Evaluation of the Mechanical, Physical, and Anti-Fungal Properties of Flax Laboratory Papersheets with the Nanoparticles Treatment

In the present study, novel mixed additives of Chitosan or Paraloid B-72 combined with nanoparticles (NPs) of Ag, ZnO, or cellulose (NCL) were examined for their effects on the mechanical, optical, and fungal inhibition properties of the papersheets produced. The highest tensile, tear, and burst indices of the papersheets were observed for flax pulp treated with additives of Paraloid B-72 + ZnO NP (1%), Chitosan + ZnO NP (3%), and Chitosan + NCL (3%) at levels of 59.93 N·m/g, 18.45 mN·m2/g, and 6.47 kPa·m2/g, respectively. Chitosan + ZnO NP (1%) added to flax pulp showed the highest fungal mycelial inhibition (FMI) (1.85%) against Aspergillus flavus. Chitosan + Ag NP (1%) exhibited the highest FMI percentage (11.48%) when added to pulp against A. terreus. Pulp treated with Paraloid B-72 + Ag NP (1%) exhibited the highest activity against Stemphylium solani with an FMI value of 3.7%. The results indicate that the technological properties of the papersheets were enhanced with the addition of novel mixtures to the pulp.


Introduction
In the pulp and paper industry, different materials in the form of nanoparticles (NPs) are used as pulp additives or for coated paper to enhance the mechanical, physical, optical, and antimicrobial properties of the produced papersheets [1][2][3][4][5][6].
Chitosan or its derivatives are added to water-suspended paper pulp mixture with non-fibrous additives furnish [7,8] or used for coating paper [9][10][11] to obtain packaging papers with improved tensile and burst strength properties. The wet and dry tensile strength values of recycled corrugated carton pulp were improved by different dosages of high-molecular-weight Chitosan [12]. Smooth surface properties and greater resistance to humidity were observed in paper manufactured with the Materials 2020, 13, 363 3 of 23 to obtain two different concentrations (1% and 3%) of ZnO/chitosan, Ag/chitosan, and cellulose/chitosan nanocomposites. A quantity of 0.1 M NaOH was added slowly to each solution with vigorous stirring until the pH reached 6.0 and the solution was kept overnight at 60 • C [41].
The paraloid nanocomposite was prepared according to Salem et al. [42]. Briefly, a co-polymer emulsion lattice with a 50/50 composition ratio of methyl methacrylate/ethyl acrylate (MMA/EA) monomers was used to produce poly(MMA-Co-EA) [43]. It was prepared by an emulsion polymerization technique with solid content of 5% in the presence of 1% or 3% nanoparticles. The polymerization was carried out according to the following procedure: in a 250 mL three-necked flask, 1 g of emulsifier, sodium dodecyl sulfate (SDS), was dissolved in a desired amount of distilled water. The desired amount of the monomer with the selected composition ratio (50/50 MMA/EA) was added and well emulsified for 30 min at room temperature using a mechanical stirrer (500 rpm) in the presence of 1% or 3% of ZnO, Ag, or cellulose NPs, separately. Then, the mixture was heated to 80 • C [44]. Next, the redox initiation system composed of potassium persulphate (PPS) (0.27 g) and sodium bisulphite (SBS) (0.416 g) dissolved in 50 mL of distilled water was added dropwise to the reaction mixture under continuous stirring for 3 h.
It should be noted that from the literature, most of the published works which chose concentrations of NPs between 1% and 3% achieved greater modifications in the required chemical and mechanical properties of chitosan and Paraloid [29,[45][46][47]. Paraloid B-72 (70 Ethyl Methacrylate (EMA)/30MA) and Chitosan were added at 4% as a constant amount when making the composite treatment.
Additionally, the polymer nanoparticles nanocomposite mixture sonicated for 15 min using pulse-echo method operating at amplitude 350 Watt and frequency of 2 MHz (central frequency of 0.7 MHz and bandwidth of 1.4 MHz). The uncertainty of the measurements is ±10 m/s using an oscilloscope (60 MHz time base oscilloscope, Philips, Eindhoven, Netherlands).

Morphological Analysis of the Prepared Nanocomposites
The morphological analyses of the prepared nanocomposites were performed via transmission electronic microscopy (TEM), where the TEM images were obtained using a JEM-1230 electron microscope operated at 60 kV (JEOL Ltd., Tokyo, Japan). Before taking a TEM image, the sample was diluted at least 10 times by water. A drop of well-dispersed diluted sample was placed onto a copper grid (200 mesh and covered with a carbon membrane) and dried at ambient temperature.

Flax Material and the Soda-Anthraxquinone Pulping Process
The flax plants grown in the North of Egypt during 2018 were used in this research with well-shaped and visually free from defects. The stems of flax plants were cut into approximately 20 mm in length, screened, and then air-dried. Preparation of the flax samples for analysis and moisture content determination were done according to T 257 and T 208 , respectively.
Prior to pulping, 200 g oven-dried flax plant was swelled for one day, filtrated, and washed several times with hot water. The soda-anthraquinone pulping experiment was carried out using a stainless steel vessel of 3 L capacity, equipped with a rotating and heating oil bath and temperature and pressure monitor (0.7 MPa) devices. The pulping properties were active alkaline 17%, 170 • C temperature, 180 min reaction time, 0.15% anthraquinone based on the oven-dried weight (3 mL dosage from a solution of 10% anthraquinone dissolved in ethyl alcohol), and liquor ratio of 10:1 (liquid to solid). After pulping, the solid residue was defibrated, refined, and then washed with hot water and cold water to a neutral pH. The washed pulp was screened in a Valley flat screen (machine manufactured in Germany) with 0.25 mm slots.

Sheet Formation and Papersheet Testing
The flax pulp was beaten in a valley beater according to T 200 , then 1.6 g (oven-dry) stock was placed on a paper sheet cylinder to make standard sheets of 80 g/m 2 with an area of 200 mm 2 (T 205 sp-02) [48]. For determination of the dry strength properties, the samples were conditioned at 50% ± 2% RH and 23 ± 1 • C according to T 402 sp-98 for at least 4 h [48]. The strength properties of the papersheets were measured (T 218 and T 220 ). The produced paper sheets were tested for their tensile index (T 403 ), tear index (T 414 ), burst index (T 405 ), double fold (T 423 ) and the percentage of brightness [49] (Table 2).

In Vitro Inhibition of Fungal Infestation
Three fungi, Aspergillus flavus AFl375, A. terreus Ate456, and Stemphylium solani Ssol382, deposited in Genbank under accession numbers MH355958, MH355953, and MH355956, respectively, were used to study their growth inhibition around the produced papersheets with different additives (Table 1). Seven-day-old Potato Dextrose Agar (PDA) cultures from each fungus were prepared. Paper discs of papersheets 9 mm in diameter were put directly over the inoculated media with a disc (5 mm diameter) of each fungus in petri dishes for 14 days at 25 ± 1 • C. The inhibition percentage of fungal linear growth was measured using the following formula [33]: where A c and A t represent the average diameters of the control and treatment fungal colonies, respectively. To prevent cross contamination between samples in dishes, firstly, the paper samples were sterilized in the autoclave before being placed on the medium and secondly, the infection was done inside the laminar flow, in the presence of the UV-lamp, to complete sterilization. In addition, each dish contains only one fungus.

Scanning Electron Microscopy
At the end of the incubation period of the produced paper sheets with the fungi, the symptoms or inhibition of fungal infestation on the manufactured flax papersheets with different additives were examined using a Scanning Electron Microscope (SEM). The papersheet samples were coated with gold in a fine coat and examined via SEM-JEOL (JFC-1100E Ion sputtering device, model JSM-5300, JEOL Co., Tokyo, Japan) at 8 kV.

Statistical Analysis
Values of the mechanical, physical, and fungal inhibition properties of the flax papersheets as affected by different pulp additives were statistically analyzed with analysis of variance (ANOVA) using the Statistical Analysis System (SAS) [50], and compared with the control treatments using Duncan's Multiple Range Test. TEM images represent spread of NPs over fibrous shape of chitosan in case of chitosan/NP nanocomposites, but in case of Paraloid B-72/NP nanocomposites, the NPs spread over spherical shape of Paraloid B-72. All the prepared samples are in one dimensional shape. Because both of thin film and fibrous shape are from one dimension (single dimension) nanomaterials.
The addition of Chitosan and Paraloid B-72 at 4%, as well as their combinations with Ag NPs, ZnO NPs, and NCL, led to considerable increases in the papersheets' mechanical properties compared with the control (pulp without additives). Significant enhancements in the tensile and tear indices and in the brightness percentages were observed in pulp treated with Paraloid B-72 (4%) compared to that with Chitosan 4%, while no differences were found for burst index, fold number, and grammage.
Compared to the control treatment, pulp additives significantly enhanced (p < 0.05) the mechanical and physical properties of the produced flax papersheets (Table 3).
Based on the visual observations of fungal growth as well as the antifungal activities, the samples chosen for SEM measurements clearly exhibited different degrees of growth of the tested fungi. Dense and huge fungal mycelial growth of A. terreus ( Figure 5) was clearly visible on the examined flax paper sheets produced without pulp additives (Figure 5a,b), with 4% chitosan (Figure 5c,d), and with 4% Paraloid B-72 (Figure 5e,f). Dense mycelial growth of A. terreus was found on the tested paper sheets manufactured with pulp additives of Chitosan + ZnO NP (3%) (Figure 5g,h). On the other hand, the fungal mycelial growth of A. terreus over paper samples produced with pulp additives of Chitosan + Ag NP (3%) (Figure 4i), Paraloid B-72 + Ag NP (3%) (Figure 5j), and Paraloid B-72 + NCL (3%) (Figure 5k)  Based on the visual observations of fungal growth as well as the antifungal activities, the samples chosen for SEM measurements clearly exhibited different degrees of growth of the tested fungi. Dense and huge fungal mycelial growth of A. terreus ( Figure 5) was clearly visible on the examined flax paper sheets produced without pulp additives (Figure 5a,b), with 4% chitosan (Figure  5c,d), and with 4% Paraloid B-72 (Figure 5e,f). Dense mycelial growth of A. terreus was found on the tested paper sheets manufactured with pulp additives of Chitosan + ZnO NP (3%) (Figure 5g,h). On the other hand, the fungal mycelial growth of A. terreus over paper samples produced with pulp additives of Chitosan + Ag NP (3%) (Figure 4i), Paraloid B-72 + Ag NP (3%) (Figure 5j), and Paraloid B-72 + NCL (3%) (Figure 5k) decreased. The same trend was found with the growth of A. flavus, where huge hyphae growth was observed over the papersheets produced without pulp additives (Figure 6a,b), with 4% chitosan (Figure 6c,d), and with 4% Paraloid B-72 (Figure 6e,f). Flax papersheets produced with pulp additives of Paraloid B-72 + Ag NP 1% (Figure 6g) and Chitosan + Ag NP 3% (Figure 6h) showed reasonable The same trend was found with the growth of A. flavus, where huge hyphae growth was observed over the papersheets produced without pulp additives (Figure 6a,b), with 4% chitosan (Figure 6c,d), and with 4% Paraloid B-72 (Figure 6e,f). Flax papersheets produced with pulp additives of Paraloid B-72 + Ag NP 1% (Figure 6g) and Chitosan + Ag NP 3% (Figure 6h) showed reasonable decreases in the hyphae growth of A. flavus, while dense growth was observed in pulp treated with Paraloid B-72 + ZnO NP 3% (Figure 6i) and Paraloid B-72 + Ag NP 3% (Figure 6j). decreases in the hyphae growth of A. flavus, while dense growth was observed in pulp treated with Paraloid B-72 + ZnO NP 3% (Figure 6i) and Paraloid B-72 + Ag NP 3% (Figure 6j). Dense growth of S. solani was observed in flax paper sheets manufactured without pulp additives (Figure 7a,b), with 4% chitosan (Figure 7c,d), and with 4% Paraloid B-72 (Figure 7e,f). Furthermore, dense growth was observed in pulp treated with chitosan + ZnO NP 3% (Figure 7g), Paraloid B-72 + ZnO NP 1% (Figure 7h), and chitosan 4% + NCL 1% (Figure 7i).
Control treatments did not exhibit any antifungal activities against the growth of A. flavus, A. terreus, or S. solani. This indicates that the antifungal properties of flax pulp were significantly improved when the flax pulp was treated with nanocomposite additions of chitosan 4% or Paraloid B-72 4% combined with Ag NP or NCL at 3%. Dense growth of S. solani was observed in flax paper sheets manufactured without pulp additives (Figure 7a,b), with 4% chitosan (Figure 7c,d), and with 4% Paraloid B-72 (Figure 7e,f). Furthermore, dense growth was observed in pulp treated with chitosan + ZnO NP 3% (Figure 7g), Paraloid B-72 + ZnO NP 1% (Figure 7h), and chitosan 4% + NCL 1% (Figure 7i).
Control treatments did not exhibit any antifungal activities against the growth of A. flavus, A. terreus, or S. solani. This indicates that the antifungal properties of flax pulp were significantly improved when the flax pulp was treated with nanocomposite additions of chitosan 4% or Paraloid B-72 4% combined with Ag NP or NCL at 3%. (e,f) with 4% Paraloid B72; (g) with chitosan 4% + ZnO NP 3%; (h) with Paraloid B72 4% + ZnO NP 1%; (i) with chitosan 4% + NCL 1%. Arrows refer to the growth of hyphae.

Discussion
The TEM images showed the prepared of nanomaterials, where the aggregation of NPs on the chitosan surface increased from Ag to ZnO, and high aggregation was observed in the case of NCL, which could be related to the increasing particle size of NCL relative to Ag and ZnO NPs [51]. Additionally, the NPs were uniformly distributed on the surface of the polymer matrix, which confirmed the successful preparation of Paraloid B-72 nanocomposite with Ag NPs, ZnO NPs, and NCL [29].
According to the chemical analysis of flax plant, the lignin content was much lower than that in hardwood (25%-30%), but the ash content was higher than values obtained from hardwood species (0.2%-1.5%) [52,53].
Pulp additives were significantly affected the mechanical properties of the manufactured flax papersheets. Tensile index values are lower than those values reported from soda-AQ pulping of bagasse (77.8-73.8 N·m/g) [40]. Our results are in agreement with those by Jahan et al. [54] who found that the average value of the tensile index of papersheets produced from soda-AQ pulping of Acacia auriculiforms reached 45.1 N·m/g. The present values of tear index are higher than those reported from papersheets produced from soda-AQ pulping of bagasse, with values ranged from 5.7 to 6.0 mN·m 2 /g [40] and from A. auriculiformis (3.7 to 6.7 mN·m 2 /g) [54]. Comparative to other lignocellulosic plants, burst index values are higher than those reported in the literature (kPa·m 2 /g) for stems of Stipa (e,f) with 4% Paraloid B72; (g) with chitosan 4% + ZnO NP 3%; (h) with Paraloid B72 4% + ZnO NP 1%; (i) with chitosan 4% + NCL 1%. Arrows refer to the growth of hyphae.

Discussion
The TEM images showed the prepared of nanomaterials, where the aggregation of NPs on the chitosan surface increased from Ag to ZnO, and high aggregation was observed in the case of NCL, which could be related to the increasing particle size of NCL relative to Ag and ZnO NPs [51]. Additionally, the NPs were uniformly distributed on the surface of the polymer matrix, which confirmed the successful preparation of Paraloid B-72 nanocomposite with Ag NPs, ZnO NPs, and NCL [29].
According to the chemical analysis of flax plant, the lignin content was much lower than that in hardwood (25-30%), but the ash content was higher than values obtained from hardwood species (0.2-1.5%) [52,53].
In the present study, the treatments of Chitosan + NCL (1% or 3%) and Paraloid B72+ZnO NP (1%) increased the tensile strength of the paper sheets. These results are in agreement with Vikele et al. [15] who found that micro-nanochitosan increased the tensile index and concluded that micro-nanoparticles fill the submicroscopic voids of the porous paper structure and create additional bonds. NCL, with its high surface area and flexibility, increases the strength of the network [62,63] by increasing the number of hydrogen bonds between each fibril and fibers [64]. Pulp additives with 6% (dry weight) NCL showed a resulting increase in the tensile strength of the produced 60 g/m 2 papersheets by 26% to 30% [65], and the same trend was found by Bilodeau and Bousfield [66], Hamann [67], and Madani et al. [68]. NCLs enhanced the fiber-fiber bond strength; subsequently, a strong reinforcing effect in paper and board products occurred [69].
Previously, Chitosan in its free polymer form was proven to exhibit potential antifungal activity against A. niger, A. alternata, Rhizopus oryzae, Phomopsis asparagi, R. stolonifera, Botrytis cinerea, and F. oxysporum [71][72][73][74]. In the present study, pulp with Chitosan applied as an additive showed intense growth of fungi. However, other pulp additives that showed superior or stronger results for preventing fungal growth can be seen in Table 4 and Figure 4a-c. Chaetomium globosum growth significantly affects the dry mass as well as the tensile elastic modulus of some tested natural fiber mats and composites including non-woven flax fibers [75].
Overall, the enhancing effects of additives on the technological properties are much greater than the antifungal activities of the produced flax papersheets.

Conclusions
In this study, additives were used to enhance the mechanical, optical, and antifungal properties of paper sheets manufactured from flax pulp. Remarkable enhancement in the tensile index was found in pulp treated with Paraloid B-72 + ZnO NP 1% and Chitosan + NCL (1% or 3%) compared to control treatments (pulp without additives, with chitosan 4%, or with Paraloid B-72). Addition of Chitosan + ZnO NP (1% or 3%) and Paraloid B-72 4% increased the Tear index values. Furthermore, the burst index values of the paper sheets were enhanced with the addition of Chitosan + NCL (3%), Paraloid + NCL (3%), and Paraloid + ZnO NP (1%), while the double fold number was improved with the addition of Chitosan + NCL (3%), Paraloid B-72 + ZnO NP (1%), Chitosan 4%, or Paraloid B-72 4%. Pulp additives significantly affected the optical properties of the produced papersheets. The novel combination treatments can be considered to produce antifungal papersheets when compared to the huge growth of Aspergillus flavus, A. terreus, and Stemphylium solani that was observed over papersheets produced with pulp with additives of Chitosan and paraloid B-72 at 4% as well as pulp without additives.