The Biofungicide Activity of Some Plant Essential Oils for the Cleaner Production of Model Linen Fibers Similar to Those Used in Ancient Egyptian Mummification

: In this work, the essential oils (EOs) from Eriocephalus africanus leaf, Vitex agnus-castus leaf and fruit, Cymbopogon citratus leaf, and Rosmarinus officinalis leaf were used as antifungal agents against isolated Aspergillus flavus , Cladosporium cladosporioides , and Penicillium chrysogenum from an ancient Egyptian child’s mummy. The isolated fungi were used to colonize the samples of linen fibers. The best oil was used as a novel natural product for the cleaner production of model linen fibers similar to those used in ancient Egyptian mummification. Standard and original linen fibers were compared with the infected Linen samples using Fourier transform infrared (FTIR) and X-ray diffraction (XRD) analyses. The FTIR revealed the changes in the molecular structure of the cellulose, hemicellulose, and lignin of the infected linen fibers. The cellulose crystallinity indices decreased to 64.61%, 52.69%, and 54.63% in the linen inoculated with A. flavus , C. cladosporioides , and P. chrysogenum compared to the control sample (72.08%), thereby affecting the chemical properties of the cellulose. The mycelia inhibition percentages of the three fungi reached 100% after the leaf EO from V. agnus-castus was applied, followed by C. citratus . The V. agnus-castus leaf EO applied at contraptions of 250, 500, 50, 1000, and 2000 µL/mL showed 100% inhibition for A. flavus and P. chrysogenum and reached 100% against C. cladosporioides at concentrations of 500, 750, 1000, and 2000 µL/mL. C. citratus leaf essential oil applied at concentrations of 500, 750, 1000, and 2000 µL/mL showed 100% inhibition to the growth of A. flavus and C. cladosporioides and reached 100% inhibition against the growth of P. chrysogenum at concentrations of 750, 1000 and 2000 µL/mL. This inhibition could be related to the main compounds of caryophyllene (23.13%), eucalyptol (20.59%), sabinene ( β -thujene) (12.2%), γ -elemene (9%), and β -farnesene (6.14%) identified in V. agnus-castus leaf EO or due to the main compounds of β -citral (43.63%) and geranial (41.51%), as identified in the leaf EO of C. citratus by GC/MS. The morphological changes in the hyphae of the fungi were observed via SEM examination, where V. agnus-castus leaf EO, the best active oil, showed potent inhibition to fungi grown on the model linen fiber. In this way, the morphology and the structure of the hyphae were effectively changed. Our findings prove that the designed model linen fiber standard, original, and linen fibers infected with fungi. Five EOs extracted from E. africanus leaf, V. agnus-castus leaf and fruit, C. citratus leaf, and R. officinalis leaf were tested for their antifungal activity. V. agnus-castus leaf oil produced the best inhibition of fungal mycelial growth. The effectiveness of the innovated and treated model linen fibers with V. agnus-castus leaf EO was achieved, and SEM examination showed inhibited growth of the fungal mycelium, as well as changes in the morphology and structure of the hyphae.


Isolated Fungi from the Ancient Egyptian Child Mummy and Colonization Test
We used the following three fungi, Aspergillus flavus, Cladosporium cladosporioides, and Penicillium chrysogenum (under the accession numbers LC325160, LC325159, and LC325162, respectively), previously isolated from the mummy of a child aged 6 to 8 years and identified using internal transcribed spacer (ITS) sequencing [36].
For the colonization test, standard linen samples cut into 2 × 2 cm squares using a scalpel and saturated with liquid media and maintained on a malt extract agar (MEA) (Sigma-Aldrich, Darmstadt, Germany) with 5% NaCl (Sigma-Aldrich, Darmstadt, Germany) were inoculated with a spore suspension from each of the three fungi, according to the recommendations of Miller et al. [37], Mansour [36], and Mohamed et al. [7].

FTIR Analysis of the Standard, Original, and Infected Linen Samples
Three months after the inoculation of the linen fibers with each of the three fungi, an Fourier transform infrared (FTIR) spectrometer (Model 6100 Jasco, Tokyo, Japan) was used to analyze the samples. This analytical technique was performed on both the used linen fiber (2 cm × 2 cm) and on a linen sample from the original mummy (small fragments).

X-Ray Diffraction Analysis (XRD) for the Crystallinity Index
The band positions of the crystalline and amorphous cellulose forms of the studied control and the samples colonized with A. flavus, C. cladosporioides, and P. chrysogenum were measured using a Philips Analytical X-ray (Philips-Eindhoven -the Netherlands) by means of Match 3 + PDF-4 2015 software program (Kreuzherrenstr, Bonn, Germany) diffractometer type: PW 1840, with a Cu tube anode, a generator tension of 40 kV, and a generator current of 25 mA). The crystallinity index (CI) was measured according to Segal et al. [38] with the following formula:  (1) where I002 is the diffraction intensity at the 002 peak position (2Ɵ ≈ 22.5°) and Iam at 2Ɵ = 18° (angle of amorphous cellulose).

Plant Material and Essential Oil Extraction
The Eriocephalus africanus leaf, Vitex agnus-castus (leaf and fruit), Cymbopogon citratus leaf, and Rosmarinus officinalis leaf, were collected in 2017 from the nursery of the Department of Floriculture, Ornamental Plants and Garden Design, Faculty of Agriculture, Alexandria University, Alexandria, Egypt. The plants were identified and authenticated by the co-author Dr. Mervat El-Hefny. The essential oils (EOs) were extracted via the water distillation method using a proper essential oil trap (Clevenger's apparatus) [39]. The EOs were kept dry in sealed Eppendorf tubes at 4 °C to wait for the chemical analysis.

GC/MS Analysis of the EOs
A Trace GC Ultra/Mass spectrophotometer ISQ (Thermo Scientific) instrument equipped with a flame ionization detector and a DB-5 narrow bore column (Agilent, Palo Alto, CA, USA) was used. The temperature program of the column oven and the other specifications of the operating system can be found in a previously published work [40]. Briefly, The GC-MS was equipped with a ZB-5MS Zebron capillary column (length 30 m × 0.25 mm ID, 0.25 µm film thickness; Agilent, Palo Alto, CA, USA). Helium, the carrier gas, with an average velocity of 39 cm/s, was used. Firstly, the temperature of the oven was held at 45 °C (2 min); then, the temperature was increased from 45 to 165 °C at 4 °C/min and 165-280 °C at 15 °C/min. the mass spectra were measured and collected under electron impact ionization at 70 electron volts and scanned from m/z 50-500 at five scans/second. The identification of the chemical compositions was performed based on an MS library search [41] and confirmed by measuring the Standard and Reverse Standard Indices [7,9,42,43].

Preparation of Oil Concentrations and Antifungal Activity
Firstly, the stock solution was 20 mL, wherein the oil amount was diluted in 10% dimethyl sulfoxide (2 mL Dimethyl sulfoxide (DMSO) diluted in 18 mL sterilized distilled water) (Sigma-Aldrich, Darmstadt, Germany). EOs were prepared at concentrations of 62, 125, 250, 500, 1000, and 2000 µL/mL by dissolving the EO in DMSO 10%, and a few drops of Tween-80 detergent were added to each EO concentration to emulsify the carrier oils in water. The inhibitory effects of the tested EOs on the linear growth of three fungi (Aspergillus flavus, Cladosporium cladosporioides, and Penicillium chrysogenum) were estimated. The measurement was performed in triplicate [1,44,45]. The mycelial growth inhibition (%) was calculated according to the following equation: where the MGI is mycelia growth inhibition, and AC and At are the average diameters of the fungal colonies of the control (10% DMSO + few drops of Tween-80) and the treatment, respectively.

Model Linen of Mummification
Firstly, the linen samples were vapour treated with concentrated EO prepared at 0 (control), 125, 250, 500, 750, 1000, and 2000 µL/mL using the evaporation method [44,46,47] with minor modification. Linen samples were placed in petri dishes containing eight layers of Wattman No. 1 filter papers (Loba Chemie Pvt. Ltd., laboratory reagents & fine chemicals, Mumbai, India) overlaid by a polyethylene spacer mesh (Hebei Hightop Metal Mesh Co., Ltd., China). Dishes were autoclaved at 121 °C for 20 min and then left to cool. Each concentration of the EO was impregnated over the filter papers and kept for 48 h. This period allow the EO (fumigant) to evaporate and subsequently become absorbed by the linen samples.
A linen piece 15 cm long and 3 cm wide layer was fumigated with the best active concentrated EO resulting from the antifungal bioassay test. The sample model of the mummy was then produced and wrapped using three layers of linen pieces 15 cm long and 3 cm wide in the form of a roll and put in the Petri dish (15 cm) ( Figure 1). The linen layers with infected each fungus were put between the treated linen layers with the best active EOs and the linen layers without any treatment in the form of a wide roll. Similarly, the fumigated linen pieces (2 × 2 cm) with different concentrations of the best EO were incubated in Petri dishes and inoculated with the spores of each fungus.

Scanning Electron Microscope (SEM) Examination of Inoculated Linen with Fungi
SEM analysis was used to examine the linen inoculated with the three fungi. The SEM model was a FEI Quanta 200 SEM FEG (FEI Company, Eindhoven, The Netherlands), which was used to study changes in the surface morphology and colonization of the fungi in both the inoculated and non-inoculated linen samples.

Statistical Analysis
The mycelial growth inhibition (%) of the fungi, as affected by the five EOs with 6 concentrations, was analyzed using a two-way ANOVA, while the mycelial growth inhibition (%) resulting from the best EO applied to the model mummification linen fiber was analyzed using a one-way ANOVA.

FTIR Analysis of the Linen
The changes in the wavenumbers of the functional groups of the main chemical structures of the linen fibers (standard, original, and infected with fungi) are shown in Figure 2a-c. The fungal spores possess a high absorption ability in the short-wavelength spectral region (1533 cm −1 ) due to chitin in the cell wall, as well as the high content of proteins [48]. Moreover, dramatic decreases of the band to a maximum at 1500 cm −1 were found, indicating a typical lignin [49]. Fungi have high absorption rate in the long-wavelength spectral region (1500-500 cm −1 ) due to the high content of carbohydrates [50].
Due to their chemical composition (especially cellulose), historical textile materials can easily decay via P. chrysogenum and C. cladosporioides when exposed to high humidity [28,32,34]. The mycelia of cellulolytic fungi lead to the loss of fibre strength as the fungi penetrate the cellulosic materials [30,31].
The assignment of OH stretching appeared at 3300-3400 cm −1 , which is characteristic of OH stretching in alcohols and phenols of cellulose, lignin, or hemicellulose [11,44,[51][52][53][54][55]. The band at 3423 cm −1 (-OH groups bending) in the spectrum of decayed and original samples became wider than the standard linen spectrum, and its peak shifted to a high wave, wherein hydroxyl groups on the backbone of the hemicelluloses were found.
Changes in the wavelengths at 1424 cm −1 and 1512 cm −1 confirmed the ability of fungi to decompose the lignin [55,56]. The bands' relative intensities of absorption at 1510-1267 cm −1 increased significantly. The lower absorption peaks of the lignin [57] occurred under hydrolysis, oxidation, and the breaking of intermolecular bonds, which were caused by fungi. It was reported that the FTIR technique might be applied to detect mound fungi growth on materials [58]. This means that the primary functional groups that reoccurred in the infected linen and the original sample were attacked by microorganisms. The crystallinity degree of native cellulose is between 60% and 90% [59,60]. Furthermore, the cellulose fibers collapsed, which means that the samples decayed [61].

Cellulose Crystallinity of Linen Fibers
The results of the XRD (

Antifungal Activity of the EOs
The antifungal activities of the EOs of E. africanus leaf, V. agnus-castus leaf and fruit, C. citratus leaf, and R. officinalis leaf are presented in Table 1. The results of the mycelial inhibition percentages show that the leaf EO from V. agnus-castus possessed the best activities against the growth of A. flavus, C. cladosporioides, and P. chrysogenum, as most of the studied EO concentrations showed a 100% inhibition of fungal mycelial growth, followed by C. citratus EO.

Antifungal Activity of the Model Linen Treated with V. agnus-castus Leaf EO
The antifungal activity of the studied EOs illustrates that the leaf EO from V. agnus-castus has the highest and best activity against the three fungi. The samples of the linen fibers were incubated with each fungus in petri dishes containing concentrated oil (Figure 4). The results of the antifungal activity after the application of V. agnus-castus leaf EO as a fumigant agent to the model linen fibers similar to those used for the mummy are shown in Table 6. The lowest concentrations of EO from the V. agnus-castus leaf when the linen pieces were fumigated caused a 100% growth inhibition of the three fungi (1000 µL/mL, 500 µL/mL, and 500 µL/mL for A. flavus, C. cladosporioides and P. chrysogenum, respectively).

SEM Examination
The model linen pieces treated with the lowest concentrations of the V. agnus-castus leaf EO caused 100% inhibition to the growth of fungi and, to compare them with the control treatment, were subjected to SEM examination. Figure 5a-d shows the SEM images with different magnifications of the linen fibers after treatment with EO without fungi. These photos show the distribution of the V. agnus-castus leaf EO spots over the fibers. As shown in Figure 5i,j, the linen fibers not treated with the EOs feature significant growth of fungal mycelium from A. flavus (e,f), C. cladosporioides (g,h), and P. chrysogenum (i,j).   Previous studies have indicated the significant growth of fungal mycelium. Pulp paper from Whatman, cotton, or chemical pulp showed deterioration symptoms when inoculated with Trichoderma harzianum and Paecilomyces variotii [70]. Papyrus strips treated with some additives decreased the growth of A. flavus, A. niger, and C. gloeosporioides compared to the control treatment [10]. An erosion of fiber morphology occurred in Linen pulp fibers when inoculated with A. niger [11], but with the addition of some plant source additives, this growth decreased.
Finally, according to the SEM examination of linen fibres treated with EO and inoculated with the fungi, the V. agnus-castus leaf EO inhibited the growth of mycelium and was able to effectively destroy the morphology and the structure of the hyphae. This study suggests a novel eco-friendly treatment of model linen fibers similar to the method used in the ancient Egyptian mummification of the child mummy using the essential oil of V. agnus-castus leaf. This oil offered potent activity against the growth of A. flavus, C. cladosporioides, and P. chrysogenum, which were previously isolated from the child mummy and inoculated into the designed model linen fiber for mummification.

Conclusions
Isolated fungi A. flavus, C. cladosporioides, and P. chrysogenum from an ancient Egyptian mummy were examined for their roles as biodetergent agents in the linen fibres that were used. The analytical methods of FTIR and XRD showed that these three fungi affected the chemical composition of the linen samples through changes in the wavenumbers of the functional groups of the main chemical structures of the standard, original, and linen fibers infected with fungi. Five EOs extracted from E. africanus leaf, V. agnus-castus leaf and fruit, C. citratus leaf, and R. officinalis leaf were tested for their antifungal activity. V. agnus-castus leaf oil produced the best inhibition of fungal mycelial growth. The effectiveness of the innovated and treated model linen fibers with V. agnus-castus leaf EO was achieved, and SEM examination showed inhibited growth of the fungal mycelium, as well as changes in the morphology and structure of the hyphae.

Conflicts of Interest:
The authors declare no conflicts of interest.