Antioxidant, antibacterial, and molecular docking of methyl ferulate and oleic acid produced by Aspergillus pseudodeflectus AUMC 15761 utilizing wheat bran

Secondary metabolites (SMs) are the primary source of therapeutics and lead chemicals in medicine. They have been especially important in the creation of effective cures for conditions such as cancer, malaria, bacterial and fungal infections, neurological and cardiovascular problems, and autoimmune illnesses. In the present study, Aspergillus pseudodeflectus AUMC 15761 was demonstrated to use wheat bran in solid state fermentation (SSF) at optimum conditions (pH 7.0 at 30 °C after 10 days of incubation and using sodium nitrate as a nitrogen source) to produce methyl ferulate and oleic acid with significant antioxidant and antibacterial properties. Gas chromatography-mass spectrometry (GC–MS) analysis of the crude methanol extract revealed eleven peaks that indicated the most common chemical components. Purification of methyl ferulate and oleic acid was carried out by column chromatography, and both compounds were identified by in-depth spectroscopic analysis, including 1D and 2D NMR and HR-ESI–MS. DPPH activity increased as the sample concentration increased. IC50 values of both compounds obtained were 73.213 ± 11.20 and 104.178 ± 9.53 µM, respectively. Also, the MIC value for methyl ferulate against Bacillus subtilis and Staphylococcus aureus was 0.31 mg/mL, while the corresponding MIC values for oleic acid were 1.25 mg/mL and 0.62 mg/mL for both bacterial strains, respectively. Molecular modeling calculations were carried out to reveal the binding mode of methyl ferulate and oleic acid within the binding site of the crucial proteins of Staphylococcus aureus. The docking results were found to be well correlated with the experimental data.

Phylogenetic analysis based on ITS sequencing was employed to confirm the identification of the strain.The final ITS data set contained 20 sequences that produced 616 characters, of which 505 characters could be correctly aligned, 46 characters were counted as variable, and 8 as informative.The Tamura 3-parameter using a discrete Gamma distribution (T92 + G) was the perfect model used to represent the relationship among taxa.The maximum Parsimony method yielded 10 trees, the most parsimonious of which (Fig. 2) has a tree length of 61, the highest log likelihood of − 1180.74,consistency index of 0.733333, retention index of 0.818182, and a composite index of 0.600000 is shown in Fig. 2.

Production of secondary metabolites by Aspergillus pseudodeflectus AUMC 15761 utilizing lignocellulosic wastes
The five lignocellulosic wastes (barley bran, date palm leaves, orange peels, rice straw, and wheat bran) used in SSF were fermented in different proportions by A. pseudodeflectus AUMC 15761.Wheat bran produced the most powerful crude extract that exhibited the largest inhibition zone against the examined strains.Following Escherichia.coli in terms of severity of impact were Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis (Fig. 3).

GC-MS analysis
The GC-MS analysis of the methanol extract was carried out to evaluate its potential components since wheat bran extract was determined to be the most promising.Based on the retention time, molecular weight, and fragmentation pattern of the most prominent chemicals, the current results revealed eleven peaks.These were shown to have retention times of 23.222, 27.429, and 29.769 min, respectively, for trans-Ferulic acid, 3-(2, 5-Dimethoxyphenyl) propionic acid, and oleic acid (Table 1; Fig. 4).

Optimization of production conditions of the bio-active secondary metabolites using wheat bran
Based on a one factor at a time (OFAT) analysis, the results obtained revealed that A. pseudodeflectus AUMC 15761 could produce the most bio-active secondary metabolites with the greatest effect against the tested bacteria Figure 2. The most parsimonious phylogenetic tree obtained from a heuristic search (1000 replications) of the ITS sequence of A. pseudodeflectus AUMC 15761 (in blue) compared to other closely similar ITS sequences belonging to genus Aspergillus: section Usti in GenBank.Bootstrap support values for ML/MP ≥ 50% are indicated near the respective nodes.The tree is rooted to Aspergillus creber BRRL 5892 as an outgroup (in red).at pH 7.0 using sodium nitrate as a nitrogen supply after 10 days of incubation at 30 °C.These optimal conditions were found to cause the greatest inhibition of the four tested bacterial strains.The inhibition zones were 20.5 ± 1.3, 37.9 ± 1.7, 27.0 ± 1.8, and 19.0 ± 1.7 mm for B. subtilis, E. coli, S. aureus, and S. epidermidis, respectively (Fig. 5).

HR-ESI MS and NMR spectroscopic analysis
Compound 1 was isolated from A. pseudodeflectus AUMC 15761 as an off-white powder (8.0 mg), suggesting a molecular formula of C 11 H 12 O 4 as deduced from its HR-ESI-MS spectrum (Fig. 6) which exhibited a [M−H] − peak at m/z 207.0664 and a [M−H + H 2 O] − peak at m/z 224.9995.The APT NMR spectrum of 1 (Table 3; Fig. 7), along with the HSQC analysis, confirmed the presence of 11 carbon atoms.These showed four quaternary        , along with the rest three quaternaries, revealed the presence of a tri-substituted benzene ring (Table 3; Fig. 9).The olefinic proton resonates at δ H 7.28 and showed 1 H-1 H cosy correlations to the olefinic proton at δ H 7.63 (Table 3; Fig. 10) and HMBC correlations with both the carbonyl carbon at δ C 167.8 and the aromatic carbon at δ C 127.1, indicating that 1 is a cinnamic acid derivative (Table 3; Fig. 11).The HMBC correlations between the methoxy protons [δ H 3.79 (3H, s)] and the carbonyl carbon (δ C 167.8) confirmed that 1 is a cinnamic acid methyl ester derivative.The other methoxy group at δ H 3.92 (3H, s) showed a HMBC correlation      4; Fig. 14) exhibited a protons signal at δ H 5.34 (2H, m, H-9, H-10) and δ H 2.01 (4H, m, H-8, H-11), assignable to olefinic protons and allylic protons, respectively.The spectrum also revealed a methylene group α to carbonyl functionality (δ H 2.34, 2H, t, J = 7.6, H-2).This was also substantiated by the APT NMR spectrum of 2 (Table 4; Fig. 15) which revealed the presence of a carbonyl carbon signal (δ C 180.4,C-1), two olefinic carbon signals (δ C 130.2 and 129.9, C-9, C-10), a group of methylene carbons resonances at δ C 22.8-34.2,and finally a primary methyl group signal (δ C 14.3, C-18), all of which were in agreement with a monounsaturated fatty acid.Thus, by comparing the 1 H, 13 C-NMR, and mass data of compound 2, oleic acid was therefore determined to be the substance involved (Fig. 12).

Antioxidant activity of methyl ferulate and oleic acid produced by Aspergillus pseudodeflectus AUMC 15761
The results of the current study revealed two antioxidant substances-methyl ferulate and oleic acid.The DPPH activity increased as the sample concentration increased (Fig. 16A), with IC 50 values of 73.213 ± 11.20 and 104.178 ± 9.53 µM were significantly greater (p < 0.05) respectively, for both substances, as compared to the value (60.299 ± 4.769 µM) of ascorbic acid (Fig. 16B; Table 5).

Antibacterial activity of methyl ferulate and oleic acid
In the present study, the antibacterial tests against B. subtilis and S. aureus demonstrated a significant antibacterial impact for both methyl ferulate and oleic acid.The MIC of methyl ferulate against the two bacterial strains was 0.31 mg/mL, while the MICs of pure oleic acid against both strains were 1.25 and 0.62 mg/mL, respectively (Tables 6, 7; Fig. 17).R = zero (no linear association between the variables or no consistent linear component to that relationship); R = 1 (perfect positive linear relationship between the variables); 0 < R < 1 (positive linear trend and the sampled individuals are scattered around the line of best fit; the smaller the absolute value of R the less well the data can be visualized by a single linear relationship.M = mean; %MB = mean value divided by the standard value.

Docking computations
In order to recognize target proteins for the antibacterial activity of the two compounds obtained in the present study, four various protein targets of Staphylococcus aureus were investigated, including dihydrofolate reductase (PDB code: 5ISP 32 ), pyruvate kinase (PDB code: 5OE3 33 ), and sortase A (PDB code: 2MLM 34 ).Before generation of data, the performance of the AutoDock 4.2.6 software was evaluated the of the co-crystalized inhibitors towards their targets.Based on the data illustrated in Fig. 18, the binding modes were essentially identical to their native structures with RMSD values less than 1.0 Å, displaying the strong accuracy of the utilized technique.

Discussion
Secondary metabolites created by fungi have been shown to be a wonderful source of new drugs, biofuels, industrial chemicals, food additives, and feed additives 35 .Penicillins, lovastatin, echinocandin B, and cyclosporine A offer examples of how important it is to investigate fungal sources for novel pharmaceuticals 36 .Aromatic compounds, amino acids, fatty acids, butanolides, butenolides, cytochalasans, macrolides, naphthalenones, pyrones, and terpenes are only a few of the structural types of metabolites that fungi produce 36,37 .There have been 15600 fungal metabolites identified from species of Alternaria, Aspergillus, Claviceps, Fusarium, and Penicillium 38 .
In addition, several bio-active secondary metabolites have been produced by many species of fungi in SmF or SSF 47 , such as penicillins and cephalosporins isolated from Penicillium and Acremonium, respectively 48,49 ; feruloyl esterase produced by Aspergillus terreus GA2 using maize bran 50 ; methyl ferulate from the fruiting bodies of Coriolopsis aspera 40 ; festuclavine 2 produced by A. fumigatus 51 ; cyclosporine 41, which exhibits broad spectrum of antifungal activity 52 , derived from Toplypocladium inflatum 53 ; geranylgeraniol, farnesol, hexacosanol, oleic acid, and squalene synthesized by Colletotrichum coccodes 54 ; 1-Octacosanol produced by Phyllosticta capitalensis 55 ; a set of peptaibols, that are extremely potent growth inhibitors of several species of fungi, including the plant pathogens Alternaria alternata, Phoma cucurbitaceum, Fusarium spp., as well as the human pathogen A. fumigatus, have been reported from Trichoderma reesei 56 ; and Echinocandin B 38 from Aspergillus nidulans 57 .
The most potent antibacterial crude metaboliate from Aspergillus pseudodeflectus AUMC 15761's was produced after 10 days of fermentation utilizing wheat bran in SSF at pH 7.0 and 30 °C, using sodium nitrate as a nitrogen source.The pH of the growth medium and other physical factors, such as the incubation temperature, were found to have a substantial impact on the production of secondary metabolites, with synthesis rapidly decreasing on either side of an optimal point.By changing the degree of dissociation of different molecules in the media, the quantity of hydrogen or hydroxyl ions may have direct or indirect effects on a cell.As a result,  One of the unsaturated fatty acids formed by the reaction of palmitic and stearic acids is oleic acid.Moreover, enzymatic activity can convert saturated fatty acids such as stearic acid and palmitic acid into oleic acid 60 .In the present study, the methanolic extract of wheat bran fermented by A. pseudodeflectus AUMC 15761 was utilized to isolate oleic acid.Saturated fatty acids and monounsaturated fatty acids, including palmitic and oleic acids, occurred in abundance in the fatty acid profiles of Mucor circinelloides URM 4140, M. hiemalis URM 4144, and Penicillium citrinum URM 4126 61 .Oleic acid, also known as 9-octadecenoic acid, is a healthy kind of omega-9 unsaturated fatty acid that is very useful for people's health 22 .Unsaturated fatty acids do indeed lower cholesterol by activating cholesterol acetyltransferase, as is widely known.Cancer, cardiovascular, autoimmune, Parkinson's, Alzheimer's, inflammatory, and hypertensive illnesses are all treated effectively with fatty acids.These compounds have been employed as an anticancer treatment because they may cause cancer cells to undergo apoptosis and regulate the cell membrane 22 .
Aspergillus terreus, Claviceps purpurea, Tolyposporium sp., Mortierella alpina, and Mortierella isabellina are a few examples of species of fungi that may collect lipids.Although several fungi may produce lipids, the majority of fungi are studied primarily for their capacity to create specific lipids such as docosahexaeneoic acid (DHA), gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) 62 .
Bio-guided isolation of the secondary metabolites of the methanol extract of A. pseudodeflectus AUMC 15761 led to the purification of two active compounds-methyl ferulate and oleic acid.Their structures were determined by comparing their NMR and HR-EI-Ms data with that available from the literature 15,39,42 .It is noteworthy that this is the first report of producing oleic acid and methyl ferulate from A. pseudodeflectus.
Methyl ferulate and oleic acid in this study were found to have a significant antibacterial potential against gram positive bacteria, showing the best activity against B. subtilis and S. aureus, while E. coli and S. epidermidis were not affected by either compound in the present study.In the study, the MICs for methyl ferulate and oleic acid against B. subtilis and S. aureus were 79% and 69%, respectively, compared to the bacitracin standard, with MICs of 79% and 69%, and 58% and 40%, respectively.Similarly, the antibacterial activity of methyl ferulate against Shigella putrefaciens was determined 63 .Certain oils, including rose essential oil, have been proven to have antibacterial properties against S. aureus, and the efficacy against gram positive S. aureus was observed to be less than that of rifampin and gentamicin, with negligible MIC values 64 .Because of the observed sensitivity of gram-positive bacteria to the presence of one phenolic hydroxyl group in methyl ferulate, its antibacterial mechanism was taken into consideration 42,65 .Although the exact mechanism of the antibacterial activity of fatty acids is unknown, it is thought that their functional nature is connected to the permeability, membrane disruption, and fatal changes in the bacterial cytoplasm.As a result, rupture or alteration of the membranedependent conduction systems may occur 22,66 .Escherichia coli, a normally resistant bacterium, becomes very susceptible to the bactericidal effects of fatty acids if the lipopolysaccharide outer membrane is destroyed using ethylenediaminetetraacetic acid.As gram-negative bacteria, they are protected by their outer lipid membrane from the corrosive effects of oleic acid 67 .
Methyl ferulate has been reported to have antioxidant activity (% DPPH), with IC 50 values of 73.213 11.20 µM, respectively 41 .Considering that, the principal mode of action of phenolic antioxidants is believed to be the scavenging of free radicals 68 .Due to the presence of one phenolic hydroxyl group in methyl ferulate, its antioxidant mechanism was taken into consideration 42,65 .Antioxidant activity (% DPPH) of OA with IC 50 values of 104.178 ± 9.53 µM has been reported in the literature 69 .Methy ferulate is a new natural antibacterial agent with strong efficacy and low toxicity.It has great potential for applications in food preservation 63 .Oleic acid, which accounts for about 80% of the total fatty acids in virgin olive oil, has recently become an often used substance to protect food from oxidizing 24 .
Oleic acid and methyl ferulate both had positive docking results against several Staphylococcus aureus protein targets, based on the docking results.The development of hydrogen bonding interactions with the active site of the Staphylococcus aureus target proteins under investigation may be responsible for the high docking scores of these two compounds.These findings shed additional light on the significance of the chemicals that have been identified as potential candidates for antibacterial medication.

Materials and chemicals
For the extraction, fractionation, and column chromatography, the organic solvents used were supplied by El-Nasr Pharmaceutical and Chemical Co.(ADWIC), Egypt.The deuterated chloroform (CDCl 3 ) used for NMR analysis was purchased from Sigma-Aldrich.TLC pre-coated plates (F 254 & PF 254 ) and silica gel for column chromatography (70-230 and 230-400 Mesh) were provided by Merck (Darmstadt, Germany).

Strain isolation and preservation
Using the dilution plate technique 70 , the strain for this investigation was isolated from a soil sample collected from Egypt's Aswan Governorate.Before adding Czapek's Dox agar (CzA) to Petri plates, the soil solution was appropriately diluted.The cultures were then maintained for two weeks at 25 °C.To create pure cultures of the fungus, the developed colonies were purified on CzA utilizing the single spore isolation technique 71 .For morphological identification of the strain of Aspergillus, the fungus was inoculated on Malt Extract Agar (MEA), Czapek's Yeast Autolysate Agar (CYA), and CzA 72 , and incubated for 7 days at 25 °C.The fungus in this study was morphologically identified on the basis of its macroscopic and microscopic characteristics, following the relevant key of Aspergillus: section Usti 73 .This strain was deposited and designated as AUMC 15761 in the culture collection of the Assiut University Mycological Centre.DNA isolation was carried out 74 , and the PCR reaction was performed by SolGent Co., Ltd (Daejeon, South Korea) using SolGent EF-Taq and the universal primers ITS1 and ITS4 75 .DNASTAR (version 5.05) was used to generate the contiguous sequences of the species of Aspergillus used in this investigation.There are 20 sequences in the overall ITS dataset that were used for phylogenetic analysis, consisting of one outgroup sequence for Aspergillus creber NRRL 58,592 (NR_135442), the sequence for Aspergillus pseudodeflectus (AUMC 15761 in this manuscript), and 18 sequences from the genus Aspergillus: section Usti acquired from GenBank.MAFFT (version 6.861b) 76 with the default settings was used to align all sequences.Optimization of the alignment gaps and sparse uninformative characters was conducted by BMGE 77 .Maximum-likelihood (ML) and maximum-parsimony (MP) phylogenetic analyses were carried out using MEGA X (version 10.2.6) 78 , and 1000 replications 79 were employed to assess the robustness of the most parsimonious trees.The ideal nucleotide substitution model for ML analysis was identified using Modeltest 3.7's Akaike Information Criterion (AIC) 80 .After editing, the tree was saved in TIF format 81 .

Solid state fermentation (SSF) of lignocellulosic wastes
In order to determine how the selected isolate of A. pseudodeflectus produced an antibacterial chemical, five samples of agricultural waste were studied.The fermentation materials included barley bran (BB), date palm leaves (DPL), orange peels (OP), rice straw (RS), and wheat bran (WB).They were purchased from local marketplaces in Egypt's Assiut Governorate.Before being reduced to a size of 500 µm, they were cleaned with tap water to get rid of dirt and other impurities.As part of the pretreatment procedure, the samples were treated with 1.0% NaOH, thoroughly filtered, and then washed with tap water.They were then dried at 60 °C83 .In order to determine how the selected A. pseudodeflectus produced an antibacterial chemical, five samples of agricultural waste were studied.Separate Erlenmeyer flasks (500 mL) were filled with 10 g of the lignocellulosic material, and the residue was then wetted down by 88% with a citrate buffer (pH 5.0).The flasks were next incubated for seven days at 30 °C.

Extraction of bio-active compounds
Following the incubation time, the flask contents underwent 48 h of oven drying at 60 °C.The mycelial mat and solid substrate were stirred in 50 mL of methanol for 2 h at 180 rpm in each flask.The clear supernatant was obtained after centrifugation (10,000 rpm at 4 °C for 10 min).The volume of the methanol extract was then reduced by a rotary evaporator (Heidolph: Model reddot winner 2020; Germany).The sample was lyophilized into a powder using a freeze dryer (VirTis: Model 6 KBTES-55, NY; USA) 84 .

Antibacterial effect of the crude extracts
The agricultural waste-derived crude extract residue from each sample was dissolved in dimethyl sulfoxide (DMSO) at a 10% concentration.The antibacterial efficacy of the crude extract was assessed using the agar well diffusion technique 85

GC-MS analysis
This analysis was carried out at the Analytical Chemistry Unit (ACAL), Faculty of Science, Assiut University, Egypt.Before being injected into a GC-MS device (7890A-5975B; Thermo Scientific GC/MS; model ISQ; USA), with a nonpolar HP-5MS Capillary Standard column (30 × 0.25 × 0.25) mm, 0.5 g of the sample residue was dissolved in 5 mL of methanol and centrifuged for 15 min (10,000 rpm and 5 °C).The following was the cycle's parameters: oven program on at 120 °C for 5 min, 30 °C/min rising to 265 °C for 25 min, then 50 °C/ min increased to 280 °C for 5 min; run duration 48 min; post run 260 °C for 2 min; flow program 0.5 mL/min.for 10.9 min., and then 1 mL/min for 30 min.Equilibration time was 0.5 min, and the maximum temperature 280°C 84 .

Optimization of bio-active secondary metabolites production by A. pseudodeflectus AUMC 15761
For maximization of secondary metabolites production, the respective pH (4.0, 5.0, 6.0, 7.0, and 8.0), temperature (20, 25, 30, 35, and 40 °C), nitrogen source (peptone, yeast extract, sodium nitrate, ammonium chloride, and ammonium sulphate, each at 0.2%), incubation time (2, 4, 6, 8, up to 14 days) were adjusted using one factor at a time (OFAT) 84 .For the testing, a 10 g amount of wheat bran was placed in 500 mL Erlenmeyer flasks.Following the incubation time, the bio-active Secondary metabolites were extracted as described above and then used in further tests.Ten g portions of wheat bran were separately placed into 500 mL Erlenmeyer conical flasks, and the spore suspension (1.5 × 10 8 spore/mL) was prepared from a seven-day-old A. pseudodeflectus AUMC 15761 culture and added to each flask in a volume of 5.0 mL were used to inoculate the residue.The flasks were then kept under optimal production cultural conditions.Following the incubation time, the flask contents were extracted, and the dried extract was applied throughout the purification process.

Sample preparation for column chromatography
The residue was combined with an equivalent weight of silica gel powder and a trace amount of methanol was added to create a slurry prior to each step of purification.The extract was placed onto a vacuum liquid chromatography (VLC) column for fractionation after being dried and slurred.The last stage of purification was carried out by adding the obtained fractions to silica gel column chromatography 84 .

Vacuum liquid chromatography (VLC)
With 900 g of silica gel (230-400 Mesh), the entire crude extract was fractionated using a VLC column (5.0 × 120 cm).The n-hexane, Dichloromethane (DCM) and 0-100% gradients of DCM in MeOH (by adding 10.0% MeOH each time) were used in the fractionation process.A low-pressure evaporator was used to dry out the solvents after collecting a total of 250 mL of elutes.Fractions that produced the strongest antibacterial properties and have comparable spots were combined, condensed, and dried for use in the further purification process.

Thin layer chromatograph (TLC)
TLC was carried out on pre-coated silica gel F 254 plates.A series of solvents of increasing polarities were used for developing the spots.For visualization of the spots, the plates were subjected to a UV inspection (at 365 and 254 nm) and then sprayed with 10% (v/v) H 2 SO 4 in methanol, dried using a hot air drier, and heated to 110°C 84 .

Final purification of the active secondary metabolites
The fraction containing active chemicals was subsequently chromatographed on an open column (1.0 × 100 cm) equipped with a 35 g silica gel (70-230 Mesh).It was eluted in n-hexane gradients with 0-20% EtOAc (adding 1.0% EtOAc each time).TLC was used to detect 25 mL elutes using three mobile systems (n-hexane: EtOAc: 95: 5, 90: 10, and 80: 20).After combining and drying similar elutes, nine subfractions were obtained.The subfractions containing the most active compounds were uploaded over a second column (0.5 × 25 cm) packed with 15 g silica gel and a solvent system of n-hexane: acetone (95: 5) was used for elution.A 15 mL elutes were collected, subjected to TLC, and those containing nearly pure compounds were combined.Following the method of Siddiqui, et al. 86 , they were finally purified using preparative TLC plates (60 PF 254 ).The antibacterial test for the purified compound was performed as described above.

Spectroscopic NMR
The analysis was completed at the Micro-Analytical Unit (MAU) of Cairo University's School of Pharmacy in Giza, Egypt.Bruker Avance III HD 400 and 100 MHz spectrometers (Bruker Biospin, Rheinstetten, Germany) and NMR software Topspin 3.2 pl 6 were used to produce the 1 H and 13 C-NMR spectra.The internal reference standard was tetramethylsilane (TMS).An LTQ Orbitrap XL spectrometer was used to obtain the HR-ESI-MS data (Thermo Fisher Scientific; USA).

DPPH radical scavenging activity
Using the methods described by Yen and Duh 87 , a freshly prepared methanol solution containing 0.004% (w/v) of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was refrigerated at 10 °C in the dark.Pure compound concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µM methanol were utilized for the pure synthesized sample, respectively.The absorbance at 515 nm was estimated using a 40 µL fraction of the sample-containing methanol solution combined with 3 mL of the DPPH solution (Milton Roy, Spectronic 1201; Canada).The decrease in the absorbance at 515 nm was monitored continuously until it stabilized, with data obtained at 1 min intervals for 16 min.The absorbance of the DPPH radical without an antioxidant was measured, as was the absorbance of ascorbic acid, a reference chemical.The percentage inhibition (PI) of the DPPH radical was estimated using the Eq. ( 1): where AC is the control absorbance at time zero and AT is the sample absorbance plus DPPH at 16 min.The dose-response curve graphic plots were used to estimate the 50% inhibitory concentration (IC 50 ), which is the concentration necessary to inhibit the DPPH radical by 50%.The experiment was carried out in triplicate.

Computational methods
The crystal structures of dihydrofolate reductase (PDB code: 5ISP 32 ), pyruvate kinase (PDB code: 5OE3 33 ), and sortase A (PDB code: 2MLM 34 ) were obtained and utilized as templates for all in-silico computations.To prepare these proteins, all inhibitors, ions, and water molecules were removed.Modeller software was employed to construct all missing residues 88 .In addition, the H + + website was utilized to inspect the protonation states of the studied targets 89 .All missing hydrogens were added.The structure of the investigated inhibitors was manually

Figure 3 .
Figure 3. Antibacterial activity (as inhibition zone), of the crude extract of different lignocellulosic wastes fermented by A. pseudodeflectus AUMC 15761 under SSF.Mean values (± SD) on the bars of the graph with different letters are significantly different (p ≤ 0.05; n = 3).

Figure 4 .
Figure 4. GC-MS chromatogram of the methanol extract wheat bran fermented by A. pseudodeflectus AUMC 15761 under SSF.

Figure 5 .
Figure 5. Optimization of fermentation conditions of the bio-active secondary metabolites using wheat bran fermented by A. pseudodeflectus AUMC 15761 under SSF.Mean values (± SD) for bars on the graph with different letters are significantly different (p ≤ 0.05; n = 3).

Figure 18 .
Figure 18.(i), Superimposed structures of the experimental mode (in cyan) and the anticipated docking pose (in grey) (ii), 2D representation of the predicted binding modes of the co-crystalized ligands with the active site of different protein targets of Staphylococcus aureus.

Figure 19 .
Figure 19.2D molecular interaction of the obtained compounds towards the active site of different protein targets of Staphylococcus aureus.

Table 1 .
GC-MS spectral analysis of the chemical compounds detected in the methanol extract of wheat bran fermented by A. pseudodeflectus AUMC 15761 under SSF.

Table 2 .
The antibacterial potential of the fractions obtained by VLC column of wheat bran fermented by A. pseudodeflectus AUMC 15761 under SSF.*Mean values (± SD) with different letters are significantly different (p ≤ 0.05; n = 3).

Table 6 .
Antibacterial activity of pure methyl ferulate produced by Aspergillus pseudodeflectus AUMC 15761 from wheat bran in SSF compared to the Bacitracin antibacterial standard.For the data above, r equals 0.92, 0.98.This represents a very strong positive correlation.M = mean; %MB = mean value divided by the standard value.

Table 7 .
Antibacterial activity of pure oleic acid produced by A. pseudodeflectus AUMC 15761 from wheat bran in SSF compared to the Bacitracin antibacterial standard.For the data provided above, r equals 0.833, 0.883.This represents a very strong positive correlation.

Table 8 .
Computed docking scores of the obtained compounds towards different protein targets of Staphylococcus aureus.

and molecular identification of the Aspergillus strain
, using 50 µL in each 5 mm well for Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, and Staphylococcus epidermidis ATCC 12228.Bacitracin (10 U) and Piperacillin/Tazobactam 10: 1 (110 µg/disc), served as a positive control.This test was performed after each stage of purification.