Ph.D. in Chemical Engineering Presentation

Improvement of conventional leather making processes to reduce the

environmental impact

Eduard Hernàndez Balada

Doctoral Thesis directed by

Dr. José Costa LópezDr. Jaume Cot Cosp

Programa de Doctorat d’Enginyeria del Medi Ambient i del ProducteBienni 2006-2008

Barcelona, 5 de febrer 2009

FACULTAT DE QUÍMICADEPARTAMENT D’ENGINYERIA QUÍMICA

The research included in this presentation was developed in its totality at the United States Department of Agriculture (USDA), Eastern Regional Research Center (ERRC) (Wyndmoor, PA).

Length of stay: From July 2005 thru October 2008

Research Unit: Fats, Oils and Animal Coproducts (FOAC)

Research Leaders: Dr. William N. Marmer and Dr. Daniel K.Y. Solaiman

CRIS (Current Research Information System) projects assigned:

1. New and efficient processes for making quality leatherProject number: 1935-41440-013-00DLead Scientists: Dr. William N. Marmer and Dr. Cheng-Kung LiuObjectives: 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: 1935-41440-014-00DLead Scientist: Dr. Eleanor M. BrownObjectives: 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 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.

http://cris.csrees.usda.gov

TABLE OF CONTENTS

1. NEW AND EFFICIENT PROCESSES FOR MAKING QUALITY LEATHER

1.1. Introduction

• Hides and skins

• Preservation of raw hides and skins

1.2. Stratrigraphic study

1.3. Mathematical model

1.4. Degreasing study

2. SUSTAINABLE TECHNOLOGIES FOR PROCESSING OF HIDES, LEATHER, WOOL AND ASSOCIATED BYPRODUCTS

2.1. Introduction

• Fillers in the leather industry

2.2. Biopolymer study

2.3 Filler study

• Raw hides and skins are byproducts of the meat industry, and in turn are the raw material of the leather industry.

• Hide, hair, bones and organs account for an approximate 23% of the animal’s weight.

• The tanner employs the word hide to refer to the skin covering large animals, (e.g. cows, steers, horses) and the word skin is mostly used to refer to smaller animals (e.g. sheep, goats, pigs).

Schematic cross section of a bovine hideComposition of hide

Collagenous fiber

Fibril

Microfibril Collagen molecule

Polypeptide chain

Introduction – Hides and skins

Hide preservation. Treatment given to raw hides or skins just removed from the carcass of the animal to minimize putrefaction.

Beamhouse (Pretanning). Processes in the tannery that take place between the removal of the skins or hides from storage and their preparation for tanning.

Tanning. Process by which the pelt is converted into a stable material which is resistant to microbial attack and has enhanced resistance to heat.

Post tanning operations. Includes wringing, splitting, retanning, coloring, fatliquoring, setting out, drying.

Finishing operations. The act of making completely tanned leather more attractive, serviceable and durable.

Raw hides and skins

Preserved hides and skins

Wet blue leather

Crust leather

Finished leather

Pelt


Conversion of hides and skins into leather


Conversion of hides and skins into leather

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

Sta

ges

Un

it O

per

atio

ns

Ou

tcom

e

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

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

Economic figures on the American market of hides and skins

Source: U.S. Leather Industry Statistics (2006 edition)


• The United States is a hide exporting nation.

• An 80% of the hides produced in the U.S. in 2008 were exported, half of which were shipped to China and Hong Kong (Source: United States Hide Skin & Leather Association).

[Note: 1,000 hides]

[Note: 1,000 hides]

Goal → To temporarily prevent deterioration of raw hides and skins 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.

• Physical damage. Occurs before the slaughter of the animal. Includes tears, scratches, cuts, hook marks, contamination with dirt, insect attack, etc.

• Putrefaction. 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.

Factors that affect the growth of bacteria:

• Food

• Water

• pH

• Temperature

Introduction – Preservation of raw hides and skins


Methods

• Drying → Earliest method of preservation of raw hides and skins.

• Cooling and chilling

• Cooled air treatment → 1 hour at 3 to 5 ºC. Storage time: 12 h.

• Addition of ice → Causes a drop of about 20 ºC in the hide. Storage time: 24 h.

• Dry ice → Cools hides to temperatures of -35 ºC. Storage time: 48 h.

• Freezing → Medium term preservation technique. Requires high investment.

• Freeze drying → Operational costs too high for common use.

• Gamma irradiation → Successful salt-less preservation technique. High cost capital investment required.

• Electron beam irradiation → Successful salt-less preservation technique. High cost capital investment required.


Advantages Drawbacks

Formaldehyde• It is effective in low amounts.

• Toxicity.

Potassium chloride

• Production of less excess salt.• Possible use as a plant fertilizer.• Rapid kinetics on uptake of salt.• Elimination of halophilic bacteria.

• More expensive than sodium chloride.• Low solubility of potassium chloride at low temperatures.

Boric acid

• Reduction of Total Dissolved Solids and chlorides in the effluents.

• Higher cost than the traditional brine curing.

Silica gel

• Reduction of Total Dissolved Solids and chlorides in the effluents.• Strong dewatering of the hides.• No impact on the quality of the final product.

• Production cost of silicate is double than that of common salt.

Chemicals

• Sodium chloride (NaCl) is the most popular and inexpensive material used to preserve hides and skins. • It reduces the water content of the hide and lowers the water activity of the remaining moisture.

1. Salt pack curing (Green salting)• The more antique method of salt preservation.• Consists of sprinkling solid salt onto the flesh surface of the hide • Not common in the United States or Europe but extensively applied in India and other Asian countries.

2. Brine curing• Extensively used in American and European hide processing facilities. • Huge vat containing an almost saturated solution of sodium chloride (brine) where hides are suspended for a minimum of 18 h.

• Concentration of brine in the raceway is monitored with a salometer, which scale ranges from 0 ºSAL (pure water) to 100 ºSAL (saturated brine).


Preservation of raw hides and skins with common salt

29%

4%63%

2%

1%

1%

CollagenOther proteinsWaterFatsMineral saltsOther substances

Composition of the hide before the cure

29%

4%

49%

2%15% 1%

CollagenOther proteinsWaterFatsMineral saltsOther substances

Composition of the hide after the cure


Brine curing of raw hides and skins

H2O H2O H2O H2O

Hair side

Flesh side

NaClNaCl NaCl NaCl


63.5

53.4

10.4

0.248.5

14.0

Source: J.A.Chittenden EPA (1976)

Sat. ~ 90%Sat. ~ 60%Sat. ~ 1%


• The degree of curing of a hide can be assessed by means of the salt saturation level.

100359.0

1

%

%%

Water

SaltSaturation

• A standard of 85% salt saturation was established by the U.S. Hide, Skin & Leather Association. That means that the moisture that remains in the hide after the cure has to be 85% saturated with salt.

Advantages

• Relatively inexpensive ($0.11/kg).

• Capacity to process a high volume of hides and skins (various thousands per day).

• No high tech knowledge involved.

• Usage of safe chemicals.

Disadvantages

• Water pollution. Release of about 50% of the total dissolved solids of the whole leather making

process during the soaking.

• The large amount of salt required.

• Increasing commodity prices for sodium chloride (a 10-15% increase over the past few years).

• Batch process. Hides might be removed from the vat in a different order that they were put in.

• Vat overload. Leads to a slower diffusion rate of salt into the hide.

• Red heat damage. Caused by bacteria that grow in a concentrated salt environment (halophiles).



Red heat damage on salted skins

Stratrigraphic study – Experimental

Split the hide in three layers (grain, middle, flesh)

Piece of ~ 100 g

Cure with 95 °SAL brine (initial concentration) for various time intervals (0.5, 1, 2, 4, 8, 16, 24 h) and 500% float

Squeeze out the excess moisture

% moisture

% ash

% salt saturation

Wash raw hide @ 6 rpm for 2 hours, 100% float (with 0.15% Boron TS and

0.10% Proxel GXL)

100)·/(% hidebrine VVFloat

(ρhide=1 g/cm3)

Flesh

Middle

Grain

Flesh and adipose tissue

Junction of grain and corium and epidermis

Majority of the corium

• Water is desorbed very rapidly through both flesh and grain surfaces.

• The diffusion of salt takes place in its majority from the flesh side.

• The flesh split is the first to reach an 85% saturation level, followed by the middle and grain splits.

35

40

45

50

55

60

65

70

75

0 4 8 12 16 20 24

Flesh layerMiddle layerGrain layer

Hid

e m

oist

ure

con

tent

(%

)

Time (h)

Stratrigraphic distribution of water

0

5

10

15

20

0 4 8 12 16 20 24

Flesh layerMiddle layerGrain layer

Hid

e a

sh c

onte

nt (

%)

Time (h)

Stratrigraphic distribution of ash (salt)

0

20

40

60

80

100

0 4 8 12 16 20 24

Flesh layerMiddle layerGrain layerH

ide

salt

satu

ratio

n (%

)

Time (h)

Stratrigraphic distribution of salt saturation

Stratrigraphic study – Results

15min 30min 1h 2h 3h 4h 5h 28h 48hFlesh

Hair

CoroNa Green dye is a sodium ion indicator that exhibits an increase in green fluorescence emission intensity upon binding Na+ with little shift in wavelength.

Fluorescence imaging

CoroNa Green Na+ indicator

Fluorescence emission spectra of the CoroNa Green indicator

MW=586 Da

Brine concentration: 30% (w/v) NaCl

500% (v/v) float

5 μM CoroNa Green

Stereomicroscope equipped for epifluorescence

Samples irradiated with blue light (470/40 nm)

Magnification: 2.5x


Back-scattered/Low Vacuum Scanning Electron Microscope (SEM-BSE)

• Backscattered electrons consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume. • The brightness of the BSE image tends to increase with the atomic number. • High-quality atomic number contrast images are produced.• No conductive coating is required.• Wet samples can be imaged directly as received.

SEM Settings• Low Vacuum Mode ON (0.98 Torr)• HV: 25.0 kV• Magnification: 50x• 3.0 Spot Size• Contrast: 88.0• Brightness: 44.9


Brine curing of raw hides and skins - Variables

• Concentration of brine

• Length of curing

• Float percentage

• Mechanical action

• Temperature of curing

• Degree of fleshing of the hide

• Usage of additives and/or biocides

Mathematical Model

Mathematical model – Experimental

Lab scale drum

Piece of ~ 100 g

Cure with brine of various initial concentrations or floats (drum, 6rpm)

Wash raw hide @ 6 rpm for 2 hours, 100% float (with 0.15% Boron TS and

0.10% Proxel GXL)

• 300% (v/v) float at initial brine concentrations of 64, 80, 96 and 100 ºSAL.

• Initial brine concentration of 96 ºSAL at float percentages of 300, 500, 750 and 1000 % (v/v)

Collection of residual brine at different times

Filtration

Determination of chloride concentration (classical Mohr titration)

Continuous reaction model to describe the diffusion of NaCl from the bath containing brine solution to the surface of the solid phase (hide).

Assumptions• Salt will further diffuse into the hide’s inner volume where it will form a non-stationary concentration field. • Diffusion takes place only into the flesh side.• Hide parameters such as thickness, surface and properties of both hair and flesh sides will remain constant throughout the whole process.

0),0( x

c0)0,( xc

pcc 00 )0(

)(),( 0 cbc

),(),( 02

2

00

FXX

CFX

F

C

)(),1( 000 FCFC

)(),1( 00

00 F

F

CNaF

X

C

0),0( 0

FX

C

0)0,( XC

1)0(0 C

pc

cC

0

pc

cC

0

00

b

xX

2b

DFo

V

VNa 0

Na

Na

cc

ccC

P

P 12

)(

)()(

00

000

Mathematical model – Results

)(),( 00

c

Vbx

cDS

Non-stationary one dimensional concentration field (Fick´s second law)

Mass balance (closed system)

Boundary conditions

Dimensionless parameters

10 X

00 F

),(),(2

2

xx

cDx

c

bx 0

0

22

'

a

D

b

D

Dimensionless model

Transport coefficient

4,85

13,3

8,8

12,7

0

5

10

15

300 650 1000

% Float (v/v)

Dif

fusi

on

Co

effi

cien

t x

101

0 (

m2/s

)

Effect of initial brine concentration and %float on the diffusion coefficient

Note: assuming an initial hide thickness of 5 mm.


The values of λ may be affected by:

• Thickness of the hide

• The pore length

• Dry matter content of the hide

• Temperature

• Measurement error

Transport Coefficient λ for Various Conditions of Initial Brine Concentration

(c0p) and Soaking Number (Na)

Na = 3 C0p = 96 ºSAL

C0p (º SAL) λ·105 (s-1) R2 Na λ·105 (s-1) R2

64 4.2 0.835 3 5.3 0.949

80 3.8 0.921 5 9.0 0.887

96 5.3 0.949 7.5 4.1 0.905

100 10.7 0.776a 10 8.6 0.876 aR2 < critical value for α = 0.05.

1

9,44

20,4

4,855,71

0

5

10

15

20

25

65 75 85 95

Initial Brine Concentration (Salometer)

Dif

fusi

on

Co

effi

cien

t x1

010

(m

2/s

)

Initial brine conc. (º SAL)

Float (%) 85% Saturation?

64 - X

80 - X

96 440 √

100 280 √

Conclusions of the mathematical model

• The usage of saturated brine (100 ºSAL) as well as floats ≥ 500% (v/v) yielded higher values of the transport coefficient λ, and therefore higher diffusion rates.

• An 85% salt saturation level is attained in shorter times as the %float increases (and initial brine concentration permitting).

• The established 85% salt saturation in the hide plays a critical role in the search for optimum conditions of curing.


• Concentration of brine

• Length of curing

• Float percentage

• Mechanical action

• Temperature of curing

• Degree of fleshing of the hide

• Usage of additives and/or biocidesUsage of commercial degreasers and microbial biosurfactants

Brine curing of raw hides and skins - Variables

Degreasing study – Introduction

Grease distribution in raw hide and wet blue

Removal of fat

Increase of NaCl uptake

Reduction of turn around times in the raceway

Creation of additional curing capacity

• Non-ionic.

• NPE-free. Growing concern regarding the environmental persistence of NP-related compounds as well as their toxicity.

• Excellent wetting and grease removal properties.

• Moderate foaming.

• Rapidly biodegradable.

Commercial degreasers

Nonylphenol ethoxylate (NPE)

Alkylphenol ethoxylate (APE)Alkylphenol (AP)

Nonylphenol (NP)Ethoxylation

Primarily used as surfactants

Hydrophobic Hydrophilic

The European Union severally restricted marketing and use of NPEs effective January 2005

Alternative: Linear Alcohol Ethoxylates (LAEs)

0 12 2014

Greater attraction for fatty matter

More hydrophilic in nature

Hydrophilic-Lipophilic balance (HLB)


Ethoxylation

• Microbial biosurfactants produced by the yeast Candida bombicola using renewable (non-petroleum) feedstocks. Typically grown in a medium composed of two different carbon sources (usually sugar and oil) and a nitrogen source (frequently yeast extract).

• Disaccharide sophorose, typically with acetylated 6’ and 6’’ hydroxy groups.

• A fatty acid (FA), linked to the disaccharide through a glycosidic bond. The FA chain length varies between 16 to 18 carbons, which may be saturated or unsaturated.

• The carboxylic acid portion of the fatty acid can be lactonized to the disaccharide ring or remain as a free acid.

• They are non-toxic and biodegradable.

• They have antimicrobial properties.

• They have a great potential for large scale commercialization.

• They are currently used in the cosmetic industry, in the formulation of high value products, and as an active ingredient in detergent composition.

Sophorolipids (SLs)

Lactonized form of C18:1 SLFree acid form of C18:1 SL


Piece of ~ 100g

Cure with 100 °SAL brine (initial concentration) for 16 h @ RT and 6 rpm,

500% (v/v) float

Squeeze out the excess moisture

% moisture

% ash

% salt saturation

% fat content

Addition of commercial degreasers OR sophorolipid

• Commercial degreaser 1, 2 and 3 at 0.5% (w/w), with respect to combined weight of solution plus hide.

• Commercial degreaser 1 at a 0.25, 0.5 and 1% (w/w) concentration level.

• Sophorolipid at 0.5% (w/w), filtered or unfiltered.

Wash raw hide @ 6 rpm for 2 hours, 100% float (with 0.15% Boron TS

and 0.10% Proxel GXL)

Degreasing study – Experimental

Commercial Degreasers - Results

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)

Effect of commercial degreasers on brine curing [0.5% w/w]



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


Degreasing study – Results

Fat Removal (%)/Degreaser As it is Moisture free basis (MFB)

Moisture and ash free basis (MAFB)

Degreaser 1 51.8 48.3 42.5

Degreaser 2 9.5 11.5 10.9

Degreaser 3 48.7 47.8 46.1

SL-filtered 36.9 32.3 27.2

SL-unfiltered 36.6 38.5 40.4

Commercial degreasers and SL: 0.5% (w/w)

Effect of sophorolipid on brine curing



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



Sophorolipids - Results

Conclusions of the degreasing study


• The addition of a 0.5% (w/w) of a commercial degreaser made of a blend of nonionic surfactants to the brine, significantly decreased the fat content of the hide and significantly enhanced the uptake of salt as well.

• The composition of the degreaser was a critical parameter for the purpose of defatting the hide. It is likely that different values of HLB amongst the various commercial degreasers affected their activity onto the hides.

• 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) make it an attractive choice of surfactant.

Future work – Preservation of raw hides and skins

• 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%.

• 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.

• A non-destructive rapid test method for cure validation is needed.

Introduction – Fillers in the leather industry

Veins

• Easily recognizable.

• Caused by many factors: age, diet and breed of the animal, climate, period of slaughter, improper bleeding…

• Most likely in belly and neck areas.

Grain break

• Characterized by the wrinkles formed on the surface of leather when it is bent grain inward.

• Naturally occurring characteristic of the skin, although it can be influenced by processing.

• Most likely in belly and shoulder areas.

Coarse break Fine break

Veiny wet blueRegular wet blue


Filler

• Give more body to the leather.

• First fillers: extracts of vegetable tanning agents, barium compounds, glucose, flour…

• More recently, they were obtained from conventional petroleum feedstocks.

Utilization of waste proteins

Price ($/lb)

Sodium caseinate 5.80

Gelatin 2.60

Whey 0.31

Whey protein isolate 1.05

Becoming increasingly expensive


Property β-Lg α-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

Whey

• Whey is a by-product of cheese production.

• Whey is composed by lactose (70-75%), minerals (12-15%) and whey proteins (8-10%).

Beta-lactoglobulin (ß-Lg)

Alpha-lactalbumin (α-La)

Bovine serum albumin (BSA)

Immunoglobulins (Ig)

Lactoferrin (LF)


Gelatin• Obtained by the partial hydrolysis of collagen (acidic, alkali or enzymatic).

• The capability to form heat-reversible gels is one of the most unique properties of gelatin solutions.

• The Bloom strength gives an indication of the strength of a gel formed from a gelatin solution.

• It can be isolated from solid tannery waste (e.g. chrome shavings).

Hydrolysis

Bloom strength Average molecular weight (kDa)

50 – 125 (Low Bloom) 20 – 25

175 – 225 (Medium Bloom) 40 – 50

225 – 325 (High Bloom) 50 – 100


Substrate Effect of transglutaminase

Gelatin, caseinate, soy protein isolate, egg yolk

• Increase in breaking strength of gels

Caseinate• Increase of gel forming ability, modification of breaking strength, strain and cohesiveness of the gels formed.

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.

Soy protein• Increase in solubility, decrease in surface hydrophobicity, improvement of emulsifying and foaming properties, reduction of bitterness.

Gluten • Improvement of solubility, foaming, emulsifying and surface functional properties.

β-lactoglobulin • Enhancement of heat stability, increase of viscosity and gel forming ability.

Microbial Transglutaminase (mTGase)• Enzyme capable of forming inter- or intra-molecular crosslinks in many proteins.• Catalyzes an acyl transfer reaction between the γ-carboxamide group of peptite-bound glutamine

residues as acyl donors and primary amines as acceptors.

Acyl donor

Acyl acceptor

ε-(γ-glutamyl)lysine crosslink

Biopolymer study – Experimental

And the next day…

CharacterizationIncubation @ 45 ºC for 5 h

Addition of mTGase solutions in selected samples

10 min @ 90 ºC

Cool to RT 17 h @ 10 ºC

• Gel strength

• Viscosity

• Rheology

• SDS-PAGE

Whey protein isolate (from 1 to 10% w/w)

Type B Gelatin (from 1 to 10% w/w)

10 mg Dithiothreitol (DTT)/g protein

Swell Heated @38 ºC 1 h

Cool to RT

Adjustment of pH to 7.5

Store @ 4 ºC

Biopolymer study – Results

Biopolymer study – Gel strength results

G: Gelatin (from 0 to 3% w/w)W: Whey protein isolate (10% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Enzyme. Reacted with 2 U mTGase/g protein

• Increasing gel strength with increasing gelatin concentration.

• Significant effect of the reductant DTT at gelatin concentrations > 6% (w/w).

• Significantly lower gel strength values in diluted (1 to 5% w/w) gelatin solutions reacted with the enzyme mTGase, with respect to samples of gelatin alone.

G: Gelatin (from 1 to 10% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Enzyme. Reacted with 10 U mTGase/g protein

• Very weak gels for gelatin alone or blended with 10% (w/w) WPI, with or without mTGase in a non-reducing environment.

• Dramatic increase in gel strength for mTGase-treated WPI-gelatin blends in a reducing environment.


Biopolymer study – Viscosity results

G: Gelatin (from 1 to 10% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Reacted with 10 U mTGase/g proteinViscosity measured at 60 ºC

W: Whey (from 1 to 10% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Reacted with 10 U mTGase/g proteinViscosity measured at 25 ºC

G: Gelatin (from 0 to 3% w/w)W: Whey protein isolate (10% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Reacted with 2 U mTGase/g proteinViscosity measured at 25 ºC

• No effect of neither mTGase nor DTT alone on gelatin viscosity.

• Dramatic increase in viscosity of gelatin solutions of concentrations ≥ 7% (w/w), reacted with mTGase in the presence of DTT.

• Significant effect of mTGase on the viscosity of a 10% (w/w) WPI solution under reducing conditions.

• Significantly higher viscosity values of WPI-gelatin blends than those of gelatin alone, only in a reducing environment.


Biopolymer study – Rheology results

W: WPI (10% w/w)G: Gelatin (3% w/w)R: Reducing conditions (10 mg DTT/g protein)E: Reacted with 10 U mTGase/g protein (for WE and WRE); or with 2 U mTGase/g protein (for WGE and WGRE)

2.5 mm

Time sweep measurements to study the evolution of the storage modulus (G’) as a function of time.

• Different behavior between a sample of WPI treated with mTGase in a reducing or non-reducing environment.

• Exponential increase of G’ in a WPI-gelatin blend incubated with mTGase in a reducing environment.

• The increase of G’ is associated to the formation of a permanent and reticulated network that becomes more stable with time.


Biopolymer study – SDS-PAGE results

1 2 3 4 5 6 7 8 90 Da

200 kDa

Gelatin WPI mTGase DTT Lane 2 √ × × × Lane 3 √ × √ √ Lane 4 × √ × × Lane 5 × √ √ × Lane 6 × √ √ √ Lane 7 √ √ × × Lane 8 √ √ √ × Lane 9 √ √ √ √

Inter-protein crosslinking was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE)

Lane 1: molecular weight markersLane 2: gelatin 10% (w/w)Lane 3: gelatin 10% (w/w) after treatment with mTGase (10 U/g) under reducing conditionsLane 4: WPI 10% (w/w)Lane 5: WPI 10% (w/w) after treatment with mTGase (5 U/g)Lane 6: WPI 10% (w/w) after treatment with mTGase (5 U/g) under reducing conditionsLane 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)Lane 9: WPI 10% (w/w) with gelatin 1.5% after treatment with mTGase (2 U/g) under reducing conditions


• 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.

• 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 bands due to inter-protein crosslinking in SDS-PAGE gel patterns than for either gelatin or WPI treated separately were observed.

• The reducing environment partially unfolds the whey proteins, increasing access to glutamine andlysine side chains, favoring the gelatin chains to 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.

Biopolymer study – Conclusions

Filler study – Experimental

Mechanical properties• Tensile strength• Young modulus• Tear strength

Subjective evaluation• Handle• Fullness• Grain tightness (break)• Color• General appearance

Extraction of aliquots (Protein determination assay)

All weights calculated on the basis of the weight of wet blue

Whey protein isolate

Type B Gelatin

Dithiothreitol (DTT)

Swell in 200% float

Heated @38 ºC 1 h

Cool to RT

Adjustment of pH to 7.5

Store @ 4 ºC

Addition of WPI + gelatin (200% float)

Retan-Color-Fatliquor

Drain 1 h @ RT

5 h @ 45°C

Neutralization with 4% (w/w)

NaHCO3

Addition of mTGase solution

(200% float)

Wet Blue

Drain

Wash (x2) Dry Analyses

Shoe upper

Upholstery

Filler study – Results

0

10

20

30

40

50

60

0 10 20 30 40 50 60

5% mTGase2.5% mTGase

Time (min)

Shoe upper wet blue – Protein uptake results

0

20

40

60

80

100

0 1 2 3 4 5 6

5% mTGase2.5% mTGase0% mTGase

Time (h)

Proteinaceous blend: 5% (w/w) WPI + 0.5% (w/w) gelatin, with respect to weight of wet blue

% protein uptake % washed out protein

0% mTGase 98% 8%

2.5% mTGase 86% 6%

5% mTGase 83% 0%


Upholstery wet blue – Protein uptake results

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)

mTGase offer: 2.5% (w/w)

Proteinaceous blend:2.5% (w/w) WPI + 0.25% (w/w) gelatin, with respect to weight of wet blue

% protein uptake % washed out protein

0% mTGase 90% 5%

2.5% mTGase 100% 4%


Protein uptake results – Uptake rate coefficients

One may consider that the absorption of protein by the wet blue follows a first order reaction kinetics, where k is the reaction rate coefficient.

Wet blue % mTGasea % WPIa Gelatina k (h-1) R2

Shoe upper 0 5 0.5 0.608 0.994

Shoe upper 2.5 5 0.5 0.413 0.851

Shoe upper 5 5 0.5 0.323 0.950

Upholstery 0 2.5 0.25 0.365 0.830

Upholstery 2.5 2.5 0.25 1.377 0.956

Uptake rate coefficient k for various treatments

x 0.68

x 0.78x 0.53

x 3.77

y = 0.3653x - 1.7999

R2 = 0.8297

y = 1.3767x - 2.4396

R2 = 0.9559-7.00-6.00-5.00-4.00-3.00-2.00-1.000.001.002.003.00

0 1 2 3 4 5 6

Time (h)

LN

(C

once

ntratio

n)

0% mTG

2.5% mTG

Linear (0% mTG)

Linear (2.5% mTG)

Upholstery wet blue

y = 0.3233x - 3.2307

R2 = 0.95y = 0.4133x - 2.6956

R2 = 0.8513

y = 0.6081x - 3.0909

R2 = 0.99370.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 1 2 3 4 5 6

Time (h)

LN

(C

once

ntratio

n)

5% mTGase 2.5% mTGase 0% mTGase

Linear (5% mTGase) Linear (2.5% mTGase) Linear (0% mTGase)

Shoe upper wet blue


Shoe upper wet blue – Mechanical properties

Three dimensional regression plots of the mechanical properties of the belly area with respect to % WPI and % mTGase.

Upholstery wet blue – Mechanical properties


Shoe upper wet blue – Subjective properties

0% mTGase 2.5% mTGase 5% mTGase

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

All test samples treated with 5% (w/w) WPI + 0.5% (w/w) gelatin, with respect to weight of wet blue


Improvement

No effect

Worsening

Shoe upper wet blue – Subjective properties (Grain break)

Control sample – Belly area

Pretreated with 2.5% mTGase followed by treatment with 5% WPI + 0.5% gelatin (all weights with respect to weight of wet blue).

Test sample – Belly area



Shoe upper wet blue – Subjective properties (Color)

Test: Pretreated with 2.5% mTGase followed by treatment with 5% WPI + 0.5% gelatin (all weights with respect to weight of wet blue).


Belly Butt Neck

Con

trol

Tes

tShoe upper wet blue – SEM images

Magnification: x1000

Test: Pretreated with 2.5% mTGase followed by treatment with 5% WPI + 0.5% gelatin (all weights with respect to weight of wet blue).

50.0 μm


Upholstery wet blue – Subjective properties

2.5% mTGase

Butt

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

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall

2.5% mTGase

0% mTGase

0% mTGase

0% mTGase

2.5% mTGase

2.5% mTGase

2.5% mTGase

All test samples treated with 2.5% (w/w) WPI + 0.25% (w/w) gelatin, with respect to weight of wet blue

Filler study – Conclusions

• A blend composed by whey protein isolate along with small amounts of gelatin was 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 was significantly improved over the samples that did not undergo the treatment with the WPI-gelatin blend.

• The grain break for shoe upper and upholstery leather fared markedly better when samples were pretreated with mTGase.

• The pretreatment of the samples with a solution of mTGase affected the protein uptake ratio k and the trend observed was reverse for the cases of shoe upper and upholstery leather.

• Filled samples were a little stiffer and presented slight lower tear strength than the control. However, the various treatments did not negatively affect the mechanical properties of crust leather.

• It opens up the possibility of exploring even cheaper sources of protein as a raw material for bio-based leather products.

Future work – Renewable biopolymers as filling agents 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. By doing so, a lesser concentrations of reactants and volume of float would be needed due to a stronger mechanical action.

• It would be interesting to make the filler from the tannery's solid 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 be increasing the value of low quality hides that will be treated with the product.

Acknowledgements

Universitat de Barcelona – Facultat de Química

• Dr. José Costa

• Dra. Carme González

• Blaia Martínez

• Manoli Mora

Consell Superior d’Investigacions Científiques

• Prof. Jaume Cot

• Dr. Agustí Marsal

• Dr. Albert Mª Manich

• Sr. Josep Carilla

• Dra. Merche Catalina

• Dr. Fernando Fernández

• Elena Bautista

Eastern Regional Research Center

• Ellie Brown, PhD

• Ms. Maryann Taylor

• Ms. Laurelie Bumanlag

• William Marmer, PhD

• Cheng-Kung Liu, PhD

• Daniel K.Y. Solaiman, PhD

• Peter Cooke, PhD

• John Phillips, PhD

• Mr. Nick Latona

• Ms. Renée Latona

• Mr. Joe Lee

• All the visiting scientits I met during my stay at the ERRC.

Tomas Bata University (Czech Republic)

• Prof. Karel Kolomazník

• Michaela Uhlirova, PhD

My Family

THANK YOU

Technology

Ph.D. in Chemical Engineering Presentation