tot inferior al 200%, a causa d’una major
acció mecànica. També es important destacar que les propietats mecàniques dels cuirs
sotmesos al procés de rebliment, tot i ser una mica més rígids, no es van veure
significativament afectats.
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