Bioactive Fibers and Polymers - ACS Publications - American

Chapter 16

Biodeterioration of Wool by Microorganisms and Insects Jeanette M. Cardamone Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 26, 2015 | http://pubs.acs.org Publication Date: August 10, 2001 | doi: 10.1021/bk-2001-0792.ch016

Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038

Wool is stable through alpha-keratin peptide linkages and a highly crosslinked network of disulfide bonds. Microbes and insects can digest wool by secreting extracellular keratinolytic enzymes that catalyze the hydrolysis of peptide linkages releasing polypeptides and soluble sulfhydryl-containing amino acids that disrupt disulfide bonds. Ensuing mechanical damage opens the fiber's outer cuticle to attack of the inner cuticle layers and the lipid-rich complex of membrane cells (CMC), whereby the innermost structural elements, the cortical cells, become vulnerable to enzymatic digestion. Wool keratin can be the ideal substrate for enzymatic attack by proteases, esteroses, lipases, and those that act specifically on disulfide bonds. The principal causes of accelerated microbial growth are prolonged high moisture, ambient temperature, and assimilable sources of nutrients for the specific organism. Enzymatic digestion is assisted if carbohydrates, fats, and additional sources of nitrogen are present in culture media, soaps, suint, and products of chemical attack on wool. Wool's heterogeneous and composite structure confounds the elucidation of the mechanisms of biological decay, consequently correlations among chemical and structural modifications are difficult. Biological decay can be documented with microscopy inspection to show damage to the edges of the distal scales on the cuticle and disaggregation with fiber fibrillation and fraying to the cortical cells. The role of wool's morphological fiber components (including constituent fatty acids of wool's lipids as sole sources of carbon and energy) to resist microbiological attack by bacteria, fungi, and mildew is examined, as are chemical modifications to protect wool from insect attack.

f

Mention of brand or firm name does not constitute an endorsement by the Department of Agriculture above others of a similar nature not mentioned. © 2001 American Chemical Society

In Bioactive Fibers and Polymers; Edwards, J. Vincent, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

263

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 26, 2015 | http://pubs.acs.org Publication Date: August 10, 2001 | doi: 10.1021/bk-2001-0792.ch016

Introduction

For 12,000 years, wool, the "Golden Fleece," has provided not only warmth and comfort, but has been and is a vital commodity in world trade. With the advent of wool spinning in 3500 B . C . men could expand the frontiers of exploration, thereby spreading civilization, with sheep at their side to provide food and clothing suitable for changing climates. Throughout history, the sheep industry spread from Central Asia to Europe by way of Greece, to Rome, Persia, North Africa, and to Europe where all contributed to improvements in the breeds. The Spaniards who colonized California and New Mexico introduced the first sheep into America in 1519. By 1698 wool production had become a colonial initiative and essential enterprise for export and trade. Soon spinning and weaving became acts of patriotism to allay the threat of British imperialism. By the 19 century millions of sheep grazed in the Southwest where Spanish sheep were mixed with the sheep from Eastern herds brought by pioneers seeking grazing lands in the West and Northwest. Although wool's current position in various end-use markets is low as a percentage of fiber production worldwide, it is stronger than the overall data might suggest because wool is positioned at the quality, high value end of the market. In these sectors wool's position is stronger than in the market as a whole. th

Wool's physico-chemical structure combines strength, elasticity, and resiliency with hygroscopicity for durability and comfort. As wool absorbs moisture, heat is generated, causing a net slowing of the transmission of body heat; hence, wool is warm in winter, (i) Wool fiber consists of a thin outer covering of overlapping, scales for moisture penetration into inner spindle-shaped cortical cells at the fiber core. These cortical cells are longitudinally divided into distinct sections, their relative proportions and arrangement varying with fiber diameter. (2) They exhibit differential behavior to chemical and physical stimuli, and are responsible for wool's crimp that contributes to warmth and insulation. Paradoxically, wool provides coolness by slowing heat transmission from the atmosphere to the body and trapping body heat for slow cooling through evaporation. The complex nature of wool and its unique properties suggest a myriad of end uses that continue to inspire scientific investigations. The hydrophobic nature of the surface of wool keratin fibers suggests that a waxy or oily lipid material is present. Apparently and observably, the surface and bulk portions of wool exhibit different mechanical and chemical properties. Lipids, present both in external grease originating from sebaceous gland secretions (wool grease) and in internal structural material, were proposed to originate as lipoprotein membranes of once living follicle cells (3). There is evidence of amide and ester covalent linkages between lipid and protein in the protein membranes (4). The three major lipid classes in wool with their approximate amounts are: sterols (40%) consisting of cholesterol and desmosterol (2:1), polar lipids (30%) consisting of ceramides and glycosphingolipids, cholesterol sulfate, and fatty acids (25%) consisting of stearic, palmitic, 18-methyleicosanoic acid, oleic, and myristic acids (4). Only small amounts of phospholipids have been found. The composition of raw wool from fine Merino and coarse Lincoln breeds is reported in Table I.


265 Table I. Composition of Raw Wool (5) Component Moisture Grease Suint" Sand and dirt Clean fiber Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 26, 2015 | http://pubs.acs.org Publication Date: August 10, 2001 | doi: 10.1021/bk-2001-0792.ch016

3

Merino 16.8 20.2 9.3 7.2 46.5

Lincoln 17.1 6.7 2.3 5.1 68.8

a

An ester of high molecular weight fatty acids - a saponifiable portion contains esters of cerotic, stearic, and palmitic acids with some free fatty acids, and an unsaponifiable portion containing cholesterol (C27H45OH). Dried perspiration consisting of a mixture of potassium salts of organic acids (oleic, palmitic, and acetic). b

Wool is a member of the alpha-keratin family of proteins, as are horn, fingernails, skin, and feathers. Its fiber diameter ranges from 18 microns (fine-grade) to 40 microns (coarse-grade). The morphology of wool is highly complex because its composite structure includes an outer layer of grease that sometimes exceeds the weight of protein, and a covalently bound surface layer surrounding differentiated cellular structures. Each fiber is made up of three cellular structures: the cuticle, cortex (90% by weight), and the medulla (hollow cells that are absent in fine wool fibers) (6). Cuticle cells or scales are flat, rectangular, plate-like structures that are arranged to overlap in a manner similar to roofing tiles, to completely cover the cortex. They compose the outermost regions of the wool fiber and can be removed by ball milling, grinding, scraping, slicing, or enzymatic treatment. Within each cuticle cell there are three isolatable layers: the exocuticle A-layer, ( 1 0 0 ° A thick) being outermost that forms sacs on the outer surface, the exocuticle B-layer that is more readily digested by enzymes, and the innermost layer, the endocuticle (7). During the keratinization process, the living cell membranes form a new structure commonly referred to as the Cell Membrane Complex (CMC) that exists underneath the cuticle and between all regions in assemblies of keratinized tissues, for example, within the intercellular spaces between the underlying cortical cells of the wool fiber. Essentially, the cuticle and cortical cells are held together by a continuous, chemically-resistant proteinaceous C M C membrane. Under transmission electron microscopy two regions were identified as comprising the C M C : an intensely stained gamma-layer consisting mainly of protein and a beta-layer containing lipids. (8). The lightly crosslinked protein material is referred to as "globular protein cement." The lipid component is susceptible to enzymatic attack. Although only 3% by weight of fiber, it has an inordinate effect on mechanical and chemical properties. Current thinking is that the adhesive strength of the C M C derives from intercellular linkages formed between intermediate filament proteins of neighboring cells bridged by proteins remnant from the membrane keratinization process (9). The C M C contains a similar amino acid composition as wool but with lower cysteine content (1.3% compared to 10% for whole wool and 11.9% for the epicuticle), thus, it contains less



266

crosslinked disulfide bonds (10). Consequently, it is easily swollen and easily modified. Underneath, the cortical cells (100 microns long and 4 microns thick) are arranged lengthways along the fiber axis as closely packed elements that develop near the base of the fiber follicle. They elongate, keratinize, and form cross-links through the amino acid, cysteine. The differentiation of the cortex into ortho- meso-, and para cells leads to a bilateral structure (one side referred to as ortho-cortex and the other as paracortex) that is responsible for the crimp in wool fibers. Whiteley and Kaplin have shown that in the fine (Merino sheep) fibers, those with low-crimp frequency differ from those with high crimp frequency by the substitution of more mesocortex for paracortex on the central paracortical side of the fiber (11). In Figure 1, the mesocortex is shown at the boundary of the bilateral structure as a highly ordered component that is differentiated from the paracortex. In a wool follicle the paracortex is found on the inside of a crimp curvature and is thought to be richer in cysteine amino acids, thus it reacts differentially to environmental changes (12).

Figure 1. Cross-section of a Merino woolfibershowing the structure at prog magnifications (Produced by H. Roe from a drawing by Dr. RDB Fraser, Freughelman, M., Mechanical Properties and Structure of Alpha-keratin Fi University of South Wales Press, Sydney, Australia, 1997). At a higher level of order, the proto-, micro-, and macrofibrillar structures of wool are made up of the cortex cells containing spindle-like crystalline proteins surrounded by the amorphous C M C matrix that is rich in high-sulfur and high



267

glycine/high tyrosine globular proteins. The assembles of microfibrils, rich in lowsulfur proteins containing lysine, leucine, and aspartic and glutamic acids, favor the alpha-helix configuration (13). Each microfibil can be approximately 7nm in diameter and at least one micron long (14, 15). The protein chains are associated through hydrogen bonding and polar forces of intramolecular attraction and cystine disulfide groups are associated through covalent bonds of intermolecular attraction. Protofibrils pack to form microfibrils that build to form higher-ordered macrofibrils. A cortical cell, composed of a bundle of 500-600 microfibrils as the primary structural element in keratin fibers, has been described as "a bundle of keratin intermediate filaments, embedded in keratin-associated proteins" (76). These filaments aggregate in discrete domains to form the microcrystalline structure of alpha-keratin wool fibers. In 1950, X-ray data confirmed that wool's molecular architecture is one of associated helical configurations. It was postulated that "there are 3.6 to 3.7 amino acid residues per turn of the helix and the nitrogen atom of each planar peptide is bonded to the oxygen atom of the third peptide beyond it in the chain by a hydrogen bond with length 2.79°A" as shown in Figure 2 below (17).

Figure 2. Helical molecular arrangement of wool (The International W Secretariat, Heusden, The Netherlands) Wool keratin is of amphoteric nature, exhibiting acidic and basic functionalities that contribute to ionic forces of attraction or salt linkages between helical chains, shown in Figure 3 below. Through hydrolysis wool decomposes into the various amino acids shown in Table II. In Bioactive Fibers and Polymers; Edwards, J. Vincent, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

In Bioactive Fibers and Polymers; Edwards, J. Vincent, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001. 89.10

117.15

131.18

165.19

Alanine (Ala)

Valine (Val)

Leucine (Leu)

Phenylalanine

(Phe)

75.07

MoLWt.

Glycine (Gly)

Amino Acids

3.75

11.30

4.72

4.40

6.50

a

NP

aromatic

NP

NP

NP

NP

% of Wool Chemical (g/WOg) Nature

I

^H

3

CH—CH*—Ç—COO"

Continued on next page.

OHcr

C H/

\H— — i c

CH —Ç—COO" NH, + 3

H—(j C O O " NH ν

1

Structure

Table Π. Amino Acid Composition of Wool Keratin (Molecular Weight 68,000 kD) (18,19)


In Bioactive Fibers and Polymers; Edwards, J. Vincent, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001. thiol NP

cyclic NP

6.76

5.80

119.12

181.19

Threonine (Thr)

Tyrosine (Tyr)

phenolic Ρ

hydroxyl Ρ

hydroxyl

9.41

105.09

Serine (Ser)

c

NP

121.16

0.71

6.75

% of Wool Chemical (z/lOOg) Nature sulfhydryl 12.72

Mol.Wt.

149.21

115.13

Cysteine (Cys)

Amino Acids

(Met)

Methionine

Proline (Pro)

s

7

3

3

f

x

2

3

H —Ç—COO"

Ç—COO

I NHi

CH3—ç

m

+

HO—CH^—Ç—COO" NH +

ι

*H

HS—CH^—C—COO*

Structure

NH +

cn —s—CHf—CHr-c—coo-

H

Hi



204.23

133.11

147.13

174.20

Tryptophan (Trp)

Aspartic Acid (Asp)

Glutamic Acid (Glu)

Arginine (Arg)

Table II. Continued

10.40

15.27

7.27

0.70

basic (+)

acidic (-) Ρ

acidic (-) Ρ

indole NP

r

2

2

2

C—CH —C—COO

Η

f— CH —CH —Ç—COO"

Ό \

Η


κ»


155.16

Histidine (His) 0.70

3.30

imidazole Ρ

basic (+) Ρ

Ψο of Wool Chemical (R/WOR) Nature

Structure

J

Η

HN^NH

NH,

r

2

+

NH

3

H3N—CH-j—CHj—CH2—CH —C—COO"

disulfide groups through intermolecular crosslinking as shown in Figure 3.

146.19

Lysine (Lys)

Amino Acids Mol Wt.


272

Υ>

α/

•ai

\ »

W ai


Ni/

-a/

œ — -a/ W NH

-CH

œ

œ

Μ/ Χ

οο

C>tinelii*age

W

a> W mL—

ΛΗ

œ W M /

GhÉamcackl

Lysine

Sai Linkages

œ V»

Figure 3. Molecular Structure of Wool (Von Bergen, W., American Wool H Text and Reference Book for the Entire Wool Industry. NY: Textile Book Pu 1948).

Wool's chemical composition is 50% carbon, 22-25% oxygen, 16-17% nitrogen, 7% hydrogen, and 3-4% sulfur (19). The salient features of wool's composition leading to its unique set of physical and chemical properties are the following: a great number of protein configurations enable great flexibility; a large number of highly polar peptide linkages give rise to inter- and intramolecular bonding and high reactivity; large side chains pendant from the polypeptide backbone prevent close packing of the protein molecules leading to a high amorphous content accessible to the environment; forces of association through side chains including intramolecular hydrogen bonding and intermolecular covalent bonding through cystine's disulfide linkages contribute stability and strength. Reagents that alter the stability of the disulfide bond will alter and destroy wool's physical structure (19).


273


Microbial Contamination Wool as fleece on the sheep, and through many stages of manufacture and wear, is subjected continually to contamination by microorganisms. Pathogenic organisms may be contained within this microorganic flora, as well as nonpathogenic microbes, capable of multiplying rapidly under favorable conditions. The problem becomes visually apparent when wool develops mildew staining that can accompany strength loss or other forms of deterioration. Mildew will form in wool storage when bales become wet or in fabric storage when occluded starch or glue becomes wet. A comparison of the amounts of mold and bacteria found in wet and dry wool has been reported as shown in Table III.

Table III. Bacterial and Mold Counts per gram in Wool (19) Raw Wool Shaken Wool Scoured Wool Wet Molds Bacterial, all types Bacterial spores

2,700 1,200,000 190,000

36,000 17,000,000 210,000

Scoured Wool Dried 300 3,400,000 110,000

300 65,000,000 100,000

Shaking spreads molds and bacteria and results in a wider distribution of spores over the fiber surfaces. In the case of molds, scouring removed substantially most of the contaminant; but led to higher bacterial counts because of redistribution over a greater area. Only scouring followed by drying with heat was effective in inactivating bacterial contamination. Modern nitrogen scours with ethoxylate detergents at 65C have been found to kill most organisms before they reach the peroxide bath (20) In simulated storage studies, the mean numbers of bacteria and fungi in 30 scoured wools were recorded before and after storage in TablelV.

Bacteria Fungi

Table IV. Number of Micro-organisms on Scoured Wool (Mean Counts /gram) (21) After Storage Before Storage 10 10 - 10" 10

Bioactive Fibers and Polymers - ACS Publications - American

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