Materials and Reagents

The bentonite raw material (NB) was collected from a quaternary sedimentary basin situated in Sokolac which is located near Šipovo, in Bosnia and Herzegovina (44° 16′ 31.08″ N, 17° 2′ 21.12″ E). Non-woven textile (NT) with a surface mass of 37.00 g m^–2, made from 100% polyester yarns, and knitted fabric (PL) with a surface mass of 117.60 g m^-2, made from 100% viscose bamboo rayon (Dubicotton, Kozarska Dubica, Bosnia and Herzegovina) were used. All chemicals used were of analytical grade: NaCl, CuSO₄٠5H₂O, ZnSO₄٠7H₂O, methylene blue dye, Na₄P₂O₇, H₂SO₄, and Na-alginate were purchased from Lach-ner (Neratovice, Czech Republic). Nutrient agar (NA) and Muller–Hinton agar (MHA) were purchased from Liofilchem (Roseto degli Abruzzi, Italy). Antibiotic discs of erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), and ampicillin (10 μg) were from the Mast Group (Bootle, UK). Twice-distilled (tdw) or demineralized water (dmw) from Water System Mihajlov (Srbobran, Serbia) was used for preparation of all solutions.

Preparation of Modified Bentonite Samples

NB was dried for 24 h at 60°C, ground, and sieved. A fraction with particle size <0.2 mm was used for further experiments. Modified bentonite samples were prepared by a partially modified method of Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al. (2017). In short, Na-bentonite (NaB) was prepared by dispersion of 10.0 g of NB in 100 mL of 1 M NaCl solution (58.44 g NaCl/L dmw). The suspension was placed on an ARE 5 magnetic stirrer (Velp Scientifica, Usmate, Italy) and stirred for 24 h at room temperature (600 rpm), filtered through a Büchner funnel, and the filter cake was rinsed with dmw until a negative reaction to Cl^– ions occurred. The NaB obtained was dried for 24 h at 60°C, ground, and sieved to the particle size <0.2 mm. Cu-bentonite (CuB), Zn-bentonite (ZnB), and Cu/Zn-bentonite samples were prepared by dispersion of 10.0 g NaB in 100 mL of: 1 M CuSO₄ solution (249.685 g CuSO₄٠5H₂O/L dmw), 1 M ZnSO₄ solution (287.547 g ZnSO₄٠7H₂O/L dmw), and 1 M of mixed CuSO₄ and ZuSO₄ solutions, in ratios of Cu:Zn = 1:1, 1:2, or 1:4, respectively. Suspensions were placed on the ARE 5 magnetic stirrer and stirred for 24 h at room temperature (600 rpm), then sieved through a Büchner funnel. Upon rinsing with dmw multiple times, the filter cake was dried for 24 h at 60°C, ground, and sieved to the particle size <0.2 mm. Modified bentonite samples with CuSO₄ and ZuSO₄ solutions were marked: Cu/Zn-B1 (Cu:Zn = 1:1), Cu/Zn-B2 (Cu:Zn = 1:2), and Cu/Zn-B3 (Cu:Zn = 1:4), respectively.

Printing of Textile Materials with Selected Modified Bentonite Samples

Antibacterial treatment of the textile materials with selected modified bentonite was done using a printing process with a screen-printing semi-automatic machine S-300 (CENTRO MAŠINE, Sremski Karlovci, Serbia). Each sample of the textile material was printed in two passes with a 10 line sieve. The sieve line was determined by the granulation of the selected modified bentonite sample. The printing paste was prepared by adding Na-alginate and the selected modified bentonite sample in various percentages to dmw and mixing with a stick mixer until a homogeneous and consistent structure of the printing paste was achieved. Stereomicroscopic images of the textile materials were taken with 30× magnification using a TM-505 microscope (Mitutoyo, Kanagawa, Japan) and a high-resolution Moticam camera (5MP) (Motic, Hong Kong, China), before and after the printing process with paste containing various percentages of modified bentonite. Stereomicroscopy provides a good representation of changes on printed and dyed samples (Reference Grujić, Savić, Topalić-Trivunović, Jevšnik, Rijavec and GorjancGrujić et al., 2015; Reference Amir, Hasany and AsgharAmir et al., 2023; Reference Wilson, Laing, Tan, Wilson, Arachchige, Gordon and Fraser-MillerWilson et al., 2023). The structures of the textile materials (non-woven textile (NT) and knitted fabric (PL)), the preparation of the printing paste, the printing process, as well as the labels and the appearance of the printed samples obtained using a stereomicroscope, are illustrated in Fig. 1.

Fig. 1 The process of printing modified bentonite sample on textile materials

Characterization of the Bentonite Samples

The methylene blue adsorption method was used for the determination of the cation exchange capacity (CEC) of NB (Reference Aprile and LorandiAprile & Lorandi, 2012; Reference PejonPejon, 1992). A methylene blue solution was prepared in a glass flask by adding 1.5 g of dye to 1 L of dmw and shaking thoroughly to obtain a homogeneous solution. 2 g of NB was placed in a 50 mL beaker containing 10 mL of dmw and stirred vigorously. The methodological assay began by adding 0.5 mL of the methylene blue solution to the beaker containing NB. After 3 min, a drop of the suspension material was removed with a glass rod and deposited on filter paper. When a light blue halo around the dark patch of NB appeared on the filter paper, the test was complete. Equation 1 was used to calculate the CEC:

(1) $\text{CEC}=\frac{V\times C\times 100}{M}$

where, CEC is in mmol_c kg^–1, V is the volume consumed of the methylene blue solution (mL), C is the concentration of the methylene blue solution, and M is the mass of dry NB (kg).

The methylene blue test was used for the quantification of Mnt content of NB (VDG P69, 1999). From the dried NB, 0.5 g was weighed on a KB 2400-2N balance (KERN & SOHN, Balingen, Germany) with an accuracy of 0.01 g and poured into an Erlenmeyer flask, to which 50 mL of dmw and 5 mL of a saturated solution of Na₄P₂O₇ had previously been added. The suspension thus prepared was boiled for 5 min, cooled, and 2 mL of 5 N H₂SO₄ was added and mixed for 30 s. The suspension was titrated with the methylene blue solution, and at the same time mixed vigorously for 2 min. With a glass rod, a drop of the suspension was deposited on filter paper until the end of the titration (a blue circle in turquoise blue color appeared around the solution on the paper). After the blue circle was detected, the solution was stirred in the flask for another 2 min and deposited again on filter paper. If the blue circle appeared again, then it was the end of the titration, and if it did not appear, the titration continued. Equation 2 was used for the calculation of the Mnt content in NB:

(2) $\%MM=\text{mL}\text{MB}\times 2$

where, %MM = Mnt content; mL MB = mL of methylene blue used for titration.

The chemical composition of the bentonite samples was determined by energy dispersive X-ray fluorescence spectrometry (ED-XRF) using an 8000P ED-XRF spectrometer (Shimadzu, Kyoto, Japan). The instrument was equipped with an X-ray tube with a rhodium anode. Measurements were performed at 50 kV and 1000 μA. A 10 mm collimator and silicon drift detector were used. The PCEDX Navi software was used for measurement and data processing.

The phase compositions of bentonite samples were determined by XRPD using a Bruker D4 Endeavor diffractometer (Billerica, Massachusetts, USA), using CuKα radiation (λ=0.1541 nm) operated at 40 kV and 35 mA over the range 4–60°2θ with a step size of 0.02°2θ.

The morphological properties of bentonite samples were recorded by scanning electron microscopy (SEM). Samples of 1 cm×1 cm size were stuck to the carrier across double-sided adhesive carbon tape and coated with gold in a BAL-TEC SCD005 device (Balzers, Liechtenstein) for cathode coating for 4 min from the distance of 50 mm, at 30 mA, which created a conductive surface. The recording was performed using JEOL JSM-5300 SEM (Tokyo, Japan). The observations were performed at an accelerating voltage of 20 kV. SEM analyses were performed with magnifications of 5,000× and 10,000×.

FTIR analysis of bentonite samples was performed using an IRSpirit ATR-FTIR spectrophotometer (Shimadzu, Kyoto, Japan) in the range from 4000 to 400 cm^–1. Specific surface area (SSA), total pore volume (Vp), and mean pore diameter (d) of bentonite samples were determined by N₂ physisorption at 77 K in a Gemini VII analyzer (Micromeritics, Norcross, Georgia, USA). The specific surface area (SSA) of the samples was calculated using the Brunauer–Emmett–Teller (BET) method (SP _BET). The total volume of mesopores, V _mp, as well as the mean diameter of mesopores, d _mp, were determined on the basis of the adsorption branch of the isotherm according to the BJH method (Reference Barrett, Joyner and HalendaBarrett et al., 1951). The total micropore volume (V _micro,t), external specific surface area (SP _ext,t), and micropore surface area (SP _micro,t) were determined using the t-method (Reference Lippens and de BoerLippens & de Boer, 1965). Prior to measurement, the samples were dried for 2 h at 200°C, then degassed for 1 h under a nitrogen stream at 140°C.

(level2) Determination of Water Absorption Capacity of Textile Fabrics (WAC)

The water absorption capacity of the textile materials was tested according to the standard DIN 53923:2022 DE (Testing of textiles–determination of the water absorption capacity of textile fabrics). Dry weight of fabric (10 cm×10 cm) was measured, then it was immersed in a bath of tdw for 5 min. Then, the fabric was hung vertically until no water droplet dripped for 30 s. At that time, the fabric was weighed again and the water absorption capacity was calculated using Eq. 3.

(3) $\text{Water}\text{absorption}\text{capacity}\left(\%\right)=\frac{wetweight-dryweight}{dryweight}\text{x}100\left(\%\right)$

Antibacterial Activity

Four bacteria were used in this study: Escherichia coli ATCC 25922 (E. coli), Pseudomonas aeruginosa ATCC 10145 (P. aeruginosa), Staphylococcus aureus ATCC 25923 (S. aureus), and Bacillus cereus ATCC 7004 (B. cereus). The bacteria were grown on NA for 24 h at 37°C. After the incubation period, the colonies were prepared for application by a direct suspension of colonies in the logarithmic phase (Reference Ortez and CoyleOrtez, 2005). Suspension density was determined spectrophotometrically (OD 625 nm) with spectrophotometer V-110 (Wagtech Projects, Thatcham, UK), using the 0.5 McFarland standard (1.5×10⁸ cfu/mL) for comparison. The cultures were diluted in the physiological solution and their densities were set to 1×10⁶ cfu/mL.

The antibacterial activity of the bentonite samples was determined by the agar dilution method with certain modifications (Reference Magana, Quintana, Aguilar, Toledo, Angeles-Chavez, Cortes, Leon, Freile-Pelegrin, Lopez and Torres SanchezMagana et al., 2008). Before usage, the ground bentonite samples were sterilized for 30 min in a thin layer under the UV lamp (at 254 nm), then weighed under sterilized conditions and added to 2 mL of tdw. Bentonite was held in water at room temperature for 2 h, with occasional shaking, and a specific amount of melted MHA cooled at 50°C was added. The prepared medium was then homogenized and poured into sterile Petri dishes. The media obtained contained the following concentrations of the bentonite (in mg/mL): 0.94, 1.875, 3.75, 7.5, 15, 30, 60, and 100. After cooling the media, 10 μL drops of bacterial cultures were applied at the surfaces of all media and incubated for 24 h at 37°C. MIC values were determined in all the Petri dishes with the lowest concentration of the bentonite sample with no visible growth of microorganisms. All the Petri dishes without visible growth were loop-inoculated, in a way that all the spots containing drops of cultures were picked up with a sterile loop and transferred on sterile MHA. After incubating for 24 h at 37°C, the MBC values were read at MHA, where the growth of microorganisms was not spotted. As a positive control, the media without bentonite were used. As a first negative control, the salt solutions (CuSO₄٠5H₂O and ZnSO₄٠7H₂O) in the agar medium were used, where the salt concentration was (in mg/mL): 0.25, 0.5, 1 2, 4, 8, 16, or 32. The other type of negative control was in the form of antibiotic discs (erythromycin 15 μg; gentamicin 10 μg; ciprofloxacin 5 μg, ampicillin 10 μg).

The antibacterial activity of the textile was tested by the Parallel Streak Method (AATCC TM 147-2004). Specimens of the test materials were placed in direct contact with the agar surface which had previously been streaked with an inoculum of a test bacterium. After incubation, a zone of inhibition (ZOI) (clear area of interrupted growth underneath and along the side of the test materials) was measured in mm. If a zone of inhibition was present, the streaks stopped at the edge of the sample and no growth was seen below the sample, it is defined as contact inhibition. This condition was defined as contact inhibition and the sample was reported as pass. Each measurement was determined in triplicate. After incubation, Eq. 4 was used to calculate the size of the zones of inhibition:

(4) $Zi=\frac{T-D}{2}\left(\text{mm}\right)$

where, Zi = width of zone of inhibition, T = width of sample + zone of inhibition, D = width of sample (mm).

Characterization of Bentonite Samples

Natural bentonite (NB) from the Šipovo deposit in Bosnia and Herzegovina with an average particle size <0.2 mm was used. Mineralogical analysis revealed that the NB contained ~90% of Mnt (Eq. 1), with a CEC of 67.08 mmolM⁺/100 g (Eq. 2).The chemical composition of NB and modified bentonite samples determined by the ED-XRF method (Table 1) revealed that Si and Al were the main components of NB, and a larger amount of Al was indicative of a higher concentration of Mnt. NB contained larger amounts of Fe and Mg, a medium amount of Ca, while K, Ti, and P were barely present. As NB does not contain Na, this type is classified as Ca-bentonite. The chemical analysis results of NB are in line with previously published results (Reference Petrovic, Dugic, Aleksic, Begic, Sadadinovic, Micic and KljajicPetrović et al., 2014, Reference Petrović, Levi and Penavin-ŠkundricPetrović et al., 2019). Si and Al were the main components of NaB. Also, NaB contained more Fe and Na, medium amounts of Mg and Ca, while K, Ti, and S were barely present. Modification of NaB with copper and zinc ions caused the reduction of Ca, Mg, and K, and the complete absence of Na, which implies that an ion exchange occurred. The highest concentration of copper and zinc was found in Cu/Zn-B2. The adsorption behavior of the bentonite toward zinc and copper ions in aqueous solutions depends heavily on pH. When the pH is between 3 and 7, the basic mechanism that controls the adsorption properties of bentonite is ion exchange and specific adsorption (Reference Aldayel, Alandis, Mekhemer, Hefne and Al-RaddadiAldayel et al., 2008; Reference Kaya and ÖrenKaya & Ören, 2005). The concentration of TiO₂ in all bentonite samples was practically constant and showed that the Ti⁴⁺ cation is not exchangeable. A significant increase in the S concentration in the modified bentonite samples, in comparison to NaB, is the result of incomplete rinsing of the samples after modification.

Table 1 Chemical compositions of bentonite samples expressed as concentrations of metal oxides (in mass percentage)

XRD patterns of the modified bentonite samples (Fig. 2) revealed a diffraction peak at 7.16°2θ in the NaB, corresponding to d ₀₀₁ = 1.23 nm for Mnt. Upon modification with copper and zinc ions, the diffraction peak moved to higher angles of 7.08, 7.16, and 7.23°2θ (Cu/Zn-B1, Cu/Zn-B3, CuB, respectively), which corresponded to the smaller basal spacings of 1.25, 1.23, and 1.22 nm, respectively. As far as Cu/Zn-2 and ZnB are concerned, the reflection appeared at the lower angles of 6.84 and 6.64°2θ, respectively, which corresponded to an insignificant increase in the basal spacings to 1.29 and 1.33 nm, respectively. It is well known that bentonite consists mostly of Mnt. Modification of NaB with copper and zinc decreased the basal spacings of Cu/Zn-B1, Cu/Zn-B3, and CuB, but increased it for Cu/Zn-B2 and ZnB. Basal spacings of 1.24 and 1.34 nm for Cu- and Zn-Mnt, respectively, were reported by Reference Kozák, Praus, Machovič and KlikaKozák et al. (2010). The spacing depended on the initial concentration of the metal ion and the balance of pH, according to Reference Németh, Mohai and TóthNémeth et al. (2005). In the case of a large initial concentration of copper and low pH; Cu-Mnt was reported to have a basal spacing of ~1.25 nm, which implied that the copper was inside the interlamellar space of Mnt with one layer of water. The basal spacing of Zn-Mnt increased progressively as the pH decreased, until it reached the permanent value of 1.40–1.50 nm at neutral pH, which implies that zinc has a tendency to exist with two layers of water inside the interlamellar space of Mnt. At lower pH, the interlayer zinc exists with two water layers. Bearing in mind the modification conditions of metal concentration and solution pH, the present results are in line with the results of Reference Kozák, Praus, Machovič and KlikaKozák et al. (2010), and are quite compatible with the results presented by Reference Németh, Mohai and TóthNémeth et al. (2005). The results of the XRPD analysis are an additional confirmation that, during the modification of NaB, the ion exchange of copper and zinc with exchangeable cations occurred.

Fig. 2 XRD patterns of the bentonite sanples

To test the morphological properties of the bentonite samples, scanning electron microscopy (SEM) was performed. NaB showed the typical layered, loosely connected structure (Fig. 3) and, upon modification, no changes were observed in the layered structure or particle size and shape.

Fig. 3 SEM images of the bentonite samples: a NaB, b CuB, c ZnB, d Cu/Zn-B1, e Cu/Zn-B2, and f Cu/Zn-B3

The FTIR spectra of the original and modified bentonite samples (Fig. 4) are almost identical. The octahedral Al²O-H stretching vibration was observed at 3629 cm^-1, while the broad band at 3377 cm^-1 (with CuB at 3169 cm^–1, and with ZnB at 3030 cm^–1), was ascribed to interlayer H₂O stretching modes (Reference Farmer and FarmerFarmer, 1974; Reference MadejovàMadejová, 2003; Reference Russell and FarmerRussell & Farmer, 1964). The OH-bending modes were observed at 1637 cm^–1. The bands at 1098 cm^–1 and 995 cm^–1 were the classical tetrahedral Si–O bands (Reference Hayati-AshtianiHayati-Ashtiani, 2012; Reference MadejovàMadejová, 2003). The bands corresponding to AlAlOH and AlFeOH bending modes which reflect a partial substitution of octahedral Al by Fe (Reference MadejovàMadejová, 2003; Reference Tyagi, Chudasama and JasraTyagi et al., 2006), were observed at 903 and 870 cm^–1, respectively. The presence of quartz in the samples (which was proved by XRPD analysis) is indicated by the bands at 780 and 694 cm^–1 (Reference Ezquerro, Ric, Miñana and BermejoEzquerro et al., 2015; Reference Tyagi, Chudasama and JasraTyagi et al., 2006), while the bands at 510 and 457 cm^–1 belong to AlOSi and SiOSi vibrations, respectively (Reference Kumar and LingfaKumar & Lingfa, 2019; Reference MadejovàMadejová, 2003). The results for the most important textural properties of the bentonite samples, i.e. specific surface area (SP _BET), total pore volume (Vp), and middle pore diameter (d) (Table 2), showed that modification of NaB resulted in significant changes in textural properties, namely, reduction in the specific surface area and total pore volume as well as moving of the middle mesopore diameter toward the larger diameters. In comparison to NaB, the greatest reduction in the above-stated textural properties was with ZnB (SP _BET = 6.55 m²/g, V _p = 0.023 cm³/g, and d = 13.41 nm), and the smallest was with Cu/Zn-B2 (SP _BET = 35.5 m²/g, V _p = 0.052 cm³/g, and d = 6.49 nm). In all samples, the value of the SP _ext,t was much greater than the SP _micro,t; V _micro,t was small, and in the case of ZnB and Cu/Zn-B1 it even had a negative value (below the limit of detection). ZnB and Cu/Zn-B1 had the most pronounced mesoporous character, while NB had the least pronounced mesoporous character. The reduction of the SSA and the total pore volume could be attributed to the presence of copper and zinc cations in the interlamellar space and pores of bentonite which inhibited the passage of nitrogen molecules and their physisorption (Reference de Araujo, Bertagnolli, da Silva, Gimenes and de Barrosde Araujo et al., 2013; Reference Tan, Zhang, Zhang, Xie and LiuTan et al., 2008). Similar results were obtained with Mnt modified with zinc and zinc/cerium ions (Reference de Araujo, Bertagnolli, da Silva, Gimenes and de Barrosde Araujo et al., 2013; Reference Tan, Zhang, Zhang, Xie and LiuTan et al., 2008). Besides that, heat treatment could influence the clay textural properties. Prior to measurement, all bentonite samples were dried for 2 h at 200°C. The loss of mass in the temperature range 50 to 200°C corresponded to dehydration or the loss of physically adsorbed water and water connected to the exchangeable cations in the interlayer of aluminosilicate surfaces, which frequently led to a reduction in interlayer distances (Reference Balek, Beneš, Šubrt, Pérez-Rodriguez, Sánchez-Jiménez, Pérez-Maqueda and Pascual-CospBalek et al., 2008). The SSA of bentonite increased as the temperature increased to 100°C, but further increase in the temperature above 100°C caused the SSA to decrease (Reference ToorToor, 2010). The increase in temperature above 100°C led to the removal of water connected to the exchangeable cations in the interlayer and to reduction of the interlayer distance. This reduction of the interlayer distance brought the particles closer and they started creating aggregates, which resulted in reduction of the specific surface area. The nitrogen adsorption isotherms for NB and modified bentonite samples (Fig. 5) and the structural properties estimated based on them (Table 2) revealed that all isotherms belong to the H type (IUPAC classification) (Reference BalciBalci, 2019; Reference Jović-Jovičić, Milutinović-Nikolić, Gržetić, Banković, Marković and JovanovićJović-Jovićič et al., 2008; Reference Ranđelović, Purenović, Matović, Zarubica, Momčilović and PurenovićRanđelović et al., 2014; Reference Vuković, Milutinović-Nikolić, Krstić, Abu-Rabi, Novaković and JovanovićVuković et al., 2005). An isotherm of this type is characteristic of solid materials that can be non-porous, mesoporous, or even microporous to a certain extent. A small gas adsorption value corresponding to the micropore area (p/p ₀ < 0.02) was observed in all of the isotherms, which indicated a pronounced mesoporosity of the samples, and they all have steep slopes in the relative pressure range of 0.98–1.0. The modification process does not affect the type of isotherms obtained so one may conclude that the modification process did not affect the mesoporous character of the samples, but it led to significant changes in textural properties.

Fig. 4 FTIR spectra: a NaB, b ZnB, c CuB, d Cu/Zn-B1, e Cu/Zn-B2, and f Cu/Zn-B3

Table 2 Overview of textural properties of bentonite samples

Fig. 5 Nitrogen adsorption isotherms for the bentonite samples

Determination of Water Absorption Capacity of Textile Materials (WAC)

The WAC is expressed as the amount of water absorbed (%) in the textile material after immersion in water at a temperature of 20°C. The WAC of the NT samples increased from 702.53 % (NT-1) to 865.27 % (NT-10) with increasing concentration of the modified bentonite in the paste (Table 3), thus proving the ability of modified bentonite to absorb large amounts of water. In the case of the PL samples, no clearly defined increase in WAC was observed. This can be explained by the yarn used in the PL samples which was obtained by the viscose process from bamboo and, thus, had a deformable structure that prevented the application of the bentonite in a precise thickness of the printing paste, whereas the opposite was true in the case of the NT samples.

Table 3 Results of testing of WAC (%)

Antibacterial Activity

The MIC and MBC values of salts (as control) and bentonite samples (Table 4) showed that CuSO₄٠5H₂O had good antibacterial activity. The antibacterial activity depended on the copper content, type of copper compound, temperature, pH, type of bacteria, etc. (Reference Ahamed, Alhadlaq, Khan, Karuppiah and Al-DhabiAhamed et al., 2014; Reference Benhalima, Amri, Bensouilah and OuzroutBenhalima et al., 2019; Reference Chen, Alexander and BakiChen et al., 2016; Reference Javadhesari, Alipour, Mohammadhejad and AkbarpourJavadhesari et al., 2019). In comparison to CuSO₄٠5H₂O, ZnSO₄٠7H₂O had a better inhibition effect on S. aureus and E. coli (MIC 0.5 and 1 mg/mL, respectively), and weaker on B. cereus and P. aeruginosa (MIC 2 and 4 mg/mL, respectively). Bacterial resilience in the presence of heavy metals depends on the type and concentration of metal, duration of exposure, type of bacteria, size of inocula, and other factors (Reference Carpio, Ansari and RodriguesCarpio et al., 2018; Reference Henao and Ghneim-HerreraHenao & Ghneim-Herrera, 2021). NaB showed no antibacterial activity, while Cu²⁺- and Zn²⁺-modified bentonite showed antibacterial activity (but at different levels). The results obtained for NaB corresponded to the data available in the literature, where high concentrations of Na-Mnt had no inhibitory effect on bacterial growth (Reference Bagchi, Kar, Dey, Bhandary, Roy, Mukhopadhyay, Das and NandyBagchi et al., 2013; Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al., 2017; Reference Tong, Yulong, Peng and ZirongTong et al., 2005). The smallest MIC value (0.47 mg/mL) was established for Cu/Zn-B2 in relation to S. aureus, while the largest MIC value (15 mg/mL) was established for Cu/Zn-B3 in relation to P. aeruginosa. Cu/Zn-B2 was applied to NT and PL due to the best antibacterial effect (Table 4). The Parallel Streak Method (ATCC TM 147) was used to test the antibacterial effect of textile samples treated with modified bentonite. With this technique, the antibacterial effect is manifested through the diffusion of the applied agent into the substrate and the appearance of a zone of inhibition or absence of bacterial growth under the sample. Untreated textile is used as a control sample (Figs. 6 and 7). Due to their surface topography and structure, NT and PL did not bind the tested bacteria or contain substances that have an antibacterial effect, or interfere with the formation of biofilms on the samples (Reference Catovic, Abbes, Barreau, Sauvage, Follet, Groboillot, Leblanc, Svinareff, Chevalier and FeuillolleyCatovic et al., 2022; Reference Ivankovic, Rajic, Ercegovic Razic, Rolland du Roscoat and SkenderiIvankovic et al., 2022). The NT samples showed better antibacterial effects than PL samples (Figs. 6 and 7), because NT is a less porous and flat textile product, so more modified bentonite remained on the surface of the samples compared to PL, which is more porous, so the modified bentonite was incorporated between the threads within yarn. Due to the smaller amount of bentonite on the surface, PL had a weaker inhibitory effect on the growth of bacteria. The lack of an inhibition zone around the samples was expected considering the small desorption of Zn²⁺ and Cu²⁺ ions into the solid substrate, so the antibacterial action is localized on the surface of the modified bentonite (Reference Hu and XiaHu & Xia, 2006; Reference Pasquet, Chevalier, Pelletier, Couval, Bouvier and BozingerPasquet et al., 2014), and occurs according to the mechanism that was previously described in the text. However, for both samples (Figs. 6 and 7), the inhibition of bacteria visibly increased with the concentration of applied modified bentonite. To test the survival of bacteria, the samples that were removed from the incubated media were again transferred to MHA and placed on the media on the side that was in contact with the bacteria, incubated for 24 h at 37°C, and removed from the media again. Much fewer bacteria were observed under the NT samples (Fig. 6), which can be explained by a larger amount of modified bentonite on their surface. Under the NT samples with modified bentonite in a concentration of >1 mg/mL, almost no bacterial growth occurred, indicating an outstanding bactericidal and bacteriostatic efficiency. The antibacterial effect of Cu- and Zn-modified Mnt is realized through direct contact of a negatively charged surface of bacterial cells and positively charged modified Mnt (Reference Hu and XiaHu & Xia, 2006; Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al., 2017; Reference Kalia, Abd-Elsalam and KucaKalia et al., 2020; Reference Tong, Yulong, Peng and ZirongTong et al., 2005). Positively charged nanoparticles of metals connected to the cell membrane increased its permeability, therefore. Nanoparticles can also release metal ions which penetrate the cells and generate free radicals (Reference Sanchez-Lopez, Gomes, Esteruelas, Bonilla, Lopez-Machado, Galindo, Cano, Espinia, Ettcheto, Camins, Silva, Durazzo, Santini, Garcia and SoutoSanchez-Lopez et al., 2020). ZnB had a better antibacterial effect than CuB in relation to S. aureus (MIC = 0.94 mg/mL) and E. coli (MIC = 1.875 mg/mL), and weaker compared to B. cereus and P. aeruginosa (MIC = 7.5 mg/mL). According to Reference Qingshan, Shaozao, Quihui, Zepeng, Yousheng and YibenQingshan et al. (2010), antimicrobial activity increased with increase in zinc content, and with 6.28% zinc they obtained MIC of 3.5 mg/mL against E. coli and of 3 mg/mL against S. aureus, but with significantly higher MBC. According to Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al. (2017), antimicrobial activity increased along with the increase in the SSA of Mnt when the size of the particles was reduced, which implied that the antimicrobial effect of Mnt did not depend only on the copper or zinc ions, but also on the surface characteristics of Mnt, as was partially confirmed by the present study (Tables 2 and 3). Using the SEM analysis, Reference Zou, Wand, Cui, Zeng, Wand, Han, Qiu, Chen, Chen, Guan and ZhengZou et al. (2019) ascertained that growing S. aureus and E. coli in the presence of Zn-Mnt causes morphological changes manifested in a rough and distorted surface, as well as cell-membrane damage which leads, in turn, to cytoplasm leakage. According to Reference Qingshan, Shaozao, Quihui, Zepeng, Yousheng and YibenQingshan et al. (2010), Reference Tan, Zhang, Zhang, Xie and LiuTan et al. (2008), and Reference Zou, Wand, Cui, Zeng, Wand, Han, Qiu, Chen, Chen, Guan and ZhengZou et al. (2019), Zn-modified bentonite and Mnt have better antimicrobial impact on Gram-positive than Gram-negative bacteria. Those authors explained their results by the presence of the outer membrane of Gram-negative bacteria, which creates an additional protective barrier against foreign matter such as Zn-Mnt. Reference Garshasbi, Ghorbanpour, Nouri and LoffimanGarshasbi et al. (2017) and Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al. (2017) stated that the zinc-modified Mnt performed better with Gram-negative bacteria due to the large negative charge of these bacteria, which enabled the contact between cells and the positively charged zinc ions. In the present experiment, the inhibiting effect of ZnB was not related to the bacteria cell wall structure. B. cereus was less sensitive than E.coli, and P. aeruginosa was less sensitive than S. aureus, probably due to the reduced sensitivity of Bacillus and Pseudomonas to heavy metals (Reference Carpio, Ansari and RodriguesCarpio et al., 2018; Reference Henao and Ghneim-HerreraHenao & Ghneim-Herrera, 2021). The Cu/Zn-modified bentonite samples had, in most cases, a better inhibiting effect on bacteria than CuB; Cu/Zn-B1 and Cu/Zn-B2 had a better inhibiting effect than ZnB, which implied a synergistic antibacterial effect (Reference Jiao, Lin, Cao, Wang, Wu, Shu and HuJiao et al., 2017; Reference Tan, Zhang, Zhang, Xie and LiuTan et al., 2008). Based on the chemical composition (Table 1), the antibacterial activity clearly increased when ZnO and Zn were present. In the present experiment, the largest amount of ZnO and Zn was in the Cu/Zn-B2, which showed the best antibacterial effect, especially to S. aureus. The fact that the amount of zinc ions in Cu/Zn-B3 was greater than in ZnB, but the antibacterial activity was less, could be because of a larger SSA and smaller nanoparticles in ZnB than in Cu/Zn-B3 (Reference Bagchi, Kar, Dey, Bhandary, Roy, Mukhopadhyay, Das and NandyBagchi et al., 2013; Reference da Silva, Abucafy, Manaia, Junior, Chiari-Andreo, Pietro and Chiavaccida Silva et al., 2019).

Table 4 MIC and MBC of salts and bentonite samples (mg/mL)

Fig. 6 Antibacterial activity of NT samples (right side of each petri dish indicates where the sample was removed from the agar plate)

Fig. 7 Antibacterial activity of PL samples (right side of each petri dish indicates where the sample was removed from the agar plate)