Antifungal efficacy of microencapsulated miR166 and miR159 oligoDNAs through whey protein concentrate (WPC) as coated protein against Verticillium dahliae

Antifungal efficacy of microencapsulated miR166 and miR159 oligoDNAs through whey protein concentrate (WPC) as coated protein against Verticillium dahliae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antifungal efficacy of microencapsulated miR166 and miR159 oligoDNAs through whey protein concentrate (WPC) as coated protein against Verticillium dahliae Mahboobeh Nouri, Mojtaba Keykhasaber, Mahdi Pirnia, Mohammad-Amin Miri, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7525311/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Verticillium dahliae Kleb. is one of the most destructive fungal pathogens, infecting susceptible plants through their roots and disrupting vascular tissues. As soil-borne diseases pose a persistent threat to agriculture, the development of sustainable biocontrol strategies has gained significant attention. Among these, biomolecule-based approaches—particularly the use of oligonucleotides—have emerged as an innovative and eco-friendly alternative for plant disease management. In this study, we evaluated the antifungal potential of microencapsulated double-stranded oligoDNAs (corresponding to microRNA166 and microRNA159) produced via electrospraying with a whey protein concentrate (WPC) polymer. The encapsulated oligoDNAs were tested against V. dahliae by mixing 10 µL of fungal spores (1×10⁶ spores/mL) with 5 µL of the microcapsule solution, followed by culturing on potato dextrose agar (PDA). Our findings demonstrate that both oligoDNA 159 and oligoDNA 166 significantly inhibit fungal growth. Notably, encapsulation with WPC enhanced their antifungal efficacy, suggesting that this technology improves oligoDNA stability and enables controlled release. These results highlight the potential of oligonucleotides as effective biocontrol agents against fungal pathogens. Furthermore, encapsulation presents a promising strategy to optimize their delivery and application in sustainable agriculture. This study provides compelling evidence for the use of microencapsulated oligoDNAs in fungal disease management, offering a viable, environmentally safe alternative to conventional chemical treatments. Future research should explore field applications and long-term effects to validate their practical use in crop protection. Antisense oligonucleotides microRNA plant disease control fungal diseases biological control Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The growing global population and limited natural resources have increased the demand for sustainable agricultural production [ 1 ]. However, the outbreak of plant diseases caused by pathogens such as fungi, bacteria, and viruses is considered one of the major challenges in this field [ 2 ]. One of the most significant fungal pathogens, responsible for vascular wilt in over 400 plant species worldwide, is Verticillium dahliae Kleb. This soil-borne fungus attacks susceptible plants through the roots, damaging their vascular tissues [ 3 , 4 ]. It causes millions of dollars in annual losses worldwide [ 5 ]. Identification of resistant cultivars/genotypes to Verticillium is one of the important strategies used in disease management. Some researchs have been focused on evaluating resistance to Verticillium and/or detecting resistance gene analogs (RGAs) in cultivars [ 4 , 6 ]. Recent studies have led to the development of novel and low-risk methods for plant disease management. Powder and liquid formulation of potent yeast strains showed high efficacy in attenuating disease severity and toxin production of Aspergillus flavus [ 7 ]. Essential oils showed great potential for controlling plant pathogens, but due to their volatility, their effectiveness is lost over time [ 8 ]. Encapsulation of essential oils in biodegradable, edible coated protein such as zein, has been shown to increase thier efficacy and reduce disease severity [ 9 , 10 , 11 ]. Moreover, the use of fungal metabolites for the green synthesis of zinc oxide nanoparticles have also raised hopes for the control of plant pathogens [ 12 ]. The development of biological control strategies based on biomolecules such as nucleic acids has emerged as an innovative and environmentally friendly approach for managing plant diseases [ 13 ]. Numerous studies have demonstrated that the topical application of dsRNA on plants can effectively prevent the spread of a wide range of plant diseases caused by fungi, oomycetes, and viruses [ 13 , 14 ]. For example, the use of dsRNA has been successful in controlling fungal diseases such as Fusarium and Botrytis in crop plants [ 15 , 16 ]. The mechanism of action of dsRNA is based on silencing essential pathogen genes, where these molecules interfere with the expression of key genes, thereby inhibiting the growth and proliferation of pathogens [ 1 ]. One of the main limitations in the practical application of dsRNA is its low stability in the environment. Environmental factors such as light, temperature, and degrading enzymes can quickly damage dsRNA and reduce its efficacy [ 1 ]. Additionally, its design and synthesis are highly complex and expensive. In contrast, the structure of DNA oligonucleotides is very similar to that of RNA molecules, which are naturally used by cells, making DNA oligos a viable low-cost alternative. Their production and purification are inexpensive and easy, and they are highly stable [ 17 ]. The DNA oligonucleotide technology was first successfully used in plant cells to modify the expression of the transcription factor SUSIBA2 [ 18 ]. The researchers' results demonstrated that antisense oligonucleotides were effectively delivered into the leaves and reached the nuclei and chloroplasts [ 18 , 19 ]. To enhance the stability of oligonucleotides when applied in plants, various methods such as encapsulation have been investigated [ 20 ]. Nanocapsulation systems create a protective coating around DNA oligonucleotides, shielding them from degrading factors and enabling their controlled release at the target site [ 20 , 22 ]. Given the sensitivity of bioactive compounds, various encapsulation methods have been developed. The electrospraying process, or microencapsulation using electrohydrodynamic processes, is a simple and effective method for preserving and enhancing the bioavailability of active compounds [ 23 ]. In the electrospraying process, various proteins—including whey protein isolate (WPI), soy protein isolate, egg albumin, collagen, gelatin, zein, wheat gluten, and casein—have been investigated and evaluated [ 23 ]. Among these, whey protein concentrate (WPC), as one of the main by-products of the dairy industry, holds particular significance. WPC contains a mixture of proteins, notably beta-lactoglobulin, alpha-lactalbumin, and serum albumin, and is recognized as a rich source of natural emulsifiers [ 24 ]. It has been proposed as a suitable candidate for encapsulation via electrohydrodynamic processes due to its excellent electrospray ability and effective performance as a carrier for bioactive compounds [ 25 , 26 , 27 , 28 ]. Previous research has established that two virulence genes in Verticillium dahliae - the Ca²⁺-dependent cysteine protease (Clp-1) and isotrichodermin C-15 hydroxylase (HiC-15) - are critical for fungal pathogenicity. These genes are specifically targeted by miR166 and miR159, respectively, which were identified in resistant cotton cultivars [ 29 ]. Building on this finding, the current study investigates the antifungal potential of microencapsulated double-stranded oligoDNAs (corresponding to microRNA166 and microRNA159) produced via electrospraying using whey protein concentrate (WPC), an edible and biodegradable polymer, for controlling V. dahliae growth. Materials and Methods Preparation of WPC Solution WPC solutions (30% w/v) were prepared by dissolving the required amount of WPC in sterile distilled water with gentle stirring at room temperature. The solutions were kept at room temperature for 24 hours, and their pH was adjusted to 6 [ 30 ]. Preparation of Oligo DNA-Loaded WPC Nanoparticles via Electrospraying To encapsulate Oligo DNA within WPC nanoparticles, 20 ng/µL of Oligo DNA was added to the WPC solution [ 13 ]. The solutions were stirred at room temperature for 30 minutes and then subjected to electrospraying. Microcapsules were prepared using an ES1000 electrospray device (Fanem Co., Iran). A 5 mL syringe was filled with the electrospray solution and mounted on the device. The needle-to-collector distance was set to 14 cm. Oligo DNA-loaded WPC microcapsules were electrosprayed at 18 kV with a flow rate of 0.5 mL/h for 10 hours at room temperature [ 30 ]. Scanning Electron Microscopy (SEM) The morphology of microcapsules was analyzed using a scanning electron microscope (EM-8000F, KYKY, Germany) after coating with a gold-palladium mixture (20 nm thickness) via sputter coating. The average diameter of electrosprayed microcapsules was determined by randomly measuring 100 microcapsules from each SEM image using ImageJ software, with an accelerating voltage of 20 kV [ 31 ]. Transmission Electron Microscopy (TEM) To confirm encapsulation and the presence of oligoDNA, samples were analyzed using transmission electron microscopy (TEM) with negative staining. In this technique, samples are coated with a high electron-density material (such as uranyl acetate or phosphotungstic acid). This staining agent penetrates the surrounding sample space, causing areas with lower electron density (such as microcapsules) to appear as brighter regions against a dark background. Consequently, the white and dark spots in TEM images represent differences in the sample's electron density [ 32 ]. In this study the morphology of microcapsules and Oligo DNA loading efficiency were evaluated using a Philips EM208S 100KV TEM (Netherlands). Particle Size Distribution and Hydrodynamic Diameter The average hydrodynamic diameter (dls) and particle size distribution of freshly diluted samples were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK) [ 33 ]. Fourier Transform Infrared (FTIR) Spectroscopy FTIR spectroscopy is used to examine the chemical structure of samples. Different peaks in the spectrum represent different functional groups in the sample [ 46 ]. Polypeptide and protein repeating units exhibit specific IR absorption bands for amides A and B, as well as amides I to VII. Among these, amide I (C = O stretching vibration at 1580–1720 cm⁻¹) and amide II (N–H bending vibration at 1450–1600 cm⁻¹) are the two key vibrational bands of the protein backbone. However, changes in secondary structure are more clearly observed in the amide I absorption bands due to the minor contributions of C–N stretching, C–C–N deformation, and in-plane N–H bending [ 34 , 35 , 36 ]. So, to study the chemical structure of WPC microcapsules loaded with Oligo DNA, FTIR analysis was performed [ 27 ]. All measurements were carried out using a Thermo Nicolet spectrophotometer (AVATAR 370 FTIR, USA) in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ [ 37 ]. X-ray Diffraction (XRD) Analysis The X-ray region of the electromagnetic spectrum lies between gamma rays and ultraviolet wavelengths. This spectral region can be used to obtain information about the structure and type of material. In this study, X-ray diffraction was employed to assess the mixing and dispersion of different compounds and to determine the physical state of Oligo DNA within the electrosprayed whey protein microcapsules. The samples were scanned using an XRD analyzer (Unisantis, XMD-300) with CuKα radiation (λ = 1.5418 Å) at an incident angle (2θ) ranging from 5° to 40° at room temperature. The X-ray diffraction spectra were evaluated comparatively [ 38 ]. Differential Scanning Calorimetry (DSC) DSC measurements were performed using a Perkin Elmer STA6000 differential scanning calorimeter at a heating rate of 10°C/min. Samples weighing 5 mg were heated under a nitrogen (N₂) atmosphere from 25°C to 400°C [ 31 ]. Thermogravimetric Analysis (TGA) TGA provides essential information on thermal stability, decomposition temperature, and residual mass of a sample by measuring weight changes as a function of temperature. Differential scanning calorimetry (DSC) is another powerful tool that offers insights into thermal transition events by measuring the heat flow associated with these processes. In this study, simultaneous thermal events were measured using both TGA and DSC techniques. Additionally, derivative thermogravimetry (DTG) was employed as a function of applied temperature to provide further data and more precise insights into thermal decomposition processes. To evaluate the thermal stability of the microcapsules, a Perkin Elmer STA6000 instrument was used. Approximately 5 mg of the sample was placed in a platinum crucible and heated under a nitrogen flow of 40 mL/min at a heating rate of 10°C/min from 25°C to 750°C. Pore Size and Volume Measurement (BET) The pore size analysis of the microcapsules was performed using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods based on nitrogen (N₂) adsorption-desorption isotherms. A BELSORP-mini II instrument was used for this analysis. This model is employed to determine the specific surface area of porous and powdered materials [ 39 ]. In this analysis the nitrogen adsorption-desorption isotherm provides information about the surface area, pore volume, and pore size distribution of the sample. The difference between the adsorption (ADS) and desorption (DES) curves indicates the presence of hysteresis, which is characteristic of a mesoporous structure [ 40 , 41 ]. Inhibition of Verticillium Fungal Growth A Verticillium spore suspension was prepared at a concentration of 1×10⁶ spores/mL. The prepared microcapsules were dissolved in water, and 10 µL of the fungal spores were mixed with 5 µL of the microcapsule solution. After 30 minutes of incubation, the mixture was cultured at the center of a Petri dish containing PDA [ 13 ]. The treatments included Verticillium spores alone (control), Verticillium spores mixed with pure WPC microcapsules, Verticillium spores mixed with encapsulated oligoDNA159 + oligoDNA166, Verticillium spores mixed with unencapsulated oligoDNA159 + oligoDNA166. The samples were monitored for 12 days, and colony growth rate was measured using ImageJ software and the results were used for t-test analysis (P = 0.05). Results Morphology and Diameter Determination by SEM SEM images of the electrosprayed structures for WPC, and WPC loaded with oligoDNA159 and oligoDNA166 are presented in Fig. 1. The images reveal that in all treatments, spherical microcapsules with smooth surfaces were successfully obtained via electrospraying from an aqueous solution containing 30% (w/w) protein concentration. The mean diameters of WPC microcapsules without oligonucleotides, WPC microcapsules containing oligoDNA159, and WPC microcapsules containing oligoDNA166 were 0.20831 µm, 0.35886 µm, and 0.26744 µm, respectively. Transmission Electron Microscopy (TEM) Analysis TEM images of WPC microcapsules with and without oligoDNA have been presented in Fig. 2. The images reveal that WPC particles without oligoDNA appear as brighter spots against the dark background. These particles exhibit spherical or near-spherical morphology with small sizes ranging approximately 50–200 nm (based on the 200 nm scale bar). These likely represent successfully formed individual microcapsules. The images demonstrate relative heterogeneity in particle size and distribution, potentially arising from intermolecular protein interactions. The simultaneous presence of individual particles and larger aggregates indicates incomplete system uniformity, consistent with DLS results. The absence of uniform layers on particle surfaces suggests no active material (oligonucleotides) in the system. WPC microcapsules containing oligoDNA also display spherical or near-spherical morphology. Negative staining causes them to appear as brighter spots against the dark background, likely representing successfully formulated individual microcapsules. Since DNA molecules primarily consist of carbon, oxygen, nitrogen, and phosphorus atoms (which have low electron density compared to staining agents), DNA-containing regions are difficult to distinguish from the dark background in TEM images. The small size of oligonucleotides (21 nucleotides) makes them nearly undetectable compared to microcapsule dimensions. Larger aggregates and particle clusters are observable in some image areas, potentially resulting from strong interactions between whey protein and oligonucleotides that lead to formation of larger structures. Increased particle size compared to normal whey protein dimensions suggests the formation of oligonucleotide-containing capsules. Some particles exhibit uniform surface layers, indicating successful loading of active material (oligonucleotides). These findings align with previous research and provide visual confirmation of the encapsulation process and oligonucleotide incorporation within the WPC microcapsules. The TEM analysis complements the DLS data by offering direct morphological evidence at the nanoscale level. Particle Size and polydispersity index (PDI) PDI is among the most important characteristics of nano-carrier systems. The mean particle size and polydispersity index of the samples are listed in Table 1 . The uniformity (homogeneity) of droplet size in emulsions can be inferred from the polydispersity index. The PDI values for pure WPC, WPC containing oligoDNA159, and WPC containing oligoDNA166 were 0.69, 0.52, and 1.0, respectively. The results indicated that nearly all emulsions exhibited heterogeneous populations with multiple particle populations in the samples. Figure 3 shows the particle size distribution histogram (number-based) obtained by dynamic light scattering. All three graphs demonstrate an asymmetric distribution with the majority of particles concentrated in the 100–1000 nm range. A gradual decrease in particle count with increasing size was observed, indicating the presence of fewer larger aggregates. This histogram confirms that most particles in the samples are small, while larger aggregates contribute very little numerically. The broad standard deviation indicates relative heterogeneity in particle size, which is consistent with the high PDI values of the samples. Considering the objective of controlled release of materials, this composition is likely suitable. The combination of predominantly small particles with limited large aggregates appears appropriate for controlled release applications, despite the observed size heterogeneity. Fourier Transform Infrared Spectroscopy (FTIR) The amide bands of WPC were studied to analyze the encapsulation of oligoDNA and its effect on the molecular organization of the capsules. In the spectra corresponding to nucleic acids, the peaks in the 1600–1800 cm⁻¹ region are typically attributed to the stretching vibrations of carbon-oxygen double bonds (C = O) in carbonyl groups. Strong peaks in this region indicate the presence of a large number of carbonyl groups in the nitrogenous base molecules of DNA. The FTIR spectra of WPC powders revealed characteristic functional groups, including Amine or N–H stretching (3433.11 cm⁻¹), Aliphatic C–H stretching (2925.86 and 2862.21 cm⁻¹), Amide I (C = O stretching) (1643.26 cm⁻¹), Amide II (N–H bending and stretching) (1525.61 cm⁻¹), C = C bending in aromatic rings of phenolic compounds (1461.96 cm⁻¹). The IR spectrum exhibited characteristic bands at 3458.19, 2075.30, and 1637.48 cm⁻¹ for oligoDNA159, at 3477.47 and 1650.98 cm⁻¹ for oligoDNA166. The observed bands at 1637.48 cm⁻¹ and 1650.98 cm⁻¹ correspond to stretching vibrations of carbon-oxygen double bonds (C = O) in the carbonyl groups of DNA nitrogenous bases. Figure 4 presents the characteristic FTIR bands of oligoDNA159, oligoDNA166, WPC, and WPC loaded with oligoDNA159 and oligoDNA166. The microcapsules showed the following diagnostic absorption bands: N-H stretching (amine) at 3435.04 cm⁻¹, Aliphatic C-H stretching at 2923.93 and 2856.43 cm⁻¹, Amide I (C = O stretching) at 1643.26 cm⁻¹, Amide II (N-H bending and stretching) at 1543.86 cm⁻¹, C = C bending in aromatic rings of phenolic compounds at 1461.96 cm⁻¹. As shown in the Fig. 4, no new spectrum was observed for WPC particles loaded with oligoDNA. However, changes in the position and intensity of the peaks were observed in the spectra of microcapsules containing oligoDNA compared to the pure spectra of WPC. The position of the amide I bands in microcapsules containing oligoDNA159 remained unchanged, whereas this band shifted toward a higher wavenumber in microcapsules containing oligoDNA166. A shift toward higher wavenumbers was also observed in the amide II band in the spectra of microcapsules containing both types of oligoDNA, indicating some molecular changes in the whey protein as a result of microcapsule formation. Additionally, some bands in the spectra of microcapsules containing oligoDNA were absent, likely due to the low concentration of oligoDNA inside the microcapsules or because they were overshadowed by the stronger peaks of the whey protein. This suggests that the oligoDNA is physically entrapped within the particles and that no strong chemical bonds exist between them. Furthermore, differences in the position and intensity of the bands were observed in the pure spectra of oligoDNA159 and oligoDNA166, which are likely due to variations in the base types present in the two sequences. Table 1 The mean particle size and polydispersity index of the samples Sample Polydispersity Index (PDI) Hydrodynamic Diameter (nm) WPC 0.690 434.2 MC-WPC oligo159 0.522 754.4 MC-WPC oligo166 1.000 2075 X-ray Diffraction (XRD) Analysis X-ray diffraction patterns are used to examine the crystalline structure of biopolymeric materials. Figure 5 shows the XRD patterns of WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. Two distinct peaks were observed in the pure WPC diffractogram at 8.51° 2θ (d-spacing = 10.3 Å) and 19.58° 2θ (d-spacing = 4.52 Å). The first peak is attributed to the interhelical packing distance, while the second peak corresponds to the d-spacing of α-helical structures. In this study, the microcapsule structure appears predominantly amorphous, with possible small nanocrystalline regions, as no sharp and well-defined peaks indicative of high crystallinity were detected. The first diffraction peaks of oligoDNA159 and oligoDNA166 in WPC were observed at 8.33° 2θ (interlayer spacing of 10.6 Å) and 8.22° 2θ (interlayer spacing of 10.7 Å), respectively, while the second diffraction peaks appeared at 19.64° 2θ (interlayer spacing of 4.51 Å) and 19.83° 2θ (interlayer spacing of 4.4 Å), respectively. The peak intensities for oligoDNA159 remained nearly unchanged, whereas those for oligoDNA166 decreased. Additionally, the first peak (2θ = 8.51°) shifted to a lower angle in both treatments, and the second peak (2θ = 19.58°) shifted to a higher angle in both treatments. Thermogravimetric Analysis (TGA) Figure 6, illustrates the thermal and degradation behavior of WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. As shown in the figure, the TGA and DSC curves for all samples are nearly identical and can be divided into several distinct stages: -First Stage (Moisture Loss): The initial weight loss in the TGA curve corresponds to moisture removal, with reductions of 7.3%, 8.32%, and 7.81%, respectively. This initial weight loss occurs at approximately 30–150°C and is attributed to the evaporation of free water and surface moisture. -Second Stage (Solvent and Bound Water Removal): The second stage in the TGA curve corresponds to the removal of solvents and bound water, with weight losses of 3.06%, 3.06%, and 2.04%, respectively. This stage occurs in the temperature range of 150–250°C and is likely associated with water bound to the protein structure and residual solvents from the production process. -Third Stage (Main Protein Structure Degradation): The major weight loss (60.85%, 62.67%, and 64.88%, respectively) occurs between 250–400°C, likely indicating the denaturation and breakdown of WPC—particularly β-lactoglobulin and α-lactalbumin—as well as the cleavage of disulfide bonds between protein chains and the degradation of encapsulated oligonucleotides. -Fourth Stage (Resistant Residue Degradation): The final weight loss (6.41%, 3.74%, and 3.06%, respectively) occurs between 400–800°C, representing the gradual decomposition of thermally resistant structures and the carbonization of remaining organic residues. The remaining approximately 22% at the end of the analysis (800°C) is likely related to inorganic compounds and highly stable carbon structures that do not decompose even at high temperatures. Samples containing oligonucleotides exhibit less weight loss (60–62%) compared to the oligonucleotide-free sample (~ 64%). Additionally, the initial moisture content and water release were similar across all samples, indicating that the thermal differences are primarily due to molecular structure and internal composition. The higher thermal stability of the oligonucleotide-containing samples is likely attributed to stronger interactions between the protein and oligonucleotides, which enhance heat resistance. Differential Scanning Calorimetry (DSC) DSC provides insights into thermal transition events by measuring the heat flow associated with these events in the sample. Figure 7 shows the DSC analysis curves for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. The temperature range of 30 to 150°C, where a small endothermic peak is observed at around 39°C. This peak indicates energy absorption for the evaporation of surface moisture and free water from the microcapsules. This finding aligns with the initial weight loss in the TGA curve within the same temperature range. The temperature range of 150 to 300°C, where the curve shifts downward (endothermic), indicating the onset of phase transitions in the protein structure. A major endothermic valley is observed at 342.8°C, 371.9°C, and 381.9°C, respectively, indicating significant endothermic processes. These include thermal denaturation of whey proteins, cleavage of chemical bonds within the protein structure, and thermal degradation of oligonucleotides. This endothermic valley closely aligns with the major weight-loss stage in the TGA analysis (observed in the 250–400°C range). A sharp increase in heat flow occurs at temperatures above 400°C, where the curve shifts continuously upward (exothermic). This trend suggests more complex thermal processes, and likely final decomposition of residual materials. This behaviour correlates with the final weight-loss stage in the TGA analysis. BET (Brunauer–Emmett–Teller) analysis The specific surface area for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules that was calculated using the slope and intercept of the BET linear plot, where a steeper slope corresponds to a higher surface area (Table 2 ). Pure WPC microcapsules exhibited a specific surface area of 4.8283 m²/g, WPC microcapsules with oligoDNA159 showed a significantly reduced surface area of 0.55338 m²/g, and WPC microcapsules with oligoDNA166 had an intermediate value of 2.9665 m²/g. The Barrett-Joyner-Halenda (BJH) model was employed to evaluate the actual pore volume of the samples. The BJH plots are presented in Fig. 8, where X-axis: Pore radius (logarithmic scale, 1–100 nm), Y-axis: Derivative of pore volume with respect to pore radius, representing the pore size distribution. All samples exhibited a dominant peak in the 1–3 nm range, indicating minimal variation in pore size across samples. This peak confirms the presence of micropores ( 50 nm), confirming a predominantly micro-mesoporous structure. The pore volume measurements revealed significant differences among the samples: Pure WPC microcapsules (without oligoDNA) exhibited a pore volume of 0.0078513 cm³/g, WPC microcapsules containing oligoDNA159 showed a dramatic reduction to 0.001442 cm³/g (81.6% decrease), WPC microcapsules containing oligoDNA166 demonstrated an intermediate value of 0.0047458 cm³/g (39.6% decrease compared to pure WPC). This substantial decrease in pore volume following oligoDNA incorporation suggests potential pore blockage by oligonucleotide molecules, structural modifications in the protein matrix, and altered packing density of the microcapsule walls. The more pronounced effect observed with oligoDNA159 (compared to oligoDNA166) may indicate sequence-dependent interactions between the oligonucleotides and whey protein components, warranting further investigation into the molecular mechanisms underlying these textural changes. Table 2 Presents the specific surface area for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules that was calculated using the slope and intercept of the BET linear. Parameter WPC (No oligoDNA) WPC + oligoDNA159 WPC + oligoDNA166 BET Surface Area (m²/g) 4.8283 0.55338 2.9665 BJH Pore Volume (cm³/g) 0.0078513 0.001442 0.0047458 Dominant Pore Size (nm) 1.64 1.21 1.85 All three samples exhibit Type VI adsorption isotherms, consistent with IUPAC classification, indicating the presence of a mesoporous structure (pores ranging from 2 to 50 nm). The presence of a hysteresis loop between the adsorption and desorption curves suggests a diverse range of pore shapes and sizes, as well as varying mechanisms for pore filling and emptying. Analysis of antifungal activity The colony growth rate of the fungus in each experimental treatment was monitored in 2 days intervals over a 12-day period after cultivation on PDA, with measurements taken using ImageJ software (Fig. 9). After 12 days, the average colony diameter was recorded, and a t-test analysis (P = 0.05) revealed that treatments containing either encapsulated or unencapsulated oligoDNA 159 + oligoDNA 166 exhibited significantly smaller colony diameters—2.5 cm and 2.8 cm, respectively—compared to the pathogen control (3.2 cm). Furthermore, the growth reduction rate was higher in treatments with encapsulated oligoDNA 159 + oligoDNA 166 compared to unencapsulated samples (Figs. 9, 10). These results demonstrate the inhibitory effect of oligoDNA on fungal growth, suggesting that encapsulation enhances efficacy—likely due to improved stability and controlled release of the oligoDNA. Discussion This study aimed to evaluate the antifungal efficacy of whey protein concentrate (WPC) microencapsulated double-stranded oligoDNAs (mimicking miR166 and miR159) against Verticillium dahliae . The oligoDNAs were prepared using electrospraying technology with WPC, an edible and biodegradable polymer matrix. The results of this study clearly demonstrate the ability of oligo DNA 159 and oligo DNA 166 to inhibit the growth of V. dahliae , which may be related to the RNA interference (RNAi) mechanism [ 42 , 43 ]. Our findings are consistent with the studies of Zhang et al. [ 29 ], as they showed that cotton plants increase the production of microRNA166 and microRNA159 in response to infection with V. dahliae and transfer these microRNAs to the fungal hyphae to perform specific gene silencing. They found that the Clp-1 and HiC-15 genes are targeted by miR166 and miR159, respectively [ 29 ]. They also observed that mutant strains of Verticillium in which the Clp-1 gene was inactivated had normal colony morphology but inhibited microsclerotia formation. In contrast, mutant strains in which the HiC-15 gene was inactivated had severely inhibited hyphal growth but normal microsclerotia formation, consistent with the findings of this study that fungal colony growth was reduced compared to the control. In a study, using the encapsulation method by incorporating dsRNA into double layer hydroxide (LDH) nanosheets known as "BioClay", viral protection was applied and the stability of dsRNA on tobacco leaf surfaces increased from 5 to 20 days [ 44 ]. Also, Whitfield et al. (45) reported that the persistence of dsRNA in soil was increased by up to 3 weeks through the use of a poly(2-(dimethylamino)ethyl acrylate) analogue [ 45 ]. WPC has been proposed as a suitable candidate for encapsulation via electrohydrodynamic processes due to its excellent electrospray ability and effective performance as a carrier for bioactive compounds [ 25 , 26 , 27 , 28 ]. In current study, the increased efficacy in oligo DNA encapsulated samples with WPC indicates that the encapsulation technology improves the stability of the oligo DNA and enables its controlled release. This results in longer access and facilitated transmission of the oligo DNA to fungal cells and consequently increased efficacy in growth inhibition. The lack of significant effect of pure WPC microcapsules on V. dahliae colony growth also confirms that the observed antifungal effect is due to the oligo DNA itself and not simply the WPC biochemical chracterestics. Overall, this research provides strong evidence for the potential of oligo DNA as a biological antifungal agent, and oligo DNA encapsulation is a promising strategy to enhance its efficacy and application in the control of fungal diseases. Declarations Funding This work was supported by the financial support of the Research Vice-Chancellor of University of Zabol under grant number UOZ-GR-9618-129. Acknowledgement We would like to acknowledge the financial support of the Research Vice-Chancellor of University of Zabol for supporting us in conducting this research. Statement of Conflicting Interests The authors have no competing interests to declare that are relevant to the content of this article. Authors’ Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mahboobeh Nouri, Mojtaba Keykhasaber, Mohammad-Amin Miri, Mahdi Pirnia, Shirahmad Sarani, and Hoseyn Kamaladini. 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Study on starch-protein interactions and their effects on physicochemical and digestible properties of the blends. Food Chem. 2019;280:51–8. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7525311","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":520689130,"identity":"52bbb469-aa91-47a0-93bd-e41726a31d45","order_by":0,"name":"Mahboobeh Nouri","email":"","orcid":"","institution":"University of Zabol","correspondingAuthor":false,"prefix":"","firstName":"Mahboobeh","middleName":"","lastName":"Nouri","suffix":""},{"id":520689131,"identity":"56c2637d-3117-4edf-a21f-6082cc1632aa","order_by":1,"name":"Mojtaba 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1","display":"","copyAsset":false,"role":"figure","size":465134,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/e8eea00ac5d473de96723d1f.png"},{"id":92324745,"identity":"67db2241-c053-4e50-85f4-64a2f1b77f62","added_by":"auto","created_at":"2025-09-27 17:30:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93434,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/410bd5fe33b4cbbc181ad2ca.jpg"},{"id":92325106,"identity":"27f004a4-750a-48a6-a80c-e69c4c7b7106","added_by":"auto","created_at":"2025-09-27 17:38:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202848,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/c9550d49eb7f21fb3385e565.jpg"},{"id":92325107,"identity":"a4926e6f-8cec-48a0-b752-446a5f46c8f6","added_by":"auto","created_at":"2025-09-27 17:38:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":208590,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/fc132ca49eb3650d1821a77d.jpg"},{"id":92325290,"identity":"bafbad36-c96c-4e60-9fc6-74f38e10c450","added_by":"auto","created_at":"2025-09-27 17:46:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87631,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/a267e93df7ceb6f0d592a4e6.jpg"},{"id":92324777,"identity":"fd144e22-4d41-49b7-9cbd-0e5f0b5943e6","added_by":"auto","created_at":"2025-09-27 17:30:53","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":938495,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/7a1f27f7923b0b778e60223c.jpg"},{"id":92324760,"identity":"6d3f36be-4bb8-43f9-a68b-f776a142ad4f","added_by":"auto","created_at":"2025-09-27 17:30:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":339220,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/7dd36207a72aba1f58939e69.jpg"},{"id":92325109,"identity":"1458202e-c1f2-46bb-bc3c-e1796fa5838c","added_by":"auto","created_at":"2025-09-27 17:38:53","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":287710,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/4d4c33473f250dcbe2191bf9.jpg"},{"id":92325291,"identity":"8a48b970-d37b-4427-8f58-dee79b58154f","added_by":"auto","created_at":"2025-09-27 17:46:53","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3936824,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/42ed87bbc796e08ce29297b1.jpg"},{"id":92324753,"identity":"55c9e2f9-2af3-4d5b-a6b3-61d7c435df2d","added_by":"auto","created_at":"2025-09-27 17:30:53","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":250866,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/d8e992b0b6e23b801ec62c99.jpg"},{"id":94489696,"identity":"651e7b1e-4df0-4956-ae16-f07509407c49","added_by":"auto","created_at":"2025-10-27 17:05:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7902387,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7525311/v1/c0d9baec-9972-4760-9df6-d0107b6f5194.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antifungal efficacy of microencapsulated miR166 and miR159 oligoDNAs through whey protein concentrate (WPC) as coated protein against Verticillium dahliae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe growing global population and limited natural resources have increased the demand for sustainable agricultural production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the outbreak of plant diseases caused by pathogens such as fungi, bacteria, and viruses is considered one of the major challenges in this field [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. One of the most significant fungal pathogens, responsible for vascular wilt in over 400 plant species worldwide, is \u003cem\u003eVerticillium dahliae\u003c/em\u003e Kleb. This soil-borne fungus attacks susceptible plants through the roots, damaging their vascular tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It causes millions of dollars in annual losses worldwide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIdentification of resistant cultivars/genotypes to \u003cem\u003eVerticillium\u003c/em\u003e is one of the important strategies used in disease management. Some researchs have been focused on evaluating resistance to \u003cem\u003eVerticillium\u003c/em\u003e and/or detecting resistance gene analogs (RGAs) in cultivars [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies have led to the development of novel and low-risk methods for plant disease management. Powder and liquid formulation of potent yeast strains showed high efficacy in attenuating disease severity and toxin production of \u003cem\u003eAspergillus flavus\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Essential oils showed great potential for controlling plant pathogens, but due to their volatility, their effectiveness is lost over time [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Encapsulation of essential oils in biodegradable, edible coated protein such as zein, has been shown to increase thier efficacy and reduce disease severity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, the use of fungal metabolites for the green synthesis of zinc oxide nanoparticles have also raised hopes for the control of plant pathogens [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe development of biological control strategies based on biomolecules such as nucleic acids has emerged as an innovative and environmentally friendly approach for managing plant diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Numerous studies have demonstrated that the topical application of dsRNA on plants can effectively prevent the spread of a wide range of plant diseases caused by fungi, oomycetes, and viruses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, the use of dsRNA has been successful in controlling fungal diseases such as \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eBotrytis\u003c/em\u003e in crop plants [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The mechanism of action of dsRNA is based on silencing essential pathogen genes, where these molecules interfere with the expression of key genes, thereby inhibiting the growth and proliferation of pathogens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne of the main limitations in the practical application of dsRNA is its low stability in the environment. Environmental factors such as light, temperature, and degrading enzymes can quickly damage dsRNA and reduce its efficacy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Additionally, its design and synthesis are highly complex and expensive. In contrast, the structure of DNA oligonucleotides is very similar to that of RNA molecules, which are naturally used by cells, making DNA oligos a viable low-cost alternative. Their production and purification are inexpensive and easy, and they are highly stable [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The DNA oligonucleotide technology was first successfully used in plant cells to modify the expression of the transcription factor SUSIBA2 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The researchers' results demonstrated that antisense oligonucleotides were effectively delivered into the leaves and reached the nuclei and chloroplasts [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo enhance the stability of oligonucleotides when applied in plants, various methods such as encapsulation have been investigated [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nanocapsulation systems create a protective coating around DNA oligonucleotides, shielding them from degrading factors and enabling their controlled release at the target site [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Given the sensitivity of bioactive compounds, various encapsulation methods have been developed. The electrospraying process, or microencapsulation using electrohydrodynamic processes, is a simple and effective method for preserving and enhancing the bioavailability of active compounds [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the electrospraying process, various proteins\u0026mdash;including whey protein isolate (WPI), soy protein isolate, egg albumin, collagen, gelatin, zein, wheat gluten, and casein\u0026mdash;have been investigated and evaluated [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among these, whey protein concentrate (WPC), as one of the main by-products of the dairy industry, holds particular significance. WPC contains a mixture of proteins, notably beta-lactoglobulin, alpha-lactalbumin, and serum albumin, and is recognized as a rich source of natural emulsifiers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It has been proposed as a suitable candidate for encapsulation via electrohydrodynamic processes due to its excellent electrospray ability and effective performance as a carrier for bioactive compounds [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious research has established that two virulence genes in \u003cem\u003eVerticillium dahliae\u003c/em\u003e - the Ca\u0026sup2;⁺-dependent cysteine protease (Clp-1) and isotrichodermin C-15 hydroxylase (HiC-15) - are critical for fungal pathogenicity. These genes are specifically targeted by miR166 and miR159, respectively, which were identified in resistant cotton cultivars [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Building on this finding, the current study investigates the antifungal potential of microencapsulated double-stranded oligoDNAs (corresponding to microRNA166 and microRNA159) produced via electrospraying using whey protein concentrate (WPC), an edible and biodegradable polymer, for controlling \u003cem\u003eV. dahliae\u003c/em\u003e growth.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of WPC Solution\u003c/h2\u003e\u003cp\u003eWPC solutions (30% w/v) were prepared by dissolving the required amount of WPC in sterile distilled water with gentle stirring at room temperature. The solutions were kept at room temperature for 24 hours, and their pH was adjusted to 6 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of Oligo DNA-Loaded WPC Nanoparticles via Electrospraying\u003c/h3\u003e\n\u003cp\u003eTo encapsulate Oligo DNA within WPC nanoparticles, 20 ng/\u0026micro;L of Oligo DNA was added to the WPC solution [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The solutions were stirred at room temperature for 30 minutes and then subjected to electrospraying. Microcapsules were prepared using an ES1000 electrospray device (Fanem Co., Iran). A 5 mL syringe was filled with the electrospray solution and mounted on the device. The needle-to-collector distance was set to 14 cm. Oligo DNA-loaded WPC microcapsules were electrosprayed at 18 kV with a flow rate of 0.5 mL/h for 10 hours at room temperature [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eThe morphology of microcapsules was analyzed using a scanning electron microscope (EM-8000F, KYKY, Germany) after coating with a gold-palladium mixture (20 nm thickness) via sputter coating. The average diameter of electrosprayed microcapsules was determined by randomly measuring 100 microcapsules from each SEM image using ImageJ software, with an accelerating voltage of 20 kV [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eTo confirm encapsulation and the presence of oligoDNA, samples were analyzed using transmission electron microscopy (TEM) with negative staining. In this technique, samples are coated with a high electron-density material (such as uranyl acetate or phosphotungstic acid). This staining agent penetrates the surrounding sample space, causing areas with lower electron density (such as microcapsules) to appear as brighter regions against a dark background. Consequently, the white and dark spots in TEM images represent differences in the sample's electron density [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study the morphology of microcapsules and Oligo DNA loading efficiency were evaluated using a Philips EM208S 100KV TEM (Netherlands).\u003c/p\u003e\n\u003ch3\u003eParticle Size Distribution and Hydrodynamic Diameter\u003c/h3\u003e\n\u003cp\u003eThe average hydrodynamic diameter (dls) and particle size distribution of freshly diluted samples were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFourier Transform Infrared (FTIR) Spectroscopy\u003c/h2\u003e\u003cp\u003eFTIR spectroscopy is used to examine the chemical structure of samples. Different peaks in the spectrum represent different functional groups in the sample [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Polypeptide and protein repeating units exhibit specific IR absorption bands for amides A and B, as well as amides I to VII. Among these, amide I (C\u0026thinsp;=\u0026thinsp;O stretching vibration at 1580\u0026ndash;1720 cm⁻\u0026sup1;) and amide II (N\u0026ndash;H bending vibration at 1450\u0026ndash;1600 cm⁻\u0026sup1;) are the two key vibrational bands of the protein backbone. However, changes in secondary structure are more clearly observed in the amide I absorption bands due to the minor contributions of C\u0026ndash;N stretching, C\u0026ndash;C\u0026ndash;N deformation, and in-plane N\u0026ndash;H bending [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. So, to study the chemical structure of WPC microcapsules loaded with Oligo DNA, FTIR analysis was performed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. All measurements were carried out using a Thermo Nicolet spectrophotometer (AVATAR 370 FTIR, USA) in the range of 4000\u0026ndash;400 cm⁻\u0026sup1; with a resolution of 4 cm⁻\u0026sup1; [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eX-ray Diffraction (XRD) Analysis\u003c/h3\u003e\n\u003cp\u003eThe X-ray region of the electromagnetic spectrum lies between gamma rays and ultraviolet wavelengths. This spectral region can be used to obtain information about the structure and type of material. In this study, X-ray diffraction was employed to assess the mixing and dispersion of different compounds and to determine the physical state of Oligo DNA within the electrosprayed whey protein microcapsules. The samples were scanned using an XRD analyzer (Unisantis, XMD-300) with CuKα radiation (λ\u0026thinsp;=\u0026thinsp;1.5418 \u0026Aring;) at an incident angle (2θ) ranging from 5\u0026deg; to 40\u0026deg; at room temperature. The X-ray diffraction spectra were evaluated comparatively [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDifferential Scanning Calorimetry (DSC)\u003c/h3\u003e\n\u003cp\u003eDSC measurements were performed using a Perkin Elmer STA6000 differential scanning calorimeter at a heating rate of 10\u0026deg;C/min. Samples weighing 5 mg were heated under a nitrogen (N₂) atmosphere from 25\u0026deg;C to 400\u0026deg;C [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eThermogravimetric Analysis (TGA)\u003c/h2\u003e\u003cp\u003eTGA provides essential information on thermal stability, decomposition temperature, and residual mass of a sample by measuring weight changes as a function of temperature. Differential scanning calorimetry (DSC) is another powerful tool that offers insights into thermal transition events by measuring the heat flow associated with these processes. In this study, simultaneous thermal events were measured using both TGA and DSC techniques. Additionally, derivative thermogravimetry (DTG) was employed as a function of applied temperature to provide further data and more precise insights into thermal decomposition processes. To evaluate the thermal stability of the microcapsules, a Perkin Elmer STA6000 instrument was used. Approximately 5 mg of the sample was placed in a platinum crucible and heated under a nitrogen flow of 40 mL/min at a heating rate of 10\u0026deg;C/min from 25\u0026deg;C to 750\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePore Size and Volume Measurement (BET)\u003c/h2\u003e\u003cp\u003eThe pore size analysis of the microcapsules was performed using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods based on nitrogen (N₂) adsorption-desorption isotherms. A BELSORP-mini II instrument was used for this analysis. This model is employed to determine the specific surface area of porous and powdered materials [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this analysis the nitrogen adsorption-desorption isotherm provides information about the surface area, pore volume, and pore size distribution of the sample. The difference between the adsorption (ADS) and desorption (DES) curves indicates the presence of hysteresis, which is characteristic of a mesoporous structure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibition of\u003c/b\u003e \u003cb\u003eVerticillium\u003c/b\u003e \u003cb\u003eFungal Growth\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA \u003cem\u003eVerticillium\u003c/em\u003e spore suspension was prepared at a concentration of 1\u0026times;10⁶ spores/mL. The prepared microcapsules were dissolved in water, and 10 \u0026micro;L of the fungal spores were mixed with 5 \u0026micro;L of the microcapsule solution. After 30 minutes of incubation, the mixture was cultured at the center of a Petri dish containing PDA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The treatments included \u003cem\u003eVerticillium\u003c/em\u003e spores alone (control), \u003cem\u003eVerticillium\u003c/em\u003e spores mixed with pure WPC microcapsules, \u003cem\u003eVerticillium\u003c/em\u003e spores mixed with encapsulated oligoDNA159\u0026thinsp;+\u0026thinsp;oligoDNA166,\u003cem\u003eVerticillium\u003c/em\u003e spores mixed with unencapsulated oligoDNA159\u0026thinsp;+\u0026thinsp;oligoDNA166. The samples were monitored for 12 days, and colony growth rate was measured using ImageJ software and the results were used for t-test analysis (P\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphology and Diameter Determination by SEM\u003c/h2\u003e\n \u003cp\u003eSEM images of the electrosprayed structures for WPC, and WPC loaded with oligoDNA159 and oligoDNA166 are presented in Fig.\u0026nbsp;1. The images reveal that in all treatments, spherical microcapsules with smooth surfaces were successfully obtained via electrospraying from an aqueous solution containing 30% (w/w) protein concentration. The mean diameters of WPC microcapsules without oligonucleotides, WPC microcapsules containing oligoDNA159, and WPC microcapsules containing oligoDNA166 were 0.20831 \u0026micro;m, 0.35886 \u0026micro;m, and 0.26744 \u0026micro;m, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eTransmission Electron Microscopy (TEM) Analysis\u003c/h2\u003e\n \u003cp\u003eTEM images of WPC microcapsules with and without oligoDNA have been presented in Fig.\u0026nbsp;2. The images reveal that WPC particles without oligoDNA appear as brighter spots against the dark background. These particles exhibit spherical or near-spherical morphology with small sizes ranging approximately 50\u0026ndash;200 nm (based on the 200 nm scale bar). These likely represent successfully formed individual microcapsules. The images demonstrate relative heterogeneity in particle size and distribution, potentially arising from intermolecular protein interactions. The simultaneous presence of individual particles and larger aggregates indicates incomplete system uniformity, consistent with DLS results. The absence of uniform layers on particle surfaces suggests no active material (oligonucleotides) in the system. WPC microcapsules containing oligoDNA also display spherical or near-spherical morphology. Negative staining causes them to appear as brighter spots against the dark background, likely representing successfully formulated individual microcapsules. Since DNA molecules primarily consist of carbon, oxygen, nitrogen, and phosphorus atoms (which have low electron density compared to staining agents), DNA-containing regions are difficult to distinguish from the dark background in TEM images. The small size of oligonucleotides (21 nucleotides) makes them nearly undetectable compared to microcapsule dimensions. Larger aggregates and particle clusters are observable in some image areas, potentially resulting from strong interactions between whey protein and oligonucleotides that lead to formation of larger structures. Increased particle size compared to normal whey protein dimensions suggests the formation of oligonucleotide-containing capsules. Some particles exhibit uniform surface layers, indicating successful loading of active material (oligonucleotides). These findings align with previous research and provide visual confirmation of the encapsulation process and oligonucleotide incorporation within the WPC microcapsules. The TEM analysis complements the DLS data by offering direct morphological evidence at the nanoscale level.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003eParticle Size and polydispersity index (PDI)\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003ePDI is among the most important characteristics of nano-carrier systems. The mean particle size and polydispersity index of the samples are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The uniformity (homogeneity) of droplet size in emulsions can be inferred from the polydispersity index. The PDI values for pure WPC, WPC containing oligoDNA159, and WPC containing oligoDNA166 were 0.69, 0.52, and 1.0, respectively. The results indicated that nearly all emulsions exhibited heterogeneous populations with multiple particle populations in the samples. Figure\u0026nbsp;3 shows the particle size distribution histogram (number-based) obtained by dynamic light scattering. All three graphs demonstrate an asymmetric distribution with the majority of particles concentrated in the 100\u0026ndash;1000 nm range. A gradual decrease in particle count with increasing size was observed, indicating the presence of fewer larger aggregates. This histogram confirms that most particles in the samples are small, while larger aggregates contribute very little numerically. The broad standard deviation indicates relative heterogeneity in particle size, which is consistent with the high PDI values of the samples. Considering the objective of controlled release of materials, this composition is likely suitable. The combination of predominantly small particles with limited large aggregates appears appropriate for controlled release applications, despite the observed size heterogeneity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\n \u003cp\u003eThe amide bands of WPC were studied to analyze the encapsulation of oligoDNA and its effect on the molecular organization of the capsules. In the spectra corresponding to nucleic acids, the peaks in the 1600\u0026ndash;1800 cm⁻\u0026sup1; region are typically attributed to the stretching vibrations of carbon-oxygen double bonds (C\u0026thinsp;=\u0026thinsp;O) in carbonyl groups. Strong peaks in this region indicate the presence of a large number of carbonyl groups in the nitrogenous base molecules of DNA. The FTIR spectra of WPC powders revealed characteristic functional groups, including Amine or N\u0026ndash;H stretching (3433.11 cm⁻\u0026sup1;), Aliphatic C\u0026ndash;H stretching (2925.86 and 2862.21 cm⁻\u0026sup1;), Amide I (C\u0026thinsp;=\u0026thinsp;O stretching) (1643.26 cm⁻\u0026sup1;), Amide II (N\u0026ndash;H bending and stretching) (1525.61 cm⁻\u0026sup1;), C\u0026thinsp;=\u0026thinsp;C bending in aromatic rings of phenolic compounds (1461.96 cm⁻\u0026sup1;).\u003c/p\u003e\n \u003cp\u003eThe IR spectrum exhibited characteristic bands at 3458.19, 2075.30, and 1637.48 cm⁻\u0026sup1; for oligoDNA159, at 3477.47 and 1650.98 cm⁻\u0026sup1; for oligoDNA166. The observed bands at 1637.48 cm⁻\u0026sup1; and 1650.98 cm⁻\u0026sup1; correspond to stretching vibrations of carbon-oxygen double bonds (C\u0026thinsp;=\u0026thinsp;O) in the carbonyl groups of DNA nitrogenous bases. Figure 4 presents the characteristic FTIR bands of oligoDNA159, oligoDNA166, WPC, and WPC loaded with oligoDNA159 and oligoDNA166. The microcapsules showed the following diagnostic absorption bands: N-H stretching (amine) at 3435.04 cm⁻\u0026sup1;, Aliphatic C-H stretching at 2923.93 and 2856.43 cm⁻\u0026sup1;, Amide I (C\u0026thinsp;=\u0026thinsp;O stretching) at 1643.26 cm⁻\u0026sup1;, Amide II (N-H bending and stretching) at 1543.86 cm⁻\u0026sup1;, C\u0026thinsp;=\u0026thinsp;C bending in aromatic rings of phenolic compounds at 1461.96 cm⁻\u0026sup1;. As shown in the Fig. 4, no new spectrum was observed for WPC particles loaded with oligoDNA. However, changes in the position and intensity of the peaks were observed in the spectra of microcapsules containing oligoDNA compared to the pure spectra of WPC. The position of the amide I bands in microcapsules containing oligoDNA159 remained unchanged, whereas this band shifted toward a higher wavenumber in microcapsules containing oligoDNA166. A shift toward higher wavenumbers was also observed in the amide II band in the spectra of microcapsules containing both types of oligoDNA, indicating some molecular changes in the whey protein as a result of microcapsule formation. Additionally, some bands in the spectra of microcapsules containing oligoDNA were absent, likely due to the low concentration of oligoDNA inside the microcapsules or because they were overshadowed by the stronger peaks of the whey protein. This suggests that the oligoDNA is physically entrapped within the particles and that no strong chemical bonds exist between them. Furthermore, differences in the position and intensity of the bands were observed in the pure spectra of oligoDNA159 and oligoDNA166, which are likely due to variations in the base types present in the two sequences.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe mean particle size and polydispersity index of the samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003ePolydispersity Index (PDI)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHydrodynamic Diameter (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eWPC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e434.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMC-WPC oligo159\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e754.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMC-WPC oligo166\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2075\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eX-ray Diffraction (XRD) Analysis\u003c/h2\u003e\n \u003cp\u003eX-ray diffraction patterns are used to examine the crystalline structure of biopolymeric materials. Figure\u0026nbsp;5 shows the XRD patterns of WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. Two distinct peaks were observed in the pure WPC diffractogram at 8.51\u0026deg; 2\u0026theta; (d-spacing\u0026thinsp;=\u0026thinsp;10.3 \u0026Aring;) and 19.58\u0026deg; 2\u0026theta; (d-spacing\u0026thinsp;=\u0026thinsp;4.52 \u0026Aring;). The first peak is attributed to the interhelical packing distance, while the second peak corresponds to the d-spacing of \u0026alpha;-helical structures. In this study, the microcapsule structure appears predominantly amorphous, with possible small nanocrystalline regions, as no sharp and well-defined peaks indicative of high crystallinity were detected. The first diffraction peaks of oligoDNA159 and oligoDNA166 in WPC were observed at 8.33\u0026deg; 2\u0026theta; (interlayer spacing of 10.6 \u0026Aring;) and 8.22\u0026deg; 2\u0026theta; (interlayer spacing of 10.7 \u0026Aring;), respectively, while the second diffraction peaks appeared at 19.64\u0026deg; 2\u0026theta; (interlayer spacing of 4.51 \u0026Aring;) and 19.83\u0026deg; 2\u0026theta; (interlayer spacing of 4.4 \u0026Aring;), respectively. The peak intensities for oligoDNA159 remained nearly unchanged, whereas those for oligoDNA166 decreased. Additionally, the first peak (2\u0026theta;\u0026thinsp;=\u0026thinsp;8.51\u0026deg;) shifted to a lower angle in both treatments, and the second peak (2\u0026theta;\u0026thinsp;=\u0026thinsp;19.58\u0026deg;) shifted to a higher angle in both treatments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eThermogravimetric Analysis (TGA)\u003c/h2\u003e\n \u003cp\u003eFigure 6, illustrates the thermal and degradation behavior of WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. As shown in the figure, the TGA and DSC curves for all samples are nearly identical and can be divided into several distinct stages:\u003c/p\u003e\n \u003cp\u003e-First Stage (Moisture Loss): The initial weight loss in the TGA curve corresponds to moisture removal, with reductions of 7.3%, 8.32%, and 7.81%, respectively. This initial weight loss occurs at approximately 30\u0026ndash;150\u0026deg;C and is attributed to the evaporation of free water and surface moisture.\u003c/p\u003e\n \u003cp\u003e-Second Stage (Solvent and Bound Water Removal): The second stage in the TGA curve corresponds to the removal of solvents and bound water, with weight losses of 3.06%, 3.06%, and 2.04%, respectively. This stage occurs in the temperature range of 150\u0026ndash;250\u0026deg;C and is likely associated with water bound to the protein structure and residual solvents from the production process.\u003c/p\u003e\n \u003cp\u003e-Third Stage (Main Protein Structure Degradation): The major weight loss (60.85%, 62.67%, and 64.88%, respectively) occurs between 250\u0026ndash;400\u0026deg;C, likely indicating the denaturation and breakdown of WPC\u0026mdash;particularly \u0026beta;-lactoglobulin and \u0026alpha;-lactalbumin\u0026mdash;as well as the cleavage of disulfide bonds between protein chains and the degradation of encapsulated oligonucleotides.\u003c/p\u003e\n \u003cp\u003e-Fourth Stage (Resistant Residue Degradation): The final weight loss (6.41%, 3.74%, and 3.06%, respectively) occurs between 400\u0026ndash;800\u0026deg;C, representing the gradual decomposition of thermally resistant structures and the carbonization of remaining organic residues.\u003c/p\u003e\n \u003cp\u003eThe remaining approximately 22% at the end of the analysis (800\u0026deg;C) is likely related to inorganic compounds and highly stable carbon structures that do not decompose even at high temperatures. Samples containing oligonucleotides exhibit less weight loss (60\u0026ndash;62%) compared to the oligonucleotide-free sample (~\u0026thinsp;64%). Additionally, the initial moisture content and water release were similar across all samples, indicating that the thermal differences are primarily due to molecular structure and internal composition. The higher thermal stability of the oligonucleotide-containing samples is likely attributed to stronger interactions between the protein and oligonucleotides, which enhance heat resistance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eDifferential Scanning Calorimetry (DSC)\u003c/h2\u003e\n \u003cp\u003eDSC provides insights into thermal transition events by measuring the heat flow associated with these events in the sample. Figure\u0026nbsp;7 shows the DSC analysis curves for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules. The temperature range of 30 to 150\u0026deg;C, where a small endothermic peak is observed at around 39\u0026deg;C. This peak indicates energy absorption for the evaporation of surface moisture and free water from the microcapsules. This finding aligns with the initial weight loss in the TGA curve within the same temperature range. The temperature range of 150 to 300\u0026deg;C, where the curve shifts downward (endothermic), indicating the onset of phase transitions in the protein structure.\u003c/p\u003e\n \u003cp\u003eA major endothermic valley is observed at 342.8\u0026deg;C, 371.9\u0026deg;C, and 381.9\u0026deg;C, respectively, indicating significant endothermic processes. These include thermal denaturation of whey proteins, cleavage of chemical bonds within the protein structure, and thermal degradation of oligonucleotides. This endothermic valley closely aligns with the major weight-loss stage in the TGA analysis (observed in the 250\u0026ndash;400\u0026deg;C range). A sharp increase in heat flow occurs at temperatures above 400\u0026deg;C, where the curve shifts continuously upward (exothermic). This trend suggests more complex thermal processes, and likely final decomposition of residual materials. This behaviour correlates with the final weight-loss stage in the TGA analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eBET (Brunauer\u0026ndash;Emmett\u0026ndash;Teller) analysis\u003c/h2\u003e\n \u003cp\u003eThe specific surface area for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules that was calculated using the slope and intercept of the BET linear plot, where a steeper slope corresponds to a higher surface area (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Pure WPC microcapsules exhibited a specific surface area of 4.8283 m\u0026sup2;/g, WPC microcapsules with oligoDNA159 showed a significantly reduced surface area of 0.55338 m\u0026sup2;/g, and WPC microcapsules with oligoDNA166 had an intermediate value of 2.9665 m\u0026sup2;/g.\u003c/p\u003e\n \u003cp\u003eThe Barrett-Joyner-Halenda (BJH) model was employed to evaluate the actual pore volume of the samples. The BJH plots are presented in Fig.\u0026nbsp;8, where X-axis: Pore radius (logarithmic scale, 1\u0026ndash;100 nm), Y-axis: Derivative of pore volume with respect to pore radius, representing the pore size distribution. All samples exhibited a dominant peak in the 1\u0026ndash;3 nm range, indicating minimal variation in pore size across samples. This peak confirms the presence of micropores (\u0026lt;\u0026thinsp;2 nm), and small mesopores (2\u0026ndash;20 nm). The decline in pore volume beyond 20 nm suggests the absence of macropores (\u0026gt;\u0026thinsp;50 nm), confirming a predominantly micro-mesoporous structure.\u003c/p\u003e\n \u003cp\u003eThe pore volume measurements revealed significant differences among the samples: Pure WPC microcapsules (without oligoDNA) exhibited a pore volume of 0.0078513 cm\u0026sup3;/g, WPC microcapsules containing oligoDNA159 showed a dramatic reduction to 0.001442 cm\u0026sup3;/g (81.6% decrease), WPC microcapsules containing oligoDNA166 demonstrated an intermediate value of 0.0047458 cm\u0026sup3;/g (39.6% decrease compared to pure WPC). This substantial decrease in pore volume following oligoDNA incorporation suggests potential pore blockage by oligonucleotide molecules, structural modifications in the protein matrix, and altered packing density of the microcapsule walls. The more pronounced effect observed with oligoDNA159 (compared to oligoDNA166) may indicate sequence-dependent interactions between the oligonucleotides and whey protein components, warranting further investigation into the molecular mechanisms underlying these textural changes.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePresents the specific surface area for WPC, WPC-oligoDNA159, and WPC-oligoDNA166 microcapsules\u0026nbsp;that was calculated using the\u0026nbsp;slope and intercept of the BET linear.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWPC (No oligoDNA)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWPC\u0026thinsp;+\u0026thinsp;oligoDNA159\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWPC\u0026thinsp;+\u0026thinsp;oligoDNA166\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBET Surface Area (m\u0026sup2;/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.8283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.9665\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBJH Pore Volume (cm\u0026sup3;/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0078513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0047458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDominant Pore Size (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll three samples exhibit Type VI adsorption isotherms, consistent with IUPAC classification, indicating the presence of a mesoporous structure (pores ranging from 2 to 50 nm). The presence of a hysteresis loop between the adsorption and desorption curves suggests a diverse range of pore shapes and sizes, as well as varying mechanisms for pore filling and emptying.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of antifungal activity\u003c/h2\u003e\n \u003cp\u003eThe colony growth rate of the fungus in each experimental treatment was monitored in 2 days intervals over a 12-day period after cultivation on PDA, with measurements taken using ImageJ software (Fig.\u0026nbsp;9). After 12 days, the average colony diameter was recorded, and a t-test analysis (P\u0026thinsp;=\u0026thinsp;0.05) revealed that treatments containing either encapsulated or unencapsulated oligoDNA 159\u0026thinsp;+\u0026thinsp;oligoDNA 166 exhibited significantly smaller colony diameters\u0026mdash;2.5 cm and 2.8 cm, respectively\u0026mdash;compared to the pathogen control (3.2 cm). Furthermore, the growth reduction rate was higher in treatments with encapsulated oligoDNA 159\u0026thinsp;+\u0026thinsp;oligoDNA 166 compared to unencapsulated samples (Figs.\u0026nbsp;9, 10). These results demonstrate the inhibitory effect of oligoDNA on fungal growth, suggesting that encapsulation enhances efficacy\u0026mdash;likely due to improved stability and controlled release of the oligoDNA.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to evaluate the antifungal efficacy of whey protein concentrate (WPC) microencapsulated double-stranded oligoDNAs (mimicking miR166 and miR159) against \u003cem\u003eVerticillium dahliae\u003c/em\u003e. The oligoDNAs were prepared using electrospraying technology with WPC, an edible and biodegradable polymer matrix. The results of this study clearly demonstrate the ability of oligo DNA 159 and oligo DNA 166 to inhibit the growth of \u003cem\u003eV. dahliae\u003c/em\u003e, which may be related to the RNA interference (RNAi) mechanism [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our findings are consistent with the studies of Zhang et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as they showed that cotton plants increase the production of microRNA166 and microRNA159 in response to infection with \u003cem\u003eV. dahliae\u003c/em\u003e and transfer these microRNAs to the fungal hyphae to perform specific gene silencing. They found that the Clp-1 and HiC-15 genes are targeted by miR166 and miR159, respectively [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. They also observed that mutant strains of Verticillium in which the Clp-1 gene was inactivated had normal colony morphology but inhibited microsclerotia formation. In contrast, mutant strains in which the HiC-15 gene was inactivated had severely inhibited hyphal growth but normal microsclerotia formation, consistent with the findings of this study that fungal colony growth was reduced compared to the control.\u003c/p\u003e\u003cp\u003eIn a study, using the encapsulation method by incorporating dsRNA into double layer hydroxide (LDH) nanosheets known as \"BioClay\", viral protection was applied and the stability of dsRNA on tobacco leaf surfaces increased from 5 to 20 days [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Also, Whitfield et al. (45) reported that the persistence of dsRNA in soil was increased by up to 3 weeks through the use of a poly(2-(dimethylamino)ethyl acrylate) analogue [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. WPC has been proposed as a suitable candidate for encapsulation via electrohydrodynamic processes due to its excellent electrospray ability and effective performance as a carrier for bioactive compounds [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In current study, the increased efficacy in oligo DNA encapsulated samples with WPC indicates that the encapsulation technology improves the stability of the oligo DNA and enables its controlled release. This results in longer access and facilitated transmission of the oligo DNA to fungal cells and consequently increased efficacy in growth inhibition. The lack of significant effect of pure WPC microcapsules on \u003cem\u003eV. dahliae\u003c/em\u003e colony growth also confirms that the observed antifungal effect is due to the oligo DNA itself and not simply the WPC biochemical chracterestics.\u003c/p\u003e\u003cp\u003eOverall, this research provides strong evidence for the potential of oligo DNA as a biological antifungal agent, and oligo DNA encapsulation is a promising strategy to enhance its efficacy and application in the control of fungal diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;the financial support of the Research Vice-Chancellor of University of Zabol under grant number UOZ-GR-9618-129.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the financial support of the Research Vice-Chancellor of University of Zabol for supporting us in conducting this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u0026nbsp;Mahboobeh Nouri, Mojtaba Keykhasaber, Mohammad-Amin Miri, Mahdi Pirnia,\u0026nbsp;Shirahmad Sarani, and Hoseyn Kamaladini. The first draft of the manuscript was written by Mojtaba Keykhasaber and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVetukuri RR, Dubey M, Kalyandurg PB, et al. Spray-induced gene silencing: An innovative strategy for plant trait improvement and disease control. 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Food Chem. 2019;280:51\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antisense oligonucleotides, microRNA, plant disease control, fungal diseases, biological control","lastPublishedDoi":"10.21203/rs.3.rs-7525311/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7525311/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eVerticillium dahliae\u003c/em\u003e Kleb. is one of the most destructive fungal pathogens, infecting susceptible plants through their roots and disrupting vascular tissues. As soil-borne diseases pose a persistent threat to agriculture, the development of sustainable biocontrol strategies has gained significant attention. Among these, biomolecule-based approaches\u0026mdash;particularly the use of oligonucleotides\u0026mdash;have emerged as an innovative and eco-friendly alternative for plant disease management. In this study, we evaluated the antifungal potential of microencapsulated double-stranded oligoDNAs (corresponding to microRNA166 and microRNA159) produced via electrospraying with a whey protein concentrate (WPC) polymer. The encapsulated oligoDNAs were tested against \u003cem\u003eV. dahliae\u003c/em\u003e by mixing 10 \u0026micro;L of fungal spores (1\u0026times;10⁶ spores/mL) with 5 \u0026micro;L of the microcapsule solution, followed by culturing on potato dextrose agar (PDA). Our findings demonstrate that both oligoDNA 159 and oligoDNA 166 significantly inhibit fungal growth. Notably, encapsulation with WPC enhanced their antifungal efficacy, suggesting that this technology improves oligoDNA stability and enables controlled release. These results highlight the potential of oligonucleotides as effective biocontrol agents against fungal pathogens. Furthermore, encapsulation presents a promising strategy to optimize their delivery and application in sustainable agriculture. This study provides compelling evidence for the use of microencapsulated oligoDNAs in fungal disease management, offering a viable, environmentally safe alternative to conventional chemical treatments. Future research should explore field applications and long-term effects to validate their practical use in crop protection.\u003c/p\u003e","manuscriptTitle":"Antifungal efficacy of microencapsulated miR166 and miR159 oligoDNAs through whey protein concentrate (WPC) as coated protein against Verticillium dahliae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-27 17:30:48","doi":"10.21203/rs.3.rs-7525311/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d51ef91f-4b55-4dca-8876-a691ec29792a","owner":[],"postedDate":"September 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T14:59:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-27 17:30:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7525311","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7525311","identity":"rs-7525311","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}