Impregnated Sheep Wool Fibers with an Antimicrobial Effect ^†

Abstract

This study proposed an approach for the enhancement of coarse wool from a Romanian sheep breed—Țurcana—for its utilization as a sustainable and efficient thermal insulation material. The process involved washing with a non-polar solvent, to eliminate lanoline and impurities from the raw sheep wool, followed by an impregnation with a binder, dolomite, and copper nanoparticles. The structural and elemental changes were analyzed using the scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) techniques. The antimicrobial effects of copper nanoparticles were analyzed on two bacterial strains. The proposed enhancement process creates a barrier against harmful biodeterioration activities by bacteria.

1. Introduction

The built environment significantly contributes to energy consumption and CO₂ emissions [1]. Using natural fibers in such a built environment, as replacers of synthetic alternatives, has garnered significant attention [2]. The increased interest in these natural fibers is due to their specific properties, cost-effectiveness, health benefits, and recyclability. Both engineering and ecological design approaches prioritize non-toxic materials, transformability, and recyclability. Prioritizing organic materials aligns with alternative resource requirements, sustainable development, and innovative green technologies [3,4].

However, exclusively focusing on thermal insulating “sealing” based on the total heat transfer is not ideal, as a low ventilation rate can lead to the accumulation of moisture, volatile organic compounds (VOCs), allergens, gases, radioactive materials, pollutants, and inorganic substances, significantly decreasing the indoor air quality [5].

Throughout history, various natural resources have provided the raw materials for clothing, and these renewable and sustainable fibers remain essential components of home textiles, technical textiles, and apparel. Farmers harvest approximately 35 million tons of natural fibers annually from various plants and animals [6]. Among these fibers, sheep wool is one of the oldest textile materials in human history.

The appeal of using sheep’s wool lies in its remarkable thermo-insulating properties, offering both warmth and breathability. In the modern era, the utilization of sheep’s wool has transcended traditional use, expanding into diverse industries, such as fashion [7] or construction [8,9]. Its inherent qualities make it an invaluable resource, effectively combatting extreme temperatures and efficient heat regulation during hot summers. This thermal insulating characteristic is attributed to its structure, characterized by the overlapping scales and crimps that create air pockets, ensuring optimal temperature control and efficient moisture management [3,6].

To unlock the full potential of sheep wool, it must undergo a series of crucial processes. Commencing with sheep wool washing, the fibers are meticulously prepared for further refinement. Subsequently, the valuable substance lanolin is extracted, contributing to the wool’s prized properties.

Under the natural conditions, the wool is impregnated with lanolin, a wax that is formed via the esterification of lanolin alcohols and lanolin acids. Depending on the sheep breed and wool quality, wool grease constitutes 5–25% of the weight of the shorn fleece [10]. Lanolin serves as a natural lubricant that shields the wool from weathering effects, like the sun, wind, and rain. The raw wool also contains considerable amounts of soil, salts, water, and foreign organic matter [11]. The optimal residual lanolin content in wool fibers should be around 2–3% by weight of lanolin per clean, dry wool. This critical parameter requires meticulous control prior to wool manufacturing, as the processing properties of wool textiles are intricately linked to the lanolin residues present in the fibers [12].

Sheep wool is also considered a particular by-product due to its potential bacterial load, as it consists of keratin proteins that act as nutrients for bacteria. Many antimicrobial agents are used, especially in the textile industry, for treating wool-based materials, such as metals and metal compounds [13].

The bioactivity of copper, both as a free ion and in the form of copper complexes, has been known for centuries, with the evidence dating back to ancient civilizations of treating various conditions or maintaining hygiene with copper-based materials. Although the human body is not highly sensitive to copper, it severely affects microorganisms, including bacteria, fungi, and micro-algae. Copper generates hydroxyl radicals that lead to the oxidation of proteins and the cleavage of DNA and RNA molecules [14,15]. Another inorganic compound with good stability and antibacterial effect is dolomite. This is a mineral that is commonly found in nature, mainly consisting of calcium magnesium carbonate, which, due to its adsorption capacity, can inhibit the growth of bacteria [16,17,18]. Dolomite also has a high thermal stability, indicating that it is heat resistant, which is an additional benefit that can be integrated to increase the fire resistance performance of products.

This study proposed an approach for enhancing the properties of local sheep breed wool to serve as a thermal insulation material. To achieve this, a solvent washing process was conducted to eliminate the lanoline and impurities, followed by the impregnation of the sheep wool fibers with the dispersion made from dolomite, copper particles, and binders in molds to ensure their uniformity. Incorporating copper nanoparticles during impregnation endows the wool with antimicrobial properties, making it highly effective in creating a protective barrier against harmful bacteria.

2. Materials and Methods

2.1. Sheep Wool Washing

The wool for the laboratory experiments was from the Romanian sheep breed Țurcana from the Câmpulung area, Argeș, Romania.

A series of wool samples were prepared through a washing process with a non-polar solvent. The sheep wool samples were accurately weighed and immersed in the respective solvents (1:10 w/w) within an airtight jar, allowing for a 24 h extraction period at room temperature. Subsequently, the washed wool was gently removed from the jars and left to dry for 10 h at 80 °C.

2.2. Preparation of Copper Particles

The component with bactericidal properties, finely divided Cu, was prepared through the reduction of Cu(NO₃)₂ in water in the presence of a triblock copolymer surfactant composed of polyethylene oxide (PEO) and polypropylene oxide (PPO). The reaction took place at 60 °C for 30 min, where an aqueous solution of 20% ascorbic acid was gradually added. After 2 h at 60 °C, copper aggregates were obtained, with particles of varying sizes between 100–680 nm obtained through dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (red badge).

2.3. Determination of Dolomite Porosimetry Characteristics

The textural characteristics of dolomite were assessed through nitrogen adsorption measurements that were performed on a NOVA 2200e-Quantachrome apparatus (Quantachrome Instruments, Boyton Beach, Florida, USA). Data processing was conducted using NovaWin software version 11.03. Prior to analysis, the samples underwent vacuum degassing at 160 °C for 4 h. The specific surface area was determined through plotting the adsorption isotherm using the BET (Brunauer–Emmett–Teller) method, while the total pore volume and pore size distribution were estimated based on the amount of N₂ adsorbed.

2.4. Preparation of Impregnated Wool

In order to obtain impregnated wool, degreased wool fibers were placed in a lid container. An aqueous dispersion based on polymers, copper microparticles, and dolomite was prepared. The dispersion obtained was applied by spraying onto wool fibers that were previously degreased with solvents. The impregnated wool was arranged in a mold and allowed to undergo the drying process. Subsequently, samples were collected from the prepared wool specimens (

Table 1. Sheep wool impregnation composition.

Sample Code	Characteristic
1	Sheep wool washed and impregnated with 5% polymer and 5% dolomite
2	Sheep wool washed and impregnated with 5% polymer and 5% dolomite
3	Sheep wool washed and impregnated with 5% polymer, 5% dolomite, and 0.5% Cu microparticles
4	Sheep wool washed and impregnated with 5% polymer, 5% dolomite, and 0.5% Cu microparticles

). Samples 1 and 2 were impregnated with dolomite and polymer, whereas samples 3 and 4 were further treated with copper particles during the impregnation process.

Samples of untreated and impregnated sheep wool were analyzed using a Hitachi TM4000plus II scanning electron microscope with a backscattered electron (BSE) detector and equipped with energy-dispersive X-ray spectrometry (EDS). Prior to analysis, the samples were coated with a thin layer of gold measuring 5 nm in thickness. Subsequently, they were attached to a carbon tape and carefully positioned within the apparatus.

2.5. Experimental Protocol for Testing the Antimicrobial Effect

The determination of the antimicrobial potential of the wool samples coded 1, 2, 3, and 4 against the reference strains Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 was conducted using the liquid medium cultivation method (Figure 1). The experimental protocol involved the following steps:

• Sterilization of the receptacles through exposure to UV radiation for 30–40 min on each side.
• Dispensing the sterilized receptacles into sterile 50 mL bottles.
• Adding 6 mL of a nutrient culture medium composed of 10 g/L peptone, 1 g/L meat extract, 2 g/L yeast extract, and 5 g/L NaCl, at a pH of 7, to each bottle.
• Inoculating the culture medium with 30 µL of bacterial inoculum, adjusted to a turbidity equivalent to the 0.5 McFarland standard, corresponding to a bacterial density of 1...2 × 10⁸ CFU/mL, resulting in an inoculum density of 5 × 10⁵ CFU/mL in each sample.
• Incubating the samples at 37 °C with agitation at 140 rpm for 24 h.
• Plating 100 μL from each sample (appropriately diluted in sterile saline) onto the surface of a solid nutrient agar medium (composed of 10 g/L peptone, 1 g/L meat extract, 2 g/L yeast extract, 5 g/L NaCl, and 18 g agar; pH 7.0).
• Incubating the plated samples at 37 °C for 24 h.
• Counting the colonies and determining the number of colony forming units (CFU) per mL in the tested samples compared to a blank sample (without wool).
• The percentage of bacterial growth reduction was calculated using Formula (1).

% = 100 − [100 × (CFU_sample/CFU_blank)

(1)

3. Results

This section shows the results of the tests that were conducted. Figure 2 illustrates the dimensions of the unwashed wool, revealing the presence of indistinct wool yarn cuticles that are likely covered with impurities. These dimensions vary within the range of 30–80 µm. Subsequent washing of the fibers with various solvents proved to be highly effective in their cleansing, as depicted in Figure 3, where the fiber cuticles are more clearly visible. Their sizes ranged from 27 µm to 61 µm.

SEM images of the impregnated samples at an accelerating voltage of 15 kV have been presented in Figure 4. The deposition of adhesives, dolomite, and copper were clearly observed as powder deposited on or around the fibers.

In order to analyze the elemental composition of the samples, energy dispersive spectroscopy (EDS) was carried out. Figure 5 and Figure 6 depict the composition of the impregnated fibers, primarily comprising carbon and oxygen.

Additionally, smaller quantities of elements, such as potassium, sulfur, calcium, magnesium, aluminum, sodium, and silicon were also found to be present. It has been noted that the spectra and percentages provided in this study do not represent the true composition of the samples due to the limited analysis area and lack of complete homogeneity.

Table 2. The elemental percentages obtained in EDS analysis.

Element	Wt %
	Sample 3	Sample 4
O	41.1	38.8
O	22.5	24.7
Cu	22.1	22.4
N	11.1	11.7
K	1.3	0.4
Ca	0.9	1.1
S	0.9	0.9
Cl	0.1	0.0

shows the percentages of the elements that were found in the impregnated wool samples corresponding to the two EDS figures.

Table 3. The specific surface and the total pore volume of the used dolomite.

Product Name	Specific Surface (m2/g)	Total Pore Volume (cm3/g)	Average Pore Diameter (nm)
Dolomite	5.108	0.0055	8.508

shows the characteristics of dolomite used in wool impregnation.

The specific surface area was 5.108 m²/g, which indicates that the used dolomite possesses a moderate to relatively high surface area per unit mass. Dolomite could contribute to the antibacterial activity due to its structure being prone to nanoparticle formation. An antibacterial material exhibits strong antibacterial activity when it includes/forms nanoparticles [16].

The results of testing the effect of samples 1, 2, 3, and 4 on the growth of the Gram-positive bacterial strain S. aureus and the Gram-negative strain E. coli are presented in

Table 4. Results on the growth of the bacterial strains.

Bacterial Strain	Sample	CFU/mL	% Reduction in Bacterial Growth Compared to the Blank Sample
S. aureus	Blank	8.75 × 1010	-
	1	4.1 × 1010	−53.1%
	2	2.92 × 1010	−66.6%
	3	5.1 × 102	−100%
	4	0	−100%
E. coli	Blank	1.06 × 1010	-
	1	5.1 × 109	−51.89%
	2	3.06 × 1010	0%
	3	0	−100%
	4	0	−100%

, and images with colonies after the incubation period are presented in

Table 5. Colonies after the incubation period in the presence of the samples.

	E. coli, Dilution 10−7	S. aureus, Dilution 10−7
Sample 1
Sample 2
	E. coli, undiluted	S. aureus, undiluted
Sample 3
Sample 4

. The following findings were observed:

• Samples 3 and 4 completely inhibited the growth of the E. coli and S. aureus strains.
• Samples 1 and 2 had either a moderate (Sample 1—S. aureus, Sample 2—S. aureus, and Sample 1—E. coli) or absent (Sample 2—E. coli) antibacterial effect.

4. Conclusions

This research shows the benefits of using ecological materials sourced from renewable or natural origins, particularly locally raw sheep wool from the Țurcana breed, as a promising option for thermal insulation. The incorporation of copper nanoparticles offers a resilient bacterial barrier without posing any harms to human health. Moreover, employing natural flame retardants, like dolomite, can improve the indoor air quality by absorbing harmful gases. This combination of eco-friendly materials and innovative approaches showcases the promising roles they can play in promoting sustainable and healthier living environments.

5. Patents

OSIM Patent Application A/00230/11.05.2023, “Thermal Insulation Material for Buildings Based on Sheep Wool and Method of Obtaining It “. Authors: Vasilievici Gabriel, Mîrț Andreea-Luiza, Ghimiș Simona-Bianca, Vlaicu Alexandru, Zaharia Emilian, Bomboş Mariana Mihaela, and Bomboş Daniel. Subsidiary contract sequence—1783/22.08.2022; applicants: National Institute of Research and Development for Chemistry and Petrochemistry (INCDCP)-ICECHIM; Atica Chemicals SRL.