Antibacterial Property of Cotton Fabric Dyed with Vaccinium bracteatum Thunb. Leaves

Abstract

Vaccinium bracteatum Thumb. leaves have a variety of nutritional values and physiological functions, and are rich in useful resources. In this study, these leaves were used as a raw material to make vegetable dyes. Cotton fabric was then dyed with them by the direct staining method. Good dyeing effects and antibacterial properties of the resulting fabrics could be obtained using an optimized dyeing process. The antibacterial properties of the fabric were directly proportional to the dye uptake (K/S value) of the fabric. To improve dyeing, mordant and chitosan were used separately to pretreat the cotton fabrics. The K/S value and antibacterial properties of the pretreated dyed cotton fabrics were improved after pretreatment.

Introduction

Due to its good wearing properties, cotton fabric is one of the main materials used in clothing. ¹ However, cotton fabrics are liable to mildew as microorganisms, which reproduce easily under humid conditions and secrete fiber enzymes and acid, can spoil and discolor cotton fiber. At present, many bio-based antibacterial finishing agents are used to treat cotton fabrics. These include ε-polylysine and prodigi-osin, chitosan, modified chitosan, and nano- chitosan, and various types of plant-based antibacterial finishing agents, including honeysuckle, aloe, Houttuynia cordata, and licorice. ²

Vaccinium bracteatum, a kind of evergreen shrub, belongs to the rhododendron family, ³ which grows widely in south China. Its leaves are rich in polysaccharides and flavonoids,^4,5 and nutrients such as various fatty acids, ⁶ amino acids, ⁷ and vitamins. It is commonly used in to cook rice with the juice of its leaves in south China. ⁸ Acting as an excellent natural dye with antibacterial and antiviral effects,^9,10 it can also be used to dye food, fabric, and hair. ¹¹ However, overall, the development and use of this plant resource is still in the early stage.

In this study, Vaccinium bracteatum dyeing experiments and antibacterial testing were conducted on the dyed cotton fabrics. Studies such as this can help to provide industry with more environmentally-responsible textile dyeing and finishing methods.

Experimental

Materials

Bleached, pure woven cotton fabric (100 g/m²) was used. Vaccinium bracteatum Thunb. leaves were picked in April 2020 at Tianmu Lake of Changzhou City, Jiangsu Province, China. leveling agent (Goon 301, Dongguan Jiahong Silicone Technology Co. Ltd.), JFC penetrating agent, Na₂SO₄, FeCl₃, chitosan, and acetic acid, were provided by Sinopharm Chemical Reagent Co. Ltd. Deionized water was used in all experiments. Escherichia coli was provided by the Jiangnan University Ecological Textile Key Laboratory of the Ministry of Education.

Preparation of Vegetable Dye

Fresh Vaccinium bracteatum Thunb. leaves were cleaned with tap water, then deionized water, dried naturally, and crushed. These dried and crushed leaves were soaked in deionized water for 30 min at a liquor ratio of 1:2.5. After filtration with gauze, the filtrate (mixed solution) was stirred with a heated magnetic stirrer (60 °C, 3 h) along with a 60% ethanol solution, followed by vacuum filtration. Lastly, the pigments of the leaves were obtained by rotary evaporation. The process is shown in Fig. 1. The Buchi R-3 Rotary Evaporator (Buchi Labortechnik AG) was used for solvent evaporation.

Cotton Fabric Treatment

Direct Dyeing

Fig. 1

The technological process of vegetable dyestuff abstraction.

Direct dyeing was used to conduct the single factor test. The dyeing conditions were as follows: liquor ratio (LR) of 1:40, dyeing time of 40 min, dyeing temperature of 80 °C, and the concentration of dye solution was 6% owf. Only one variable was changed during each dyeing experiment, with the other variables remaining fixed. Each individual experiment was repeated three times, and the results were averaged. The dyeing process is shown in Fig. 2. A Mettler Toledo AL204 Electronic Balance (Mettler Toledo International Trading, Shanghai, China) was used for weighing and a HH-4 Ther-mostat Water Bath (Jiangsu Changzhou Ronghua Instrument Manufacture Co. Ltd) was used for dyeing the fabrics.

Fig. 2

The prescription and flow of direct dyeing.

Pretreatment with Chitosan

To improve dyeing, the cotton fabric was pretreated with chitosan. Since chitosan is insoluble in water, acetic acid was used as solvent. The concentration of chitosan and acetic acid were 20 g/L and 100 g/L, respectively, and the LR was 1:40. The chitosan pretreatment process is shown in Fig. 3, followed by direct dyeing.

Fig. 3

The process of pre-treatment with chitosan.

Pre-Mordanting with FeCl₃

Firstly, the cotton fabric was wetted at 40 °C for 10 min, then the FeCl₃ mordant was added for pre-mordant treatment. The concentration of mordant was 20% and the LR was 1:30. The process flow is shown in Fig. 4. The pre-mordanted fabric was then dyed.

Antibacterial Activity

Part 3 of GB/T 20944-2008 provides for quantitative tests and evaluation methods for the determination of antibacterial properties of textiles by oscillation method, on the basis of which, quantitative determinations for antibacterial properties of dyed cotton fabrics were conducted.

Fig. 4

The process of pre-mordant with FeCl₃.

There are three main reagents need to be prepared in advance to test the antibacterial property of the fabric, with the recipes given in Table I.

Table I

Prescriptions of Tree Main Reagents

Reagents	Prescriptions
Nutrient agar	Peptone (g)	10
	Beef extract (g)	5
	NaCl (g)	5
	Agar powder (g)	12
	Distilled water (mL)	1000
Nutrient broth	Peptone (g)	10
	Beef extract (g)	5
	NaCl (g)	5
	Distilled water (mL)	1000
PBS	Na2HPO4·12H2O (g)	10.74
	NaH2PO4·2H2O(g)	2.34
	Distilled water (mL)	1500

Bacterial Suspension

The preserved Escherichia coli (E. coli) bacteria were inoculated to sterile nutrient agar medium, which was then put under constant temperature and humidity for 18∼24 h, and labeled as 1N. 1N was then inoculated to sterile nutrient agar medium, which was then put under constant temperature and humidity for 18∼24 h, and labeled as 2N. Finally, 2N was then inoculated to sterile nutrient agar medium, which was then put under constant temperature and humidity for 18∼24 h, and labeled as 3N, which was inoculated into sterilized broth (20 mL). The bacterial suspension was then kept at a constant temperature and humidity for 18∼24 h under shock. This process is shown in Fig. 5. SW-CJ-2F Clean Bench (Suzhou Purification Equipment Co. Ltd.) was used for bacterial inoculation LDZH-100KBS sterilizers (Shanghai Shenan Medical Instrument Factory) was used for sterilization of related instruments and solutions. The TS-2102C Orbital Shaker (Shanghai Tiancheng Experimental Instrument Manufacturing Co. Ltd.) provided the constant temperature and humidity environment.

Fig. 5

The process of preparation for bacterium suspension.

Bacterial Treatment of Samples

Each sample fabric (0.75 ± 0.05 g) was cut into 5 × 5 mm fragments. Each fabric was then added into a triangular flask containing phosphate buffer solution (PBS, 0.03 M, pH = 7.2 ± 0.2, 70 mL) and sterilized at 121 °C for 20 min. The E. coli suspension (A, 2 mL) was transferred into one test tube containing sterilized cooled broth (9 mL) and thoroughly mixed to give suspension B. B (1 mL) was added into another test tube containing 9 mL of sterilized cooled broth and was mixed well to give C. C (1 mL) was added into a test tube containing 9 mL sterilized PBS (0.03 M) and thoroughly mixed to give D. D (5 mL) was transferred into a triangular flask containing 45 mL sterilized PBS (0.03 M) and mixed thoroughly to give E. To inoculate the sample, the inoculant liquid should be used within 4 h to ensure the inoculant activity. The inoculant (5 mL) was respectively transferred into each sterilized cooled sample solution and shaken for 18-24 h in a constant temperature and humidity environment. The technological process is shown in Fig. 6.

Fig. 6

The process of preparation for sample solution with bacteria.

Preparation of Dilution Method

Bacteria were cultivated by the dilution method and the sample solution respectively diluted 10¹ ∼ 10⁸ times. Taking different diluted multiple sample solutions (50 μL) respectively directly into three sterile nutrient agar culture medium, they were then all put in an environment at constant temperature and humidity (37 °C, 60% RH, 18–24 h) for cultivating. At the specified time, the petri dish was taken out to select the appropriate dilution factor area for calculation. The specific process is shown in Fig. 7.

Fig. 7

The process of preparation for dilution method.

Performance Tests of Dyed Fabrics

K/S Value

The Kubelka-Munk dyeing depth equation established a certain function relationship between the absorption coefficient K and scattering coefficient S of the measured object and the concentration of colored substance C in a solid sample. The larger the K/S value obtained, the darker the surface color of the solid sample, that is, the higher the concentration of colored substances, the better the dyeing performance of the dye. The Datacolor 400 computer colorimeter was used to measure the color of the dyed fabric four times and the average value was taken.

Antibacterial Property

The antibacterial property could be represented by the antibacterial rate as calculated using Eq. 1.

$$Y=\frac{W-Q}{W}$$

Eq. 1

Y represents the antibacterial rate, W and Q are the mean value of the viable bacteria of three control samples and three test samples respectively.

Color Fastness

Color fastness to washing was determined according to GB/T 3921.1-2008 Color Fastness to Washing Test for Textiles: Test 1. The specific experiment procedure was as follow: The dyed cotton fabric sample was cut to a fixed size (100 × 40 mm), which should be stitched together with the same size interlining and weighed (1.567 g). The bath ratio was set as 1:50, with 5 g/L soap fakes (0.3925 g) and 2 g/L of anhydrous sodium carbonate (0.157 g) as detergent. The test specimen, detergent and steel ball of the stainless steel cup were all installed on the main shaft of the SW-12 Wash Fastness Tester (Dongguan SafTonny/Tomy Co. Ltd.), heating the water in the cabinet after reaching 60 °C, and driven by motor spindle rotate at a speed of 40 rpm for 30 min. Then the sample was rinsed and dried thoroughly. This operation was to simulate the daily household cleaning or commercial washing five times.

Results were determined by observing the color change of samples after washing, and evaluating the discoloration and staining grade with the Gray Scale for Evaluating Staining (AATCC).

The color fastness to friction was determined according to GB/T3920-2008 Textile Color Fastness Test to Friction. The dyed cotton fabric sample was cut to a fixed size (200 × 50 mm) and the Y571B Friction Fastness Tester (Dongguan BLD Instrument Equipment Co. Ltd.) was used to test the crock fastness of dyed cotton fabrics. The number of times of friction application was set at 10, and the dry and wet friction of the fabric were tested successively. Results were obtained by observing the color change of samples after rubbing, and evaluating the discoloration and staining grade with the Gray Scale for Evaluating Staining.

The color fastness to sunlight was determined according to GB/T8427-2008 Textile Color Fastness Test Xenon Arc Lamp to Artificial Light. The Q-Sun Xe-2 Xenon Test Chamber (Q-Lab Corp.) was used and experimental conditions were set as shown in Table II. In this experiment, the fabric sample should be checked once after every 7-8 h of irradiation. If the fading of the fabric sample did not reach level 4, the irradiation would continue. If the fabric sample fades to level 4, the irradiation was removed. Results were determined by observing the color change of the blue card, and evaluating the grade of the lighting fastness.

Results and Discussion

Influences on Dyeing Effect and Antibacterial Property

Bath Ratio

As shown in Fig. 8, the antibacterial property decreased with the increase of the dyeing bath ratio, and the best antibacterial property could be obtained when the bath ratio was 1:20 and 1:30. As a larger bath ratio means a smaller concentration of pigment in the solution, the ability of pigment bonding with the fabric reached saturation when the bath ratio was set at 1:20. If the bath ratio was decreased further, it might lead to uneven dyeing, which might affect the K/S value and antibacterial property.

Fig. 8

The process of preparation for dilution method.

Dyeing Concentration

When the dyeing solution concentration reached 8%, the K/S value was the highest (Fig. 9). Meanwhile, the highest antibacterial rate, which means the best antibacterial property was obtained. As the dyeing solution concentration increased, more pigment molecules were combined with fabrics, obtaining a better dyeing effect. When the dyeing solution concentration increased to 8%, the combination of pigment molecules and fabric achieved a balance. At greater concentrations, the dyeing depth and antibacterial property leveled off.

Fig. 9

The influence of owf on dyeing effect and antibacterial property.

Table II

Experimental Conditions of Color Fastness to Sunlight Test

Items	Conditions
Filter	7 IR
Irradiation strength	76%
Sample chamber temp	35 °C
Fan speed	700 rpm
Relative humidity	0.4
Black standard temp	Max 50

Dyeing Temperature

Fig. 10 shows that with the increase of dyeing temperature, the pigment molecules were fully released, and was conducive to the combination of pigment molecules and fabric. However, when the temperature exceeded 80 °C, the flavonoids in the dye solution decomposed, ¹² resulting in a smaller K/S value and poorer antibacterial property.

Fig. 10

The influence of dyeing temperature on dyeing effect and antibacterial property.

Dyeing Time

The best dyeing effect and antibacterial property was obtained when the dyeing time was 50 min. As shown in Fig. 11, when the dyeing time exceeded 50 min, the dyeing effect remained stable, which means the combination of pigment and fabric reached a balance. Moreover, the pigment could decompose due to the long dyeing time. ¹³

Fig. 11

The influence of dyeing time on dyeing effect and antibacterial property.

The dyeing rate of the fabric was positively correlated with the antibacterial property. Therefore, better antibacterial properties can be obtained by increasing the dyeing effect on the cotton fabric.

Effect of Various Processes on K/S Value and Antibacterial Property

Directly Dyeing

Table III shows the K/S value and antibacterial property of the extract from V. bracteatum Thunb. leaves. The content of the pigments are mainly 10-p-trans-coumaroy 1-1 S-momotrotropein (i.e., vaccinoside), which could closely combine with the fabric and color the fabric a specific color. Moreover, the direct dyeing process gave the fabric the antibacterial property (Y = 71.60%) because of the flavonoids in the V. bracteatum Thunb. leaves. ¹⁴

Table III

Comparison of K/S Value and Antibacterial Properties of Various Processes

Properties	Cotton fabric	Directly dyed cotton fabric	Cotton fabric pretreated with chitosan	Cotton fabric pretreated with FeCl3
K/S value	0.25	1.94	2.39	2.47
Antibacterial property	0	76.10%	81.40%	76.30%

Pre-Treatment with Chitosan before Directly Dyeing

It can be seen in Table III that the K/S value and antibacterial property could be improved, with the antibacterial property increasing from 76.10% to 81.4% after chitosan pretreatment, compared to the cotton fabric dyed directly with the extract from V. bracteatum Thunb. leaves. This is because chitosan is a good dye promoter for reactive dyes. ¹⁵ In addition, Fig. 12 shows that chitosan pretreatment (c) not only reduced the number of bacterial colonies, but also reduced the size of bacterial colonies, compared with the testing results of raw cotton fabric (a) and directly dyed cotton fabric (b). Cotton fabric treated with chitosan has an inherent antibacterial property, which indicates that a compound antibacterial effect on the fabric could be obtained. The inac-tivation of Escherichia coli by chitosan occurs via a two-step sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane. The similarity between the antibacterial profiles and patterns of chitosan and those of two control substances, polymyxin and EDTA, verified this mechanism. ¹⁶

Fig. 12

(a) The antibacterial property result of raw cotton fabric, (b) the antibacterial property result of directly dyed cotton fabric, (c) the antibacterial property result of directly dyed cotton fabric with chitosan pretreatment, and (d) the antibacterial property result of directly dyed cotton fabric with FeCl₃ pretreatment.

Pre-Mordanting with FeCl₃ before Directly Dyeing

Pretreatment with iron mordant (FeCl₃) greatly improved the K/S value of the dyed fabric and deepened the dyeing effect (Table III), but the antibacterial property remained virtually unchanged. Although the quantity of bacterial colonies pretreated with iron (d) showed little change when compared with (a) and (b), the size of the bacterial colonies decreased (Fig. 12). This is because iron ions can inhibit bacterial growth by catalyzing hydroxyl radical formation, ¹⁷ resulting in a compound antibacterial effect on the fabric.

Color Fastness and Antibacterial Durability

Color Fastness

A dyeing experiment was carried out based on the optimal process of directly dyeing cotton fabric, as previously discussed, to test the K/S value and dyeing fastness of the fabric. Table IV shows that the qualified (conforming to the national standard) dyeing fastness of cotton fabric could be obtained under the optimal dyeing process.

Table IV

Color Fastness Level of Dyed Cotton Fabric

Perspiration fastness		Crock fastness		Soaping fastness		Lighting fastness
Staining	Fading	Dry friction	Wet friction	Staining	Fading
3–4	3–4	3–4	3	3–4	3–4	4

Antibacterial Durability

Antibacterial durability was determined in the same manner as the test method for color fastness to washing. It can be seen from Table V that the antibacterial property of the directly dyed cotton fabric was still over 70% after 20 washing cycles, indicating that the pigment has been closely combined with the cotton fabric.

Table V

Results of Antibacterial Durability of Directly Dyed Fabric

Times of washing	0	5	10	15	20
Y (%)	86.2	81.7	78.1	75.5	71.4

Conclusions

Optimal dyeing of cotton fabric was obtained when the vegetable dye was extracted from Vaccinium bracteatum Thunb. leaves. Testing results showed that the optimal dyeing and antibacterial property were obtained when the bath ratio was 1:40, the dyeing solution concentration was 8%, and the dyeing temperature and dyeing time were 80 °C and 50 min, respectively.

Both the pretreatment of chitosan and FeCl₃ could not only increase the dyeing effect on cotton fabric, but also improve the antibacterial property of the cotton fabric as well. Chitosan had a greater impact on improving the antibacterial property compared to FeCl₃, while FeCl₃ could better improve the dyeing effect. Chitosan and FeCl₃, together with plant dyes made from leaves of V. bracteatum Thunb., can give cotton fabrics a composite antibacterial property.

A new process of cotton fabric dyeing was developed, which adds ecological and functional value to cotton fabric, greatly improving the added value of the fabric and providing new ideas for ecological textile dyeing and finishing technology.