Introduction

Paper degradation is a serious concern in libraries and archives, and it can occur due to a variety of physical, chemical, and biological causes. The basic structural component of paper is cellulose1,2. The linear condensation polymer consists of D-anhydro glucopyranose units linked by β-1,4-glycosidic links3. Fungal degradation can also cause paper hydrolysis, which produces several cellulase enzymes that break down the paper’s fundamental components4. Enzymes like amylase, xylanase, and gelatinase play an important role in the biodegradation of paper components, and fungal development creates extracellular enzymes and hyphae5, which can generate mechanical damage on paper because of extraenzymes can break down polymers in paper, such as cellulose and lignin, leading to deterioration of paper’s mechanical properties, and affect paper structure. Acids, whether created by fungi acidic sizing or derived from air pollution, play a crucial part in the degrading process of ancient manuscripts6,7,8,9,10. Degradation processes, such as acid-catalyzed hydrolysis and oxidation, demonstrate the complexity of cellulose-based materials. Hydrolysis happens when a hydrogen ion protonates a glycosidic oxygen atom, which reduces polymerization11,12.

The oxidation process in paper occurs through numerous methods, including the oxidation of the pyranose ring’s hydroxyl groups to carbonyl and carboxyl groups, as well as free radical processes. The pyranose rings’ locations determine whether oxidizing chemicals are non-specific or specific. Periodic oxidation of cellulose disrupts the connection between the C2 and C3 ring atoms, resulting in 2,3-dialdehyde cellulose13. Oxidation processes can open ring structures, increase acidity, and weaken glycosidic bonds, resulting in hydrolytic depolymerization. The development of electron-acceptor groups near glycosidic oxygen is associated with enhanced hydrolysis12. described cellulose degradation as a combination of hydrolytic and oxidative events enhanced by autocatalysis, and oxidation can also affect the interactions between ink and carrier materials during storage14,15,16. Furthermore, the high acidity of some inks, such as iron gall ink, which is formed by the interaction of iron (II) ions and gallic acid, results in the formation of an iron (II) gallate complex, which is then used to create ink, and the Arabic gum, which acts as a binder17, may cause the hydrolytic breakdown of cellulose, resulting in the production of acid. The second distinguishing trait is the presence of soluble and mobile metal ions, which can act as catalysts for cellulose oxidative degradation. This oxidation necessitates the intermediate generation of a radical (O2 y, HOOy) interaction of metal ions with HOOy in an acidic medium, which results in the production of H2O2, which can then further oxidize the paper, and sulfuric acid is released that leads to corrosion the ink and increase the acidity of paper12,17,18,19,20.

The primary goal is to preserve paper manuscripts from degradation, and it is important to use safer, more environmentally friendly, and sustainable natural products, which have led to the discovery of a diverse range of bioactive metabolites produced by plants and microorganisms, including primary and secondary metabolites. There are more than 23,000 known microbial secondary metabolites, with actinomycetes producing 42%, fungi producing 42%, and bacteria producing 16%. Some of these secondary metabolites are antioxidants with potential uses in a variety of industries21, such as endophytic fungi such as Aspergillus flavus, Aspergillus fumigatus and marine fungi such as Aspergillus wentii22. Penicillin, derived from the Penicillium fungus, is an example of a natural antimicrobial compound, paving the way for numerous therapeutic agents23. Animals are also a rich source of antimicrobial peptides and proteins that aid in their natural defense against pathogens. Various animal species, including insects, amphibians, reptiles, birds, and mammals, create AMPs that may target and damage microorganisms’ cell membranes, with broad-spectrum action against bacteria and fungi24.

Primary metabolites are needed for microorganism growth and reproduction, while secondary metabolites are formed from primary metabolites25. Secondary metabolites found in fungal extracts include phenols, flavonoids, tannins, saponins, alkaloids, polyketides, terpenoids, and hybrids26. Fungal mycelium is rich in phenolic compounds27. Phenolic and polyphenolic compounds are the primary antioxidant components generated by fungi. These compounds are vital for fungi’s growth and reproduction, as well as their color and sensational characteristics. Fungi are thought to be capable of preventing oxidation due to their ability to protect against microbial assaults and render food unattractive to predators28.

Methyl gallate (MG, methyl 3,4,5-trihydroxybenzoate) is a natural polyphenolic chemical found in fungal extracts with the molecular formula C8H8O5. It is soluble in hot water, ethanol, and diethyl ether. MG contains antioxidants, antimicrobials, and other beneficial compounds29,30.

Earlier studies have shown that antifungal agents are most effective when supplied using in direct and indirect approaches and showed that the mechanism of action of these chemicals has been proven to be preferentially absorbed onto the lipophilic surface of mycelia, with the amount of inhibition increasing with mycelial surface area31,32. It is hypothesized that they form irreversible cross-links with fungal cell membrane components, causing intracellular leakage content, and damaging fungal membranes33,34.

Because of their distinctive characteristics, nanomaterials generated interest in the field of cultural heritage. Metal oxide nanoparticles have been used to protect surfaces from the formation of biofilm, consolidate degraded materials, self-clean, improve material surface quality, and function as a biocide to prevent biodeterioration35,36,37,38. Nanoparticles produced from TiO2, ZnO, SiO2, CeO2, and Ag have been used in conservation, either alone or in combination with other additives37,38,39,40.

Cerium oxide nanoparticles (CeO2NPs) are a rare earth metal and the first element in the periodic table’s lanthanide series. It is employed in a range of sectors because nano-sized cerium has distinct features compared to bulk cerium, such as redox activity, free radical scavenging, and biofilm suppression, making it useful in several antifungal and antioxidant applications. CeO2NPs (nanoceria) have demonstrated exceptional antifungal efficacy, outperforming zinc oxide nanoparticles (ZnONPs) and titanium dioxide nanoparticles (TiO2NPs) against a diverse range of fungal strains. Moreover, nanoceria has exhibited superior antioxidant activity compared to ZnONPs and TiO2NPs in various investigations41,42. CeO2NPs can be synthesized using traditional methods like precipitation, hydrothermal, and sol-gel. Green synthesis methods, such as plant-mediated synthesis, fungus-mediated synthesis, polymer-mediated synthesis, and nutrient-mediated synthesis, use natural matrices as stabilizing agents to alleviate biocompatibility concerns and provide safer routes for preparing CeO2NPs. These methods produce smaller, more stable, fluorescent CeO2NPs with high fluorescent properties43,44,45.

To protect paper with iron gall from serious problems related to fungal deterioration and UV degradation, this study aims to create a novel evaluation of the polyphenolic compounds in the fungal extracts and CeO2NPs as nanoparticles. The best bioactive compound CeO2NPs at a concentration of 18.11 μg/mL was the most effective in acting as antifungal, antioxidant agent, and achieved both curative and preventive conservation for the samples without negatively impacting the paper properties that evaluated by using a variety of examinations and analyses including disc diffusion assay, scanning electron microscope with EDX analysis, transmission electron microscope, zeta potential, size distribution, X-ray diffraction analysis, Fourier transform infrared analysis, and HPLC analysis.

Methods

Chemicals used

Dimethyl sulfoxide (DMSO) 99.9%, tween 80, and other chemicals used in PDA media were acquired from PioChem Company. Menthol 99.9% (Sigma Aldrich), and methyl gallate 94% (ISO Company for Chemicals). Cerium nitrate (Ce (NO3)2.6H2O) and hexamethylenetetramine (HMT) were bought from Loba Chemie, India, and sodium hydroxide (NaOH) was supplied from Millipore Merck, Germany, chlorogenic acid (98.06%) was acquired from Nawah Scientific, Egypt, and caffeic acid (98.5%) was supplied from Glentham life science. Tannic acid powder (99.9%), and gallic acid (99.9%)were acquired from ISO Chem Company. These chemicals were used without further purification.

Paper preparation and artificial aging

The experimental pure cotton paper (25 g/m2) was cut into small square chips (20 × 20 mm2) and written with iron gall ink. To prepare iron gall ink formulations, 7 g of tannic acid powder, 3.3 g of Arabic gum, 14.7 g of iron sulfate, and 100 mL of water. Inks were applied to one side of an uncoated paper with soft round brushes (one for each ink to prevent contamination)46. At the National Measurements and Calibration Center, all paper samples were artificially aged at 80 °C and 65% relative humidity for 120 h to 10 days47.

Fungal isolates, DNA extraction, and ITS sequencing

Using cotton swabs, isolates from a paper manuscript dated back to 17 Muharram 1030 AH, or roughly 404 years ago, are first used to identify fungi. For seven days, the fungal isolates were cultivated on PDA plates and incubated at 25 °C48. A small amount of the fungal mycelium was scraped and suspended in 100 µL of distilled water, boiled at 100 °C for 15 min, and stored at −70 °C. Dead fungal cells were sent to SolGent Company (Daejeon, South Korea) to carry out the whole procedure from DNA extraction to the final step of DNA sequencing. Fungal DNA was extracted and isolated by Solg™ Genomic DNA Prep Kit [Column type], which extracts genomic DNA using a glass microfiber membrane. It is a kit for obtaining high-quality DNA by removing PCR inhibition substances (divalent cation and protein, etc. The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using universal primers: ITS1 (5’-TCCGTAGGTGAACCTGCG G-3’), and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). Amplification was performed using an ABI, 9700 thermal cycler.

The polymerase chain reaction (PCR) mixtures were prepared using Solgent EF-Taq as follows: 10X EF-Taq buffer 2.5 µL, 10 mM dNTP (T) 0.5 µL, primer (F-10p) 1.0 µl, primer (R-10p) 1.0 µl, EF-Taq (2.5U) 0.25 µL, template 1.0 µl, DW to 25 µL. Then the amplification was carried out using the following PCR reaction conditions: one round of amplification was performed consisting of denaturation at 95 °C for 15 min followed by 30 cycles of denaturation at 95 °C for 20 sec, annealing at 50 °C for 40 sec and extension at 72 °C for 1 min with a final extension step of 72 °C for 5 min49. Then the PCR products were purified with the SolGent PCR Purification Kit-Ultra (SolGent, Daejeon, South Korea) before sequencing. Electrophoresis on 1% agarose gel was used to confirm the purified PCR products (using a size marker).

The bands were then eluted and sequenced. Each sample was sequenced in both the sense and antisense directions using the same primers and ddNTPs (big dye). Contigs were generated from sequencing data using the CLCBio Main Workbench tool. The sequences that were obtained were further analyzed using BLAST from the National Center for Biotechnology Information (NCBI) website. The sequence obtained together with those retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov) was subjected to the Clustal W analysis using MegAlign software version 5.05 (DNASTAR Inc., Madison, Wisconsin, USA) for the phylogenetic analysis50.

Extraction of fungal metabolites

Three fungal strains Aspergillus fumigatus (AUMC 16752), Aspergillus flavus (AUMC 16751), and Aspergillus wentti (AUMC 25221) provided from the Faculty of Science, Assiut University, Egypt were chosen as sources of bioactive compounds.

These were used to assess their efficacy against the most common fungi isolated from the paper manuscripts: Aspergillus fumigatus (AUMC16601) and Aspergillus terreus (AUMC16602).

Potato dextrose agar (PDA) with discs (15 mm) holding the fungi were moved to 250 mL Erlenmeyer flasks containing 100 mL of potato dextrose broth culture media per fungus. Erlenmeyer flasks were placed on a rotary shaker for 15 days at 25 ± 2°C and 150 rpm50. Following incubation, the fermented broth was filtered using Whatman filter paper No. 1 (90 mm) and coupled to a vacuum pump. The filter paper disc holding the fungus was dried in an oven at 35 °C until it achieved a steady weight, which was then measured using an analytical balance. The evaluation was conducted in triplicate (Fig. 1).

Fig. 1: Preparation and extraction of fungal metabolites.
Fig. 1: Preparation and extraction of fungal metabolites.The alternative text for this image may have been generated using AI.
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a Aspergillus flavus, b Aspergillus fumigatus, c Aspergillus wentti under the light microscope, d preparation of fungal extracts by producing a fungal suspension on a PDA medium, e filtering it with Whatman paper No. 1, f weighing the extractions, g drying, h grinding, and i storing them in Eppendorf.

HPLC analysis of fungal extracts

The chemical compounds in the fungal biomass extract were analyzed with an Agilent 1260 series. The separation was performed using a Zorbax Eclipse Plus C8 column (4.6 mm × 250 mm i.d., 5 μm). The mobile phase was composed of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate of 0.9 mL/min. The mobile phase was programmed sequentially in a linear gradient, as follows: 0 minutes (82% A); 0–1 min (82% A); 1–11 min (75% A); 11–18 min (60% A); 18–22 min (82% A); and 22–24 min. The multi-wavelength detector was examined at 280 nm. The injection volume was 5 μL for each sample solution. The column temperature was kept at 40 °C at the aromatic and medical plants laboratory (National Research Centre, Dokki, Egypt)51.

Preparation of methyl gallate

Methyl gallate was prepared by the reaction of gallic acid with methanol, as illustrated in Fig. 2. After the solution reached the boiling point, a white precipitate from methyl gallate was formed in an Erlenmeyer; after that, the white powder was collected, filtered using Whatman filter paper No. 1 (90 mm), and ground to a fine powder. The formed methyl gallate was characterized with FTIR compared with methyl gallate standard samples52. Because DMSO oxidizes when mixed with DMSO only and turns back into the solution. It was mixed with 10% DMSO diluted with 90% distilled water at concentrations 5, 10, 25, 50, 100, and 200 μL/mL and then shaken for 2 min until completely dissolved (Fig. 2).

Fig. 2: Preparation of methyl gallate.
Fig. 2: Preparation of methyl gallate.The alternative text for this image may have been generated using AI.
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a Mixing the methanol with gallic acid to produce methyl gallate. b Subjecting the mixture to heat. c Grinding methyl gallate to fine powder.

Preparation of phenolic solutions applied to the treated sample

First, gallic acid, chlorogenic acid, caffeic acid, and methyl gallate were evaluated on paper samples using a poultice method with a solution concentration of 18.11 μg/mL. The goal was to select the best phenolic compound and apply it to the treated sample without causing damage. The solutions were prepared by combining 10% DMSO diluted with 90% distilled and shaking for 2 minutes until completely dissolved.

Synthesis of CeO2NPs

Cerium oxide nanoparticles (CeO2NPs) were prepared via a hydrothermal approach. Briefly, with 0.125 M HMT and 0.025 M Ce (NO3)3·6H2O. HMT was added dropwise to a stirred Ce (NO3)3·6H2O solution. All solution was transferred into a 50 mL autoclave for 2 h at 100 °C or 12 h at 150 °C. The cerium oxide nanoparticle powder was obtained by centrifugation and washing after the reaction solution was cooled to room temperature. CeO2NPs were synthesized as follows: 53 1 or 1.5 g Ce (NO3)3·6H2O was dissolved in water, and a NaOH solution (16 g) was rapidly added with stirring. After 30 min, the solution was transferred into a 50 mL autoclave for 24 or 36 h and hydrothermally treated at 200 °C. After the solution was cooled to room temperature, powders of CeO2NPs were obtained by centrifugation and washing53 and mixed with DMSO to obtain the concentrations of 5, 10, 25, 50, 100, and 200 μL/mL after that sonicated for 2 min by VCX 500 probe Sonicator at the Polymers lab (The National Research Centre, Dokki, Egypt).

Scanning electron microscope

The sample of CeO2NPs was observed using a JEOL JSM 5400LV EDX Link ISIS-Oxford Detector High Vacuum with Energy dispersive X-ray (EDX) at The SEM Laboratory, The British University, Egypt.

Transmission electron microscope

Morphological analysis of the prepared CeO2NPs was conducted using a Talos F200i (Thermo-Scientific) high-resolution transmission electron microscope with an accelerating voltage of 200 kV. Samples for TEM were prepared by placing a droplet of colloid suspension in the proper solvent on a Formvar carbon-coated, 300-mesh copper grid (Ted Pella) and allowing it to evaporate in the air at ambient conditions.

Zeta potential and Size distribution analysis

Zetasizer Nano ZS measured the zeta potential and size distribution of nanoparticles, and the powder of nanoparticles was dispersed in distilled water before analyzing at the laboratory of Nano Gate company, El Mokatim, Egypt.

pH measurements

The AD11 Waterproof pH-TEMP Pocket Tester with Replaceable Electrode, which was produced in Romania, was used in Cairo University’s Faculty of Archaeology’s manuscript lab. Using the cold extraction procedure, triplicate samples were divided into 1 g pieces and placed in 70 mL of cold (25 ± 5°C) distilled water for one hour54.

Fourier transform infrared analysis

All synthesized CeO2NPs and Methyl gallate powders were analyzed with an FTIR spectrometer (Model 6100, Jasco, Tokyo, Japan). The spectra were collected in transmission mode with a triglycine sulfate (TGS) detector using the ATR method and ranged from 400 to 4000 cm−1 at the Lab of Textile, The National Institute of Standards (NIS), Giza, Egypt.

X-ray diffraction analysis

An XRD pattern was obtained using an XPERT-PRO Powder Diffractometer device, with 2 thetas (10°–90°) for measuring the crystallinity index of cellulose of the paper samples, and 2 thetas (10°–45°) for the characterization of CeO2NPs, minimum step size 2Theta: 0.001, and wavelength (Kα) = 1.54614°, and X-ray diffraction applications, including reflectometry, high-resolution diffraction, in-plane grazing incidence diffraction (IP-GID), small angle X-ray scattering (SAXS), as well as residual stress and texture investigations at the Faculty of Nanotechnology, Egypt.

Antifungal activity determination of polyphenolic compound and CeO2NPs compounds using a disk diffusion assay

In order to estimate the clear zone and inhibition rate of fungi by using the inhibition equation after 7 days of incubation in comparison to the control sample, methyl gallate, a bioactive compound found in the extracts of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus wentii, as well as CeO2NPs as nanoparticles, were evaluated at concentrations ranging from 5 to 200 μL/mL inside Petri dishes containing PDA media against the fungi were isolated from historical paper manuscripts Aspergillus fumigatus and Aspergillus terreus, and the IC50 values were then determined.

After being combined with DMSO and some drops of Tween 80, these concentrations achieved 50% inhibition of the fungal attack in Petri dishes according to IC50. After treatment, all paper samples were exposed to artificial UV aging by a UVA BL-TB lamp/ F 30 watt made in Germany at 25 cm from the surface of the samples for 120 h, equivalent to 50 years of aging55.

The most effective concentration of CeO2NPs 18.1 μL/mL, which achieved 50% inhibition of the fungal attack in Petri dishes according to IC50, was evaluated. Two 9 mm Whatman papers are submerged in Petri dishes to determine the best concentrations for inhibiting fungal growth. The paper without ink and the paper with iron ink are then layered between two sheets of paper that have been saturated with the treatment agents. They are then securely closed after being covered in two layers of sterile glass slides on both sides. When they are entirely dry, they are left for a full day and then put back for four more hours.

Color change in the CIELAB system

The colors of the samples were measured with a WR10 High-precision Digital Colorimeter Color Difference Energy Meter Tester, and the evaluation was conducted in triplicate.

Statistical analysis

Data on the antifungal activity were statistically analyzed using a two-way ANOVA test in SAS version 8.1. All measurements were performed in triplicate, and the values are presented as means ± SD. Differences between variables were analyzed using Duncan’s Multiple Range Test.

Results

Sequencing results

The phylogenetic tree based on ITS rDNA sequences of Aspergillus fumigatus AUMC16601 matched closely comparable sequences of the same species obtained from GenBank (Fig. 3). This strain had 100% identity and 100% coverage with related fungal strains, including the type of material A. fumigatus ATCC 1022 (GenBank accession NR_121481). Also, a phylogenetic tree based on ITS sequences of rDNA from Aspergillus terreus AUMC16602 was linked with closely similar sequences of the same species obtained from GenBank (Fig. 4). This strain has 99.49-99.66% identity and 100% coverage with related fungal strains, including the material A. terreus ATCC 1012 (GenBank accession NR_131276).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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A phylogenetic tree based on ITS rDNA sequences from Aspergillus fumigatus isolate connected to closely related sequences of the same species retrieved from GenBank.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Phylogenetic tree of Aspergillus terreus isolate (B) strain AUMC16602 (arrowed) based on ITS rDNA sequences matched with closely similar sequences from the same species obtained from GenBank.

HPLC analysis of the fungal extracts

HPLC analysis revealed several peaks for flavonoids and phenolic compounds in the liquid extracts and crudes. Table 1 and (Fig. S1) show the compounds, their names, and concentrations. HPLC examination of the extracted crude of Aspergillus wentii indicated that its highest concentration was chlorogenic acid, which ranged in the crude extract to (2738.40 µg/g) and the liquid extract to (2.99 µg/mL). Hesperetin (467.88 µg/g), gallic acid (456.15 µg/g), and syringic acid (66.80 µg/g) are the next highest concentrations of flavonoids. Other phenolic chemicals detected in crude were caffeic acid (39.67 µg/g), ellagic acid (7.78 µg/g), p-coumaric acid (8.09 µg/g), vanillin (4.89 µg/mL), and cinnamic acid (4.13 µg/g). Due to its antioxidant and antifungal properties, the polyphenolic substance methyl gallate (13.99 µg/g) was selected as an antioxidant agent on the paper samples. The flavonoids were present at modest concentrations: kaempferol (33.68 µg/g), quercetin (13.61 µg/g), and daidzein (5.34 µg/g). The liquid extract of Aspergillus wentii had the highest concentration of chlorogenic acid (2.99 µg/mL) and gallic acid (0.66 µg/mL). The concentration of phenolic contents ranged from 0.04 µg/mL to 0.13 µg/mL, and the concentration of flavonoids ranged from 0.07 µg/mL to 0.23 µg/mL.

Table 1 HPLC analysis of compounds in fungal extracts of Aspergillus wentii, Aspergillus fumigatus, and Aspergillus flavus
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The concentration of phenolic compounds in the extracted crude of Aspergillus fumigatus was presented as the largest percentage of the concentration including chlorogenic acid (371.47 µg/g), gallic acid (315.04 µg/g), caffeic acid (85.07 µg/g), syringic acid (16.84 µg/g), ellagic acid (11.35 µg/g), coumaric acid (5.95 µg/g), vanillin (5.29 µg/g), cinnamic acid (3.76 µg/g), and ferulic acid (3.22 µg/g). Still, the low concentrations were flavonoids compounds such as hesperetin, naringenin, kaempferol, querectin, and daidzein (18.55, 10.24, 8.02, 4.00, and 81.08 µg/mL). The liquid extract of Aspergillus fumigatus appeared to have a low concentration of compounds compared to the crude with the concentration of phenolic compounds ranging from 0.03 µg/mL to 1.26 µg/mL and flavonoids ranging from 0.05 µg/mL to 0.23 µg/mL.

According to the results, the concentration of gallic acid (582.55 µg/g), chlorogenic acid (461.71 µg/g), and caffeic acid (182.43 µg/g) in the crude of Aspergillus flavus are higher than in the crude of Aspergillus fumigatus with other phenolic compounds that raged from (3.46 µg/g) to (18.81 µg/g). However, the flavonoid compounds had low concentration compared to the crude of Aspergillus fumigatus ranging from 6.58 µg/g to 21.67 µg/g. In the liquid extract of the same fungi, the concentration of hesperetin (2.97 µg/mL) was the highest compared to other liquid extracts and represented the mean compound in the liquid extract as a flavonoid compound (Table 1).

Figure 5 includes SEM images of ceria nanoparticles that have been synthesized. This image shows that the CeO2NPs have a cuboidal shape. The EDS spectra confirmed the presence of Ce and O in NPs. The elements’ atomic percentages were 62.42 ± 9.07% for O and 37.33 ± 2.13% for Ce. This finding lends support to the hypothesis of particles with stoichiometry near CeO2.

Fig. 5: Characterization of CeO2 nanoparticles.
Fig. 5: Characterization of CeO2 nanoparticles.The alternative text for this image may have been generated using AI.
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a, b SEM and TEM images, and c EDS spectra for CeO2 nanoparticles.

The TEM was used to investigate nanoparticle morphological shapes and particle sizes. The formed CeO2NPs were small in size, less than 50 nm, and cuboidal shaped. In terms of hydrodynamic average size using DLS, CeO2NPs had average diameters 232.76 nm ±34.1 by size distribution. These results are consistent with the zeta potential. The zeta potential of CeO2NPs is −16.96 ± 1.1 mv, which is considered an indication of the stability of CeO2NPs to produce nanoparticles while avoiding further agglomeration (Fig. 6a, b).

Fig. 6: Zeta potential and particle size distribution of CeO2 NPs.
Fig. 6: Zeta potential and particle size distribution of CeO2 NPs.The alternative text for this image may have been generated using AI.
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a Zeta potential and b particle size distribution for CeO2 nanoparticles.

FTIR analysis of cerium oxide nanoparticles and methyl gallate

The FTIR spectra of cerium oxide nanoparticles illustrate a wide range of signals, showing the existence of functional groups. The spectrum reveals two high peaks at 3420 cm−1 and 1621 cm−1. The wide band in the range of 3000 cm−1–3672 cm−1 relates to the O-H stretching vibration from absorbed water/hydroxyl groups. It was described that the modest absorption peaks at 2368 cm−1 and 1386 cm−1 to the bending vibration of C-H bands of the integrated surfactant residuals56. Furthermore, the absorption band about 1621 cm−1 is due to the bending vibration of the absorbed molecule water, which can be detected in all samples. The band at 858 cm−1 corresponds to the vibrational stretching mode. of H2O. The band at 1064 cm−1 has been assigned to (Ce–O–Ce) vibration (Fig. 7).

Fig. 7
Fig. 7The alternative text for this image may have been generated using AI.
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FTIR spectra of the synthesized CeO2NPs.

The FTIR spectra of methyl gallate indicated a large sharp peak at 3494 cm−1, which corresponds to the stretching vibrations of phenolic -OH groups, and the peak at 2994 cm−1 is due to vibrations from unsaturated =CH stretching. The stretching vibration of the phenyl C = C group appeared at 1688 cm−1, which is consistent with the standard methyl gallate sample, and indicated its purity52 (Fig. 8).

Fig. 8
Fig. 8The alternative text for this image may have been generated using AI.
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FTIR spectra of the synthesized methyl gallate and the standard sample.

XRD analysis of CeO2 nanoparticles

The XRD patterns of the synthesized CeO2 nanoparticles can be seen in Fig. 9. The CeO2 NPs showed diffraction peaks at 28.54°, 33.07°, 47.55°, and 56.22° are observed to correspond to (111), (200), (220), and (311) planes. Also, 59.05°, 69.26°, 76.75°, and 79.03° corresponded to the cerium oxide phase57,58. The sharp diffraction peaks show that the particles have an ordered structure. The absence of additional phases indicates the chemical purity of the CeO2NPs.

Fig. 9
Fig. 9The alternative text for this image may have been generated using AI.
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The XRD patterns of the synthesized CeO2NPs.

pH measurements

Gallic acid, which had a pH of 3 and was the lowest phenolic compound relative to other compounds, followed by methyl gallate, was one of the phenolics with the highest acidity levels, according to an initial analysis of the pH values for the different phenolic solutions. Chlorogenic acid with a pH value of 3.3, and caffeic acid with a pH value of 3.7. As a result, the following phenolic compounds reduced the pH of the paper samples from 5 in the untreated paper sample: caffeic acid 4.8, collagenic acid 4.63, methyl gallate 4.3, and gallic acid 4.16. However, the visual assessment revealed that the samples treated with collagenic acid were brittle and broke into pieces following treatment. The inked samples treated with gallic acid and caffeic acid exhibited more curvature after being compared to the untreated sample, which was more flat. In addition, treatment with caffeic acid left acid remains on the samples’ ink surfaces, and pH values ranged from 2.56 to 2.8 As a result, methyl gallate is the best of these materials.

CeO2NPs are a neutral nanomaterial in contrast to the very acidic phenolic chemicals. This is clear from the fact that the pH values of the paper samples and the others that contained iron ink remained unchanged. The pH of the paper samples treated with CeO2NPs was 5.15, whereas the iron ink samples had a pH of 2.8, indicating no significant difference from the untreated samples (Figs. 10, 11).

Fig. 10: Morphological effects of various polyphenolic compounds on iron gall ink.
Fig. 10: Morphological effects of various polyphenolic compounds on iron gall ink.The alternative text for this image may have been generated using AI.
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a The untreated control sample, and samples treated with: b methyl gallate, c CeO2 NPs, d gallic acid (notably exhibiting edge twisting), e chlorogenic acid, and f caffeic acid (characterized by both twisting and the appearance of acid radicals on the surface).

Fig. 11: Morphological examination of polyphenolic compounds on the paper samples free iron gall ink.
Fig. 11: Morphological examination of polyphenolic compounds on the paper samples free iron gall ink.The alternative text for this image may have been generated using AI.
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a The untreated paper sample following different treated samples, and samples treated with: b methyl gallate, c CeO2 NPs, d gallic acid e chlorogenic acid, and f caffeic acid.

Antifungal activity determination of fungal bioactive compounds and CeO2NPs using a Disk Diffusion Assay

The antifungal properties of methyl gallate and CeO2NPs have been evaluated against A. fumigatus and A. terreus. CeO2NPs did not reduce fungal growth in A. fumigatus, although they were more effective against A. terreus. CeO2NPs were found to have an inhibitory rate ranging from 44.5% to 69.6% at the concentration of 5 to 200 μg/mL, respectively, against A. terreus. The antifungal concentration (18.1 μg/mL) was chosen based on IC50 to be assessed as a multifunction material to be an antioxidant agent after UV aging (Table 2).

Table 2 Determination of the inhibition potential of CeO2NPs and methyl gallate at various concentrations on A. fumigatus and A. terreus
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The inhibitory rate of methyl gallate from 50 to 200 μg/mL ranged from 14.1 to 22.1% against A. fumigatus. Furthermore, methyl gallate, a bioactive compound from fungal extracts, had no inhibition potential on A. fumigatus from 5 to 25 μg/mL, failing to achieve inhibition by 50% at a maximum concentration of 200 μg/mL. The concentrations ranging from 10 to 200 μg/mL of methyl gallate inhibited A. terreus with rates ranging from 7.4 to 25%, and the PDA medium was colored black due to the oxidation of methyl gallate. The antifungal activity against A. terreus was ineffective at 5 μg/mL (Table 2).

The color change measurements of the treated paper samples

When compared to the UV-aged untreated sample, which had a color change estimated by 2.5 due to oxidation, the study of color changes in cotton pulp paper containing iron gall ink samples noticed that treatment with CeO2NPs did not affect the paper samples before or after UV-aging, indicating that ultraviolet rays did not affect the paper samples and thus did not damage the samples. Furthermore, the treated sample with CeO2NPs showed a modest color shift, which can be attributed to the pale yellow color of the CeO2NPs. Furthermore, the UV-aged treated samples with CeO2NPs did not demonstrate any obvious color change despite being exposed to ultraviolet radiation.

For the samples containing iron ink, humidity, and heat aging caused significant damage, and a noticeable color change to the aged untreated sample resulted in the samples’ black color turning brown by visual assessment and becoming fragile with increasing (L, a, and b) values compared to the control sample, so the total color change value was estimated to be 14.50. When the aged untreated samples with ink were treated with CeO2NPs using the sandwich layers method, two layers of Whatman papers were saturated by CeO2NPs, which helped to remove any traces of salts from the damage of the iron ink on the surface of the samples, which made increased (L value) in the control sample from 16.08 to 22.93 in the heat and humidity aged untreated sample that led the sample to be more bright, but after the treatment by CeO2NPs (L value) in deceased from 22.93 to 20.54 in the treated sample with CeO2NPs, so the color of iron gall ink more became black. Also, it is noticed that the total color change rate of UV-aged treated samples with CeO2NPs is like that of the UV-aged untreated samples after aging, indicating that CeO2NPs are more efficient as an antioxidant in the paper samples than in those with iron gall ink (Table 3).

Table 3 The color change measurements of the treated samples, and treated sample with CeO2NPs before and after aging compared to the control samples
Full size table

Discussion

Scanning electron and transmission electron microscopy constitute essential microstructural investigation tools because they allow researchers to evaluate material surface morphology directly59. In the study, SEM with EDX and TEM observation detected that the nanoparticles were on the nanoscale with an average diameter of less than 50 according to60,61,62 without any impurities, which reflected that the preparation of CeO2NPs by hydrothermal approach was successful, and that was also proven by XRD that appeared a sharp peak and zeta potential and size distribution analysis that showed the stability of CeO2NPs as-prepared nanoparticles without agglomeration63,64. Three distinctive peaks in the FTIR spectra of CeO2NPs at 1620, 2920, and 3350 cm−1, are attributable to C = C vibrations and O-H bonds in water molecules. This is comparable to the FTIR analysis of produced CeO2NPs: O-H stretching at 3672 cm−1, O-H band at 2368 cm−1, and C = C vibrations at 1621 cm−1 62,63. Furthermore, FTIR analysis played a vital role in characterizing methyl gallate, which appeared to have distinctive peaks similar to synthesized methyl gallate such as the peak at 2994 cm−1 of unsaturated =CH stretching, and the stretching vibration of the phenyl C = C group appeared at 1688 cm−1 at 3494 cm−1, which related to the stretching of phenolic -OH groups. Due to the aromatic ring’s tight coupling with the oxygen in the phenolic compounds, it increased the acidity of methyl gallate, caffeic acid, chlorogenic acid, and gallic acid, and all these results are consistent with the previous studies51,65,66. In addition, methyl gallate is a bioactive component in many fungal extracts30,67.

Many studies have found that fungi have a variety of beneficial secondary metabolites. As a result, fungi have emerged as an effective group of organisms for many research objectives59,68. Endophytic fungi such as Aspergillus flavus and Aspergillus fumigatus and marine fungi such as Aspergillus wentii are well-known sources of bioactive secondary metabolites69. Endophytes and marine fungi produce secondary metabolites that contain bioactive compounds that are thought to bring out a confrontation mechanism to overcome other fungi70,71,72,73. Fungal extracts contain bioactive compounds such as phenols and flavonoids at various concentrations, whether crude or liquid. According to the findings, methyl gallate was a polyphenolic compound in all crudes of A. wentii, A. fumigatus, and the liquid extract of A. fumigatus. Furthermore, the study revealed that A. fumigatus appeared to be the most isolated fungal species in the sequencing results of the historical paper manuscript. Its crude and liquid extract provided bioactive compounds such as methyl gallate, which functioned as an antifungal with CeO2NPs on paper samples with iron gall ink against A. fumigatus and Aspergillus terreus.

Aspergillus oryzae fungal spore germination on solid substrates was inhibited by CeO2 in pervious study, with a fungistatic rather than fungicidal effect. Particularly when exposed to UV light, CeO2 suppressed fungal growth and increased the generation of reactive oxygen species (ROS). CeO2’s growth-inhibitory effects and intracellular ROS levels were both decreased by tocopherol, indicating that ROS play a role in these44.

Phenolics such as p-coumaric acid, caffeic acid, vanillic acid, methyl gallate, gallic acid, ferulic acid, and syringic acid have antifungal activity against F. oxysporum, F. verticillioides, P. brevicompactum, P. expansum, A. flavus, and A. fumigatusas74,75, but methyl gallate had a slight antifungal effect against A. fumigatus and A. terreus only by 22.11% compared to CeO2NPs (18.1 μg/mL) that had antifungal and antioxidant properties after application on paper samples by poultices method. Artificial aging can lead to many changes in the physical properties of manuscripts due to oxidation that occurs during deterioration changes in brittleness, tensile strength, and color76,77. Therefore, untreated samples showed a noticeable color change due to oxidation and iron gall ink degradation, which led to changes in the chemical composition of iron gall ink by turning the black color of ink to bright reflected by making color changes on the samples, but the aged treated samples with CeO2NPs showed no significant color change as compared to the aged untreated samples. The primary function of CeO2NPs to be an antioxidant agent is to directly scavenge reactive oxygen free radicals via their surface redox activity, and the unique nanoscale effects are caused not only by their44,78,79. CeO2NPs as an antifungal agent displayed a high potential against A. terreus isolated from the historical paper by inhibiting fungal spore germination on solid substrates creating irreversible cross-links with components of the fungal cell membrane, which damages the fungal membrane and results in intracellular leakage, and inhibiting fungal growth inside Petri dishes, so CeO2NPs is considered antifungal and antioxidant agent44,80,81.

Previous research has demonstrated that antifungals function best when they are administered through a mix of direct and indirect methods. It has been demonstrated that these substances work by preferentially adhering to the lipophilic mycelial surface, where the degree of inhibition rises with mycelial surface area. They are thought to create irreversible cross-links with components of the fungal cell membrane, which damages the fungal membrane and results in intracellular leakage33,34

It is critical in the various treatment procedures of paper manuscripts to use safe and eco-friendly methodologies that do not affect the manuscripts. As a result, the purpose of this study was to compare one of the polyphenolic compounds in fungal extracts, such as methyl gallate, and nanoparticles, CeO2NPs, by determining the ability of these compounds to protect paper manuscripts containing iron gall ink from the effects of fungi and oxidation degradation. It became obvious that the prepared CeO2NPs, which were verified to be pure, free of impurities, and manufactured on a nanometer scale, were effective in inhibiting the A. terreus and preserving paper manuscripts from the effects of aging by the ultraviolet radiation compared to UV-aged untreated samples. In addition, the application of CeO2NPs by the poultice’s method helped to remove any residues on the surface of the iron gall ink, which assisted in safeguarding the ink layer when compared to the aged untreated sample, and it recommended to use on stable iron gall ink to prevent any migration of ink in case of highly fragile iron gall ink or ink can be fixed before treatment. In contrast, methyl gallate did not achieve a notable rate of inhibiting fungal growth, so CeO2NPs at a concentration of 18.11 μg/mL are effective as antioxidant and antifungal agents.