Antibacterial and antiviral chitosan oligosaccharide modified cellulosic fibers with durability against washing and long-acting activity

Abstract

The worldwide outbreak of SARS-CoV-2 has attracted extensive attention to antibacterial and antivirus materials. Cellulose is the most potential candidate for the preparation of green, environmentally friendly antibacterial and antiviral materials. Herein, modified cellulosic fibers with sustained antibacterial and antiviral performance was prepared by introducing chitosan oligosaccharide onto the fibers. The two-step method is proved to be more effective than the one-step method for enhanced chitosan oligosaccharide loadings and antibacterial and antiviral activity. In this instance, the modified fibers with 61.77 mg/g chitosan oligosaccharide loadings can inhibit Staphylococcus aureus and Escherichia coli by 100 % after contacting with bacteria for 12 h and reduce the bacteriophage MS2 by 99.19 % after 1 h of contact. More importantly, the modified fibers have washing durable antibacterial and antiviral activity; the modified fibers have 100 % antibacterial and 98.38 % antiviral activity after 20 washing cycles. Benefiting from the excellent performance of the individual fibers, the paper prepared from the modified fibers show great antibacterial (100 %) and antiviral performance (99.01 %) and comparable mechanical strength. The modified fibers have potential applications in the manufacture of protective clothing and protective hygiene products.

1. Introduction

Bacterial and virus infection endangers the safety of food, health care, and human health, it is always an urgent issue to be solved [1], [2]. Human beings have been threatened by various viruses, such as SARS-CoV-2, SARS-CoV, influenza A and B, and human norovirus [3]. It is evident that the global spread of SARA-CoV-2 has a serious impact on human life. Therefore, it is imminent to develop sustained antibacterial and antiviral materials to protect human health and keep healthcare workers safe. At present, medical protective products are typically made from hygienic non-woven materials [4]. Among them, spunlaced non-woven fabrics account for 35 % of medical non-woven fabrics. They are commonly disposable medical textiles because they lack of antibacterial and antiviral properties which can hardly guarantee medical staff's safety under intensive and long-term work [5]. To endow protective clothing with antibacterial and antiviral properties, the commonly used method is adding metal nanoparticles such as Ag, ZnO, and Cu to the cloth [6], [7], [8]. These methods are effective, but they must face the challenge of antibacterial agent shedding and toxicity. Therefore, it is of far-reaching significance to develop green antibacterial and antiviral material with sustained effect.

Cellulose is the most abundant renewable resource in nature and is widely used in fields, such as paper, hydrogel, textile and so on due to its hydrophilicity, nontoxicity, biocompatibility, biodegradability, and easy modification [9], [10], [11]. Notably, cellulosic fiber is also an important raw material for the manufacture of spunlaced non-woven fabrics. However, cellulose itself has strong water retention ability and is easy to cause bacterial breeding [12]. The introduction of antibacterial and antiviral agents is always the common method for endowing cellulose with antibacterial and antiviral properties [13], [14], [15], [16].

Chitosan, as the biomass second only to cellulose, shows fascinating antibacterial and antiviral properties [17], [18]. Several works have been performed to introduce chitosan onto fibers to enable the fibers with antibacterial and antiviral properties. Physical methods such as coating and adsorption (spraying) are commonly used, but the inevitable loss of antibacterial and antiviral agents has limited the wide application [19], [20]. Not surprisingly, chemical modification is the potential method for solving the above problem. During the reported chemical modification, the common methods are functionalized of polymer (cellulose and chitosan) and using crosslinking agent [21], [22]. Functionalization of polymer is an effective method for antibacterial finishing [23], [24], [25], however, functionalization of cellulose usually destroys the fibers' structure and reduces the mechanical strength of the fibers, thus limiting its wide application. In addition, functionalized chitosan graft to cellulose usually has a low grafting degree because of the large molecular weight and steric hindrance of the chitosan. Therefore, it is needed to find a suitable method for chitosan to be linked to fibers through the action of crosslinking agent. The commonly used crosslinking agents are glutaraldehyde and polycarboxylic acids. Glutaraldehyde is a commonly used chemical crosslinking agent (which can interact with the amine group or hydroxyl group of polymers), but it is poisonous and not suitable for medical hygiene products [26]. In recent years, polycarboxylic acids have become a good substitute for glutaraldehyde [22], [27], [28]. Polycarboxylic acids can form ester bonds between hydroxyl and carboxyl, thereby crosslinking cellulose and chitosan. Citric acid, as a natural extractive, is one of the prominent crosslinking agents because of its low price and outstanding crosslinking effect [29]. For example, Fu et al. [30] introduced chitosan derivatives into cellulose fibers through the pad-dry-cure method using citric acid as a crosslinking agent. The strategy is effective on introducing chitosan into cellulose fibers, but the antibacterial activity of the fiber is limited because of the low chitosan loadings. Differing from the pad-dry-cure method, Liu et al. [31] used the impregnation method to prepare chitosan derivatives modified cellulose fiber with citric acid as a crosslinking agent, as well as the above study, the antibacterial activity is not satisfied. For improving the antibacterial activity, Alonso et al. [22] applied UV-irradiation to pretreat cellulose and then introduced chitosan onto the treated cellulose fibers through the impregnation method. The pre-UV-irradiation could improve the chitosan loadings and therefore antibacterial activity; however, the loadings of chitosan in the fiber are still not high (27 mg/g). The relatively low chitosan loadings and the insufficient antibacterial activity might account for the low reaction effectiveness. The long chain of the chitosan may cause steric hindrance and therefore reduce the reaction effectiveness. Moreover, the heterogeneous reaction between cellulose fiber and chitosan would cause lower reaction effectiveness. Therefore, it is important to develop an effective method to prepare modified cellulosic fibers with high chitosan loadings and excellent antibacterial and antiviral activity.

In this work, chitosan oligosaccharide (COS) was used to replace chitosan to prepare cellulosic fibers with antibacterial and antiviral performance because chitosan oligosaccharide has the advantages of relatively lower molecular weight and stronger water solubility. For introducing chitosan oligosaccharide onto cellulose fiber, the impregnation method was applied in the presence of citric acid as a crosslinking agent and sodium hypophosphite as a catalyst. By successful crosslinking of the chitosan oligosaccharide onto the cellulosic fibers, the modified fibers exhibit against both Staphylococcus aureus and Escherichia coli. Besides, it shows the highly effective antiviral property against the bacteriophage MS2. More importantly, the modified fibers have excellent antibacterial and antiviral durability; 20 washing cycles have almost no effects on the above performances. The paper sheets prepared from the modified fibers also show great antibacterial and antiviral properties, exhibiting great potential application in protective hygiene products, especially spunlaced non-woven fabric production.

2. Experimental

2.1. Materials

Eucalyptus wood pulp is provided by Zhangzhou Huixin Paper Co., Ltd. (Quanzhou, Fujian, China), the information of fiber size and size distribution of the eucalyptus wood pulp was shown in supporting information (Table S1, Fig. S1, and Fig. S2). Chitosan oligosaccharide (viscosity average molar mass ≤ 1500 Da, degree of deacetylation: 86.7 %)was purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd., the degree of deacetylation of chitosan oligosaccharide was determined according to the results of elemental analysis, as shown in supporting information (Table S2). Citric acid monohydrate (AR, 99.5 %), sodium hypophosphite monohydrate (99 %), and 2,2-Diphenyl-1-picrylhydrazyl (contains 10–20 % Benzene) (≥97 %) were purchased from Aladdin Reagent Co., Ltd. (Shanghai), LB Broth, LB Agar, Escherichia coli (E. coli) (ATCC8739), Staphylococcus aureus (S. aureus) (ATCC6538) were obtained from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, Shandong, China). 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonicacidammoniumsalt) was purchased from Beijing InnoChem Science & Technology Co., Ltd. (Beijing, China). MS2 bacteriophage (ATCC 15597-B1) and Escherichia coli (ATCC 15597) are gifts from Fuzhou University. All chemicals are used without further purification.

2.2. Fiber refining

Eucalyptus wood pulp was dispersed into water and then defibered by a dissociator for 20 min. After that, 30 g dispersed pulp (based on oven dry weight) was mechanically treated for 10,000 revolutions by a PFI mill at the consistency of 10 %; whereas the beating degree of the pulp was detected.

2.3. Modification of cellulosic fibers by chitosan oligosaccharide

One-step method. Briefly, 6 % (w/v) cellulosic fibers were added to the solution mixture containing citric acid (CA), sodium hypophosphite (SHP), and chitosan oligosaccharide (COS) and were soaked at 80 °C for 2 h. Next, the sample was dried in an oven at 105 °C, and then cured at 150 °C for 3 min.

Two-step method. As different from one step method, 6 % (w/v) cellulosic fibers firstly reacted with citric acid with sodium hypophosphite as a catalyst at 80 °C for 1 h. After that, chitosan oligosaccharide was added to the impregnation solution to continue the reaction at 80 °C for another 1 h.

After finishing the reaction, the fibers were dried in an oven at 105 °C, and then cured in an oven at 150 °C for 3 min. The treated fibers are thoroughly rinsed with distilled water, and finally, freeze-dried for the subsequent analysis and experiments.

For optimizing the reaction conditions, citric acid concentration and sodium hypophosphite concentration are selected based on the bacteriostatic rate and chitosan oligosaccharide loadings.

2.4. Characterizations of modified fiber

The surface morphology of untreated fibers and modified fibers was analyzed by scanning electron microscope (Zeiss geminisem 300, Germany).

The chemical structures of fibers, chitosan oligosaccharide, and modified fibers were analyzed by Fourier Transform Infrared Spectroscopy (FTIR) (Bruker vertex 70, Germany). The characterization was carried out in the spectral range of 4000–400 cm⁻¹ with 16 scans and a resolution of 2 cm⁻¹.

The surface chemical composition of fibers and modified fibers is determined by X-ray photoelectron spectroscopy (XPS) (Thermo Scientific k-alpha), and the energy step is set to 1.0 eV.

X-ray diffraction data were collected by X-ray diffractometer (XRD) (Ultima IV, Japan). The radiation was Cu kα(λ = 0.154 nm) at 40 kV and 40 Ma with the scanning range of 5–60°. The crystallinity index (CrI) of the samples was calculated from the XRD spectra using the following equation [32].

$$\mathrm{CrI}\left(\%\right)=100\times \left(I_{002}-I_{\mathit{am}}\right)/I_{002}$$

where I₀₀₂ is the maximum intensity of the lattice diffraction and I_am is the minimum intensity at 2θ = 18° which corresponds to the amorphous part of cellulose.

The surface elements of the modified fiber were determined by the element analyzer (German elemental Vario El cube). The chitosan oligosaccharide loadings of the modified fibers was calculated according to the following equation.

$$\mathrm{COS}\text{loadings}\left(\mathrm{mg}/\mathrm{g}\right)=\left(1000\times \mathrm{N}\%\times \mathrm{M}\right)/14$$

where N% is the nitrogen content of the modified fibers, M is the relative molecular weight of chitosan oligosaccharide.

2.5. Antibacterial activity

The antibacterial property of the modified fibers is evaluated by the shake flask method according to Shi et al. [33] with a slight modification. Staphylococcus aureus and Escherichia coli were used as the test strains. In short, the strain is cultured in a shaking table at 37 °C for 18 h until the strain concentration is 10⁹ CFU/mL and then the strain is diluted to 10⁵ CFU/mL as the inoculation solution for standby. 0.1 g modified fibers (in absolute dry) were added into a conical flask containing 10 mL inoculated bacterial solution and incubated at 37 °C for 1, 3, 6, 12, and 24 h respectively. For comparison, control experiments were performed according to the above procedure; where, 0.1 g initial fibers (in absolute dry) were used. The incubated bacterial solution was diluted 10 times with PBS buffer, and 0.1 mL was taken out and coated onto the agar plate. After 24 h incubation, the colony count and strain concentration were evaluated. All experiments are made in triplicate. The bacteriostatic rate was calculated by the following equation.

$$\text{Bacteriostatic rate}\left(\%\right)=100\times \left({\mathrm{A}}_0-\mathrm{A}\right)/{\mathrm{A}}_0$$

where A₀ and A represents the bacteria number of control and experimental sample, respectively.

For the paper antibacterial test, the method was used as described by Cabañas-Romero et al. [34] with slight modifications. Firstly, the paper (60 g/m²) was cut into 1 × 1 cm² pieces to proceed with the subsequent experiments. The paper piece was put on the culture medium and then 5 μL 10⁵ CFU/mL bacterial solution was added into the paper piece. Afterward, the paper samples were cultured at 37 °C for some time. 5 mL PBS buffer was used for washing the bacteria from the culture paper pieces with the aid of vibration. The later experiments were the same as the fiber test for antibacterial activity.

2.6. Antiviral activity

The antiviral activity of the modified fibers and paper sheets (2 mm × 2 mm pieces) was evaluated by the plaque reduction assay with bacteriophage MS2 according to Oza et al. [35] with slight modifications. 1 × 10⁶ PFU/mL MS2 bacteriophage solutions were inoculated in the form of 500 μL loads on 0.1 g fibers and modified fibers. After the samples were incubated for 1 h at 37 °C, 4.5 mL phosphate-buffered saline was added and vortexed for 2 min to thoroughly wash the surfaces of the fibers for detachment of bacteriophage plaques. Then, the bacteriophage-containing suspensions were cultured on bacterium-seeded two-layer agar plates and incubated for 4 h at 37 °C. Finally, the plaques inhibition was counted as follows:

$$\text{Plaque inhibition}\left(\%\right)=100\times \left(\mathrm{A}-\mathrm{B}\right)/\mathrm{A}$$

where A is the virus concentration in the control sample and B is the virus concentration in the experimental samples.

2.7. Washing durability test

To evaluate the durability of chitosan oligosaccharide modified cellulosic fibers against repeated launderings, the modified fibers were washed according to the standard method ISO 105-C10. Briefly, modified fibers (1 g) were washed in a 150 mL mixture solution of 2 g/L sodium carbonate and 5 g/L soap at 60 °C with an agitator speed of 1000 r/min for 30 min and then rinsed with distilled water. Finally, the modified fibers were dried at 60 °C for 30 min. For the sake of simplicity, we used 5 g/L laundry detergent in this test. The washing procedure is equivalent to five home laundering cycles.

2.8. Antioxidant activity

2,2′-Azinobis (3-ethylbenzothiazoline -6-sulfonic Acid Ammonium Salt) (ABTS) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) methods were used to determine the oxidation resistance of the modified fibers [36], [37]. ABTS and DPPH radicals scavenging rate was calculated as:

$${\text{ABTS}}^{+}\text{inhibition}\left(\%\right)=100\times \left({\mathrm{A}}_{\mathrm{i}}-{\mathrm{A}}_{\mathrm{f}}\right)/{\mathrm{A}}_{\mathrm{i}}$$

where A_i is the Initial absorbance value of ABTS⁺ and A_f is the absorbance value of ABTS⁺ after being treated by modified fibers.

$$\text{DPPH inhibition}\left(\%\right)=100\times \left({\mathrm{A}}_{\mathrm{i}}-{\mathrm{A}}_{\mathrm{f}}\right)/{\mathrm{A}}_{\mathrm{i}}$$

where A_i is the initial absorbance value of DPPH and A_f is the absorbance value of DPPH after being treated by modified fibers.

2.9. Cytotoxicity evaluation

The cytotoxicity of chitosan oligosaccharide (COS), cellulose fibers, and modified fibers was evaluated by the CCK-8 assay on L929 cells [38]. The extract is obtained by soaking 10 mg of the samples (cellulose fibers and modified fibers) in a 10 mL phosphate buffer solution (PBS) for 24 h at 37 °C. The test concentrations of chitosan oligosaccharide solution were 1 mg/mL and 0.5 mg/mL, respectively. L929 cells (100 μL, 8 × 10⁴ cells/mL) were first inoculated into 96-well plates and cultured in 5 % CO₂ at 37 °C for 24 h. The sample solutions (100 μL) were added to each well respectively and cultured in 5 % CO₂ at 37 °C for 24 h. Each well was rinsed three times with PBS, then CCK-8 reagent (100 μL, 10 %) was added and cultured in 5 % CO₂ at 37 °C for 2 h. The absorbance value of each well was recorded at 450 nm using a microplate reader. All experiments are repeated three times.

2.10. Mechanical properties of paper sheet

Canadian lab tech-4001 paper sheet former and vacuum dryer were used for paper preparation (60 g/m²). The tensile strength, burst strength and tear strength of the paper and modified paper sheet are determined by the standard method (TAPPI/ANSI T 494 om-22, TAPPI/ANSI T 403 om-15, and TAPPI/ANSI T 414 om-21). All the tests were repeated for 10 times and the average data are reported.

3. Results and discussion

3.1. Optimization of preparation process

Fig. 1 describes the two methods of cellulosic fiber modification. After the modification, the chitosan oligosaccharide was introduced onto cellulosic fibers by an esterification reaction. In the one-step method, citric acid, cellulose, and chitosan oligosaccharide were mixed and took an esterification reaction with the help of the catalyst. By contrast, in the two-step method, cellulose reacted with citric acid first and then chitosan oligosaccharide was added to proceed with another esterification.

Fig. 1.

Scheme of the preparation of chitosan oligosaccharide modified cellulosic fiber.

For optimizing the reaction condition, citric acid concentration and sodium hypophosphite concentration are discussed. As shown in Fig. 2a, both in the two methods, with the increase of citric acid concentration, the bacteriostatic rate of the modified fibers increased first and then decreased; where the bacteriostatic rate reached the maximum when the concentration of citric acid was 20 %. The increase of the concentration of crosslinking agents (citric acid) promotes the possibility of the reaction between cellulose, chitosan oligosaccharide and citric acid [30]. Notably, when the citric acid concentration exceeds 20 %, much citric acid is probably consumed on self-crosslinking of chitosan oligosaccharide, causing insufficient reaction [22].

Fig. 2.

Effect of citric acid (a), sodium phosphate (b) and chitosan oligosaccharide (c) concentration on the antibacterial property of modified fibers (the antibacterial performance is evaluated by the antibacterial rate of Staphylococcus aureus), (d) effect of chitosan oligosaccharide dosage on chitosan oligosaccharide loadings of modified fibers.

As shown in Fig. 2b, the catalyst sodium phosphate also affects the reaction and therefore bacteriostatic performance. Catalyst plays a promoting role in the formation of cyclic anhydride from citric acid, while excessive sodium hypophosphite resulted in a decrease of chitosan oligosaccharide loadings due to the neutralization of free-carboxyl groups [22]. As a result, 6 % sodium hypophosphite is suitable for reaction promotion without serious competitive reaction.

As shown in Fig. 2c and d, the concentration of chitosan oligosaccharide has a great impact on chitosan oligosaccharide loadings and the antibacterial activity of modified fibers. A suitable increase of the concentration of chitosan oligosaccharide is accompanied by the substantial increase of chitosan oligosaccharide loadings and therefore antibacterial activity of the modified fibers. However, excessive chitosan oligosaccharide concentration will cause an obvious reduction of chitosan oligosaccharide loadings and antibacterial activity. Apparently, when chitosan oligosaccharide concentration is 6 %, the chitosan oligosaccharide loadings reach the maximum, 61.77 mg/g for the two-step method and 50.26 mg/g for one step method respectively. An increase of the concentration of chitosan oligosaccharide will improve the possibility of the reaction between cellulose, citric acid, and chitosan oligosaccharide; however, excessive chitosan oligosaccharide will initiate a self-crosslinking reaction between chitosan oligosaccharide itself [22], [30]. It is accepted that loadings and stability of chitosan are the key factors influencing the antibacterial and antiviral properties. Notably, the amino groups of chitosan oligosaccharide inevitably participate in the crosslinking reaction [39], resulting in the consumption of amino groups and reducing the antibacterial activity of modified fibers. However, based on the results of the antibacterial activity of the modified fibers, we can infer that the crosslinking reaction mainly occurs between citric acid and the hydroxyl group of chitosan oligosaccharide.

In addition, the results from Fig. 2 indicate that the two-step method is more effective than the one-step method for enhanced chitosan oligosaccharide loadings and antibacterial activity. In the one-step reaction, chitosan oligosaccharide, citric acid and fiber are mixed as a suspension. Among the reaction medium, chitosan oligosaccharide is soluble in citric acid solution and readily reacts with citric acid forming ester. Moreover, chitosan oligosaccharide itself is prone to take a self-crosslinking reaction. In addition, the strong electrostatic action between the positively charged amino group and the negatively charged carboxyl group will cause precipitation, resulting in the loss of chitosan oligosaccharide and citric acid [30]. The above ineffective reactions will consume some of the reagents and therefore significantly reduce the reaction effectiveness between fiber, chitosan oligosaccharide and citric acid. By contrast, as discussed above, the two-step method can minimize the contact between chitosan oligosaccharide and citric acid, reducing the ineffective reaction.

Meanwhile, the results showed that the bacteriostatic rate was positively correlated with the chitosan oligosaccharide loadings of the modified fibers. In order to obtain modified fiber with excellent antibacterial properties, a two-step method should be selected at the citric acid concentration of 20 %, sodium hypophosphite concentration of 6 %, and chitosan oligosaccharide of 6 %.

3.2. Characterization of chitosan oligosaccharide modified cellulosic fiber

As observed from the images of the fiber and modified fiber as shown in Fig. 3 , the fibers (after mechanical refining) are fibrillated with an uneven surface (a, c); after the reaction, the microfibrils almost disappeared and the surface of the modified fibers tends to be smoother (b, d). The use of acid in the modification reaction would cause cellulose acidic degradation which is preferentially occurring on the surface of the fibers. The degradation and the dissolution of the microfibrils covered on the fiber surface results in a smoother surface of the modified fibers. Moreover, the heterogeneous reaction between cellulose and chitosan oligosaccharide made the crosslinking reaction mainly occur on the surface of the cellulose fiber, which results from a distinct smoother surface [40], [41].

Fig. 3.

SEM images of the surface of fibers (a, c) and modified fibers (b, d).

The FT-IR spectra of fibers, modified fibers, and chitosan oligosaccharide are shown in Fig. 4 . The characteristic peaks -OH, C—H, and C—O of cellulose appear at the wavelengths of 3400 cm⁻¹, 2900 cm⁻¹, and 1067 cm⁻¹ respectively [42]. Chitosan oligosaccharide showed the stretching vibration peak of amide I (C=O) and the bending vibration peak of amide II (-NH) at 1627 and 1520 cm⁻¹, and the stretching group vibration peak of -NH₂ overlapped with the peak of amide II [43], [44]. By the means of citric acid, the hydroxyl groups on cellulose and chitosan oligosaccharide react with the carboxyl groups of citric acid to form ester bonds, appearing at the peak of O-C=O at 1732 cm⁻¹ [22]. Compared with chitosan oligosaccharide FTIR, a redshift of peaks of modified cellulose amide I (C=O) and amide II (-NH) are observed. Hydrogen bonding between the -NH group and the hydroxyl group of cellulose may attribute to the redshift [45]. FTIR results showed that chitosan oligosaccharide were successfully crosslinked on fibers in the form of ester bonds.

Fig. 4.

FTIR spectra of chitosan oligosaccharide, cellulose fibers, and modified cellulose fibers.

Fig. 5 shows the XPS results of the cellulose and modified cellulose. As observed in Fig. 5a, by contrast to the raw cellulosic fiber, an N peak at 399 eV appears in the modified fiber which comes from chitosan oligosaccharide, indicating that chitosan oligosaccharide is introduced into cellulosic fibers [46]. The presence of C—N at 285.9 eV and C O, C=O-O at 288 eV (Fig. 5b) confirmed the existence of chitosan oligosaccharide and citric acid. O1s spectrum of the modified fibers also shows the 531.6 eV signal peak corresponding to C O, which proves the existence of the ester bond and carboxyl group [47], [48]. In addition, there are two signal peaks of 401 eV and 399.1 eV in the N1s spectrum of the modified fibers, which is attributed to C—N and N—H [49]. Therefore, based on the results of XPS, it is concluded that chitosan oligosaccharide and citric acid are introduced into the modified cellulosic fibers.

Fig. 5.

XPS spectra of original and modified cellulose fibers: (a) survey spectra, (b)–(d) C1s spectra, O1s spectra, and N1s spectra of modified cellulose fibers, respectively.

The XRD spectra of fiber and modified fiber are shown in Fig. 6 . The fiber and modified fiber have diffraction peaks at 2θ = 16.0, 22.7, and 34.6, corresponding to 110, 200, and 004 crystal planes respectively [50], [51]. These three crystal planes are the typical crystal planes of cellulose I, which refers that the chemical reaction between cellulose and chitosan oligosaccharide could not change the crystal structure of cellulose. However, the crystallinity of the modified fiber decreases from 72.61 % to 69.21 % (one-step method), and 70.62 % (two-step method) respectively. The slight decrease in crystallinity refers to the cellulose degradation that occurs both in the amorphous region and the surface of the crystallization region [52]. It is accepted that the degradation of amorphous cellulose will cause an increase in crystallinity; only the degradation of cellulose in the crystallization region can reduce the crystallinity.

Fig. 6.

XRD spectra of original cellulose fibers and modified cellulose fibers.

3.3. Antibacterial activity of the chitosan oligosaccharide modified fibers

The antibacterial properties of modified fibers against Staphylococcus aureus and Escherichia coli are shown in Table 1 . It is observed that when the modified fibers were in contact with the strain for 1 h, the bacteriostatic rate of the modified fibers against Staphylococcus aureus is 94.4 %, which is significantly higher than that against Escherichia coli (85.9 %). Positively charged amino groups on chitosan oligosaccharide attract negatively charged bacteria, bind to the receptors of the cell wall, and cause K⁺ to flow out of the cell membrane, stimulating cell acidification and eventually leading to microbial death [53]. The higher bacteriostatic rate of Staphylococcus aureus is related to the protective effect provided by the external lipid membrane of Gram-negative bacteria and the low tolerance of Gram-positive bacteria to oxidative stress [34]. The cell wall of Staphylococcus aureus is composed of teichoic acid and other negative substances. The amino cation of chitosan oligosaccharide can flocculate and combine the anions on the surface of bacterial cells, thereby destroying the permeability and the integrity of cell membrane. The cell wall of Escherichia coli is more complex. There is a semi-permeable membrane on the peptidoglycan layer, which prevents the antibacterial agent from entering the cells [54], [55]. When the contact time between the modified fiber and the strain extends to 12 h, the bacteriostatic rate reaches 100 %. The extremely high loadings of chitosan oligosaccharide might be responsible for the excellent antibacterial performance. As compared with the other works (As shown in Table S3), the modified fibers in this work show the highest chitosan oligosaccharide loadings and bacteriostatic rate. Therefore, the modified fibers prepared in this study with ultra-high chitosan oligosaccharide loadings have greater advantages in the field of antibacterial materials.

Table 1.

Inhibition rate of modified cellulose fibers to bacterial.

Type of sample	Time (h)	Bacteriostatic rate (%)
		Staphylococcus aureus	Escherichia coli
One-step	1	90.55	76.32
	3	97.00	91.51
	6	99.96	99.19
	12	100a	99.97
	24	100a	100a
Two-step	1	94.40	85.90
	3	98.29	92.79
	6	99.98	99.56
	12	100a	99.99
	24	100a	100a

^aThe 100 % shown in the results represents that the antibacterial rate is greater than or equal to 99.999 %.

3.4. Antiviral activity of the chitosan oligosaccharide modified fiber

The bacteriophage MS2 is a non-enveloped positive-stranded RNA virus and it is a potential surrogate of human pathogenic viruses such as SARS-CoV-2, Influenza A and B, and human norovirus due to its icosahedral structure and small size [56], [57]. Chitosan has the capacity to kill viral by binding the proteins in the viral capsid and causing viral structural damage [58]. As listed in Table 2 , the modified fibers can reduce the viral load by 99.19 % after 1 h of contact, indicating that the modified fibers have a promise of antiviral performance. Similarly, the two-step method is more effective than the one-step method for inhibiting the bacteriophage MS2, this may be related to the loadings of chitosan oligosaccharide.

Table 2.

Inhibition rate of modified cellulose fibers and modified paper to the bacteriophage MS2.

Type of sample		Time (h)	Reduction (%)
Modified fibers	One-step	1	97.59
	Two-step	1	99.19
Modified paper	One-step	1	96.72
	Two-step	1	99.01

3.5. Antibacterial and antiviral durability of the modified fibers

Antibacterial durability and antiviral durability are an important requirements for antibacterial and antiviral material application. In this work, the antibacterial durability and antiviral durability of the modified fibers were evaluated by each 10 laundering cycles. As shown in Table 3 , even after 20 washing cycles, the bacteriostatic rate against Staphylococcus aureus is still up to 100 %, and the bacteriostatic rate against Escherichia coli is above 98 %. Moreover, the modified fibers washed 30 cycles were still able to kill 99.99 % of the initial Staphylococcus aureus present which was barely achieved by adsorption. Meanwhile, the modified fibers still have the ability to reduce the bacteriophage MS2 by 95.44 % after 30 washing cycles. The results indicate that the antibacterial activity and antiviral activity of modified fibers are durable, the chitosan oligosaccharide was chemically linked with cellulose by an ester bond, which is stable to the shear forces encountered in the washing system. Unfortunately, the antibacterial (against Escherichia coli) durability of the modified fibers decreased with increasing washing cycles (30 times), it may be related to the hydrolysis of ester bonds during washing [41]. The hydrolysis of the ester bond directly leads to a decrease in the loadings of chitosan oligosaccharide on the modified fibers, thereby reducing the antibacterial activity of the modified fibers. The bacteriostatic rate of the modified fibers after washing against Escherichia coli decreased more obviously, because the inhibitory activity of chitosan oligosaccharide against Escherichia coli was lower than that of Staphylococcus aureus [54].

Table 3.

The antibacterial durability and antiviral durability of the modified cellulose fibers.

Type of sample	Washing times	Bacteriostatic rate (%)		Reduction (%)
		Staphylococcus aureus	Escherichia coli	MS2
Two-step	0	100a	99.99	99.19
	10	100a	99.29	99.02
	20	100a	98.41	98.38
	30	99.99	83.21	95.44

^aThe 100 % shown in the results represents that the antibacterial rate is greater than or equal to 99.999 %.

3.6. Antioxidant performance of the modified fiber

Fig. 7 shows the scavenging efficiency of the modified fibers for ABTS and DPPH radicals. The results showed that the modified fiber has a good quenching ability for ABTS and DPPH free radicals. The free radical scavenging rates of ABTS and DPPH were 60.50 % and 44.94 % respectively, which mainly came from the existence of amino and carboxyl groups in the modified fiber. Studies have shown that citric acid and chitosan oligosaccharide are effective antioxidants. Carboxyl can effectively combine with the oxidant Fe²⁺, thus playing an antioxidant role [59]. Amino groups can form ammonium ions in an aqueous solution that can quench free radicals [60]. Benefiting from the combination effect of citric acid and chitosan oligosaccharide, modified fibers show good antioxidant performance.

Fig. 7.

Antioxidant activity of the modified cellulose fibers.

3.7. Cytotoxicity of the modified fiber

The toxicity of chitosan oligosaccharide, cellulose fibers, and modified fibers against L929 cells is shown in Fig. 8 . The results indicate that cellulose fibers and modified fibers have no obvious toxicity to L929 cells; where, the cell viability of L929 cells barely changed after being treated with the sample solution. In contrast, the chitosan oligosaccharide shows mild toxicity to the L929 cells; as shown in the toxicity experiment, the cell viability of L929 cells is 95.7 % when the concentration of chitosan oligosaccharide solution is 1 mg / mL. It is well known that the chitosan is a non-toxicity substance [61]. The chitosan molecules with a molecular weight higher than 500 are difficult to pass through the cell membrane and thus non cytotoxicity [62]. However, the chitosan oligosaccharide with lower molecular weight shows mild toxicity at concentrations of 0.5 mg / mL and 1 mg / mL for L929 cells in this study. The chitosan oligosaccharide with molecular weight o lower than 500 might be responsible for the relative weak cytotoxicity. The low toxicity of chitosan oligosaccharide is insignificant for the modified fiber with a substantial content of cellulose; namely, the modified fibers show almost nontoxicity. In this instance, the cell viability was 97.8 % when inoculated in the extract from the modified fiber. The above results indicate the modified fibers have good biocompatibility.

Fig. 8.

The cytotoxicity of chitosan oligosaccharide, cellulose fibers and modified fibers on L929 cells.

3.8. Application of the modified fibers

The antibacterial and antiviral properties of the paper prepared from the modified fiber are shown in Table 4, Table 2 . The results showed that the paper has a strong inhibitory effect on the growth of bacteria. When exposed to 18 h, the inhibition rate against to bacteria can reach 100 %. As well as the modified fibers, the inhibition of Staphylococcus aureus is higher than that of Escherichia coli. Meanwhile, the paper prepared from modified fibers shows excellent antiviral activity, the results showed that contact 1 h resulted to reduce the bacteriophage MS2 load by 99.01 %.

Table 4.

Inhibition rate of modified cellulose fibers paper to bacteria.

Type of sample	Time (h)	Bacteriostatic rate (%)
		Staphylococcus aureus	Escherichia coli
One-step	12	98.53	90.21
	18	99.90	99.62
	24	99.96	99.87
Two-step	12	99.12	93.78
	18	100a	99.84
	24	100a	99.99

^aThe 100 % shown in the results represents that the antibacterial rate is greater than or equal to 99.999 %.

In addition, the tensile strength, tear strength, and burst strength of the paper prepared from fibers and modified fibers are tested to evaluate the influence of modification on the mechanical properties of paper. As shown in Fig. 9 , the proper mechanical refining can significantly improve the strength properties of the paper including tensile, tear and burst strength. While the chemical modification of the refined fiber can destroy the individual fibers but can improve the hydrogen bonding between fibers in some extent, as referred from Fig. 9. It is accepted that acidic degradation of the fiber can make the fiber to be shorter which has an extremely adverse effect on the tear strength of the paper [63], [64]. Although the fiber damage from the acidic degradation can lead to an individual fiber strength loss, the shorter fiber with more hydroxyl groups will form a dense paper and therefore results in an improved tensile strength [65]. However, as shown in Fig. 9, the strength of the modified fiber is much higher than that of the original fiber; referring that the modified fiber has relatively high mechanical strength. The paper made from the modified fiber show excellent antibacterial and antiviral properties, it may be a potential raw material as a furnish for spunlaced non-woven fabric production.

Fig. 9.

Mechanical properties of initial fibers (A), refined fiber (B), and modified-refined fibers (C).

4. Conclusion

In this study, chitosan oligosaccharide was successfully crosslinked onto the cellulose fibers in the form of an ester bond, endowing the cellulosic fibers with antibacterial and antiviral properties. Specifically, a two-step method was proposed to accommodate more chitosan oligosaccharide, under the condition of concentration of chitosan oligosaccharide 6 %, citric acid 20 %, and sodium hypophosphite 6 %, 61.77 mg/g chitosan oligosaccharide could be integrated onto cellulosic fibers. In this case, the modified cellulosic fibers not only can inhibit Staphylococcus aureus and Escherichia coli by 100 %, but also can inhibit the bacteriophage MS2 by 99.19 %. Importantly, the modified fibers show excellent antibacterial and antiviral durability; namely, 20 washing cycles have almost no effect on antibacterial and antiviral properties. In addition, because of the combined action of chitosan oligosaccharide and citric acid, the modified fibers also exhibit antioxidant properties. Moreover, the paper prepared from the modified fiber still shows excellent antibacterial and antiviral properties without substantial mechanical strength loss. The modified fibers will find an application in medical hygiene products.

CRediT authorship contribution statement

Jinxin Lan: Methodology, Formal analysis, Writing-Original draft preparation, Investigation. Jiazhen Chen: Investigation, Validation. Ruiqi Zhu: Investigation, Validation. Changmei Lin: Investigation. Xiaojuan Ma: Conceptualization, Writing-Review & Editing, Funding acquisition. Shilin Cao: Conceptualization, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supporting Information