Characterization and bioactivity of Fusarium incarnatum Nano-chitosan

Graphical abstract

Highlights

• •Marine fungus Fusarium incarnatum optimized for chitosan production.
• •Nano-chitosan synthesized via ionic gelation, size range 10–80 nm.
• •Characterization of Nano-chitosan is confirmed by FTIR, XRD, and TEM analyses.
• •Nano-chitosan exhibits strong antibacterial and antiviral activity against HSV-1.
• •Nano-chitosan show potent cytotoxicity on Mcf7 and HepG2 cancer cell lines.

Abstract

The current study focused on the synthesis, characterization and biological activities of Nano-chitosan (NC) derived from the marine origin fungus, Fusarium incarnatum strain Yassmine. Initial screening of various media evaluated isolate 20S as a high biomass producer. Consequently, the Plackett–Burman (PB) design was employed to optimize growth conditions, leading to a maximum fungal biomass of 3.54 g/mL, which represents a 2.6-fold increase over basal conditions. The NC was obtained from the fungal chitosan via a standard chemical conversion then characterized using advanced techniques. The FTIR spectroscopy confirmed the presence of characteristic functional groups. The XRD pattern indicated a well-ordered crystalline structure, as well as the TEM micrographs revealed uniform, spherical nanoparticles (NP) with sizes ranging from 38 to 77 nm. Biological assays demonstrated that the fungal NC possesses significant bioactivities. In particular, it exhibited a promising antimicrobial activity against several bacterial pathogens and a notable reduction in HSV-1 viral activity from 100 to 65.66%. Effectively, the NC showed a strong cytotoxic effect on human breast cancer (Mcf7) and liver cancer (HepG2) cells, with IC50​values of 71.13 ± 1.47 and 110 ± 1.6, respectively. Conclusively, these findings suggest that NC from marine fungus-derived is a valuable and safe biomaterial with promising applications in biomedical field.

1. Introduction

Marine fungi are widespread in various marine habitats, including seawater, sediment, and marine organisms. They play vital ecological roles in nutrient recycling and organic matter decomposition, with some species even acting as pathogens to marine flora and fauna¹. Despite numerous isolations from different environments for natural product research2, 3, marine fungi remain a largely underexplored group within the marine ecosystem4, 5, 6.

Fungi are regarded as one of the most significant microorganism groups for chitosan production, because their derived chitosan demonstrates strong antibacterial, antifungal, and antifouling properties7, 8. Due to its unique features including biodegradability, non-toxicity, and inherent antimicrobial effects, chitosan has a wide range of applications⁹. It is used in cancer diagnosis¹⁰, tissue engineering¹¹, wound dressings¹², as drug carriers¹³, and antimicrobial agents¹⁴.

Furthermore, the quest for sustainable, non-allergic substitutes for conventional crustacean sources has sparked an active and expanding field of research into chitosan generated from marine fungi. Compared to material derived from crustaceans, fungal chitosan has clear advantages in terms of production uniformity, purity, and functional qualities¹⁵. Chitosan could also be found in the cell walls of fungi families such as Zygomycetes and Basidiomycetes. Indeed, many workers have extracted chitin from different fungal species for various purposes of applications¹⁶. Zygomycetous fungi such as Absidia coerulea, Benjaminiella poitrasii, Cunninghamella elegans, Gongrenella butleri, Mucor rouxii, Mucor racemosus, Rhizopus oryzae, and Aspergillus niger have been studied extensively17, 18.

On the other side, chitin is the main source of nano-chitosan (NC), a sustainable biomaterial that is manufactured utilizing environmentally benign green chemistry. The environmentally friendly and sustainable manufacturing of chitosan nanoparticles (CNPs) is further highlighted by their green synthesis using techniques such as microemulsion, poly-electrolyte complexation, ionic gelation, emulsification-solvent diffusion, and reverse micellization production. Furthermore, CNPs, which are distinguished by their nanoscale size and improved physicochemical properties, offer substantial benefits in biomedicine, particularly in diagnostic imaging¹⁹.

Additionally, chitosan is a key ingredient in the cosmetics industry, found in products such as hair sprays and tonics²⁰, as well as in various skin care products²¹. In addition to its use in the food processing industry due to its nontoxic and antibacterial nature²², chitosan is also widely used in manufacturing paper products and food packaging materials²³. It has been applied to nylon/silk fibers to enhance their hydrophilic and antibacterial properties²⁴. In agriculture, chitosan shows great promise as a plant disease protector by inducing resistance against a variety of microorganisms, including bacteria, yeast, and fungi²⁵. Moreover, environmentally, chitosan is a valuable tool for the remediation of organic and inorganic contaminants, such as toxic metals and dyes, in soil and water. It is utilized in adsorption, coagulation, flocculation, oxidation/reduction processes, and membrane development for contaminant sensing²⁶. Furthermore, fungal-derived CSNPs are an efficient and environmentally beneficial soil supplement that enhances soil pH stability, organic matter, nutrient retention, and enzymatic activity while supporting plant development and physiological function. Their potential for sustainable agricultural uses is highlighted by their multifunctionality and biodegradability⁸.

Therefore, this study aimed to produce and optimize chitosan from the cell walls of the marine-derived fungus Fusarium incarnatum strain Yassmine. The extracted fungal chitosan was then converted into nano-chitosan (NC), which was thoroughly characterized and evaluated for its biological activities, including antimicrobial, antiviral, and antitumor effects.

2. Materials and methods

2.1. Study area and sample collection

Marine fungi were isolated from sediment samples collected in October 2022 from four distinct locations along the Egyptian Mediterranean Sea coastline in Alexandria: Eastern Harbor, El-Shatby, Mohamed Nagib, and Abo Qir (Fig. 1). Each sample was collected at a depth between 2.0 and 2.5 m and immediately placed in a sterile, sealable plastic bag underwater to prevent contamination from airborne fungal spores. To maintain sample integrity and prevent fungal spore discharge from drying, all samples were kept on ice during transport to the laboratory for subsequent isolation, purification, and identification²⁷.

Fig. 1.

Marine sampling sites along Alexandria coastline, Egyptian Mediterranean Sea, with corresponding latitude and longitude.

Fungi were isolated from these samples using a standard procedure. A portion of the sediment was diluted, and then plated onto three different solid media: peptone yeast dextrose agar (PYDA), potato dextrose agar (PDA) and Sabouraud dextrose agar (SDA). To screen for the medium that would yield the most fungal biomass, the corresponding liquid broths were then used²⁸.

Sediment samples were diluted (1:10) using sterile seawater, and 1 mL of the diluted sample was spread onto three isolating media (PYDA, PDA, and SDA), respectively supplemented with Gentamicin 80 mg/L, Piperacillin, and Tazobactam 100 mg/L. The plates were incubated at 30°C for 7 to 10 days²⁹. All samples were carried out in triplicate⁵. Colonies appearing on the plate were purified and maintained in SDA slants for further studies. The isolated marine fungal strains were segregated based on their morphological differences and labelled with different codes.

2.2. Screening for maximum fungal biomass production

To determine which of the three media, peptone yeast dextrose broth (PYDB), potato dextrose broth (PDB), and Sabouraud dextrose broth (SDB), produced the highest biomass, each fungal isolate was inoculated into 100 mL of each medium. The cultures were incubated at 30°C for 7 days. Subsequently, the biomass was collected by filtration, and the dry weight was measured to quantify fungal growth⁵.

2.3. Phenotypic and genotypic characterization of fungal isolate

Based on the highest chitosan production, the fungal isolate 20S was cultivated on SDA for 7 days. Its phenotypic characteristics were then identified by examining both macroscopic features, such as colony color, texture, and mycelial habit, and microscopic features, including conidiophores, vesicles, and conidia. These analyses were performed using a light microscope at 400x magnification. The isolate was classified using taxonomic guidelines from Barnett & Hunter³⁰ with additional assistance from Gad et al.²⁹.

For genotypic characterization, DNA was extracted from the most potent isolate. The Internal Transcribed Spacer (ITS) regions of the ribosomal DNA were amplified via PCR using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). After purification with the AccuPrep PCR DNA Purification Kit, the PCR products were sequenced by Macrogen Incorporation (Seoul, Republic of Korea). The resulting sequence was deposited in GenBank (accession number OR552110.1) and analyzed for similarity using BLASTN on the NCBI website. Finally, a Neighbour-joining phylogenetic tree was constructed using the MEGAX_10.1.6 software. High-quality reagents from Bioneer Corporation, DNA STAR Incorporation, and Sigma-Aldrich were used for the experiments.

2.4. Optimization of cultural conditions using Plackett-Burman design

Plackett–Burman (PB) design is an efficient technique for medium-component optimization³¹. It was used to pick factors that significantly influenced Fusarium incarnatum biomass. The Plackett–Burman design model identifies the main parameters required for maximal F. incarnatum biomass. Each variable is represented at two levels, high and low, which are denoted by (+1) and (−1), respectively. Seven independent variables (pH, incubation period (IP), peptone (Pep), dextrose (Dx), sodium chloride (NaCl), temperature (Temp), and volume of culture medium (V) were screened in twelve different combinations, organized according to the PB design matrix described in the results section. All trials were performed in triplicate, and the average results of fungal biomass were used as the response. The specific design matrix and results are detailed in Table 3, Table 4.

Table 3.

Plackett–Burman design matrix for screening significant factors affecting F. incarnatum biomass.

Run no.	Variable
	pH	IP	Pep	Dx	NaCl	Temp	V	Biomass (g/L)
1	6.6(+)	3(−)	1.5(+)	1.5(−)	1.5(−)	25(−)	125(+)	1.0785 ± 0.10
2	6.6(+)	11(+)	0.5(−)	2.5(+)	1.5(−)	25(−)	75(−)	1.8628 ± 0.17
3	4.6(−)	11(+)	1.5(+)	1.5(−)	5.5(+)	25(−)	75(−)	3.2516 ± 0.12
4	6.6(+)	3(−)	1.5(+)	2.5(+)	1.5(−)	35(+)	75(−)	0.6780 ± 0.02
5	6.6(+)	11(+)	0.5(−)	2.5(+)	5.5(+)	25(−)	125(+)	1.9909 ± 0.04
6	6.6(+)	11(+)	1.5(+)	1.5(−)	5.5(+)	35(+)	75(−)	1.5900 ± 0.11
7	4.6(−)	11(+)	1.5(+)	2.5(+)	1.5(−)	35(+)	125(+)	0.3690 ± 0.02
8	4.6(−)	3(−)	1.5(+)	2.5(+)	5.5(+)	25(−)	125(+)	1.2660 ± 0.10
9	4.6(−)	3(−)	0.5(−)	2.5(+)	5.5(+)	35(+)	75(−)	0.6780 ± 0.01
10	6.6(+)	3(−)	0.5(−)	1.5(−)	5.5(+)	35(+)	125(+)	0.6570 ± 0.02
11	4.6(−)	11(+)	0.5(−)	1.5(−)	1.5(−)	35(+)	125(+)	0.4551 ± 0.01
12	4.6(−)	3(−)	0.5(−)	1.5(−)	1.5(−)	25(−)	75(−)	1.5758 ± 0.02
13 (B.M)*	5.6(0)	7(0)	1(0)	2(0)	3.5(0)	30(0)	100(0)	1.3723 ± 0.11

^*B.M: Basal medium.

Table 4.

Statistical analysis of Plackett–Burman showing estimated effect, corresponding t-values and regression coefficient of each variable for F. incarnatum biomass.

Term	Main effect	FMS	Coef.	SE Coef.	t-test	P-Value	Confidence level (%)
Constant			1.2942	0.0732	17.69
pH	0.0436	0.000	0.0218	0.0762	0.29	0.786	21.4
IP	0.5977	0.030	0.2988	0.0762	3.92	0.011*	98.9
Pep	0.1689	0.002	0.0844	0.0762	1.11	0.318	68.2
Dx	−0.2939	0.007	−0.1469	0.0762	−1.93	0.112	88.8
NaCl	0.569	0.027	0.2845	0.0762	3.74	0.013*	98.7
Temp	−1.0998	0.101	−0.5499	0.0762	−7.22	0.001*	99.9
V	−0.6366	0.034	−0.3183	0.0762	−4.18	0.009*	99.1

^*p <0.05

The analysis of the PB experiment was performed to determine the effect of each component on the response. The effect was calculated as the difference between the average response at a high level of a given component and the average response at its low level. Therefore, the effect of a given component on the response parameter (R) can be mathematically expressed as:

$$\mathit{MainEffect}=\frac{2\left[\mathrm{\Sigma }R\left(H\right)-\mathrm{\Sigma }R(L)\right]}{N}$$

Where R(H) = response parameter of an assembly in the screening design that contains the higher quantity of a given component, R(L) = response parameter of an assembly in the screening design that contains the lower quantity of a given component, and N = number of assemblies.

The response parameters used in this study were F. incarnatum biomass in g/L. The factor that had no effect would give a value of zero if no interactions existed. A main effect figure with a positive sign indicates that the high concentration of this variable is nearer to optimum, and a negative sign indicates that the low concentration of this variable is nearer to optimum. Using Minitab Statistical Software, statistical t-values for equal-pairs samples were calculated to determine the significant variable. After determining the effect of each component, a corresponding factor mean square (FMS), which is similar to the variance for the measurements of a single quantity, was calculated. This was the average squared effect of a component on the response, i.e.:

$$\mathit{Factormeansquare}=\frac{{\left(Effect\right)}^2}{N}$$

$$=\frac{4{\left[\mathrm{\Sigma }R\left(H\right)-\mathrm{\Sigma }R(L)\right]}^2}{N^3}$$

A multiple linear regression equation incorporating interactive terms was used to calculate the response as follows:

$$Y=b0+b1X1+b2X2+b3X3+b4X4+b5X5+b6X6+b7X7$$ (1)

where Y is the dependent variable (fungal biomass; g/L). b0 is the arithmetic mean response of the twelve runs; and b1, b2, b3, b4, b5, b6, and b7 are the estimated coefficients for the factors pH (X1), incubation period (X2), peptone (X3), dextrose (X4), sodium chloride (X5), temperature (X6), and volume of culture medium (X7), respectively. The main effects (X1, X2, X3, X4, X5, X6, and X 7) represent the average result of changing one factor at a time from its low to high value.

The polynomial equations can be used to conclude after considering the magnitude of the coefficient and the mathematical sign it carries (i.e., positive or negative). The high values of the correlation coefficients for the dependent variables indicate a good fit.

2.5. Model verification

The optimized conditions predicted by the model were experimentally validated. Performance under these conditions was compared to both the initial (unoptimized) baseline and to deliberately suboptimal conditions, thereby confirming the predictive accuracy and robustness of the model.

2.6. Extraction of chitosan from F. Incarnatum

Total chitosan was extracted from dried fungal biomass through a multi-step chemical process. First, the biomass underwent deprotonation by treatment with a 1 M NaOH solution at a 1:40 (w/v) ratio, followed by autoclaving at 121°C for 20 min. The resulting alkaline-insoluble material (AIM) was filtered and washed until it reached a neutral pH. Next, the AIM was subjected to deacetylation by treating it with 50% NaOH for 2 h at 110^∘C, followed by another washing step to achieve a neutral pH. The material was then treated with 10% (v/v) acetic acid at 60°C for 6 h. the fungal chitosan was then precipitated from the supernatant by adjusting the pH to 9–11 with 4 M NaOH. The precipitated chitosan was collected via centrifugation, washed sequentially with distilled water, 95% ethanol, and acetone, and finally dried at 60°C to a constant weight. The final chitosan yield was calculated as a percentage of the initial dry weight of the fungal biomass³².

2.7. Degree of deacetylation (DD) % of chitosan

To determine the DD% of chitosan, FTIR has been used and was calculated according to Affandy & Kobun¹⁶ as; DD% = [1 − (A1655 /A3450 X 1.33)] X 100]. Where A1655 and A3450 were the absorbance at 1655 cm⁻¹ of the amide-I band as a measure of the N-acetyl group content and 3450 cm⁻¹ of the OH band as an internal standard to correct for film thickness or for differences in chitosan concentration powder form. The factor ‘1.33′ denoted the value of the ratio of A1655 /A3450 for fully N-acetylated chitosan.

2.8. Synthesis of NC from F. Incarnatum cell wall

Chitosan nanoparticles were prepared according to the ionic gelation method. Briefly, 0.5 g of chitosan was dissolved in 100 mL of 1% (v/v) acetic acid solution under continuous magnetic stirring until complete dissolution. The pH of the solution was adjusted to 4.6–4.7 using 6 M NaOH. The chitosan solution was filtered, then, 1 mL of aqueous sodium tripolyphosphate (TPP) solution (25%, w/v) was added dropwise to the chitosan solution under magnetic stirring, leading to the formation of CNPs as a result of electrostatic interaction between chitosan and TPP. The formed NP were collected by centrifugation at 16,000 rpm and subsequently freeze −dried for further characterization³³.

2.9. Characterization of F. Incarnatum NC

2.9.1. Fourier Transform Infrared (FTIR) analysis

The functional groups of the nano-chitosan structure were identified using FTIR spectrometer (Jasco FT/IR-4600 plus, Japan), with samples prepared by the KBr pellet method³⁴. Briefly, the specimens were rinsed in water for 4 min to remove any unreacted acid before drying for FTIR analysis. A few milligrams of the sample were mixed with KBr at a ratio of 1:100 and pressed into a transparent pellet 1.0 mm thick, which was analyzed in the frequency range of 4000–600 cm⁻¹ for 32 scans, after background subtraction³⁵.

2.9.2. Transmission electron microscope (TEM) analysis

Basically, the TEM analysis was used to examine the morphology of the NC. The colloidal suspension of NC was first sonicated for 2 min to ensure uniform dispersion. A single drop was then placed on a carbon-coated copper grid, negatively stained with uranyl acetate, and allowed to air-dry. Images were captured at various magnifications using a JEOL JSM5410 electron microscope to analyze the particle's morphology³⁶.

2.9.3. X-ray diffraction (XRD) analysis

The XRD analysis was performed to determine the crystal structure of the NC. An X-ray diffractometer was used to generate diffractograms by measuring the intensity of scattered X-rays. The XRD spectrum was obtained with a Bruker Meas Srv (D2-208219)/D2-2082019 diffractometer operating at 30 kV, 10 mA, and a Cu tube (=1.54) with a 2 Theta (2θ) range of 10 to 80°³⁶.

2.10. Biological activity of F. Incarnatum NC

2.10.1. Antimicrobial activity

The antimicrobial activity of NC derived from F. incarnatum was evaluated using the agar well diffusion method on nutrient agar (NA) medium. Inhibition zones were measured in millimetres (mm). Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Klebsiella pneumoniae ATCC 13883, Pseudomonas fluorescens DCM 50090, and Aeromonas hydrophila NRRL 914) and Gram-positive bacteria (Bacillus subtilis ATCC 6633, Streptococcus agalactiae CCM 6187, Staphylococcus aureus ATCC 2592, and Enterococcus faecalis ATCC 29212) served as reference strains for the antibacterial assay of NC derived from F. incarnatum. In brief, agar plates were inoculated with the bacterial strains under aseptic conditions. Wells with a diameter of 6  mm were filled with 100  μL of the chitosan nanoparticle solution at the desired concentration, which was then introduced into the well and incubated at 37°C for 24  h. After incubation, the diameters of the growth inhibition zones were measured. Ciprofloxacin was used as a control³⁷.

2.10.2. Antiviral activity

The antiviral effect of the NC derived from F. incarnatum was assessed on Herpes simplex virus 1 (HSV1) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) protocol. Additionally, the cytotoxicity of the NC was evaluated on Vero cells (from Cercopithecus aethiops kidney tissue, ATCC CCL-81) to determine its safety. This method was performed according to El-Ganainy et al.³⁸.

2.10.3. Anti-tumor activity

The antitumor activity of the NC from F. incarnatum was determined by evaluating its cytotoxicity on Vero cells using the MTT protocol³⁹. In addition, the study also examined the morphological changes on the cell surfaces of two cancer cell lines: adenocarcinoma cells ATCC HTB-22 (Mcf7), derived from human breast tissue, and hepatocellular carcinoma cells ATCC HB-8065 (HepG2) obtained from human liver tissue.

2.11. Statistical analysis

All experiments were conducted in triplicate, and data are presented as mean ± standard deviation (SD). Multiple linear regression was performed in Microsoft Excel and STATISTICA 10.0 to analyze the experimental data and determine the t-value, P-value, and confidence level⁴⁰.

3. Results

3.1. Isolation of fungal isolates from a marine habitat

A total of 22 fungal isolates were collected from 20 sea-sediment samples during four trips in 2022. The samples were taken from four locations along the Egyptian Mediterranean Sea coastline in Alexandria: Eastern Harbor, El-Shatby, Mohamed Nagib, and Abo Qir. The fungi were isolated using three different media: PYDA, PDA and SDA. All isolates were chosen for further study based on their complete growth in a 10 cm Petri dish after a 7-day incubation period. Results in Table 1 showed that samples from Abo Qir yielded the highest percentage of isolates (59%), followed by Eastern Harbor (18%), Mohamed Nagib (13%), and El-Shatby (9%), respectively. The higher abundance of fungal taxa in the Abo Qir location suggests it may be more polluted than the other sites.

Table 1.

Fungal isolates distribution and percentage by location along Alexandria coastline.

Locations	Sum of fungal isolates	Fungal isolates (%)
Eastern Harbor	4	18.18
El Shatby	2	9.09
Mohammed Nagib	3	13.63
Abu Qir	13	59.09

3.2. Screening of fungal isolates for maximum biomass on different media

As shown in Table 2, the biomass of fungal isolates cultured in three different media (PYDB, PDB and SDB) were estimated. The 20S isolate yielded the highest fungal mass on all three media, followed by isolates 5S then 4S. In contrast, isolate 1S consistently showed the lowest biomass across all media.

Table 2.

Screening of fungal isolates for maximum biomass.

Isolate code	Biomass weight (gL−1)
	YPD	PDB	SAB
1S	0.309 ± 0.019	0.604 ± 0.07	0.728 ± 0.09
2S	0.375 ± 0.013	0.700 ± 0.06	0.883 ± 0.04
3S	0.201 ± 0.15	0.443 ± 0.03	0.812 ± 0.13
4S	0.391 ± 0.097	1.221 ± 0.25	1.370 ± 0.16
5S	0.447 ± 0.085	0.900 ± 0.17	0.995 ± 0.06
6S	0.354 ± 0.04	0.831 ± 0.09	1.294 ± 0.13
7S	0.421 ± 0.014	0.619 ± 0.1	0.962 ± 0.08
8S	0.293 ± 0.012	0.650 ± 0.06	1.359 ± 0.18
9S	0.402 ± 0.13	1.045 ± 0.94	0.803 ± 0.07
10S	0.372 ± 0.05	0.839 ± 0.15	1.155 ± 0.08
11S	0.419 ± 0.06	1.172 ± 0.06	1.207 ± 0.2
12S	0.261 ± 0.15	0.485 ± 0.09	0.689 ± 0.06
13S	0.299 ± 0.36	0.841 ± 0.93	1.225 ± 0.16
14S	0.308 ± 0.07	0.903 ± 0.96	1.279 ± 0.12
15S	0.259 ± 0.06	0.493 ± 0.04	0.532 ± 0.07
16S	0.226 ± 0.05	0.661 ± 0.08	0.738 ± 0.09
17S	0.318 ± 0.012	0.519 ± 0.06	0.634 ± 0.12
18S	0.431 ± 0.015	1.015 ± 0.73	1.277 ± 0.15
19S	0.416 ± 0.17	0.919 ± 0.08	1.046 ± 0.09
20S	0.507 ± 0.18	1.440 ± 0.16	2.015 ± 0.23
21S	0.371 ± 0.07	0.471 ± 0.17	0.771 ± 0.06
22S	0.422 ± 0.27	1.145 ± 0.13	1.268 ± 0.09

3.3. Identification of fungal isolate

The identification of the marine fungal isolate 20S was performed using both phenotypic and genotypic methods. Phenotypically, the isolate was characterized by its gross macroscopic features, which included a pink, cotton-like fungal colony on SDA medium (Fig. 2).

Fig. 2.

Phenotypic characteristics of Fusarium incarnatum strain Yassmine, (A) pink color cotton-like fungal colony formed on SDA. (B) hypha (Hp), polyphialide (Ph) producing macroconidium (Mc), Fusoid mesoconidia (Me), and macroconidia (Mc) slightly curved, frequently 3–5 septate. Bars, 20  μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

For genotypic characterization, the ITS sequence of the 20S isolate was registered in GenBank under the accession number ON803635.1. The sequence analysis showed a 99.82% similarity to Fusarium incarnatum strain JL5-2, which was further supported by phylogenetic tree analysis (Fig. 3). Collectively, the results from both phenotypic and genotypic analyses confirm that isolate 20S is a Fusarium incarnatum strain, which has been named Yassmine.

Fig. 3.

Phylogenetic tree showing the query strain in relation to other species of Fusarium incarnatum strain Yassmine.

3.4. Statistical optimization of nutritional factors

Particularly, the PB design for this study consisted of 12 trials, with two concentration levels for each of the seven variables. The experiments were performed in triplicate, and the average fungal biomass was recorded. The results showed significant variation in biomass, with a maximum of 3.25 g/L achieved in one trial and a minimum of 0.37 g/L in Table 3.

3.5. Statistical analysis of variance (ANOVA) for F. Incarnatum biomass

The significant variables were identified by statistical analysis of the Fractional Factorial design experiment using Minitab Statistical Software 21.2. The ANOVA results of F. incarnatum biomass (g/L) are shown in Tables 4. The sum of the squares used to estimate factors and p-values were also represented. The Pareto chart (Fig. 4) has been described as a useful tool for identifying the most important effects, displaying the magnitude of each variable and is a convenient way to view the results of PB design. In this chart, the length of each bar on a standardized Pareto chart is proportional to the absolute value of its associated regression coefficient or estimated effect.

Fig. 4.

Pareto chart for Plackett-Burman analysis rationalizing the effect of each variable on F. incarnatum biomass. The vertical line indicates a confidence level of 95% for the effects (p < 0.05).

3.5.1. Fitting into a model

The t-test for any individual effect allows an evaluation of the probability of finding the observed effect purely by chance. In this study, a value of P = 0.05 corresponded to a statistical confidence of 95% was considered in the present study, and hence, any component showing a statistical confidence level equal/or higher than 95% was considered significant. The PB analysis for F. incarnatum biomass showed that the four variables, incubation period (IP), sodium chloride (NaCl), temperature (Temp.), and volume of medium (V) were the most significant variables at a confidence level above 95% (p-value > 0.05). Meanwhile, the confidence level of pH, peptone (Pep), and dextrose was below 95% (Table 5) and was, hence, considered insignificant. This shows the relative importance of increasing IP and NaCl, while decreasing temperature and volume of medium (V) for enhancing fungal biomass, as shown in the main effects plots in Fig. 5.

Table 5.

Verification experiment for F. incarnatum biomass, grown on basal versus pre-optimized medium.

Media applied in the verification experiment	F. incarnatum biomass (gL−1)
Basal medium	1.372
Pre-optimized medium	3.54

Fig. 5.

Main effects of nutritional factors on F. incarnatum biomass based on PB design results.

The goodness of fit of the model was checked by the determination coefficient (R²). In this case, the R² value was calculated to be 0.9541, indicating that 95.41% of the total variability in the response could be explained by this model and only 4.59% of the total variation were not explained. A regression model with R² close to 1.0 is considered as having a very high correlation. Therefore, the present R² value reflected a very good fit between the observed and predicted responses and implied that the model is reliable for predicting F. incarnatum biomass. After applying the ANOVA statistical test, it was found that the first-order models for fungal biomass were satisfactory. The polynomial model equation (in uncoded units) was proposed to calculate the optimum levels of these variables for fungal biomass can be written as: Biomass (g/L) =1.2942 + 0.0218 pH + 0.2988 IP + 0.0844 Pep- 0.1469 Dx + 0.2845 NaCl- 0.5499 Temp- 0.3183  V. Where Y represents fungal biomass and A coefficient with a positive sign represents a synergistic effect of the factor on the fungal biomass, while a negative sign indicates an antagonistic effect.

3.5.2. Validation of the model

To determine the accuracy of the applied PB screening test and based on the data obtained from experimental analysis, a verification experiment was carried out in triplicate. The following medium composition (g/L) is predicted to be near optimum culture condition for maximal F. incarnatum biomass: pH, 6.6; incubation period, 11 days; peptone, 1.5; dextrose, 1.5; NaCl, 5.5; temperature, 25°C; and volume of culture medium, 75 mL The predicted near-optimum levels of dependent variables were examined and compared to the basal condition settings and anti-optimized medium, and the average of fungal biomass is shown in Table 5. The application of a near-optimum condition yielded a F. incarnatum biomass of 3.54 g/L over an incubation period of 11 days. This result represented a 2.6-fold increase in fungal biomass compared to the average of the results obtained under the basal culture condition for F. incarnatum.

3.6. Yield and DD of chitosan from F. Incarnatum

Herein, the chitosan was extracted from the F. incarnatum biomass, and its yield was 10%. In addition, the DD% of our chitosan was calculated based on the FTIR spectra as; 65%, while the standard DD value was 87.1%. Actually, we observed that low value of amide-I indicated to high DD%. Afterwards, NC was prepared according to the TPP-gelation method. The formed NC was further characterized by FTIR, XRD and TEM as follows.

3.7. Characterization of chitosan and NC from F. Incarnatum

3.7.1. FTIR analysis

The FTIR spectrum of the extracted fungal chitosan exhibited characteristic absorption bands consistent with standard chitosan (Oxoid Co., England), as shown in Fig. 6. A broad band centered in 3342 cm^⁻1 corresponds to overlapping O–H and N–H stretching vibrations, indicative of hydroxyl and amino groups. Peaks near 2900 cm^⁻1 are attributed to C–H stretching. The presence of amide I and II bands at approximately 1647 cm^⁻1 and 1563 cm^⁻1, respectively, suggests residual N-acetyl groups. Additional peaks at 1376 cm^⁻1 and 1025 cm^⁻1 are assigned to C-O stretching vibrations of primary alcohols and CH₂OH groups. Strong absorption in the 1150–1020 cm^⁻1 region confirms the presence of C-O-C and C-O linkages from the polysaccharide backbone. The close spectral alignment between fungal and standard chitosan confirms the structural integrity of the fungal-derived product, with minor differences in peak intensities likely reflecting variations in purity, molecular weight, or degree of deacetylation.

Fig. 6.

FTIR spectra; standard chitosan (A) and fungal chitosan (B) from F. incarnatum.

3.7.2. XRD analysis

The XRD profile confirms that the fungal-derived NC retains the essential crystalline features of chitosan while exhibiting structural modifications typical of nano-scale materials. The XRD spectrum exhibits a broad diffraction peak centered in 2θ ≈ 20°, which is characteristic of the semi-crystalline nature of chitosan (Fig. 7). This broad peak indicates a predominantly amorphous structure with limited crystalline domains, which is commonly observed in nano-sized chitosan due to its high surface area and disordered molecular arrangement. The absence of sharp and well-defined crystalline peaks further supports the nano-scale and less ordered morphology of the material.

Fig. 7.

XRD spectrum of NC extracted from F. incarnatum cell wall.

3.7.3. TEM analysis

The NC particles prepared from F. incarnatum in the experiment exhibited a fine white powder shape. The TEM micrographs of the NC particles showed that they were approximately uniform spheres. The unmodified NC particles are composed of clusters of NP with nanosize ranging from 38 to 47 nm. This finding is shown in Fig. 8.

Fig. 8.

TEM Micrographs of NC from F. incarnatum (50 KM).

3.8. Biological activities of F. Incarnatum NC

3.8.1. Antibacterial activity

The synthesized NC from F. incarnatum strain demonstrated significant inhibitory activity against several Gram-positive and Gram-negative bacteria (Table 6). At a concentration of 100  μg/well, the NC produced larger inhibition zones than ciprofloxacin (CIP, 100 U/disc) against S. aureus ATCC 2592 (33 ± 0.12  mm vs. 25 ± 0.2  mm), P. aeruginosa ATCC 9027 (39 ± 0.08  mm vs. 26 ± 0.08  mm), and B. subtilis ATCC 6633 (37 ± 0.06  mm vs. 25 ± 0.06  mm), indicating a superior or comparable antibacterial efficacy against these strains. Interestingly, the NC showed no activity against several other strains, including E. coli ATCC 8739 and E. faecalis ATCC 29212, even though ciprofloxacin remained effective against all tested strains. This selective activity may be attributed to differences in cell wall structure, membrane permeability, or resistance mechanisms among the bacterial species. A moderate inhibition zone (15 ± 0.14  mm) was observed against K. pneumoniae ATCC 13883, suggesting partial susceptibility, which could be due to a protective capsule or efflux systems that reduce the NC's uptake.

Table 6.

Antibacterial activity of F. incarnatum NC.

Reference bacteria	NC conc. (100 μg /well)	Ciprofloxacin (100 U/disc)
Staphylococcus aureus ATCC 2592	33 ± 0.12	25 ± 0.2
Escherichia coli ATCC 8739	−	26 ± 0.1
Pseudomonas aeruginosa ATCC 9027	39 ± 0.08	26 ± 0.08
Bacillus subtilis ATCC 6633	37 ± 0.06	25 ± 0.06
Enterococcus faecalis ATCC 29212	−	27 ± 0.09
Klebsiella pneumoniae ATCC 13883	15 ± 0.14	27 ± 0.09
Pseudomonas fluorescens DCM 50090	−	27 ± 0.1
Streptococcus agalactiae CCM 6187	−	27 ± 0.06
Aeromonas hydrophila NRRL 914	−	27 ± 0.05

3.8.2. Antiviral activity

The results shown in Fig. 9 demonstrate a clear dose-dependent cytotoxic effect of the fungal NC. At the highest concentration tested, 1000  μg/mL, cell viability sharply dropped to approximately ∼ 7.8%, indicating high toxicity. As the concentration decreases, cell viability improves significantly. At 250  μg/mL, viability was around 48.4%, which is close to the calculated IC50-value of 246.07 ± 3.27  μg/mL. This value confirms that 246.07  μg/mL is the concentration required to inhibit 50% of cell growth. Importantly, at concentrations of 125  μg/mL and below, cell viability remained high, staying above 97%. This minimal toxicity at lower concentrations suggests that NC is a promising candidate for therapeutic applications, where low dosages are needed to minimize harm to healthy cells, while still being effective.

Fig. 9.

Cytotoxic effect of F. incarnatum NC on Vero cells.

The antiviral activity of the fungal NC against HSV-1 was evaluated and found to have a clear dose-dependent response (Fig. 10). At the highest concentration tested, 125  μg/mL, the compound achieved its peak antiviral effect with approximately ∼ 34.3% viral inhibition. This efficacy was accompanied by moderate cytotoxicity (∼34.1%), indicating a balance between therapeutic benefit and potential host cell toxicity at this concentration. As the concentration decreased, the antiviral effect diminished sharply, becoming almost negligible at 31.25 and 15.62  μg/mL. These results highlight the NC's potential as an antiviral agent but emphasize the critical need to optimize its dosage to maximize therapeutic efficacy, while minimizing harm to host cells.

Fig. 10.

Antiviral effect and cytotoxicity of F. incarnatum NC on HSV-1-infected Vero cells.

3.8.3. Anti-tumour activity

Obliviously, NC demonstrated a potent cytotoxic effects against both Mcf7 and HepG2 cells. Data showed a clear dose-dependent response, with higher concentrations leading to a significant decrease in cell viability. The IC50​ values of 71.13 ± 1.47 for Mcf7 cells and 110 ± 1.6 for HepG2 cells confirm its strong antitumor potential. Furthermore, the treatment with the NC significantly reduced HepG2 viability as determined by MTT assay. The IC50 value for NC was found to be 110 ± 1.6, which indicates that NC has a strong cytotoxic effect on HepG2 cells. Collectively, the data are shown in Fig. 11.

Fig. 11.

Anti-tumor activity of F. incarnatum NC on McF7 and HepG2 cells.

4. Discussion

Basically, the usage of natural products, such as biological metabolites or organisms or organs, is thought to be an efficient, secure, and biocompatible formulation for medication ingerdients⁴¹. In particular, chitosan is a natural polysaccharide, which is commercially resulted by deacetylation process of the chitin building the shrimp and crab shells via strong alkali at high temperature for long time. The supplies of raw materials are variable and seasonal and the process is highly cost. Therefore, the biotechnology through fermentation offers a substitute resource of chitosan from fungi⁹. Mainly, the cell walls of different fungal classes contain chitin for maintaining the integrity, strength and shape of cell structure beneficially, fungi can be grown in simple media then the chitosan can be extracted from their cell walls. Actually, the chitosan production has been investigated in several species of fungi18, 19.

The use of homogeneous solid substrates in solid-state fermentation offers advantages like better process control and easier biomass recovery. However, to the best of our knowledge, there are no available reports on chitin/chitosan production using inert substrates. Conversely, using natural solid substrates can lead to mass transfer limitations, making process control and biomass recovery more challenging42, 43.

Microscopic examination at 400x magnification revealed the presence of specific macroconidia that were slightly curved, frequently 3- to 5-septate, with a curved and tapering apical cell. These characteristics are consistent with Fusarium incarnatum as previously reported by Song et al.⁴⁴. The use of ITS sequence variability is a widely accepted method for phylogenetic studies in fungi, as it provides a reliable genetic marker for species identification⁴.

The PB design offers good and fast screening procedure and mathematically computes the significance of large number of factors in one experiment, which is time saving and maintains convincing information on each component. This indicates the effectiveness of the PB design as a tool for elucidating the most important variables affecting the response. This design is recommended when more than five factors are under investigation⁴⁵. Similarly, when El –Far et al.⁴⁶ applied PB design for P. chrysogenum MZ723110, it produced a 3.1-fold increase in the chitosan yield after statistical optimization. This showed that the type of fungal culture, peptone concentration, and incubation period were the most effective factors.

The Pareto chart has been described as a useful tool for identifying the most important effects, display the magnitude of each variables and is a convenient way to view the results of a PB design⁴⁷. In addition, the t-test for any individual effect allows an evaluation of the probability of finding the observed effect purely by chance. Collectively, some investigators find that confidence levels greater than 85% are acceptable48, 49. Other investigators find that confidence levels greater than 90% are acceptable50, 51 or even levels greater than 95% to be a significant effect on the response⁵².

Based on our experimental approach for extracting chitosan from fungal species aligns well with the findings of El- Far et al.⁴⁴, Goycoolea & Vázquez⁵³ and Koraym et al.⁵⁴, who successfully extracted chitosan from various fungi. So, our findings, fungi represent a potential alternative source for chitosan production. In particular, the percentage yield of the Fusarium sp. chitosan estimated by Aisah et al.⁴³ as 16.7%. Koraym et al.⁵⁴ conducted that different chitosan yields extracted from Fsarium equiseti, F. moniliform, F. fusarioides, F. Oxysporum, F. chlamydospum, and F. solani were 2.1, 1.15, 1.01, 1.01, 1.26, and 1.07, respectively, while chitosan from mushrooms and shrimp yielded 15 and 25%, respectively.

The DD% of chitosan obtained by Affandy & Kobun¹⁶, from mushroom, ranged from 19.15 to 81.94%. Sathiyabama & Akila³² also estimated the DD of chitosan isolated from the A. solani mycelium as 81.06%. Amazingly, Aisah et al.⁴³ prepared chitosan from the Fusarium sp. biomass with a DD of 97.77%.

The F. incarnatum NC was physically characterized involving size, morphology, crystallinity, and surface charge using techniques such as; FTIR, XRD and TEM. Specifically, the functional groups of a CNP consist of amide (NH₂) and hydroxyl (OH) group, C∙H, C∙N, C∙O, and P∙O stretching⁵⁵. Basically, the confirmatory peak for the formation of CNPs is often the FTIR peak at 3000–3500 cm⁻¹, which is attributable to the OH and NH₂ groups⁵⁶.

However, Sharma et al.⁵⁷ obtained a wider peak of the OH group (3200–3600) that caused them to determine that hydrogen bonding is enhanced in CNPs. Choudhary et al.⁵⁸ also observed a band at 3424 cm⁻¹ for CNP, which explained the stretching vibration of the combined peaks of the OH and ∙NH₂ groups. Additionally, Kain & Kumar⁵⁹ found peaks at 3317.48, 2139.29 and 1638.46 as a confirmation of the formation of CNPs. As the same, Boruah & Dutta⁵⁵ conducted a strong and broadband at 3250 cm⁻¹ confirming the stretching between OH and NH₂ groups. Moreover, they have detected peaks at 2865 cm⁻¹, 1182 cm⁻¹, 1642 cm⁻¹, and 1182 cm⁻¹ attributing to C∙H stretching, asymmetric C-O stretching, stretching between C-O and N∙H banding, and P∙O.

Further, the X-ray diffraction analysis confirms that our fungal NC contains both crystalline and amorphous regions. In fact, the presence of OH and NH₂ groups, which create the intermolecular hydrogen bonds required to construct ordered crystalline areas, enhances this crystalline formation. However, the XRD analysis by Koraym et al.⁵⁴ exhibited a key diffraction peak at 20.0^∘ for nano-shrimp chitosan, besides sharper peaks at 25.91^∘, which were detected in the nano-shrimp, mushroom and nano mushroom chitosan. This signifies a denser crystalline structure.

To TEM analysis, the results of Darwesh et al.⁶⁰ showed that the size of NC ranged between 7 and 13 as well as from 3 to 6 nm with spherical shape for shrimp and fungal sources, respectively 3 and 6 nm with spherical shape for fungal source. According to Ishkeh et al.⁶¹, CNPs typically have a size range of 15–150 nm. Moreover, Aisah et al.⁴³ found that the nano-chitosan characterization of Fusarium sp. 20CB07 revealed a nanoparticle size of 127.2 nm.

Upon the applicable level, many investigations confirmed that NC exhibits promising potential in combating clinical bacterial resistance because its nano-sized particles enhance interactions with bacterial cells, disrupting their membranes and inhibiting growth⁶². Additionally, it, being a cationic polymer, interacts positively with the microbial surface, causing changes in membrane permeability, inhibiting microbial growth, and leading to microbial death (increasing vulnerability to death)⁶³. Rezaei & Hama⁶⁴ also stated that chitosan can bind (positive charge) to the bacterial cell wall (negative charge), leading to damage to the cell wall and increased membrane permeability.

Numerous studies have confirmed that NC shows great potential in combating bacterial resistance. Its nano-size enhances its interaction with bacterial cells, allowing it to disrupt their membranes and inhibit growth54, 63. As a cationic polymer, NC positively interacts with the negatively charged microbial surface, altering membrane permeability and ultimately leading to microbial cell death63, 64. Some studies suggest that NC has broad-spectrum antimicrobial activity, as it is effective against both Gram-positive and Gram-negative bacteria58, 64. For instance, Darwesh et al.⁶⁰ found that the antimicrobial activities of different concentrations of chitosan and NC had high activity against the tested pathogens⁶⁵. However, other researches indicate a more selective effect, with NC showing greater potency against Gram-negative bacteria like E. coli and K. pneumoniae66, 67. Despite of, NC exhibited an antibacterial activity against both P. aeruginosa and S. aureus exhibiting greater selectivity towards inhibiting P. aeruginosa over S. aureus in the study carried out by Aisah et al. (2024)⁴³. This selectivity is attributed to differences in bacterial cell wall structure; the cell wall of Gram-negative bacteria is more susceptible to degradation by fungal nano-chitosan⁴⁴. This specificity holds promise for developing targeted treatments that combat infections caused by specific pathogens while sparing beneficial bacteria⁶².

Fungal NC has shown promise in combating viral diseases by overcoming the limitations of conventional antiviral drugs. Nanotechnology-based approaches can enhance the selectivity of these therapeutics towards viruses and infected cells, as highlighted by Chakravarty & Vora⁶⁸. The unique physicochemical properties of chitosan, particularly its positive charge, make it an effective nano-medicine⁶⁹. This positive charge allows for favorable electrostatic interactions with the negatively charged protein domains or surfaces of viruses, thereby inhibiting their ability to bind to and infect host cells. Moreover, chitosan's degree of polymerization enables it to act as a shield, protecting host cell binding sites from viral attachment. In addition to direct antiviral activity, chitosan has been studied for its potential in targeted drug delivery. Loutfy et al.⁷⁰ and other researchers have explored the formulation of CNPs to deliver therapeutics for viruses like HIV and hepatitis B and C, aiming to improve drug efficacy and reduce side effects.

Chitosan and its nano-derivatives can increase the anticancer effects by regulating the antioxidant defense mechanism, angiogenesis, apoptotic pathways, and cellular enzymatic functions⁶⁴. Jiang et al.⁷¹ found that CNPs have an important role in CNPs-induced cancer cell death, especially for hepatocellular carcinoma cells (SMMC-7721). The cytotoxicity investigation showed significantly a superior anticancer effect against HepG2 cell lines by Albalawi et al.⁷². Elzoheiry et al.⁷³ also observed that NC, combined with other bioactive and metal substances, improved liver fibrosis and is promising for hepatic cancer treatment. Recently, Jankovi et al.⁷⁴ confirmed the anticancer evaluation of NC, showing a higher selectivity towards HeLa and A549 cells. A cell cycle analysis indicating the NPs ability to induce mitotic arrest in cancer cells. Early, Seo et al.⁷⁵ explained that the antitumor activity of low molecular weight chitosan was due to activation of murine peritoneal macrophases to kill the tumor cells in the presence of IFN⁷⁶.

5. Conclusions

Nowadays, the majority of commercial chitosan is made from chitin that is recovered from marine waste, especially from crustacean shells. However, there are disadvantages to this supply, such as reliance on fishing seasons, variations in product amount and quality due to the varied diets of crustaceans, and hazardous waste produced during chitin production. Fungal biomass, particularly in vitro cultures cultivated in specialized, regulated conditions, provides an alternate source of chitin. Thus, our study was suggested to extract and characterize chitosan from the marine fungus Fusarium incarnatum strain Yassmine and converted it into a NC using PB statistical design, the fungal growth conditions were optimized, leading to a maximum biomass of 3.25 g/L, a 2.6-fold increase over the basal culture condition. The resulting NC, a versatile biomaterial, was characterized using different techniques including FTIR, XRD, and TEM. The fungal NC exhibited significant bioactivity. It demonstrated promising antibacterial activity against several pathogens and effectively reduced the activity of the HSV-1 virus. Furthermore, it showed a potent cytotoxic effect on Mcf7 and HepG2 cells. These findings collectively support application of the fungal NC as a valuable biomaterial in biomedical field.

CRediT authorship contribution statement

Yassmine Maher: Writing – original draft, Formal analysis, Data curation. Nevine B. Ghanem: Writing – review & editing, Validation, Supervision, Investigation, Formal analysis, Data curation, Conceptualization. Hoda Yusef: Writing – review & editing, Validation, Supervision, Methodology, Formal analysis, Data curation, Conceptualization. Hassan A.H. Ibrahim: Writing – review & editing, Validation, Supervision, Formal analysis, Data curation, Conceptualization. Ahmed M. Gad: Writing – review & editing, Supervision, Methodology, Formal analysis, Data curation. Dalia El Badan: Writing – review & editing, Validation, Project administration, Methodology, Formal analysis, Data curation, Conceptualization.