The antimicrobial potential of plant-based natural dyes for textile dyeing: A systematic review using prisma

3.1.1 Number of publications

The quantity of research studies produced serves as a pivotal statistical indicator for gauging the advancement of a research field. As depicted in Figure 2, research on the antimicrobial properties of the textile plant dyes has shown a continuous upward trend since the publication of the first paper in 2004, spanning nearly two decades until 2024. Particularly since 2018, there has been a significant increase in the number of publications. From 2018 to 2024, there were a total of 100 publications, accounting for nearly 76% of the whole publications. By and large, the growing number of publications underscores the increasing significance and influence of this field, with a growing cohort of researchers dedicating themselves to related investigations.

Figure 2

Annual number of publications.

3.1.2 Analysis of research hotspots and frontiers

Keywords serve as direct reflections of the research content within the literature, offering insights into the primary themes [27]. Figure 3 displays the keyword co-occurrence network of the literature, comprising 285 nodes representing keywords and 1,217 links, with a network density of 0.0301. According to Table 3, it can be observed that in addition to the keywords “natural dye,” “natural dyes,” “antibacterial activity,” and “antimicrobial activity,” other keywords such as “optimization,” “cotton,” “wool,” “silk,” “antioxidant,” “adsorption,” “fabrics,” “extract,” “UV protection,” “fastness,” “chitosan,” “textiles” are relatively more prominent. Among them, “cotton,” “wool,” “silk,” “fabrics,” and “textiles” indicate that the research centers on the textile industry, with a main concentration on natural fabrics. The frequent appearance of keywords like “antioxidant” and “UV protection” underscores a growing interest in multifaceted functional aspects of plant dyes with regard to improving the health and protective qualities of fabrics. Additionally, the recurring presence of keywords such as “optimization”, “extract,” “adsorption,” “fastness,” and “chitosan” indicates ongoing advancements and refinements in plant dye extraction and dyeing techniques. While plant dyes offer safety and health advantages over synthetic dyes, challenges persist due to the complexity of extraction and dyeing processes, alongside issues of low color fastness. Particularly, the antimicrobial performance is closely correlated with the dye assimilation level onto the fiber. Therefore, it is indispensable to improve the binding dye capacity to the fiber, thus continuous technological advancements are essential to fully capitalize on the benefits of plant dyes for broader application and dissemination.

Figure 3

Co-occurrence network of keywords.

“Burst words” are high-frequency keywords and show the development of hot issues and related fields during some period [22]. The burst keywords are exhibited in this domain in 2004–2024 with the red line showing the burst period, as shown in Figure 4.

Figure 4

Keywords with the strongest citation bursts between 2004 and 2024.

Based on recent keywords, it can be inferred that the research focus and future trends of plant dye antimicrobial properties have gradually shifted towards the following areas: (1) Antimicrobial mechanism research: anthocyanins belong to flavonoids, which have a wide range of biological activities and pharmacological properties. However, the antimicrobial mechanisms of different plant chemical components vary. Although there have been many studies published on the antimicrobial activity of plant dyes, the specific mechanism is still not fully understood [27]. Therefore, they can be better applied by further understanding the mechanism of plant dyes in inhibiting microbial growth. (2) Development of functional textiles: Currently, there is relatively more research on dyeing and antimicrobial treatment using cotton fabrics as carriers, because cotton is the most widely used natural textile fiber, with advantages such as soft touch and good water absorption, making it suitable for various types of textiles [3]. Especially in the field of medical textiles, antimicrobial cotton fabrics have tremendous application prospects [3]. Nevertheless, natural dyes have a lower affinity for cotton [2]. As a result, researchers are actively exploring new methods to solve this problem, like utilizing mordant dyeing, fabric modification, adjusting process parameters, etc. to improve the binding effect between natural dyes and cotton, and to find more environmentally friendly and sustainable dyeing techniques. In addition, in the future, it may be further expanded to develop new functional textiles by combining plant dyes with other biomaterials to achieve better antimicrobial performance and other functions and apply them in various fields. (3) Extraction technology improvement: Traditional plant dye extraction methods have some problems, such as being time-consuming and having low yield. Therefore, the optimization and innovation of extraction methods have become the current research hotspot, such as ultrasound-assisted extraction, which can improve the yield and biological activity of plant dyes. Future research may focus on developing new green extraction technologies, seeking more efficient, environmentally friendly, and economically viable extraction processes. (4) Research on the combination with new technology: Silver nanoparticles have become a hot topic in research, demonstrating researchers’ interest in combining traditional plant dyes with modern nanotechnology to enhance antimicrobial effects. Silver nanoparticles are increasingly being used for functional treatment of textiles due to their excellent antimicrobial properties. In the future, the research trend combining modern technology and green chemistry will bring more innovative possibilities for the application of plant dyes in the field of antimicrobials.

3.2.1 Qualitative antimicrobial tests

Qualitative testing measures the size of the zone of inhibition (ZOI) formed on an agar medium, which helps to evaluate the antimicrobial activity of stained samples. Among the 132 articles included in this study, qualitative antimicrobial test methods such as AATCC 147 and ISO 20645 were employed. AATCC 147, also known as the parallel streak method, involves streaking the microbial culture across an agar plate to form parallel stripes. Samples are inoculated onto these stripes, and after 24 h, the breadth of the ZOI around the stripes is observed. Samples with antimicrobial activity show no microbial growth around them, whereas microbial growth can be observed under non-antimicrobial fabrics [28].

ISO 20645 is also referred to as the agar diffusion plate test. In this method, test strains are first evenly inoculated onto the surface of agar medium, and then textile samples are placed on the nutrient medium. Subsequently, they are incubated at 37°C for 18–24 h to observe microbial growth and compare the size of the clear zone around the sample with that of the control [29].

Both AATCC 147 and ISO 20645 qualitative testing methods evaluate antimicrobial properties based on microorganism proliferation in the contact area between agar and the experimental sample, with AATCC 147 assessing the width of inhibition zones and ISO 20645 measuring the diameter of clear zones.

The advantage of qualitative detection methods is that they are relatively simpler and faster to perform than quantitative methods, and the results are more intuitive. However, there are also certain limitations. First, these methods are applicable mainly to antimicrobial agents that can permeate fabrics and kill microorganisms. If the antimicrobial agent is “binding type” and does not permeate, this method may not be able to detect its antimicrobial activity. Second, it cannot provide specific quantified data on antimicrobial effectiveness [28].

3.2.2 Quantitative antimicrobial tests

Quantitative testing evaluates antimicrobial effectiveness by determining the percentage inhibition of microbial growth. Among the 132 articles, quantitative antimicrobial test methods include AATCC-100, ASTM E2149, and GB/T 20944.3.

The AATCC-100 method is also known as the suspension method [30]. In the experiment, the sample was placed in a container containing a microbial suspension to effectively adsorb the suspended microorganism and ensure that no residual liquid remained in the container. Then, a neutralizing solution was added, and the samples were incubated on a rotary shaker at 37°C ± 2°C for 24 h. Afterward, the sample was removed from the mixture and diluted before being inoculated onto agar media for further incubation at 37°C ± 2°C for 24 h. Finally, the antimicrobial effect was evaluated by counting colonies on agar plates. The antimicrobial effect was calculated by the following formula: R = 100 × (B − A)/B. R represents the percentage reduction in microbial, A represents the colony counts in treated samples after inoculation and cultivation, and B represents the colony counts in samples without contact time [27,31]. Additionally, the microbial reduction rate can also be calculated by comparing colony counts between stained samples after microbial cultivation and unstained samples under identical incubation conditions. Specifically, when this formula is used, A refers to the colony counts in stained samples after 24 h of cultivation; B refers to the colony counts in unstained samples cultivated under similar conditions [28,32].

ASTM E2149 is a quantitative antimicrobial testing method conducted under dynamic contact conditions [33,34]. In this method, the test sample and control sample are separately placed in containers containing a certain concentration of microbial mixture. The mixture was then incubated in a shaker incubator at 35°C ± 2°C with agitation for 1 h ± 5 min. Subsequently, dilution and inoculation were performed on agar plates, followed by further incubation at 35°C ± 2°C for 24–48 h. After incubation, the colony counts on the culture dishes were calculated to determine the reduction rate of microorganisms [35,36]. The antimicrobial effect was calculated by the following formula: R = 100 × (B − A)/B. R represents the percentage reduction in microbial, A represents the colony counts recovered from the treated stained fabric samples after inoculation, and B represents the colony counts recovered from the untreated control fabric samples after inoculation [37,38]. Alternatively, it can also be calculated by a “0” contact time method, where A refers to the colony counts of inoculated treated samples after cultivation and B refers to the colony counts of samples with 0 contact times [35]. GB/T 20944.3 and ASTM E2149 are based on similar types of testing methods, specifically the oscillation method. In this method, oscillation is carried out for a certain period of time (18–24 h) at a temperature of 24°C ± 1°C, followed by dilution and inoculation on agar plates and further cultivation at 37°C for 18–24 h. After incubation, the colony counts on culture dishes were calculated to determine the reduction rate of microorganisms. The antimicrobial effect was calculated by the following formula: R = 100 × (B − A)/B. R represents the percentage reduction in microbial, and A and B represent visible colony counts from unstained samples and stained samples, respectively [39,40]. ASTM E2149 is an American standard, while GB/T 20944.3 is a Chinese standard. Besides the standards, differences exist in sample specifications, microbial concentrations, and inoculation quantities.

Compared with qualitative testing methods, quantitative detection methods have significant advantages in terms of accuracy of results. Specifically, they can provide specific antimicrobial performance data and quantify the antimicrobial effects of samples. They are suitable for most textiles, including both leachable and non-leachable antimicrobial fabrics. Additionally, these methods simulate the contact conditions of textiles in actual use. Therefore, more researchers choose quantitative testing methods to evaluate the antimicrobial properties of textiles. However, the testing process is time-consuming and expensive because of its complexity and requirement for multiple equipment and materials. In summary, while quantitative testing methods can provide accurate antimicrobial performance data, they are time-consuming and costly; however, qualitative testing methods are simple and fast but lack quantification ability with lower accuracy. Furthermore, according to a review of the literature, AATCC 100 is the most commonly used testing method.

3.2.3 Differences in the effects on different microbial strains

In all the studies, the bacterial species most commonly used for testing were Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The main reasons for the use of these two bacteria as the most common test species are their wide presence in the environment and their potential harm to human health. S. aureus is a major cause of cross-infection in hospital, commercial, and household laundry practices. It causes skin and tissue infections, sepsis, endocarditis, and meningitis. E. coli can cause urinary tract infections, gastrointestinal infections, wound infections, and hospital-acquired infections [41]. Additionally, E. coli is a gram-negative bacterium (G −ve), whereas S. aureus is a gram-positive bacterium (G +ve). By simultaneously using these two bacterial species, the effectiveness of antibacterial treatments on different types of bacteria can be evaluated to fully demonstrate the antibacterial capability of the materials.

According to the summary of literature, sensitivity to surface sterilization by the tested microorganisms can be ranked from high to low as follows: G +ve > G −ve. This difference is likely due to the single-layer structure of peptidoglycan in the cell wall of G +ve bacteria [42,43], compared to the multi-layered structure defined by lipoproteins, phospholipids, and lipopolysaccharides in the outer membrane of G −ve bacteria (Figure 5), which presents a potential barrier to foreign molecules [44,45,46].

Figure 5

G +ve and G −ve cell wall structure comparison diagram. (a) G +ve. (b) G −ve.

However, some studies have shown better inhibitory effects on E. coli [32,47]. S. aureus, a gram-positive bacterium, has a thick peptidoglycan layer that often contains structures cross-linked by pentaglycine, increasing the stability and tolerance of its cell wall. On the other hand, E. coli is a gram-negative bacterium with a relatively thin peptidoglycan layer and does not possess this highly cross-linked structure [47]. In addition, compared with those of Gram-positive bacteria, the cell walls of gram-negative bacteria are more negatively charged and hydrophilic, resulting in stronger electrostatic interactions between cationic dyes and negatively charged cell walls and greater antibacterial activity [48]. Therefore, these structural differences make E. coli less permeable to certain substances but also more sensitive to certain antibacterial components in certain situations.

Compared with the validation of antibacterial effects, research on antifungal capacity is relatively scarce. The fungal strains involved primarily include two genera: Candida and Aspergillus. Among these, the inhibitory effects on Candida species, particularly Candida albicans, have been studied more extensively. Candida albicans is a common opportunistic pathogenic fungus that typically resides in the human body, especially in the digestive and reproductive systems [49]. When the immune system is compromised or its balance is disturbed, this fungus can lead to various health issues [6]. Currently, many studies have validated the inhibitory effects of various plant dyes against Candida albicans and other species within the Candida genus. In contrast, research on antifungal activity against Aspergillus species has yielded somewhat inconsistent results. In a study by Flax et al., dyes extracted from barberry, bittersweet, and wine berry did not demonstrate any inhibitory effects against Aspergillus niger [56]. Conversely, research conducted by Do et al. utilizing dyes extracted from madders revealed promising antifungal activity against white mold (Aspergillus spp.) [57]. Additionally, Mohamed et al. employed various agricultural waste materials (including onion outer skin, rose, eucalyptus, lemon, grape, and peach leaves) for dyeing and eco-printing silk fabrics. Antimicrobial testing revealed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, as well as Candida and Aspergillus fungi [54].

3.3.1 Chemistry of antimicrobial plant dyes

Natural plant dyes originate in numerous plant parts, including bark, roots, fruits, flowers, seeds, along with leaves. Their antimicrobial activity stems from the presence of active biological molecules, including phytochemicals. The phytochemicals can be divided into different categories; therefore, their modes of action will also correspondingly differ. The primary antimicrobial phytochemicals pertain to the group of phenolic syntheses [58,59], which structurally encompasses one or more hydroxyl groups (–OH groups) [58]. Polyphenolic compounds are a specific type of phenolic compound characterized by multiple hydroxyl groups [60]. Phenolic compounds can inhibit bacteria by disrupting the bacterial enzyme and biomembrane systems, thus affecting energy metabolism and selective substance assimilation [61]. The phenolic compounds can be further divided into various classes such as flavonoids, tannins, quinones, and curcuminoids, based on their different chemical structures [62].

Flavonoids, a diverse group of phenolic compounds, are widely distributed throughout the plant kingdom. Flavonoids include numerous subclasses, such as flavones, flavonols, and anthocyanins [62,63]. For example, Cyanidin, Delphindin, and Pelargonidin are flavonoids with an anthocyanin structure. Studies have identified that they are the main reasons for the good antimicrobial properties of the dyed fabric in Hibiscus sabdariffa [58]. Flavonoids, with their diverse biological activities [64], including antimicrobial properties, have long been a subject of intensive research and development efforts.

Plant tissues extensively contain tannins, complex polyphenolic compounds famous for their astringent taste [65]. They encompass two sub-classes: hydrolyzable tannins and condensed tannins [66,67]. Tannins exhibit a broad-spectrum antimicrobial effect against various pathogens, and owning to the adequate hydroxyl groups and other appropriate functional groups (i.e., carboxyl groups), they can formulate efficient robust complexes with protein chains and other macromolecules on special occasions. As a result, tannins are the most common natural mordants [68].

With high reactivity features, quinones are widely founded in nature, and they are aromatic rings showing two ketone substitutions [69]. Quinones can be classified into subclasses such as anthraquinones and naphthoquinones based on their structural features. Naphthoquinones, including lawsone from henna [62], juglone from walnut [65], shikonin from alkanet [70], and alkannin, have demonstrated antimicrobial effects. Widely distributed in nature, anthraquinones serve as important natural pigments, with Alizarin and purpurin from Madder being notable examples. Many research studies indicate that Madders exhibit significant antimicrobial activity when used for fabric dyeing [62,29].

Curcuminoids, primarily composed of curcumin [71], are predominantly derived from the rhizomes of turmeric [72]. Research indicates that curcumin is environmentally safe, gentle on the skin, imparts a yellow hue, and demonstrates remarkable antioxidant and antimicrobial properties. However, due to its relatively small size and hydrophobic nature, curcumin molecules exhibit poor wash and rub resistance when applied to textiles [73]. The antimicrobial curcumin mechanism is commonly attributed to hydroxyl and methoxyl groups. However, the use of conventional colorfastness agents can diminish the availability of free hydroxyl groups, thereby compromising curcumin’s antimicrobial efficacy [74]. Consequently, numerous studies have focused on developing techniques to enhance both the colorfastness and antimicrobial attributes of curcumin in textiles. One such method involves tannic acid fixation treatments, which have been shown to significantly improve dye retention while preserving curcumin’s antimicrobial activity [73,75].

Besides the typical phenolic compounds, berberine, sourced from plants like barberry and Coptis chinensis, demonstrates notable antimicrobial properties. Berberine belongs to the alkaloid compound category and offers a diverse array of pharmacological activities, making it valuable for medical applications [76]. Additionally, other compounds such as glycosides [77], have exhibited antimicrobial effects. These bioactive compounds bind to fibers through a dying process, thereby endowing the fabric with antimicrobial properties.

3.3.2 Mechanisms of antibacterial activity

Table 4 is a compilation of the antibacterial mechanisms of different categorical phytochemicals mentioned in the literature.

In this table, the literature about the main antibacterial mechanism of different phytochemicals is summarized. Although antibacterial mechanisms show certain differences as they are based on different compounds, in general, they can be divided into the following several categories: cell membrane destruction, enzyme activity inhibition, elevated oxidative stress response, DNA damage, protein structure damage, and nutrient uptake site competition. The schematic diagram of antibacterial mechanisms is shown in Table 5. In addition, in the specific antibacterial process, multiple mechanisms often work together to finally kill the bacteria (Figure 6).

Table 5

Summary of antibacterial mechanisms

Antibacterial mechanism	Schematic drawing	Phytochemicals related to this mechanism
Cell membrane destruction: Lead to the leakage of its internal material, bacterial dysfunction and decreased viability		Polyphenols, Flavonoids, Tannins, Quiones, Curcumin, Berberine, saponins, steroids
Enzyme activity inhibition: Bacteria fail to perform normal metabolic and biochemical reactions		Polyphenols, Flavonoids, Tannins, Glycosides, Curcumin
Elevates oxidative stress response: Disrupt the physiological function and metabolic processes of bacteria		Polyphenols, Quiones
DNA damage: Bacteria fail to replicate and repair normally, resulting in their life blocked or even death		Phenolic acids, Polyphenols, Flavonoids, Quiones, Curcumin
Protein structure damage: The normal function and metabolic processes of bacteria can be affected, resulting in their failure to survive or reproduce		Polyphenols, Flavonoids, Tannins, Quiones, Curcumin, Berberine
Nutrient uptake site competition: Limit the essential nutrients needed for bacteria and interfere with their growth and reproduction		Flavonoids, Tannins, Quiones

Figure 6

Mechanisms of antibacterial action.

Finally, according to the available results, the exact mechanism of bacteria inhibition has not been fully confirmed. Meanwhile, in the actual antibacterial effect, multiple phytochemicals usually interact with each other [77]. In addition, existing research focus more on the resistance of plant dyes to bacteria, and antimicrobial experiments and analysis on fungi or other microorganisms are relatively few, requiring further research.

3.4.1 Metal mordants

Metal mordants are among the most common mordants, as they can form metal complexes with dyes and fibers. These complexes consist of dye molecules, metal ions, and the functional groups of fibers, creating an insoluble compound [62]. The formation of this insoluble compound enhances the dye adhesion to the fiber, thereby enhancing color fastness. Thus, metal mordants enhance the antimicrobial properties of plant dyes on fabric by promoting their complexation and adsorption, and this results in the fabric maintaining good antimicrobial performance even after multiple washings and exposure to light [62]. Additionally, according to the concept of Overtone and Tweedy’s chelation theory, the creation of a chelate complex between dye molecules and metal ions increases the liposolubility of the bacterial cell membrane, thereby enhancing the cell’s permeability. This makes the bacterial cells more susceptible to the bioactive compounds present in the dyes [63,117,118].

In addition to their synergistic effects with dyes, metal ions themselves also exhibit good antimicrobial properties. Their antimicrobial mechanism primarily involve the bond of metal ions to proteins or the generation of reactive oxygen species (ROS). In the first scenario, metal ions bond covalently to the –SH groups in cellular enzymes, disrupting their function and modifying bacterial metabolism, leading ultimately to cellular demise [36,62,119,120,121]. The second scenario revolves around the pro-oxidant behavior of metal ions, whereby highly reactive oxygen radicals produced during the reaction damage and eventually disintegrate bacterial structures [58,62]. The antimicrobial effects of metal mordants have been well demonstrated, for example, in research conducted by Mirjalili and Karimi, turmeric was employed as a natural dye, and ferric sulfate, cupric sulfate, and potassium aluminum sulfate, as mordants for dyeing polyamide fabrics. The results showed a significant increase in the antimicrobial features of the dyed specimens mordanted with three metal mordants compared to the samples dyed without mordants [71].

However, some studies have also indicated that the use of metal mordants can diminish the antimicrobial activity of plant dyes. This can be interpreted by the structural modifications of colored compounds formed owing to the complexation of some dye hydroxyl groups through metal mordants, which subsequently reduce the availability of functional groups for interactions between the dye and microorganisms [45,70,120,122]. Nevertheless, in terms of durability, by virtue of the high chelating capacity of metal mordants, the dye molecules are more strongly fixed to functional groups on the fibers. Therefore, in terms of maintaining antimicrobial efficacy, fabrics mordanted with metal outperform fabrics dyed directly with plant dyes [45,120].

Metal mordants are crucial for improving dye effects and antimicrobial properties; however, they also pose potential hazards, such as causing skin allergies and environmental pollution [83,123]. To find more sustainable alternatives for plant dyeing, researchers have been exploring replacements for metal mordants with other materials.

3.4.2 Plant-based mordants

Biological mordants present superior biocompatibility with ecosystems and eliminate disposal concerns, making them a compelling alternative to traditional metal mordants. These strengths contain environmental sustainability, durability, biodegradability, and non-toxicity to humans, however, lack of allergenic responses. The benefits align with the worldwide trend in the textile industry toward eco-friendly practices [95]. As detailed in the appendix, the bio-mordants used in the literature can be categorized into plant-based mordants and chitosan. Plants serve as mordants primarily due to their rich phytochemical content, such as tannins, terpenoids, flavonoids, and so on. These compounds are rich in phenolic hydroxyl groups, making them create strong bonds between dye molecules and fabrics through hydrogen bonds and ionic interactions with the carboxyl and amino groups of fibers and hydroxyl groups of dye molecules, facilitating dyeing and color fixation [43,93,98,124]. Additionally, tannins impart carboxyl (–COOH) groups to fibers, enhancing dye absorption through increased ionic interactions between COO– anions and cationic dyes. The carboxyl groups in tannins can also esterify with the hydroxyl groups of fibers, resulting in more durable dye–fiber bonds. This naturally enhances the fabric’s antimicrobial properties by enabling better dye adsorption [42].

This naturally enhances the fabric’s antimicrobial efficiency by adsorbing more dye. Furthermore, as discussed in Section 3.3, these phytochemicals possess excellent antimicrobial properties on their own. When used as mordants, they can synergize with plant dyes, thereby boosting the overall antimicrobial effect [98]. For example, Singh et al. utilized the Tamarind seed coat, which is rich in tannins, as a biological mordant and the Kapok flower as the dye to color wool. The mordanted fabric exhibited improved antibacterial properties, which increased with higher concentrations of the mordant [75]. In addition, Somayeh Baseri’s study using tamarind hulls as a bio-mordant and Origanum vulgare as a dye on cotton demonstrated a doubling in antibacterial activity compared to non-pretreated samples [95]. And another study involved adopting date seeds as a bio-mordant and Trachyspermum coptiocum (Zenian) as a dye for wool, which showed enhanced antibacterial properties after pretreating the wool yarn with date seeds [43]. Gallic acid is a natural polyphenol found in fruits, vegetables, and herbs that shows potential as a promising bio-mordant for cellulose materials. El-Zawahry and Gamal used 10% gallic acid combined with 2% gelatin for pre-mordanting cotton fabric, followed by dyeing with Haematoxylum campechianum, demonstrating high antibacterial activity against Staphylococcus aureus [55]. Safapour et al. employed gallic acid and ascorbic acid separately to mordant wool before dyeing with Millettia laurentii sawdust, revealing enhanced antimicrobial activity for both organic acids but better efficacy with gallic acid [47].

In addition, some plants contain different metal ions [124], such as various Symplocos species plants which are hyperaccumulators of aluminum (Al) compounds. These plant-based mordants with metal components accumulate high concentrations of aluminum compounds and form stable chemical bonds during the dyeing process, ensuring that the dye can firmly adhere to textiles [125,126]. Furthermore, these metal ions in these plants contribute to their antimicrobial properties [87].

3.4.3 Chitosan

Chitosan, a polysaccharide made from the copolymerization of glucosamine and N-acetyl-glucosamine, sources from the alkaline deacetylation of chitin, which comes from the exoskeletons of arthropods and crustaceans [127,128]. As a novel mordant, chitosan possesses multiple chemical properties that allow for multifunctional textile finishes and exhibits remarkable antimicrobial capabilities, making it a promising green and efficient biological material for textile modification [129].

On one hand, the amino groups (−NH₂) in chitosan can formulate hydrogen bonds through the hydroxyl groups (−OH) in the dye structure. Furthermore, the interaction between chitosan and fibers, through van der Waals forces and hydrogen bonds, provides sufficient binding strength ensuring that the dye–chitosan complexes are closely attached to the fabric surface, enhancing color fastness [57]. On the other hand, fibers pretreated with chitosan have protonated amino groups deposited on the surface of chitosan molecules, which can electrostatically bind to negatively charged dye molecules, increasing dye adsorption [69,93]. Therefore, chitosan demonstrates outstanding performance in increasing the color fastness of fabrics and dye absorption. Studies indicate that the combination of chitosan with natural plant dyes exhibits excellent antimicrobial effects [130,131].

In addition, chitosan itself also possesses antimicrobial properties. The antimicrobial effect of chitosan arises from the interplay between its positively charged structure and the negatively charged surface of microorganisms. This interaction leads to significant alterations and damage to the bacterial surface, inhibiting bacterial metabolism and ultimately resulting in bacterial death [69,132]. Specifically, the amino groups in chitosan attach to bacteria, thereby hindering their progress [133]. Furthermore, chitosan obstructs the uptake and utilization of external nutrients by bacteria and disrupts their internal metabolic pathways, preventing normal growth and reproduction [134,135,136,137]. The verification of chitosan’s enhancement of antimicrobial properties has been extensive. For instance, a study by Tian et al., the extract of V. bracteatum Thunb. leaves, adopted as a natural dye, was treated with chitosan and FeCl₃ as mordants, and their effects were compared. The results indicated that chitosan was more effective than FeCl₃ in enhancing antibacterial performance, increasing it from 76.10 to 81.4% [39].

3.4.4 Novel environmental mordants

Additionally, the exploration of novel eco-friendly materials, such as choline chloride-ethylene glycol natural deep eutectic solvent (NADES) used as a mordant, is advancing in research from Zhejiang. This research has shown that textiles dyed with natural dyes and this innovative mordant exhibit excellent antibacterial, UV-resistant, antioxidant, and flame-retardant properties, alongside good fastness and structural integrity. Notably, the residual solvent demonstrates significant reusability, and the NADES reduces water usage, chemical reagent usage, energy consumption, along with cost, while enhancing the biodegradability of dye wastewater, suggesting a reduced environmental impact [138].

In Somayeh Baseri’s study, whey protein isolate was utilized as a bio-mordant, pomegranate rind served as the dye for cotton dyeing. A comparison with cotton fabrics dyed solely revealed a higher antibacterial efficiency, maintaining efficacy even after multiple washings [89].

Chitosan is employed in combination with dendrimers to create new environmentally friendly composite bio-mordants, such as chitosan-poly(amidoamine) dendrimer hybrid (Ch-PAMAM) [139] and chitosan–polypropylene imine dendrimer hybrid (CS-PPI) [33]. Pre-treating textiles with chitosan dendrimer hybrid not only introduces new functionalities to the textiles but also generates new functional groups on textiles, thereby enhancing the assimilation sites of dye molecules on fibers, improving dye uptake and antibacterial activity of the textiles [139].

In summary, mordants utilized in plant dyeing fall into four categories: metal mordants, plant-based mordants, chitosan, and novel environmental mordants. Metal mordants are widely used due to their economic and simple application but can lead to metal pollution. In contrast, plant-based mordants, chitosan, and novel environmental mordants offer environmental benefits and show strong potential as replacements for metal mordants, presenting significant developmental value.

3.5.1 Enzyme treatment

Enzymes can change the surface properties of fibers without damaging them, improving hydrophilicity and thus better absorbing natural antimicrobial extracts. Moreover, due to their biodegradable nature, enzymes are considered environmentally friendly for use in textile processing [91]. Garg et al. used laccase enzyme to polymerize phenolic compounds from Camellia sinensis directly on wool. The enzyme-catalyzed polymerization enhanced the wash resistance between dye and fiber, and this improvement guaranteed a significant quantity of phenolic polymer. This provided more antibacterial activities [61]. Vajpayee et al. used cellulase enzymes for wet processing of lotus fabric, resulting in a more durable antibacterial effect on the material. Even after three wash cycles, the fabric maintained its antibacterial properties, while untreated lotus fabric lost its antibacterial effectiveness [91]. Samant et al. applied three enzymes (xylanase, neutral cellulase, and acid cellulase) to improve the dyeing and functional properties of cotton fabrics. Accordingly, it is indicated that the total three enzymes can effectively enhance antibacterial activity, with xylanase showing the most outstanding effect [102].

In addition, enzymes can be used to catalyze the polymerization of dyes. In the study by Baek et al., horseradish peroxidase was used to catalyze the synthesis of antibacterial gallic acid-g-chitosan derivatives, which were then used for dyeing textiles. This enzymatic catalysis method simplified the pre-treatment process of chitosan and significantly enhanced the antibacterial performance of dyed fabrics [140].

3.5.2 Crosslinking agent treatment

Citric acid, a natural compound with three carboxy groups [76], acts as a multi-carboxylic cross-linking agent that responds with the hydroxyl groups of cellulosic chains in cotton, forming ester linkages that cross-link with the fiber [35,108]. This treatment not only improves the physical performance of the fabric but also enhances its antimicrobial properties. The antimicrobial activity of citric acid is ascribed to the decrease in pH value and the destruction of enzyme activity. Another possible reason could be that carboxylic acid groups of CA-treated cotton can chelate with metal ions in cell walls, potentially preventing the assimilation of key nutrients and triggering destroy and cell death [108]. In addition, when dyeing with cationic dye berberine, CA can increase the capacity of the negative charge of the fabric. This is useful to adsorb a large amount of berberine to enhance the exhaustion of the dye, thereby enhancing the antibacterial activity [76].

Habib et al. used citric acid cross-linking to permanently combine curcumin and nano-silver onto the fabric surface, preparing textiles with excellent antibacterial activity and wash fastness. This is because the hydroxyl groups of the antibacterial curcumin dye and the hydroxyl groups of the cellulose fabric form ester bonds through citric acid, resulting in better bonding. Moreover, after citric acid treatment, the carboxylic acid functional groups on the surface of the naturally dyed fabric can develop coordination bonds through the silver ions on the surface of nano silver. This coordination stabilizes the nano silver particles, preventing their aggregation, ensuring their uniform dispersion in the substrate, and improving their adhesion to the textiles, thus enhancing durability and antibacterial effects [72].

However, there are studies suggesting that citric acid cross-linking with chitosan might negatively impact antibacterial properties. Mirshahi et al. used citric acid as a cross-linking agent to fix chitosan on cotton fabrics. During the cross-linking process, the carboxyl groups in citric acid can respond with the hydroxyl or amino groups of chitosan, formulating ester or amide bonds. Since amino groups are the source of chitosan’s positive charge, reacting with citric acid can reduce antibacterial performance. Nonetheless, increasing the concentration of chitosan can enhance antibacterial properties [35].

In addition to citric acid as a crosslinking agent, Sadeghi-Kiakhani et al. used cyanuric chloride as a cross-linking agent to graft chitosan onto cotton structures. Compared to the samples processed through chitosan alone, samples processed through the Chitosan–Cyanuric Chloride hybrid showed enhanced antibacterial efficiency and durability, owing to the development of robust covalent bonds with the cotton fabric [141].

3.5.3 Fabric modification

In the treatment of fabric modification, the most important is cationic modification. Cationic modification technology involves chemically treating fabrics to introduce cationic groups on the fiber surface before dyeing with natural dyes. This improves the bonding ability between dyes, mordants, and fibers, thereby enhancing dyeing effects and antimicrobial activity [142]. For example, Alebeid et al. modified cotton with 2,3-epoxypropyltrimethylammonium chloride (EP3MAC), which reacted with the hydroxyl groups in cotton fibers to form cationic sites on the fiber surface. These cationic sites reacted with the negatively charged carboxyl groups in Lawsonia dye, allowing the dye to directly bind to the cotton fibers without adding electrolytes. This modification enhanced the affinity of cotton fibers for the dye, improving dye absorption efficiency and thereby helping to enhance antibacterial effects [107]. In a study by Gedik et al., cotton fabrics were firstly changed with cationic agents and subsequently combined with mordants and natural dyes to achieve optimal antibacterial activity [142]. In their study, El-Sedikd et al. modified cotton fabrics using 3-chloro-2-hydroxypropyltrimethylammonium chloride and then dyed them with curcumin. Compared to untreated fabrics and solely cationized fabrics, the cationic dyed fabrics exhibited significantly enhanced antibacterial ability [106].

In addition, Szadkowski et al. modified bamboo fibers using 3-aminopropyltriethoxysilane, followed by binding with lawsone dye. The aminosilane activated surface of the bamboo fibers has functional groups that react actively with organic dyes (lawsone), allowing for the formation of chemical bonds between the surface and silane as well as lawsone. This treatment method enables the modified compound to anchor onto the surface, enhancing the antibacterial activity of lawsone [34].

In the study by Zhu et al., zinc ions were used to modify the surface of Angora wool using high-voltage electrospray technology, in order to enhance its binding ability with natural dyes. Subsequently, tannic acid was employed as a mordant, which not only achieved complete coordination of the metal ions but also imparted extremely high antibacterial performance to the fabric (with a bacteria reduction rate exceeding 90%) due to the synergistic effect of dye, metal ions, and mordant [144].

3.5.4 Plasma treatment

The reason why plasma treatment technology can improve antimicrobial effectiveness is mainly through modifying the fabric surface, improving the affinity between natural dyes and fibers, and enhancing the dyeing rate, so that the treated fiber surface can adsorb more antimicrobial dye molecules [111,145]. In addition, plasma treatment enhances the sample wettability by etching the hydrophobic surface layer of the fibers, enhancing the attachment of media dyes or other auxiliaries to the fiber surface and promoting their absorption [36]. Thus, compared to plasma treatment alone, its combination with other materials can further enhance antimicrobial activity.

Haji et al. employed atmospheric plasma treatment to alter the surface properties of nylon 6 fabric, dyed with berberis extract, using copper sulfate as a mordant to compare antibacterial effects. Results showed that untreated dyed samples did not exhibit antibacterial activity, samples mordanted through copper sulfate alone exhibited a low antibacterial activity, plasma-treated dyed samples exhibited moderate antibacterial activity, and plasma-treated samples mordanted through copper sulfate exhibited high antibacterial activity [111]. In another study by the same author, low-temperature oxygen plasma was adopted to improve wool fibers’ assimilation of β-cyclodextrin (β-CD) and dyeing with berberine. Due to β-CD’s ability to increase wool fibers’ adsorption of antibacterial dye (berberine) and its composite effect with the dye, dyed samples exhibited a 99.9% decrease rate for E. coli and S. aureus [113].

3.5.5 Ultrasonic-assisted treatment

Ultrasonic-assisted treatment can be used for the extraction of dyes and dyeing processes. Compared to traditional water-based extraction methods, ultrasonic-assisted extraction is more effective and faster. This is because the high power (20–100 kHz) of ultrasound can be utilized to enhance and intensify the extraction process. After ultrasound irradiation, microbubbles grow and vibrate rapidly, even violently bursting under high pressure. These microbubbles disrupt the solid surface and create microchannels and pores. Additionally, by disrupting cell walls, heat and mass transfer are facilitated in the liquid phase near particles through these channels [146]. Due to improved extraction efficiency, the concentration of antimicrobial active components in the final extract also increases [147], thereby enhancing its antimicrobial effect. Furthermore, many compounds in plants are sensitive to high temperatures, which may lead to their degradation with prolonged boiling [148]. Ultrasonic extraction typically does not require higher temperatures [149], helping protect thermosensitive active components in plants from being destroyed during the extraction process.

During the dyeing process, the use of ultrasound assistance can improve the utilization rate of dyes. The cavitation effects of ultrasound enhance the diffusion and penetration of dyes by disrupting aggregated dye particles, allowing dyes to penetrate deeper into the fibers and increase their binding capacity [96,150]. Therefore, ultrasonic dyeing allows fabrics to absorb more antimicrobial ingredients in dyes compared to traditional dyeing methods, thereby enhancing their antimicrobial effectiveness.

3.5.6 Dye modification

In the research of Alebeid et al., Acacia nilotica pods were applied to extract Henna dye to enhance the dye color intensity as well as the affinity between the dye and fibers. Results show that the wool fabric dyed with modified dyes has excellent antibacterial performance [151].

Rehan et al. prepared azo dyes by coupling modification of peanut red skin extract with p-nitroaniline. The prepared dyes were better bound to the fabric surface through electrostatic interactions and non-electrostatic interactions with functional groups on the fabric, exhibiting excellent antibacterial performance [152].

3.5.7 Optimization of dying process

In addition to the more extensively studied methods mentioned above, the literature also highlights other approaches to effectively improve the antibacterial features of plant-dyed fabrics. For example, as found by Ibrahim et al., a treatment sequence of salt treatment → alkali treatment → oxidation treatment was adopted to fulfill in situ deposition of metal mordant oxides, and then natural dyeing. This method demonstrated excellent antibacterial properties and wash fastness [44].

Additionally, some plant chemicals exhibit different antibacterial activities at different pH levels, primarily due to structural changes in the compounds responsible for antibacterial activity. Ren’s research revealed that tea-dyed wool fabrics exhibited the highest antibacterial efficacy at a dye bath pH of 5.5, mainly due to structural changes in catechins, the primary antibacterial substances in tea. When the dye bath is acidic or alkaline, particularly under alkaline conditions, the oxidation and polymerization of tea polyphenols reduce the amount of –OH groups, significantly decreasing antibacterial activity [78]. Ren et al. also proposed an in situ polymerization dyeing method for cellulose fibers based on the oxidation and polymerization of catechins, achieving brown coloration, good color fastness, and antibacterial characteristics for cotton structures without chemical mordants [83].

In summary, the aforementioned methods primarily enhance antibacterial efficacy by improving the adsorption rate of dyes or mordants on fabrics, allowing the antimicrobial components to function more effectively. Additionally, in situ synthesis involving silver nanoparticles and metal oxide nanoparticles can effectively improve fabric antimicrobial properties. However, this is mainly based on the fact that these nanoparticles themselves act as antimicrobial agents [53,153,154].