Fibers and Polymers 2013, Vol.14, No.11, 1826-1833

1826

Antibacterial Finishing of Cotton Fabrics Using Biologically Active

Natural Compounds

Danko Abramiuc*, Luminita Ciobanu, Rodica Muresan, Magda Chiosac1, and Augustin Muresan

Textile-Leather and Industrial Management Faculty, “Gheorghe Asachi” Technical University, Iasi 700050, Romania1National Institute of Research and Development for Microbiology and Immunology “M. Cantacuzino”,

Iasi 700495, Romania

(Received January 4, 2013; Revised March 22, 2013; Accepted May 5, 2013)

Abstract: The paper discusses a method to functionalize cotton fabrics using biologically active natural compounds toachieve the antibacterial characteristics required for medical application. The biologically active natural compounds includepropolis, beeswax, and chitosan. Three 100 % cotton knitted fabrics with different degrees of compactness were impregnatedin the emulsions containing the active ingredients and fabric variant G3 with the highest degree of impregnation wasconsidered for the evaluation of the antibacterial properties and comfort characteristics. The results show that the treatedcotton fabric had high antibacterial activity against both gram positive bacteria Staphylococcus aureus and Streptococcus βhaemolytic, and gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The presence of the biologicallyactive natural compounds on the cotton substrates modified the surface of the textile fibers as seen in the SEM images. Thetreatment also improved fabric comfort properties, the cotton substrates became less air permissive and more hygroscopicafter the treatment. The experimental results indicated that propolis, beeswax and chitosan can be applied as an emulsion tofunctionalize cotton textile materials. The antibacterial performance of the functionalized fabrics suggested that the cottonfabrics treated with those biologically active natural compounds have the potentials to be used in medical fields.

Keywords: Beeswax, Propolis, Chitosan, Cotton, Antibacterial finishing

Introduction

Cotton materials have different medical and healthcare

applications due to their advantages such as biodegrad-

ability, softness, affinity to skin and sweat absorption [1,2].

Bacterial contamination leading to infection is a common

problem in hospitals. Therefore it is mandatory to reduce the

transmission of microorganisms by developing medical

textile fabrics with antibacterial properties [3]. The antibacterial

treatment of textiles depends on the application of these

functionalized materials; permanent treatments will ensure

durable antibacterial effect (including after washing cycles),

while single use treatments do not grant this durability.

The propolis known as the bees ‘glue’ is a substance used

to seal off the beehive openings in order to avoid air

currents. It is also a mean of defence against bacteria and

mould; other insects, killed when entering the beehive, are

isolated in propolis in order to prevent decay [4,5]. Qualitative

and quantitative chemical composition of propolis varies

according to the geographic area where the beehives are

placed. Up to now, over 180 components have been identified,

including flavanoids, phenolic acids and their esters,

phenolic aldehides, chetones, etc. [6]. Propolis is known for

its antibacterial, anti-inflammatory, hepatic-protective, antioxidant,

and allergenic characteristics [7,8]. Experimental results

show that the capheic acid and quercetine have no influence

on the production of antibodies in the organism, but are

responsible for the antibacterial activity, thus the phar-

maceutical characteristics of propolis are determined by the

natural blend between components and their combined

action [9]. The commercial propolis ethanolic solutions are

often used in treating minor lacerations, angina, or skin

infection. Further positive aspects of propolis refer to non-

toxicity and lack of secondary effects although isolated cases

of allergies to propolis have been reported, caused by certain

allergens in the plants [10].

Chitosan β-(1-4) linked 2-amino-2-deoxy-D-glucose, a

polysaccharide obtained from the alkaline deacetylation of

chitin attracts special interest due to its antibacterial and

immuno-enhancing characteristics, non-toxicity, and bio-

degradability [11] and can be used to inhibit fibroplasias in

wound treatment and enhances tissue regeneration, thus

making it usable in the field of medical textiles [12].

Chitosan can be dissolved using methods previously described

in literature [13-15]. The antibacterial effect is stronger for

the chitosan with low molecular weight, under 10 kDa [16].

The antibacterial effect can be explained through two

possible mechanisms. The former refers to the interaction of

the anionic groups with the surface of the microbial cell,

forming a film that blocks the transfer of nutrient substances

for the cell; the latter mechanism involves penetrating the

cell nucleus and RNA and protein synthesis inhibition [17].

Beeswax is water repellent, presents specific emulsion

properties, creates good lubrication and improves the touch

of textile fabrics [18]. Like propolis, beeswax composition is

different according to geographic region [19] and consists

mainly of esters of higher fatty acids and alcohols [20]. It has*Corresponding author: dabramiuc@tex.tuiasi.ro

DOI 10.1007/s12221-013-1826-4

Antibacterial Finishing Using Natural Compounds Fibers and Polymers 2013, Vol.14, No.11 1827

also been reported that beeswax contains small quantities of

hydrocarbons, acids, a number of other substances and

approximately 50 aroma components.

The literature survey conducted by the authors has revealed

the absence of any studies regarding the use of these three

natural substances applied as an emulsion on a textile

substrate, with antibacterial effect, for non-implantable medical

applications. This paper deals with the development of several

emulsion variants and studies the comfort characteristics and

bacterial activity of the treated cotton materials. The bacteria

(gram-positive and gram-negative) considered for the study

were selected because they are known to be the most common

cause of hospital infections.

Experimental

Materials

The textile substrates were produced on a circular knitting

machine Mesdan Lab Knitter, gauge 10E, using three levels

of density. The yarn count was Nm 60/1. The structural

parameters of the finished fabrics (horizontal density Do and

vertical density Dv, stitch length, and fabric weight (M/m2)

are presented in Table 1. An important advantage in using

knitted fabrics is that such materials present significantly

less transfer of fibers on the human skin as compared to

traditional woven materials. The transfer is practically eliminated

when the fabrics are impregnated with the emulsion considered

for the present study.

The chitosan solution was obtained by solving of chitosan

(Fluka Chemie GmbH, Switzerland, molecular weight

100,000-300,000 and degree of deacetylation 85 %) in acetic

acid 1 % solution (in order to ensure the complete solving of

chitosan). The solution was stirred for 24 h at room

temperature and filtered in order to remove impurities. 1 %

(w/v) chitosan solution was used for experiments.

The polysorbate 80 “Tween 80” was supplied by Merck,

Germany, the glycerol anhydrous by SC. Comecom SRL

Bucure ti, the ethanol pro analysi by Chemical Company

Romania, and the buffer solution pH 7.01 came from Hanna

Instruments, Hungary.

Propolis ethanol extract (EEP) solution 30 % (w/v) was

prepared from raw propolis with ethanol. The extraction

took place at 25oC, in a dark environment, for 48 h.

Beeswax and raw propolis were procured from a private

apiary in the North-East region of Romania.

Emulsion Development and Fabric Treatment

The emulsions were obtained by the mixing under agitation

at 80oC: beeswax, chitosan, EEP, glycerol, non-ionic surfactant

Tween 80, and water. In order to identify the influence of

each of the main ingredients, a number of seven emulsion

variants were prepared, varying within a predetermined

range the concentration of beeswax, EEP, and chitosan. The

composition of each variant is defined in Table 2. The pH of

the emulsion was determined to be 4, but the pH value can

be raised to 6 without influencing the amount of emulsion

retained by the knitted substrate. A higher pH value leads

to phase separation and the emulsion is no longer usable.

The fabrics were impregnated with the emulsion at 50 oC,

using a Benz padding machine adjusted to a wet pickup of

150 %. After padding, the samples were dried for 5 min, at

50 oC.

Evaluation of Treated Cotton Fabrics

Comfort Characteristics

When considering wound dressings and bandages, comfort

properties are very important in defining the patient’s well-

being. The comfort characteristics of the knitted substrate

are modified by the presence of the substances in the system.

Therefore, comfort indices related to vapor and air permeability

and hygroscopicity were determined. All samples were

conditioned in a conditioning room at 25oC, 60 % relative

humidity for 24 h. The measurements were done in triplicate,

the average values are presented in the charts.

The air permeability was measured according to SR EN

ISO 9237, on a METEFEM apparatus (Hungary), using

Δp = 10 mm water column, testing area 10 cm2.

The relative water vapor permeability was measured using

a Permetest apparatus (Sensora, Czech Republic), using a

method similar to ISO 11092. Relative water vapor permeability

of the textile sample pwv was determined with:

(1)

where us=heat losses of the free wet surface; u0=heat losses of

the wet measuring head (skin model) with a sample.

The hygroscopicity of the treated samples was determined

according to the following method: the samples were weighed

before and after they were kept in a desiccator filled with

sç

pwv

us

u0

----- 100 %⋅=Table 1. Values of the structural parameters

Fabric

type

Do

(wales/5 cm)

Dv

(rows/5 cm)

Stitch length

(mm)

M/m2

(g)

G1

G2

G3

58

54

50

90

72

58

2.91

3.40

4.25

124

110

105

Table 2. The formulations of the emulsions used for the treatment

Variant

Compound1 2 3 4 5 6 7

Beeswax g/l 12.5 25 37.5 25 25 25 25

Glycerol ml/l 100 100 100 100 100 100 100

Tween 80 ml/l 30 30 30 30 30 30 30

Chitosan 1 % (m/v) ml/l 200 200 200 100 300 200 200

EEP 30 % (m/v) ml/l 75 75 75 75 75 25 125

1828 Fibers and Polymers 2013, Vol.14, No.11 Danko Abramiuc et al.

distilled water (humidity 90 %) for 24 h. The hygroscopicity

was calculated as:

(2)

where w1=sample weight at 90 %RH and w0=sample weight

at 45 %RH.

Antibacterial Properties of the Fabrics

The antibacterial effect was studied on the following

bacteria: Staphylococcus aureus ATCC-6538, Escherichia

coli ATCC – 10536, Pseudomonas aeruginosa ATCC-27853,

and Streptococcus β haemolytic ATCC-10556. The samples

used for testing had the G3 knitted textile substrate treated

with all emulsion variants in order to point out the effect in

accordance with the concentration of the natural components. To

grow cultures with the bacteria mentioned above, the following

mediums were used: Blood AGAR - Staphylococcus aureus

and Streptococcus β haemolytic; CLED - Escherichia coli

and Pseudomonas aeruginosa; Chapman - Staphylococcus

aureus. The Chapmann and CLED culture mediums were

selected due to their inhibiting substances that prevent

contamination with other bacteria. The resulting cultures

were used for antibacterial testing, based on the Kirby-Bauer

method. Incubation took place in a thermostat environment

at 37 oC for 24 h. The bacteria dilution factor was 11.8 UOI.

After the incubation period, the inhibition zones were

identified and measured.

Measurement of the Amount of Dried Emulsion on the

Textile Substrate

The quantity of emulsion retained by the fabric after

drying (W) was determined with the formulae:

(3)

where w0=the weight of the fabric before impregnation and

w1=the weight of the fabric after impregnation, drying at

50oC and conditioning at 65 % RH.

Time Release for Propolis

An amount of 0.1 g of sample, corresponding to each

treatment variant, was used for extraction in 10 ml solvent

(2:1 buffer/ethanol ratio), at 37oC, under mechanical agitation.

At preset intervals, 2 ml of substance were extracted,

filtered, and analyzed using a Camspec M501 Single Beam

Scanning UV/Visible Spectrophotometer at λ=313 nm. The

quantity of propolis extracted was determined using the

calibration curve. The constant volume of solution was

maintained by adding 2 ml of solution after each extraction.

Color Measurements

In order to determine how the presence of propolis affects

the color of the cotton substrate, color measurements were

carried out using a Spectraflash SF300 DATACOLOR

apparatus and Micromatch 2000® software. The intensity of

the color was expressed based on the Kubelka-Munk

equation [21]:

(4)

where R=spectral reflectance.

Results and Discussion

The Amount of Emulsion Retained by the Cotton Knitted

Fabric

The quantity of emulsion retained by the textile material

after drying depends on the fabric parameters (stitch density)

and on the content of the emulsion. To determine the influence

of the fabric parameters the samples were impregnated with

emulsion variant 2 and the amounts of emulsion determined

for the fabrics were: 23 % for variant G1, 27 % for variant

G2, and 34 % for variant G3. As a result, variant G3 was

chosen for further characterization.

To determine the influence of the concentration of the

component upon the amount of emulsion retained by the

Hw1−w0

w0

---------------- 100 %⋅=

Ww1−w0

w0

---------------- 100 %⋅=

K

S----

1−R( )2

2R-----------------=

Figure 1. Components concentration influence on the amount of

emulsion retained by the fabric variant G3.

Antibacterial Finishing Using Natural Compounds Fibers and Polymers 2013, Vol.14, No.11 1829

cotton substrate, the G3 fabric impregnated with all variants

was analyzed and the results are presented in Figure 1. After

the samples were dried, there was a significant loss of water

through evaporation that increased with the concentration of

chitosan carbohydrate polymer (see Figure 1(A)). When the

concentrations of beeswax and EEP were increased while

maintaining the concentration of chitosan constant the

emulsion retained by the textile substrate also increased (as

illustrated in Figure 1(B) and 1(C)). The presence of glycerol

prevents the total drying of the polymer, maintaining its

elastic state on the textile fibers, therefore preserving the

emulsion wet. The stickiness level is slowly increasing with

humidity.

Visual Characterisation of Treated Cotton Fabrics

SEM pictures of the fabrics before and after impregnation

(Figure 2) were compared to observe how the system was

laid on the textile substrate. The pictures presented in Figure

2(B) and 2(D) show that the emulsion is introduced in the

fabric as a film covering the fibers/yarns and not the surface

of fabric. This explains why lower stitch densities improve

the amount of emulsion, as the free spaces within each stitch

allow it to better cover the yarns.

Figure 2(D) shows how the polymer coats the fibers,

sometimes creating a film between them, but without affecting

the aspect of the yarn, as illustrated in Figure 2(B). The

small formations visible at yarn level are beeswax particles.

The higher concentration of beeswax also has a negative

effect on the emulsion as it tends to form these small

particles that are not embedded in the chitosan polymer,

leading to an unpleasant touch.

Due to the presence of propolis, the color of the impregnated

knitted fabric became yellow-brown. The intensity of the

colour (K/S) for the witness sample is 0.12, while for the

treated samples the intensity varies with the concentration of

propolis from K/S=0.69 for 25 ml/l to K/S=2.11 for 125 ml/l.

Antibacterial Performance of the Treated Cotton Fabrics

The circular samples were cut at a 5 mm diameter. After

the incubation period, the diameter of the inhibition zone

was determined for each sample. The visual aspects of the

antibacterial tests are presented in Figure 3 and the inhibition

diameters are compared in Table 3. The results in Table 3

show that most of the treated samples presented an antibacterial

effect. Best results were obtained against the S. aureus bacteria.

The higher concentration of propolis in relation to chitosan

for samples 4 and 7 amplified their antibacterial effect.

The inhibition area identified for sample 5 (blood AGAR

medium) illustrates the antibacterial effect of chitosan

together with propolis. For the same bacteria (S. aureus) but

in another culture (Chapmann) the best results were obtained

for treatment variants 3, 4, and 7, emphasizing the influence

of the concentration of propolis in relation to the concen-

tration of chitosan. In the case of P. aeruginosa and S. β

haemolytic, the highest inhibition area was determined for

the highest concentration of propolis (treatment variant 7).

Gram negative E. coli bacteria were inhibited on the entire

surface of the Petri dish due to the cumulated action of all

treatment variants. Therefore it was not possible to determine

individual areas of inhibition.

Figure 2. SEM aspect of treated (B, D) and untreated (A, C) cotton substrate; scale bar=100 μm.

1830 Fibers and Polymers 2013, Vol.14, No.11 Danko Abramiuc et al.

Comfort Characteristics

Air Permeability

Figure 4 shows the influence the components of the emulsion

have on the air permeability of the textile substrate. The

slight increase of air permeability with the variation of chitosan

(Figure 4(A)) could be explained through the tendency of

the chitosan to bond the fibers determining bigger stitch

dimensions (lower stitch density). The graphs show that an

increase of beeswax and propolis in the system leads to a

sealing of the free zones in the stitch geometry and a decrease

in air permeability for the treated fabrics, as illustrated in

Figure 4(B) and 4(C). The increase in EEP and beeswax

concentration leads to a thicker chitosan film, thus limiting

the free zones in the textile substrate and subsequently the

volume of air penetrating the samples.

Vapor Permeability

The tests conducted on a Permetest apparatus have shown

that the relative vapor permeability of the treated samples

depends strongly on the relative humidity of the environment.

Figure 3. Visual aspects of cultures after incubation. (A) blood

AGAR, Staphylococcus Aureus, (B) blood AGAR, Streptococcus

β Haemolytic, (C) CLED medium, E-coli, (D) CLED medium,

Pseudomonas Aeruginosa, and (E) Chapmann, Staphylococcus

Aureus.

Table 3. Antibacterial testing - results from antibiograms

Culture mediumBacteria Diameter of the inhibition zone (mm)

Treatment variants 1 2 3 4 5 6 7

Blood AGARStaphylococcus Aureus 6 6 6 10 10 7 10

Streptococcus β Haemolytic 6 7 8 6 6 7 8

CLEDE-coli The entire surface of the dish represents the inhibition area (see Figure 3C).

Pseudomonas Aeruginosa 0 7 6 6 6 6 8

Chapmann Staphylococcus Aureus 7 8 10 10 7 7 9

Figure 4. The influence of the concentration of emulsion

components on air permeability (dotted line=untreated cotton

substrate).

Antibacterial Finishing Using Natural Compounds Fibers and Polymers 2013, Vol.14, No.11 1831

The samples were conditioned for 48 h in a conditioning

room at: φ1=20 %, θ1=20 oC and φ2=65 %, θ2=20 oC.

After conditioning, the samples were tested. The experi-

mental results for relative vapor permeability are illustrated

graphically in Figure 5, in comparison with the values

obtained for non-treated samples conditioned in the same

way. The graphics show that the fabrics present a similar

behavior; the differences between the samples are placed in

a narrow interval for both tests. The influence of the

environment of humidity is emphasized by the values of the

treated samples in relation to the ones of the non-treated

samples: for φ1=65 %, the relative vapor permeability is

increased, while for φ2=20 % the treated samples present

decreased vapor permeability.

The different vapor permeability for the treated samples in

comparison with the untreated ones for different levels of air

humidity is explained through the fact that at low humidity

(20 %) the textile material absorbs water from the environment

and limits the vapor transfer. At 65 % humidity, the textile

material allows the water vapors to pass through the fabrics

structure. The chitosan polymer decreases the vapor

permeability (Figure 5(A)) due to the amino and hydroxil

groups that bond with water molecules.

The beeswax is hydrophobic and its presence has no

significant influence of the vapor permeability. An increase

of the concentration of beeswax can slightly reduce the

surface of the chitosan film (a decrease in the amount of

hydrophilic groups), so that higher values for the vapor

permeability are observed (see Figure 5(B)).

A small influence on vapor permeability was also observed

for propolis, mainly due to the hydrophilic groups in the

alcoholic extract propolis (Figure 5(C)). Even if the natural

active components have little influence, the vapor permeability

is strongly influenced by the hydrophilic glycerol.

Hygroscopicity

The presence of amino and hydroxyl groups in chitosan

favors hygroscopicity, so higher concentrations of chitosan

lead to its significant increase. Increase of beeswax concen-

tration will strongly reduce the hygroscopicity due to the

Figure 5. Relative vapor permeability. Figure 6. The influence of the components on hygroscopicity

(dotted line=untreated cotton substrate).

1832 Fibers and Polymers 2013, Vol.14, No.11 Danko Abramiuc et al.

hidrophobic nature of waxes (Figure 6(B)). The variation of

propolis concentration has little influence on the hygroscopicity;

the increased amount of hydroxil groups in the alcohol

determines a slight increase of the hygroscopicity (Figure

6(C)). The presence of glycerol in the system, which has a

high humidity absorbtion rate determines highy hygroscopicity

values than those for non-treated samples.

Time Release of the Active Substance

The results for the diffusion of propolis from the textile

substrate are presented graphically in Figure 7, illustrating

the variation of the concentration of each component. The

chitosan polymer solution is slightly soluble and will favor

the release of the substances embedded in the polymer. For

all treatment variants, EEP is released in the first 30 min

from the beginning of the extraction, as shown in Figure

7(A), 7(B), and 7(C). The differences refer only to the

amount of released substance and depend on the treatment

variant. The chitosan concentration is mainly responsible for

the emulsion stabilization and for embedding EEP in the

polymer. For a low concentration of chitosan the emulsion is

not stable, after 7 days the phases become separated and the

ethylic alcohol evaporates. Therefore the emulsion cannot be

stored for long periods of time. A higher concentration

improves the emulsion stability, but going over a certain

level will cover the propolis with a thick film of chitosan and

the antibacterial effect is reduced. Figure 7(C) shows that a

much higher amount of EEP was released for higher con-

centrations. For the treatment variant 3, with the highest

concentration of beeswax, the substance release increases

significantly after 80 min, due to the fact that beeswax takes

longer times to solve. In the first 80 min, the release observed

refers to the propolis contained in the emulsions.

Conclusion

The experimental work presented shows that biologically

active natural substances can be included in emulsions that

can be applied on a textile substrate, functionalizing them

for medical applications. The best type of textile substrate

proved to be jersey knitted fabrics variant G3, the amount of

emulsion retained by the textile substrate increasing with the

looseness of the fabric (decreased stitch density).

Antibacterial behavior was determined using several bacteria,

gram-positive and gram-negative, that were grown in different

mediums. The best results concerning the inhibition of

bacteria cultures were obtained for S. aureus and E. coli. The

presence of propolis in the emulsion seems to increase the

antibacterial effect. The chitosan polymer showed a certain

antibacterial effect for S. aureus, blood AGAR culture

media, but its inhibiting activity was recorded in the

presence of propolis, so a degree of influence is to be

expected. The propolis is released from the emulsions

applied on the textile substrate in the first 30 min; the

amount released is not influenced by the other components,

it depends only on its own concentration.

The impregnated chitosan-propolis-beeswax system modified

the comfort behavior of the fabric. There is a slight decrease

in air permeability and a significant increase of hygroscopicity,

due to the presence of glycerol in the emulsion. Glycerol

also influences vapor permeability, as it absorbs water from

the atmosphere. Tests showed that air relative humidity has a

strong influence on vapor permeability - for dryer environments,

the presence of the emulsions decreases vapor permeability

(the emulsions “seal” the fibers, as the system covers the

yarns, and less vapors pass through the fabrics).

Acknowledgements

This paper was realized with the support of Posdru

Cuantumdoc “Doctoral Studies for European Performances

Figure 7. Controlled release of propolis on the textile substrate.

(A) variation of chitosan concentration, (B) variation of beeswax

concentration, and (C) variation of EEP concentration.

Antibacterial Finishing Using Natural Compounds Fibers and Polymers 2013, Vol.14, No.11 1833

in Research and Inovation” ID79407 project funded by the

European Social Found and Romanian Government.

The authors are grateful for the support provided by the

research team from DWI an der RWTH Aachen-Germany

with SEM analysis.

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