Investigation into the Application of Natural Dyes Obtained from Annatto Seeds and Eucalyptus Leaves in Dyeing Textile Substrates Using Biomordants

Abstract

This study evaluated dried eucalyptus leaf extract and annatto seed extract as natural dyes for cotton, polyamide, and polyester knit fabrics. The eucalyptus leaf extract was obtained by aqueous boiling extraction, while the annatto seed extract was prepared in an alcoholic medium at 60 °C. Dyeing was carried out on fabrics mordanted with lemon juice and soy milk, using a cup dyeing machine with infrared (IR) heating at 98 °C for 30 min. SEM and FTIR analyses assessed the results regarding color intensity and color fastness. The findings indicate that both extracts can serve as sustainable alternatives for textile dyeing.

1. Introduction

Public policies and growing environmental awareness are driving a revolutionary shift in the textile sector toward sustainability. At the heart of this movement is the search for environmentally friendly alternatives to conventional synthetic dyes and mordants, which often pose serious risks to human health and the environment. Natural dyes and biomordants offer significant environmental potential, as they are sustainable and have a lower ecological impact than synthetic dyes [1].

Natural dyes have numerous advantages: they are environmentally friendly, safe, easily sourced from renewable resources, visually appealing, and non-hazardous to human health, while presenting no significant disposal issues [2]. Based on their chemical composition, natural dyes are classified into several groups, such as tannins, carotenoids, quinoids, alkaloids, anthraquinones, flavonoids, and indigoids [3]. Each category comprises a wide variety of compounds with unique properties that contribute to their dyeing capability.

Fruits and vegetables contain molecules responsible for specific colors, making them an environmentally attractive alternative to synthetic dyes in food and non-food applications [4]. Vegetable dyes have a long history of use across cultures for coloring fabrics, crafts, and artwork. These dyes are derived from various parts of plants—including roots, leaves, stems, flowers, and fruits—and provide a broad color palette. In terms of application, dyeing with crude extracts is, in all cases, the most environmentally friendly approach [4].

One study showed that microwave-assisted extraction of annatto dye significantly improves the process, facilitating the release of the orange-red bixin pigment, whose molecule can be seen in Figure 1b. The dye yielded excellent color strength when applied to silk with an aluminum salt mordant and a pomegranate extract biomordant [5]. For polyester, optimal dyeing conditions with annatto seed extract were achieved at 115 °C and pH 4.17, resulting in excellent color fastness [6]. Other studies have explored eucalyptus leaves or extracts as natural dye for cotton and wool fabrics. According to the literature, the colors obtained from eucalyptus dyeing ranged from yellow to brown, depending on the dyeing concentration, dyeing process, and choice of mordant [7]. Another study demonstrated that 100% cationic-dyed cotton fabrics with good color and light fastness can be produced using Eucalyptus globulus leaf extracts as natural dyes [8], and the molecule of this pigment can be seen in Figure 1a.

Mordants play a crucial role in natural dyeing processes by enhancing color fastness. In textile dyeing, two types of mordants are commonly used with natural dyes: chemical and biomordants. Biomordants, primarily composed of hydroxyl (-OH) groups derived from phenolic compounds, form additional hydrogen bonds with the fabric, resulting in a broader range of hues and tones [9]. Chemical mordants—such as potassium alum, sodium chloride, ferrous sulfate, calcium chloride, and tannic acid—enhance or darken the hue by forming metallic dye complexes in the fabric [9]. However, metallic mordants can also lighten, darken, or otherwise alter the final color of the fiber and have a detrimental environmental impact due to the release of heavy metal ions into dye effluents. Therefore, replacing traditional metal ions with natural mordants (biomordants) is important in advancing sustainable textile dyeing [10].

Various natural substances have been identified as biomordants, including lemon juice [1] and soy milk or protein [11]. Soy protein interacts chemically with cellulose and natural dyes to form a soy–cellulose complex during mordanting, which facilitates binding between the dye and the fabric during dyeing [12]. One study argues that soy milk acts more as a binder than a true mordant, as it does not chemically bond with dyes and fibers [13]. However, another study demonstrated that soy milk as a mordant for silk fabrics improved dye uptake and fastness when dyed with extract from Basella alba fruits [14].

Considering the above, it is justified to investigate dyeing with natural dyes aided by mordants. This study evaluated the application of two mordants—lemon juice and soy milk—and two natural dyes—eucalyptus leaves and annatto seeds—both abundant in nature, which yielded promising results for textile dyeing. Dyeing cotton fibers with synthetic dyes is typically performed at neutral or alkaline pH, while most synthetic fibers are dyed at acidic pH. This study aims to investigate the interaction of natural dye molecules with cotton, polyamide, and polyester fibers using two biomordants with pHs ranging from acidic to neutral. This study aims to understand whether, in addition to the functional groups present in these biomordants, pH variation can influence the interaction between dyes and fibers.

2. Materials and Methods

2.1. Materials

This study used water, mature Eucalyptus grandis leaves (sourced from a farm in city of Apiúna, state of Santa Catarina, southern Brazil), annatto seeds (purchased from a local store, city of Blumenau, state of Santa Catarina, southern Brazil), 92% ethyl alcohol (Alphatec), commercial soy milk (ADES), natural lemon juice (purchased from a local store), and samples of half-mesh fabric made of 100% polyamide, 100% pre-bleached cotton, and 100% polyester (all purchased from a local store).

2.2. Obtaining Eucalyptus Extract

Mature eucalyptus leaves were selected and oven-dried at a controlled temperature of approximately 50 °C (±2 °C) for 2 h. After drying, the leaves were ground using a food processor (Oster 10,000W) and a grinder (Oster–OMDR110) to obtain a fine, homogeneous powder. The powder was then sieved through a sieve with a 1.65 mm opening to remove larger impurities, and the resulting material is shown in Figure 2. To prepare the aqueous extract, 20 g of the powder was mixed with 500 mL of water, brought to a boil (approximately 98 °C), and simmered until the total volume was reduced to 250 mL. After cooling, the extract was vacuum-filtered to remove solid residues and stored in an amber bottle in a refrigerator, protected from light. The extract was obtained using water, as described in the study by [15].

2.3. Obtaining Annatto Extract

To prepare the aqueous extract of annatto, 20 g of annatto seeds were added to 100 mL of 92% ethyl alcohol, and the mixture was placed in a cup dyeing machine with IR heating, operating for 120 min at a controlled temperature of 60 °C. The mixture was then filtered using a vacuum pump, and the extract was stored in an amber bottle in the refrigerator, protected from light. The methodology for obtaining the annatto seed extract was adapted from the study by [16].

2.4. Mordanting Process of Fabrics

Fabric samples of each fiber type (cotton, polyamide, and polyester) were cut and standardized to a weight of 0.5 g each. One set of samples was mordanted with pure lemon juice (without added water, pH between 2 and 3), another set was mordanted with pure soy milk (without added water, pH between 6.5 and 7), and a third set was left untreated, serving as a purged control. The mordanting process was carried out with a bath ratio 1:10, immersing the samples in their respective mordant for 40 min at room temperature, 25 °C. After this period, the samples were passed through a foulard at 2 bar pressure and a cylinder speed of 2 m/min. The wet samples were then transferred to a drying oven at 40 °C until completely dry.

2.5. Dyeing of Samples

2.5.1. Dyeing of Samples with Eucalyptus Leaf Extract

Samples of biomordanted fabric (0.5 g each) and unmordanted fabric, previously purged with a 1 g/L solution of nonionic detergent at 80 °C for 30 min, were dyed in a 1:20 dye bath ratio and placed individually in cups containing the aqueous extract of dried eucalyptus leaves. Dyeing was carried out in a cup dyeing machine with IR heating, operating for 30 min at a temperature of 98 °C. The pH of the samples mordanted with soy milk was approximately 6, while the pH of those mordanted with lemon juice was approximately 4. After dyeing, the fabrics were removed from the cups, rinsed under running water, and dried in an oven at approximately 50 °C.

2.5.2. Dyeing of Samples with Annatto Extract

Biomordanted fabric samples (0.5 g each) and unmordanted samples, previously purged with a 1 g/L nonionic detergent solution at 80 °C for 30 min, were dyed in a 1:20 dye bath ratio and placed individually in cups containing a mixture of 20% annatto seed alcoholic extract and 80% distilled water. Dyeing was carried out in an IR-heated cup dyeing machine, operating for 30 min at a controlled temperature of 98 °C. The pH of the soy milk-mordanted samples was approximately 6, while that of the lemon-mordanted samples was approximately 4. After dyeing, the fabrics were removed from the cups, rinsed under running water, and oven-dried at approximately 50 °C.

2.6. Evaluation of the Color Intensity of Dyed Fabrics

Reflectance spectroscopy was performed using a Datacolor^® Spectrum 500 reflectance spectrophotometer to analyze the color intensity of the samples after dyeing. Measurements were carried out in the CIELAB color space, along with evaluating the absorption and scattering coefficient (K/S) to quantify the color intensity across the different processes. In addition, color strength, color differences ΔL * (lightness difference), Δa * (red–green difference, with positive values indicating more red and negative values more green), Δb * (yellow–blue difference, with positive values indicating more yellow and negative values more blue), and ΔE * (total chromatic difference) were determined, as well as variations in color strength and K/S values, enabling a detailed analysis of the chromatic changes resulting from the applied treatments.

2.7. Characterization of Biomordanted Fabrics by Scanning Electron Microscopy (SEM)

Samples of untreated fabrics and fabrics mordanted with lemon juice and soy milk were analyzed by scanning electron microscope (SEM) using a JEOL Neoscope JCM-7000 scanning electron microscope. The samples were observed at a magnification of 500×.

2.8. Fourier Transform Infrared Spectroscopy-FTIR

To verify the presence of elements from soy milk and lemon juice adhered to the textile substrate, Fourier-transform infrared spectroscopy (FTIR) was used to examine the interaction of characteristic functional groups. The analyses were performed using a CARY 600 Series FT-IR Spectrometer (Agilent Technologies, Wilmington, DE, USA) in ATR mode, with readings in the 4000–400 cm⁻¹ range and a resolution of 1 cm⁻¹. All measurements were carried out under standard environmental conditions (55–60% relative humidity, 25 ± 1 °C).

2.9. Color Fastness to Washing

The wash fastness test was carried out according to the ISO 105-C06 B2S standard. After washing, the samples were rinsed manually with two separate portions of water at room temperature and then oven-dried at 60 °C. Finally, the color change in the samples and the color transfer to the adjacent fabrics were evaluated using the gray scale specified in ISO 105-A02 (to evaluate color change) and ISO 105-A03 (for color transfer evaluation), where a score of 1 indicates significant color change (poor fastness) and a score of 5 indicates no perceptible color change (excellent fastness). The gray scale is a visual tool used to evaluate color change (color shift) and color transfer (staining) in fabrics after various types of fastness tests, such as washing, rubbing, exposure to light, etc.

3. Results

3.1. Analysis of Colorimetric Results

The samples dyed with eucalyptus leaf and annatto seed extracts are shown in Figure 3 and Figure 4.

In the cotton samples dyed with eucalyptus leaf extract, soy milk was used as the mordant, resulting in a higher color intensity. In contrast, lemon juice was used as the mordant, resulting in uneven coloration, with localized spots of more intense color. One possible explanation for this result is that the more acidic medium created by the lemon juice mordant reduces the dyeing efficiency on the cellulose fiber.

In the case of polyamide, no significant color differences were observed between the untreated and mordanted samples, suggesting that the dye–fiber interaction is primarily related to the fiber’s inherent cationic functional groups rather than to the mordants.

For polyester samples, an increase in color intensity was observed when the fabric was biomordanted with soy milk, which was also confirmed by the color intensity measurements. This result may support the concept proposed by [13], who suggests that soymilk acts as a binder on the textile substrate, promoting dye adhesion to the surface but not necessarily enhancing dye adsorption and diffusion within the fiber.

The samples dyed with the extract obtained from annatto seeds showed a color characteristic of the seeds themselves, without significant changes in hue with the application of the different biomordants. The cotton fabric showed greater color intensity with the sample mordanted with soy milk, indicating a better interaction of the dye with the protein at a pH closer to neutral.

Synthetic polyamide and polyester fibers demonstrated similar behavior to dyeing with synthetic dyes, that is, the dyeing yield was higher in samples mordanted with lemon juice, where dyeing was carried out at a more acidic pH.

After visual evaluation, the dyed samples were subjected to spectrophotometric evaluation.

Table 1. Coloristic values of samples dyed with eucalyptus leaf extract.

Cotton
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	124.05
Lemon juice	2.73	0.69	1.03	1.43	85.65	120.29
Soy milk	−7.32	0.33	−2.86	3.76	150.91	155.99
Polyamide
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	104.61
Lemon juice	0.76	0.33	0.80	0.79	92.70	90.83
Soy milk	−6.01	−0.09	−2.36	2.85	139.10	108.55
Polyester
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	22.15
Lemon juice	−2.53	0.57	−1.72	1.95	116.53	24.38
Soy milk	−16.05	1.59	−3.03	7.19	295.31	56.52

and

Table 2. Coloristic values of samples dyed with annatto seed extract.

Cotton
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	32.28
Lemon juice	−5.63	4.15	12.58	7.2	221.21	79.87
Soy milk	−10.06	8.65	17.24	9.56	366.16	124.02
Polyamide
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	229.26
Lemon juice	−0.63	2.77	1.57	1.61	114.32	251.24
Soy milk	2.87	−0.69	−0.54	1.30	77.80	175.27
Polyester
Mordant	ΔL	Δa	Δb	ΔE (CMC)	Color Strength	K/S	Color
Crude					100	35.06
Lemon juice	−6.59	10.58	13.65	7.52	257.81	91.64
Soy milk	−0.68	2.96	6.52	3.14	130.76	46.66

show the results of the substrates dyed with different extracts and mordants. Other than K/S and strength, the other data for the raw samples are not presented, as these serve as a standard against which the other samples are compared.

Table 1 presents data demonstrating the dyeing potential of dried eucalyptus leaves on fabrics made of cotton, polyamide, and polyester fibers. For the cotton fabric, the substrate mordanted with soy milk exhibited higher K/S values and greater color strength with increased brightness. In contrast, the substrate mordanted with lemon showed lower results than the untreated sample.

A similar trend was observed in the polyamide samples, where the highest color intensity was recorded in the sample mordanted with soy milk. In contrast, the substrate mordanted with lemon showed a lower K/S value than the untreated substrate. These results indicate that the interaction between the fiber and the dye is largely independent of the mordant, a characteristic typical of fibers with cationic functional groups.

The polyester fabrics showed lower K/S values compared to the other fibers. However, when analyzing color strength, an increase of more than 100% was observed in the fabric mordanted with soy milk compared to the untreated fabric. This finding supports the concept proposed by [13], which suggests that soy milk is deposited on the fiber surface, enabling bonding between the mordant and the dye, but without molecular interaction with the fiber itself.

Table 2 presents the colorimetric data of cotton, polyamide, and polyester fabrics dyed with the extract obtained from annatto seeds.

Similar to the results obtained with eucalyptus leaf dyeing, cotton dyed with annatto seed extract and mordanted with soy milk showed higher color intensity than both untreated cotton and cotton mordanted with lemon juice. The study by [11] also reported positive results when dyeing cellulose fibers mordanted with soy protein using natural dye.

Synthetic fibers, such as polyamide and polyester, exhibited a similar yet distinct dyeing behavior compared to cotton, with the highest color intensity observed in samples mordanted with lemon juice. This result is consistent with the behavior of these fibers when dyed with synthetic dyes, which typically occurs in an acidic medium, the same condition created by using lemon juice as a mordant.

3.2. Fabric Characterization by Scanning Electron Microscopy Analysis-SEM

Morphological analysis of the samples using Scanning Electron Microscopy (SEM) allowed observation of the fiber surface, highlighting structural changes resulting from applying different mordants. The images (Figure 5) reveal minor variations in fiber topography, with differences between the treated and raw samples.

When examining the raw and mordanted cotton samples, the raw cotton exhibits a smooth surface with no residue deposits. In contrast, the lemon-mordanted cotton shows a thin film adhered to its surface. The soy milk-mordanted sample displays localized spots, indicative of agglutination caused by the soy protein. The same behavior was observed in the polyamide and polyester samples.

These observations suggest that there was superficial deposition of the mordants on all the fibers studied, regardless of their composition. This is likely due to the impregnation application process, in which the mordant adheres to the fabric by mechanical forces rather than electrostatic interactions. Nevertheless, even when superficially adhered, the mordants contributed to improved color outcomes in most dyeing processes.

3.3. Characterization of Mordanted Fabrics by Fourier-Transform Infrared Spectroscopy (FTIR)

To complement the characterization of the mordanted substrates and assess whether any chemical changes occurred after mordant application, FTIR was performed, and the results are presented in Figure 6, Figure 7 and Figure 8.

Figure 6 shows the FTIR spectra of raw and mordanted cotton fabric samples. In the raw sample, a band was observed around 3300 cm⁻¹, corresponding to the stretching vibrations of OH in cellulose, and a band at 2891 cm⁻¹, corresponding to CH stretching. The peak at 1634 cm⁻¹ can be attributed to C = C stretching. The C–OH groups of secondary and primary alcohols appeared at 1159 cm⁻¹ and 999 cm⁻¹, respectively, while the C–O–C bonds of the β-(1→4) glycosidic linkages were identified at 897 cm⁻¹, as reported by [17]. A peak at 1745 cm⁻¹ was observed in the sample mordanted with soy milk, which can be attributed to the C = O bond. For the sample mordanted with lemon juice, the peaks at 1715 cm⁻¹ and 1653 cm⁻¹ may indicate the presence of C = O and C = N groups, respectively.

Analyzing the spectrum of polyamides in Figure 7, characteristic bands of this fiber are noted, such as the amide group (N-H) at 3299 cm⁻¹ due to stretching vibrations; the bands in the region 2939 cm⁻¹ and 2870 cm⁻¹, associated with the asymmetric and symmetric stretching vibrations of the CH₂ group, respectively; and the peak at 1637 cm⁻¹, the characteristic band of amide I, resulting from the stretching vibrations of the amide-carbonyl bond (C = O) [18]. It is possible to note a difference in the FTIR of the polyamide mordanted with soy milk compared to the spectra of the others at 1755 cm⁻¹, referring to the C = O group. In the sample mordanted with lemon, a band is observed at 1709, representing the presence of C = O, according to [19].

Figure 8 shows the FTIR spectra of the polyester fabrics. It can be observed that the spectra before and after mordanting are very similar. Peaks were identified at 1714, 1098, and 968 cm⁻¹, indicating the C = O, CO, and C = C groups, respectively [6]. The peaks around 1712 cm⁻¹ and 1100 cm⁻¹ were attributed to the C = O and CO groups. In this fiber mordanted with soy milk, a peak at 2856 cm⁻¹ was identified, indicating the presence of a CH group, and in the fiber mordanted with lemon juice, no differences in the spectrum could be observed.

3.4. Color Fastness

An important criterion for determining whether a substance can be used as a textile dye—beyond its dyeability—is its permanence on the substrate after dyeing and subsequent use or washing. To evaluate this property and determine the viability of the dye under study, wash fastness tests were performed on the dyed substrates.

Table 3. Notes on color fastness to color change and transfer of samples dyed with dye obtained from eucalyptus leaves.

Sample	Change Fastness	Transfer Fastness
Crude cotton	4	4/5
Cotton treated with lemon juice	3	3
Cotton treated with soy milk	4	4/5
Crude polyamide	4	4
Polyamide treated with lemon juice	3	4
Polyamide treated with soy milk	4/5	4/5
Crude polyester	4/5	4/5
Polyester treated with lemon juice	3/4	4/5
Polyester treated with soy milk	4/5	4/5

and

Table 4. Notes on color fastness to color change and transfer of samples dyed with dye obtained from annatto seeds.

Sample	Change Fastness	Transfer Fastness
Crude cotton	3/4	4/5
Cotton treated with lemon juice	3	2/3
Cotton treated with soy milk	3	4
Crude polyamide	3/4	4/5
Polyamide treated with lemon juice	4	4
Polyamide treated with soy milk	4/5	4
Crude polyester	4	5
Polyester treated with lemon juice	3/4	4
Polyester treated with soy milk	3/4	3/4

present the wash fastness results, showing both the initial color change and the dye transfer to a control fabric. The results are expressed using the grayscale rating. Dyeing processes using eucalyptus leaf and annatto seed extracts—applied to untreated fabrics and to fabrics mordanted with soy milk or lemon juice—were evaluated.

The fastness results in Table 3 indicate that color change and dye transfer to the control fabric were relatively low, with grayscale scores of 4 and 4–5 for the raw cotton and soy milk–treated cotton fabrics, respectively. Soy milk acted as an effective and stable mordant, suggesting that its presence enhanced the interaction between the dye and the fiber—an effect not observed in the samples mordanted with lemon juice. Grayscale fastness scores equal to or greater than 4 are widely accepted in the textile industry. This indicates that the dyed samples do not release significant amounts of dye onto lighter-colored fabrics after washing. Similar results were observed for the polyamide and polyester samples, although the scores for the polyester fabrics were slightly higher than those for cotton and polyamide. Table 4 presents the fastness results for the fabrics dyed with annatto seed extract.

For the dyeing performed with annatto seed extract, the polyamide sample mordanted with soy milk showed the best result for color change fastness, while the raw polyester sample, which achieved the maximum score, exhibited the best performance for color transfer fastness.

Regarding polyester fiber, it is known that dyeing carried out at 115 °C with the natural dye obtained from annatto seeds presents excellent color fastness results [6]. The color fastness results obtained on polyester fabric dyed at 98 °C are 4 in samples with the lemon juice mordant and 5 without the mordant, indicating results accepted in most commercially available articles and with less energy used in the dyeing process, which makes dyeing with this dye potentially sustainable.

4. Final Remarks

This study evaluated the dyeing performance of raw cotton, polyamide, and polyester fabrics mordanted with soy milk and lemon juice, using dried eucalyptus leaf extract and annatto seed extract as natural dyes. The evaluation parameters included color intensity, SEM, FTIR and color fastness. The results highlight both the advantages and challenges of using natural dyes and mordants.

The highest color intensity was observed in cotton dyed with annatto seed extract and mordanted with soy milk, showing a 279.0% increase, followed by polyester dyed with eucalyptus leaf extract, with a 155.2% increase. Using soy milk as a mordant for silk fabrics has enhanced dye absorption and fastness with Basella alba extract [14]. Overall, the soy protein mordant demonstrated a positive effect on the dyeability of cotton fabrics [11], possibly due to nitrogen groups in the soy protein molecule facilitating binding with the dye.

When lemon juice was used as the mordant, polyester dyed with annatto seed extract exhibited a 161.4% increase in color intensity, followed by cotton and polyamide dyed with the same extract, though with a gain of 147,4 and approximately 10%, respectively. These results demonstrate the potential of natural dye extracts combined with different biomordants to produce vibrant and intense colors.

SEM and FTIR confirmed minor surface modifications in fibers treated with the mordants. Colorfastness tests showed results comparable to conventional synthetic dyeing processes, with consistent wash fastness scores—except for cotton dyed with both eucalyptus leaf and annatto seed extracts mordanted with lemon juice—suggesting that these dyes can meet industrial quality standards.

Overall, dyeing with natural dyes and biomordants emerges as a sustainable alternative to synthetic dyes, offering safer and more environmentally friendly formulations. Further research is needed to optimize these processes for industrial application, including achieving greater color intensity with lower extract concentrations and improving fastness performance toward a maximum score of 5.

This study makes important contributions to research on natural extracts, as in the past most mordants used were primarily compounds containing metal ions. It examined natural elements with different molecular structures and pH levels, demonstrating that, in addition to pigments, mordants can also be natural, offering sustainable alternatives for textile dyeing in certain colors.