Antifungal activity assessment of nanocomposites of natural chitosan and gelatin with a mahogany plant extract for conservation of historical textiles

Fungi and bacteria are important causes of damage to historical textiles. Many methods are used to resist innate damage in historical textiles. The study aim was to use an innovative method that loaded a mahogany plant extract onto natural chitosan and gelatin nanocomposite polymers to prepare chitosan / mahogany plant extract composite and gelatin mahogany plant extract nanocomposite and evaluate their potential for protecting historical textiles from biological damage. The fungi and bacteria found on historical textile samples were identified by biochemical methods. We performed an antifungal activity assessment of the mahogany—natural chitosan and mahogany—gelatin polymers to study the effect of these materials on the mechanical, chemical, and optical properties of dyed linen textiles. New linen fabrics dyed with madder, turmeric, and pomegranate were mordanted with alum, copper, and iron mordants. These materials were applied to dyed linen fabrics, and then the treated linen was artificially aged. The mechanical, chemical, and optical characteristics of the dyed linen fabric were examined by scanning electron microscopy, Fourier-transform infrared spectroscopy, CIELab, the tensile strength and elongation test, and the air permeability test. Mahogany – chitosan was more effective than mahogany – gelatin as an antifungal and antibacterial treatment of dyed linen and caused fewer changes in the mechanical, chemical, and optical characteristics. The mahogany – chitosan composite is recommended for preservation of historical linen textiles.


Introduction
Historical textiles collections in museums are an important part of our cultural heritage. However, these historical textiles in museums or excavation sites are exposed to microbiological damage caused by fungi-and bacteria, and humidity, that can depend on the chemical composition of the fibers, and the natural composition of the historical textiles [1][2][3]. For example, historical linen textiles consist mainly of vegetable fibers, which are primarily made of cellulose (cotton > 90%, flax 60-70%, and hemp up to 77%) [4,5].
Various aspects of microbiological damage on historical textiles can be observed in several ways, such as their loss of durability, change in color gradients, and complete loss of historical textiles subjected to extreme degrees of biological damage. The fungi and bacteria found on textiles and historical organic materials include (Staphylococcus aureus, Candida albicans, Aspergillus niger, and Aspergillus fumigatus) [6,7].
Chemical methods are used to protect historical textiles from bacterial fungal damage. It has been observed that chemical preservation methods show different degrees of toxicity to humans as well as changes in the characteristics of historical textiles. Several other preservation methods such as inert gases and radiation, are being studied [8,9].
The main aim of this study was to use innovative method that loads a to that loads a mahogany plant extract onto either natural chitosan or gelatin nanocomposite polymers to prepare a chitosan-mahogany plant extract composite and a gelatin-mahogany plant extract composite and evaluate their potential for protecting historical textiles from biological damage.
The effects of these composites on the mechanical, chemical, and optical properties of dyed linen fabrics with natural dyes were also examined. Because early identification of fungi and bacteria would be necessary prior to any preservation treatments, a secondary goal was to identify the fungi and bacteria found on Egyptian textiles in the museum environment.

Plant extraction
Five grams of dry powdered plant extracted materials were subjected to successive organic solvent extraction by refluxing in Soxhlet apparatus each for 10 h. In this study, the 80% aqueous methanol was used as solvent.
All of the extracts were concentrated by oven drying. Each fraction was collected when no further elution of compounds was observed. The collected extracts were then distilled followed by drying in incubator. The dried extracts were stored in sterile containers in a refrigerator until further analysis [14].

Microbiological examination:
Historical textile samples were collected from different Egyptian museums to identify the type of fungi and bacteria to help establish the most common types of fungi and bacteria on Egyptian historical textiles and guide the experimental part of this study that evaluated the resistance of these types to potential preservation treatments. A sample was inoculated onto different microbiological broth media including brain heart infusion and Sabouraud dextrose broth. A loopful was streaked on blood agar, mannitol salt agar, MacConkey agar and Sabouraud dextrose agar (SDA). Broth and solid media were incubated at 37 °C for 24-48 h. The pure isolates were identified biochemically by conventional methods [15]. The samples are very small fibers dropped from the historical textiles object; these separate fiber samples cannot be returned and reattached to the historical textiles objects is and were about 0.5 cm in length.

Dye extraction
The linen fabrics in the wet state were placed into the mordanting or dyeing bath. This action led to the liquor being taken up evenly. It is preferable not to expose the fiber to high temperature suddenly. The fibers were first placed in the cold solution slowly heated and then dyed with turmeric, madder, and pomegranate dyes according to the following steps.
3. Soak the dyes in distilled water for 12 to extract the color solution from the dyes powder. 4. Heat the extract for 1.5 h at 70 °C with continuous stirring. Add water to compensate for the evaporated water during the heating process. 5. Cool and filter the extract many times to obtain a clear colored solution.

Dyeing procedures
The dyeing process was performed using the exhaustion method and a liquor ratio (LR) of 1:20 (1 g of goods placed in a dye bath volume of 20 ml). The dyeing process was achieved in beakers in which linen fabric was used a cellulose fiber substrate and gently stirred at 70 °C for 1.5 h. Mordants, such as alum Al 2 (NH 4 ) 2 (SO 4 ) 4 .24H 2 O (Fluka), FeCl 2 , and CuSO 4 , were added as concentrated solution (50 g L) to give a final dyebath concentration of 2.5 g L or 5 g L of mordant. The mordanting bath continued for 30 min at temperature 60 °C. After dyeing, the unfixed dyestuff was removed by rinsing three times with cold water (5 min, room temperature, LR 1:20) [16,17]. The characterizations of linen fabrics (color values, tensile strength and elongation) after dyeing are presented in Table 1.

Preparation of polymer-mahogany extracts composites
The desired amounts of extracted materials from mahogany were sonicated in a natural polymer solution (chitosan-alginate and chitosan-Alginate mixture) for 20 min using sonication dose 350 watts. Then the solution was stirred at room temperature for 8 h and washed with ultra-pure water by ultracentrifugation to remove unbound chitosan and then collected and dried at room temperature to obtain chitosan-mahogny, gelatin-mahogny and chitosan / aliginate-mahogny nanocomposites.
Transmission electron microscopy (TEM) images of a gelatin-plant extract nanocomposite, and chitosanmahogny nanocomposite are shown in Fig. 1.

Antimicrobial effect
The antimicrobial activity of the chitosan-mahogny, gelatin-mahogny, and chitosan/aliginate-mahogny nanocomposites prepared by a nano-technique were determined using an agar-disk diffusion assay and compared with that chitosan, gelatin, penicillin and miconazole 2% (w/v) against different microorganism isolates using the method described by Raeisi et al. [18].
One hundred microliters of bacterial suspensions (1.5 × 10 8 CFU/ mL) was spread on Muller-Hinton agar plates and then filtered through paper disks (6 mm in diameter). Then 10 μL of the collected material was studied. The plates were incubated at 37 °C for 24-48 h according to the tested bacteria. The diameters of the inhibition zones were measured in mm. All experiments were performed in triplicate.

Thermal ageing
The Thermal ageing experiment was comparable to that used by Feller (1994) in maturation tests for preservation materials. Accelerated thermal ageing is used to study the behavior of materials used in historical textile conservation, and to study the mechanical, chemical and optical properties of historical textiles in museums. Some previous studies have demonstrated that the quickened temperature restriction of 100 0 C degrees for 72 h is equivalent the type of ageing of historical materials that occurs in a gallery over long periods [19,20].
The treated linen fabrics samples were hang in a temperature-controlled oven (Herous -Germany). The treated linen fabrics samples were thermally aged separately at a temperature of 100 °C for 72 h and 144 h.

Digital microscope
Many magnified images of the experimentally aged materials were taken under a digital microscope to examine the surface of the linen cloth and study the homogeneity of the treatment applications (Digital Microscope, VHX-6000 Series-KEYENCE, USA) [21].

Morphological study
Scanning Electron Microscopy (SEM) is very useful for studying the surface morphology of treated and untreated fibers. Different fibers were taken from different areas of the treated fabric and examined by a HITACHI-SU-1500 Scanning Electron Microscope [22]. The linen samples were placed and adhered directly without any preparation on the SEM samples holder, and then the holder was placed in the SEM chamber.

Color measurement
The CIE Lab values of the color change of the treated and untreated dyed linen fabrics were measured using a double beam Optimatch spectrophotometer (Datacolor International Spectraflash -SF450-UK). The literatures refers to the (L*) corresponding to the brightness (100 = white, 0 = black), and to the (a*) corresponding to the red -green coordinate (positive sign = red, negative  [23,24].

Fourier-transform infrared spectroscopy (FTIR)
The change in chemical structure of the treated and untreated dyed linen fabrics were monitored by spectroscopy FTIR (BRUKER'S VERTEX 70 with attenuated total reflection (FTIR-ATR) at a resolution of 4 cm −1 [25,26]. The textile samples were placed directly on the sample holder of FTIR-ATR devise, this type of investigation is consider as nan-destructive technique. Therefore, no preparation step was done on the sample before investigation.

Mechanical measurements
Mechanical properties (tensile strength and elongation) of the treated and untreated dyed linen fabrics were tested using a Shimadzu Universal Tester type S-500 (Japan). The measurements were performed according to the ASTM 2000, D 3822-96 standard test method for tensile properties of single textile fibers [27]. The spacing of initial jaw was 50 mm and the speed of the test was 25 mm/ min, at temperature 23 °C, and R.H. 65%.

Air permeability test
The air Permeability of the treated and untreated dyed linen fabrics was tested by using a FX3300 Air Permeability SDL tester at 65 ± 2% humidity and 20% ± 2 °C according to the EN ISO 9237 standard. First, the FX3300 was calibrated by using a calibration check plate [28].

Microbial identification
Culture detection revealed small golden-yellow colonies on mannitol salt agar, different colony shape on SDA, including cream white colored raised colonies or an initial white growth becoming black later to give a "salt and pepper appearance"; and blue-green powdery colonies that were and pale yellow on reverse. Biochemical analysis revealed that Staphylococcus aureus, Candida albicans, Aspergillus niger, and Aspergillus fumigatus were the main microorganisms. These microorganisms are known to cause damage and decomposition of historical textiles, which threatens complete loss of these textiles. Therefore, we first identified the microorganisms, and then designed natural nanomaterials, and finally evaluated safety and efficacy for protection\preservation. The goal was to use suitable nanomaterials to protect real historical textiles.

Visual examination
Visual examination of the treated linen fabrics was the first important step in the examination process. Changes in the physical appearance can often be observed by the naked eye [29,30]. It was clear by the visual examination that the linen fabric treated with mahogany-chitosan was softer than the linen fabric treated with mahoganygelatin. In addition, the surface of the linen fabric treated with mahogany-chitosan was smoother than the surface treated with mahogany/gelatin.  the application to the fiber surface was more homogeneous with chitosan than with gelatin. The linen fabric surface appearance is clearly shown after treatment with mahogany -chitosan but treatment with mahoganygelatin caused what appeared to be a layer that partially coved the actual surface of the linen fabric. In addition, the treatment with mahogany-gelatin caused bridging of the interfaces between the warp and the weft threads. These results of the treated linen fabrics were confirmed by SEM. Figure 3 shows (SEM) images of the surface of the dyed fabrics treated with mahogany plant extract applied onto the natural chitosan-gelatin. The images clearly show that the application of the mahogany plant extract applied to the natural chitosan was more homogeneous and the surface structure was smoother. The shapes of the linen fibers after treatment are shown in Fig. 3A-C. It can be seen that treatment with the mahogany plant extract applied onto the natural gelatin has coated the surface of the treated linen fabrics as shown in Fig. 3D-F. The woven structure of the linen fabric treated with mahogany -gelatin is not clear after treatment.   Figure 5 show the obtained data of the effects of treatment Effect of treatment with mahogany -chitosan and with mahogany-gelatin on mechanical properties of treated linen fabric. It is clear that the application of mahogany led to a very slight change in the mechanical properties of the treated linen fabric. There is a slight increase in tensile strength and elongation of the linen fabric treated with mahogany-chitosan and with mahogany-gelatin. Application of treatment with mahogany-chitosan and with mahogany-gelatin forms a film on the surface of the linen fabric and lesser amounts can penetrate into the inter-fiber regions. The application with mahogany-chitosan and with mahogany-gelatin can affect load-bearing capability of the fabric and the symmetrical distribution of the load. There was also reduction in strength due to thermal ageing a finding is in agreement with Chattopadhayay [31].

Effect of treatment with mahogany -chitosan and with mahogany-gelatin on chemical properties
The FTIR spectra of linen fabrics treated by mahogany-chitosan nanocomposite are shown in Fig. 6A. New peaks at 803.19 cm −1 , 1262.34 cm −1 , 1315.08 cm −1 , 1374.02 cm −1 , and 2900.36 cm −1 appear in the FTIR spectra of the treated linen by mahogany-chitosan. The FTIR spectra of linen fabrics treated by mahoganygelatin are shown in Fig. 6B. New peaks at 812.48 cm −1 , 1522.91 cm −1 , 1563.24 cm −1 , 1544.63 cm −1 , and 1653.2 cm −1 appear in the FTIR spectra of the treated linen by mahogany-gelatin. The new peaks were attributed to the amino group of chitosan and the amide group of gelatin, respectively. These findings are in agreement with those of kweon et al. and Yang et al. [32][33][34].

Effect of treatment by with mahogany -chitosan and with mahogany-gelatin on the air permeability of linen fabric
The rate of the air stream through a known zone of texture was balanced to secure an endorsed pressure differential between the two textile surfaces within the test region; from this stream rate, the air permeability of the texture was calculated. Air permeability was measured according to standard BS 5636. This system measures air permeability with to a precision of + 3%. The following testing parameters were used test area of 38 cm 2 ; test pressure of 200 pa; unit of measurement of 1/m 2 / second) [35]. Table 3 shows the effects of mahoganychitosan and mahogany-gelatin on the air permeability of dyed linen fabric before and after treatment. It can be seen that the air permeability was slightly decreased after treatment by mahogany-chitosan and mahogany -gelatin. The reason for the decreased permeability in the treated linen fabrics can be attributed to the lower and decreased interfaces and spacing in the fabric structure between the warp and weft threads. The decrease in the air permeability was less for the linen treated with mahogany-chitosan than for linen treated linen with mahogany-gelatin. This indicates that the amount of coverage of the interfaces and spacing in the fabric structure was lower using mahogany-chitosan than when using mahogany-gelatin.

Conclusion
This study identified the first time to identify the most important types of microorganisms that attack and cause damage and decomposition of historical LINEN textiles in Egyptian museums. We used a biological chemistry method to identify S. aureus, C. albicans, A. niger, and A. fumigatu as the primary microorganisms on historical Egyptian textiles. Mahogany-chitosan and mahoganygelatin nanocomposites were prepared, and the effectiveness for preventing microbial growth in extracts of the nanocomposites was evaluated.
SEM images were showed that the homogeneity of the application on the fiber surface was better with mahogany -chitosan than with mahogany-gelatin. The surface appearance of the linen fabric treated was more clearly shown after treatment with mahogany-chitosan. A slight change in color of the linen fabrics treated with with mahogany-chitosan and with mahogany-gelatin of < 1 CIELab unit was measured, but this change is too small to be observed with the naked eye. Some new peaks appeared in the FTIR spectra after treatment which confirmed that the linen was grafted successfully.
There was also a slight increase in tensile strength and elongation of the linen fabric treated with mahogany-chitosan and with mahogany-gelatin. The air permeability was slightly decreased after treatment with mahogany-chitosan and with mahogany-gelatin. The antifungal and antibacterial effectiveness was greater for mahogany-chitosan treatment than for mahogany-gelatin treatment of the dyed linen fabrics.
Fewer changes in the mechanical, chemical, and optical properties of the dyed linen fabrics were observed after treatment with mahogany-chitosan than with mahogany-gelatin. The study results support the use mahogany-chitosan for preservative treatment of historical linen textiles in Egypt.