Description:FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of food preservation technologies, more particularly the present invention refers to Gossypetin Encapsulated Silk Fibroin Nanocomposite Based Edible Bio-Preservative Coating to extend the shelf life of fruits and vegetables using dip coating technique. The present disclosure also discloses a process for preparing said composition.

BACKGROUND OF INVENTION

[002] The shelf life of fruits and vegetables is a critical concern in the food industry, particularly for local farmers and small-scale vendors who face challenges in preserving their produce. Traditional preservation methods such as chemical preservatives, cold storage, dip-coating, and advanced systems like ultralow oxygen storage are effective but often expensive and difficult to implement.

[003] Thin-film edible coatings are widely recognized for their effectiveness in food preservation. These coatings are classified into three primary categories based on their composition: hydrocolloids or natural gums (proteins, polysaccharides, alginates), lipids (fatty acids, acylglycerols, waxes), and composite formulations combining multiple components. Each category exhibits distinct mechanical, physicochemical, and barrier properties, providing protection against ambient gases, moisture loss, and microbial contamination.

[004] Edible coatings serve as protective barriers, slowing respiration and oxidation rates while enhancing shelf life. Due to the limitations of individual components, modern edible coatings frequently incorporate plasticizers and active ingredients to improve flexibility, permeability, and antimicrobial properties. Common plasticizers such as polyols (glycerol, mannitol, propylene glycol, Honey, sucrose) enhance coating strength, while bioactive compounds like curcumin, rosemary oil, chitosan, and ascorbic acid provide antioxidant and antibacterial benefits.

[005] Advancements in nanotechnology have introduced innovative edible coatings with enhanced preservation capabilities, utilizing biopolymers such as chitosan, starch, alginate, pectin, xanthan, guar gum, Silk fibroin, and poly(vinyl alcohol). Various techniques, including dip coating, spray coating, and electrospun nanofiber application, have been employed to apply edible coatings to perishable fruits and vegetables. Studies demonstrate that coatings utilizing biopolymer composites significantly improve shelf life—for example, carrageenan-based coatings on bananas extend freshness by six days (Dwivany, F.M., Aprilyandi, A.N., Suendo, V., Sukriandi, N., 2020. Carrageenan Edible Coating Application Prolongs Cavendish Banana Shelf Life. International Journal of Food Science 2020, 1–11. https://doi.org/10.1155/2020/8861610), while plasticized poly (vinyl alcohol)-based films prolong banana shelf life from 9 to 19 days (Senna, M.M.H., Al-Shamrani, K.M., Al-Arifi, A.S., 2014. Edible Coating for Shelf-Life Extension of Fresh Banana Fruit Based on Gamma Irradiated Plasticized Poly(vinyl alcohol)/Carboxymethyl Cellulose/Tannin Composites. MSA 05, 395–415. https://doi.org/10.4236/msa.2014.56045).

[006] Post-harvest preservation of fruits like apples and bananas has been explored through techniques such as 1-methylcyclopropane (1-MCP) treatment and ultra-low oxygen storage systems (ULO). While effective, these technologies are costly and complex, posing challenges for local farmers and small-scale vendors.

[007] To address these limitations, the development of natural, cost-effective edible coatings has gained significant attention. Therefore, there is a high unmet need in the art to provide a nanocomposite based edible bio-preservative coating based on natural compounds that would enhance the shelf life of fruits and vegetables through accessible dip-coating techniques.
SUMMARY OF THE INVENTION

[008] In an aspect of the present disclosure, there is provided a Gossypetin Encapsulated Silk Fibroin Nanocomposite Based Edible Bio-Preservative Coating for enhancing the shelf life of fresh fruits and vegetables comprising: 1 mL of Silk fibroin aqueous solution (1% w/v), 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0), and 5% Honey, relative to the Gossypetin-Silk fibroin solution volume.

[009] In another aspect of the present disclosure, there is provided a process for preparing a Gossypetin Encapsulated Silk Fibroin Nanocomposite Based Edible bio-preservative coating for enhancing the shelf life of fresh fruits and vegetables comprising: 1 mL of Silk fibroin aqueous solution (1% w/v), 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0), and 5% Honey, relative to the Gossypetin-Silk fibroin (GTIN-SFNPs) solution volume, said process comprising:
a. Injecting 1 mL of fibroin aqueous solution (1% w/v) into 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0) to obtain a GTIN-SFNPs solution.
b. Adding 5% Honey (w.r.t. GTIN-SFNPs volume) to the GTIN-SFNPs solution.
c. Stirring the Silk fibroin nanoparticles solution, loaded with Gossypetin and Honey by a magnetic stirrer for 10 minutes at room temperature, the complete dissolution of Honey and Gossypetin.
d. After 40 minutes, injecting the composite dip-coating solution into the 5 mL on medium (fruits and/or vegetables).
e. Covering the medium on all sides by shifting position
f. Dipping the model fruits and vegetables in the solution for 30-40 seconds per cycle, repeating the process three times at five-minute intervals.
g. Finally, allow the coated food to hung dry at room temperature.

[010] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[011] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[012] Figure 1 depicts the UV-Visible analysis depicting the maximum wavelength of GTIN-Honey-SFNPs.

[013] Figure 2 depicts the FTIR depicts the functional groups associated with the GTIN-Honey-SFNPs.

[014] Figure 3 depicts the XPS survey spectrum of (a) GTIN-Honey-SFNPs nanocomposite and corresponding high resolution XPS survey spectrum of (b) C 1s, (c) N 1s, (d) O 1s.

[015] Figure 4 depicts the Field Emission Scanning Electron Microscopy images of (a) Silk fibroin (b) SFNPs and (c)-(d) GTIN-Honey-SFNPs nanocomposite. Scale bar: 2μM and 1μM.

[016] Figure 5 depicts the Transmission Electron Microscopy images of the lyophilized nanoparticles: (a) SFNPs Scale bar: 100 nm; (b) Gossypetin-loaded Silk fibroin nanoparticles prepared using a GTIN/SFNPs ratio of 1:40 (w/w). Scale bar: 200 nm; (c) Threshold of image b using imageJ (d) particle size distribution of histogram.

[017] Figure 6 depicts the Thermogravimetry curves of (a) SFNPs, (b) GTIN-Honey-GTIN

[018] Figure 7 illustrates time-lapse imaging of tomato preservation under different treatment conditions. The figure comprises:
(a) Tomatoes without any coating (Control),
(b) Tomatoes coated with GTIN-SFNPs nanocomposite, and
(c) Tomatoes coated with GTIN-Honey-SFNPs nanocomposite.

[019] Figure 8 illustrates time-lapse imaging of Ivy gourd preservation under different treatment conditions. The figure comprises:
(a) Ivy gourd without any coating (Control),
(b) Ivy gourd coated with GTIN-SFNPs nanocomposite, and
(c) Ivy gourd coated with GTIN-Honey-SFNPs nanocomposite.

[020] Figure 9 illustrates time-lapse imaging of Eggplant preservation under different treatment conditions. The figure comprises:
(a) Eggplant without any coating (Control),
(b) Eggplant coated with GTIN-SFNPs nanocomposite, and
(c) Eggplant coated with GTIN-Honey-SFNPs nanocomposite.

[021] Figure 10 illustrates time-lapse imaging of Green Banana preservation under different treatment conditions. The figure comprises:
(a)Green Banana without any coating (Control),
(b)Green Banana coated with GTIN-SFNPs nanocomposite, and
(c) Green Banana coated with GTIN-Honey-SFNPs nanocomposite.

[022] Figure 11 depicts themicrobiological characterization of selected bacterial and fungal strains:
(a)colony morphology of Bacillus subtilis,
(b) positive Gram staining result of Bacillus subtilis (magnification 40×),
(c) colony morphology of Escherichia coli,
(d) negative Gram staining result of Escherichia coli (magnification 40×),
(e) colony morphology of Aspergillus flavus, and
(f) microscopic analysis of Aspergillus flavus cell morphology (magnification 40×)

[023] Figure 12 illustrates Zone of inhibition of the Antimicrobial Activity of GTIN and Honey-Loaded SFNPs against Bacterial Pathogens (a) Escherichia coli and (b) Bacillus subtilis.

[024] Figure 13 depicts the Zones of inhibition of the antifungal activity of GTIN-Honey-SFNPs against Aspergillus flavus.

[025] Figure 14 depicts the minimum inhibitory concentration (IC50) representing scavenging activity for antioxidant properties, as determined using the DPPH assay for GTIN-Honey-SFNPs.

[026] Figure 15 depicts the In-Vitro Hemolytic Assessment of GTIN-Honey-SFNPs and GTIN-Honey-SFNPs-Tween Formulations
(a) Hemolysis rate (%) at varying concentrations of GTIN-Honey-SFNPs.
(b) Microscopic images of red blood cells (RBCs) treated with GTIN-Honey-SFNPs or GTIN-Honey-SFNPs-Tween.
Data are presented as mean ± SE (n = 3); ***P < 0.000.

[027] Figure 16 depicts the Fluorescence Imaging of C. elegans Treated with Nanocomposite Formulations
Fluorescence visualization of C. elegans exposed to:
(I) SFNPs, (II) GTIN, (III) GTIN-SFNPs, and (IV) GTIN-Honey-SFNPs, synthesized and imaged at specific excitation wavelengths.
(a) Untreated control group fed with E. coli OP50,
(b) C. elegans treated with 0.5 mg/mL of each formulation,
(c) C. elegans treated with 1 mg/mL of each formulation,
(d) C. elegans treated with 2 mg/mL of each formulation.

[028] Figure 17 depicts the Cytotoxicity Assessment of GTIN-Honey-SFNPs Nanocomposite on HCT-116 Cells.

[029] Figure 18 depicts the Fluorescence-Based Staining of HCT-116 Cells for Cytotoxicity Evaluation
Microscopic imaging of cells subjected to various fluorescence staining techniques:
(a–b) Dual staining for viability assessment,
(c–d) Propidium iodide exclusion and live/dead cell detection,
(e–f) Nuclear staining.
Panels a, c, and e represent control (untreated) cells; panels b, d, and f show cells treated with GTIN-Honey-SFNPs nanocomposite.
Scale bar: 20 μM.

DETAILED DESCRIPTION OF THE INVENTION

[030] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[031] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[032] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[033] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[034] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[035] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[036] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[038] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[039] As discussed in the background section of the present disclosure, conventional food preservative coatings cause problems depending on their composition, application method, and interaction with food. Among such methods, wax-based coatings are widely utilized for the preservation of fruits and vegetables due to their moisture-barrier and gloss-enhancing properties. However, wax coatings pose several disadvantages. These include the incorporation of chemical additives such as morpholine and oleic acid derivatives, which have been scrutinized for potential links to colorectal cancer. Furthermore, wax coatings may trap pesticide residues and restrict respiration rates, resulting in accelerated spoilage under certain conditions and raising safety concerns for prolonged human consumption.

[040] In order to overcome the aforementioned limitations, the present discloses focuses on the development of edible preservative coatings. These coatings offer a sustainable approach to extending food freshness by reducing moisture loss, preventing microbial contamination, and maintaining quality without reliance on synthetic additives. Among various biomaterials, the present invention nanoparticles have emerged as a promising solution due to their biocompatibility, non-toxicity, high stability, and strong mechanical strength. Their incorporation into edible coatings can enhance barrier properties, prolong shelf life, and provide a viable preservation method accessible to farmers and vendors.

[041] In an embodiment of the present disclosure, there is provided a Gossypetin Encapsulated Silk Fibroin Nanocomposite Based Edible Bio-Preservative Coating comprising Silk Fibroin protein nanoparticles, Gossypetin, functioning as an antimicrobial and antioxidant agent, and Honey, serving as a natural moisturizer, collectively formulated to enhance the shelf life of fresh fruits and vegetables.

[042] Preferably, the present invention an edible bio-preservative nanocomposite coating comprising of 1 mL of Silk fibroin aqueous solution (1% w/v), 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0), and 5% Honey, relative to the Gossypetin-Silk fibroin solution volume.

[043] The Silk fibroin protein nanoparticles as used in the present invention Edible Bio-Preservative Coating, are utilized as the base biomaterial and are derived from a naturally occurring polymer, specifically from a Silk fibroin protein solution sourced from Fibro Heal Wound Care Pvt. Ltd. The Silk fibroin protein nanoparticles provide a multifunctional platform for edible coating applications in fresh fruits, combining biocompatibility, non-toxicity, and biodegradability with excellent performance characteristics. The said protein forms micrometer-thin, transparent films that serve as effective moisture barriers, minimizing water loss and preserving fruit firmness during storage.

[044] Additionally, the Silk fibroin coatings regulate gas exchange by controlling oxygen and carbon dioxide permeability, thereby delaying spoilage and microbial proliferation. The coatings exhibit superior mechanical integrity and flexibility, enhancing durability without compromising sensory qualities such as texture and appearance.

[045] The Gossypetin in the present invention of edible bio-preservative nanocomposite coating, is used as an active agent and are naturally derived flavonoid present in various plant species such as Moringa oleifera leaf, hibiscus, cotton plants and so on (Muthumanickam et al., 2025). Specifically, the Gossypetin used herein is procured from Sigma-Aldrich and the said Gossypetin has multifunctional bioactivity in food-grade applications. Structurally akin to quercetin, Gossypetin exhibits robust antioxidant capabilities by neutralizing free radicals and inhibiting oxidative degradation of organic matter. When incorporated into edible coatings, it helps preserve the visual appeal, texture, and nutritional integrity of fresh fruits throughout postharvest storage. Its molecular structure also provides natural UV protection, shielding fruits from photodegradation that can lead to discoloration and quality loss.

[046] In addition to its antioxidative properties, Gossypetin demonstrates strong antimicrobial efficacy against bacteria and fungi commonly responsible for spoilage. This makes it a potent candidate for enhancing shelf life when used as an active ingredient in bio-based coatings, particularly those formulated with carriers like Silk fibroin or chitosan. Gossypetin’s plant-based origin and alignment with GRAS standards also support its safety and edibility in food contact applications. Moreover, its synergistic potential allows it to reinforce the mechanical strength and functional stability of composite coatings, making it valuable for eco-friendly, consumer-safe preservation technologies.

[047] In the present invention of edible bio-preservative nanocomposite coating, the Honey used is Dabur Honey and it act as a natural moisturizer that enhances the surface hydration of coated fruits and vegetables, thereby contributing to improved freshness and visual appeal during storage. Honey is a naturally occurring, multifunctional substance and its composition is rich in sugars, enzymes, amino acids, and polyphenols, so it also acts as a preservative and a bioactive enhancer in postharvest applications. Other benefits of using Honey are it acts as a natural humectant, helping retain moisture and prevent desiccation of fruit surfaces. Its antimicrobial properties, primarily due to hydrogen peroxide and low pH, inhibit the growth of spoilage-causing bacteria and fungi. Additionally, Honey contributes to antioxidant protection, delaying oxidative browning and preserving the sensory quality of fruits. Honey can be blended with biopolymers to form composite coatings with improved mechanical strength and barrier properties. These formulations are typically applied enhances shelf life without altering taste or texture of fruits or vegetables.

[048] In another embodiment, the present disclosure provides a process for preparing an edible bio-preservative nanocomposite coating composition configured to prolong the shelf life of fresh produce. The process comprises:

Step 1: Synthesis of Silk Fibroin Protein Nanoparticles (SFNPs)
SFNPs are synthesized via a desolvation-based protocol wherein 1 mL of an aqueous Silk fibroin solution (1% w/v, pH 7.0) is gradually added to 1 mL of ethanol containing 1 mg of Gossypetin (GTIN). Ethanol acts as a desolvating medium, promoting the self-assembly of Silk fibroin molecules into uniform, structurally stable nanoparticles, which serve as carriers in the coating formulation.

Step 2: Preparation of GTIN-Honey-Loaded Coating Solution
Following nanoparticle formation, Honey is incorporated at 5% concentration relative to the total volume of the GTIN-SFNP dispersion. The blend undergoes magnetic stirring at ambient temperature for a total duration of 50 minutes initially for 10 minutes to ensure homogeneity, followed by 40 minutes to achieve coating readiness.

Step 3: Application via Dip-Coating Technique
Fresh fruits and vegetables are subjected to a three-cycle immersion process, with each cycle lasting approximately 30–40 seconds and spaced at five-minute intervals. Post-application, the coated samples are air-dried under ambient conditions, facilitating uniform deposition and stabilization of the preservative layer.

[049] An additional embodiment discloses an optimized loading approach for GTIN onto SFNPs through incubation. Parameters examined include solvent type, mass ratio of GTIN to SFNPs (1:10, 1:20, 1:40), and incubation duration (1, 6, 24, or 48 hours). GTIN is initially dissolved in absolute ethanol and then added to SFNPs dispersed in distilled water. The resulting mixtures are rotated at 30 rpm under ambient conditions while shielded from light using aluminum foil.

[050] Post incubation, the suspensions are centrifuged at 16000g for 25 minutes to isolate nanoparticle-bound GTIN. A 200 µL aliquot of the supernatant is analyzed spectrophotometrically to evaluate encapsulation efficiency and drug loading. The pellet is then washed with ultrapure water to eliminate unadsorbed GTIN and lyophilized, yielding a pale green powder indicative of GTIN-SFNP formation.

[051] In yet another embodiment, the drug-loading content (DLC) and encapsulation efficiency (EE) of GTIN in SFNPs are evaluated to determine the optimal formulation characteristics. The desolvation-based loading approach yields encapsulation efficiencies of 44.8 ± 0.59%, 65.3 ± 0.17%, and 81.95 ± 0.28% for GTIN/SFNPs ratios of 1:10, 1:20, and 1:40 (w/w), respectively. The formulation exhibiting the highest EE and loading content corresponds to a 1:40 ratio after a 48-hours incubation period, as detailed in Table 1.

 
[052] Table 1: Encapsulation Efficiency and Loading Content
S.No GTIN/SFNPs Ratio (w/w) Encapsulation Efficiency (%) Loading Content (%)
1 1:10 44.8 ± 0.59 0.448 ± 0.57
2 1:20 65.3 ± 0.17 0.653 ± 0.78
3 1:40 81.95 ± 0.28 0.8175 ± 0.49

The 1:40 GTIN/SFNPs formulation is utilized for subsequent coating applications due to superior encapsulation performance.

[053] Preparation of GTIN- Honey loaded SFNPs based dip coating:

[054] In a related embodiment, the preparation of GTIN- Honey loaded SFNPs based dip coating solution is achieved through desolvation technique. A 1 mL aliquot of the GTIN-SFNPs dispersion (1:40 ratio) is blended with 5% Honey by volume. The resulting mixture is stirred magnetically for 10 minutes at room temperature for initial homogenization, followed by an additional 40-minute stirring period to ensure formation of a stable coating matrix.

[055] The final dip-coating solution is transferred to a 5 mL container, and fresh fruits and vegetables are coated by immersion three times, with five-minute intervals between each cycle. Each immersion lasts approximately 30–40 seconds, and produce items are repositioned during the process to achieve uniform coverage. Post-treatment, the samples are air-dried at ambient temperature to solidify the bio-preservative layer.

[056] In another embodiment, the physicochemical characterization of the GTIN-Honey-SFNPs nanocomposite is presented (Refer to Table 2). Spectroscopic validation was performed using UV, FTIR, and XPS analyses, while morphological confirmation was established through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM). Thermal stability was assessed via Thermogravimetric Analysis (TGA).

[057] Table 2: Physicochemical Characterization of GTIN-Honey-SFNPs Nanocomposite
Technique Key Observations Inference
UV–Visible Spectroscopy Absorption peak at 480 nm for GTIN-Honey-SFNPs; native SF/SFNPs show peaks at 270 nm and 420 nm Confirms successful GTIN loading onto SFNPs with distinct optical signature
FTIR Spectroscopy Composite shows characteristic amide I–III peaks, O–H, C=C, C–O, and C=O vibrations Validates integration of GTIN and Honey within the Silk fibroin matrix
XPS – Elemental Survey Presence of C1s, N1s, and O1s peaks indicating chemical diversity Confirms presence of organic moieties associated with protein and flavonoids
XPS – High Resolution C1s: C–C (293.24), C–O (294.50), C=O (295.14); N1s: C–N–C, C–N, C–O; O1s: 530.89 Reveals functional bonding environments; indicates successful chemical binding
FE-SEM Imaging Bead-like random morphology; mean diameter ~91.797 ± 43.51 nm, with the highest value reported being 198.459 nm revealed the actual morphology and average size.
TEM Imaging Globular particle morphology; moderate aggregation; particle size <200 nm Supports controlled dispersion and compatibility with food surfaces
Thermogravimetric Analysis (TGA) Progressive weight loss at 75–85°C and 240–310°C indicating staged degradation Highlights Honey’s moisture retention and confirms composite thermal behaviour.

[058] 1. Spectroscopic Validation
• As depicted in Figure 1, UV–Visible spectroscopy identified a prominent absorption peak at 480 nm, distinct from native Silk fibroin and SFNPs, confirming optical transition due to GTIN loading.
• FTIR analysis, shown in figure 2, confirmed overlapping peaks for Silk fibroin amide bands and GTIN-associated functional groups (C=C, C=O, C–O, O–H), indicating successful encapsulation and interaction within the matrix.
• Referring to Figure 3, and as detailed in Table 2, XPS analysis confirmed the surface chemical states and elemental interactions, indicating the presence of covalent and hydrogen bonding of GTIN and Honey.

[059] 2. Morphological Confirmation
[060] 2a. Field Emission Scanning Electron Microscopy (FE-SEM)
The surface morphology of GTIN-Honey-SFNPs nanocomposite was examined by field emission scanning electron microscopy. FE-SEM analysis revealed that the nanocomposites were in a random bead-like pattern, which ensured the enhanced hydrophobic characteristics. The average size (calculated from 55 particles taken from images) of GTIN-Honey-SFNPs nanocomposite was calculated using Image J software and was found to be 91.797 ± 43.51 nm, with the highest value reported being 198.459 nm. The inset image of the histogram represents the GTIN-Honey-SFNPs nanocomposite diameter range against the number of counts, as shown in Fig. 4a & b.

[061] 2b. Transmission Electron Microscopy (TEM):

The size and morphology of SFNPs and GTIN-Honey-SFNPs were examined by TEM. The TEM observations (Fig.5a and 5b) showed globular granules for SFNPs and GTIN-SFNPs with some aggregation. The aggregation observed for lyophilized GTIN-SFNPs was 148nm, probably due to the effect of GTIN on the surface, while nanoparticle sizes were less than 200nm. (Fig.5c and 5d) showed the threshold of image b using ImageJ and the particle size distribution of the histogram.

[062] 3. Thermal Stability
[063] 3a.Thermogravimetric analysis (TGA):

The chemical decomposition behavior of the GTIN-Honey-SFNPs nanocomposite as a function of time was investigated using thermogravimetric analysis shown in Fig.6, which represents the comparative thermogram of the GTIN-Honey-SFNPs nanocomposite. The GTIN-Honey-SFNPs nanocomposite undergoes more weight loss between the temperature ranges of 75–85°C. Further, the weight residue percentage falls sharply after 240–310°C, due to the degradation of SFNPs and GTIN. Such a difference in weight loss is due to the presence of water content in Honey released from the glucose, fructose, and other sugar molecules while heating at a very high temperature, which confirms that Honey acts as a natural moisturizer that prevents the peel of fruits and vegetables from dehydration and makes the skin of the fruit stiff.

[064]The combined analytical results affirm that GTIN and Honey are efficiently loaded onto Silk fibroin nanoparticles via physical adsorption and chemical entrapment. The nanocomposite exhibits structural homogeneity, thermal robustness, and functional chemical compatibilityrendering it a promising candidate for natural, non-toxic fruit and vegetable preservation.

[065] EXAMPLES
[066] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure 10 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

[067] Example 1:

[068] Shelf-Life Evaluation of Fresh Produce Using Time-Lapse Imaging:

In one embodiment of an experimental comparative study involving fresh produce, include vegetables (tomato, ivy gourd, eggplant) and fruit (green banana), the shelf life and changes in external appearance, stiffness, taste, and texture were monitored over time using time-lapse photography.

[069] Geographical Source:
The substantially identical fresh produce models include vegetables (tomato, ivy gourd, eggplant) and fruit (green banana) and the aforesaid fresh produce were procured from a Oooravayal, Karaikudi (Taluk), Tamil Nadu, India.

[070] The samples were divided into three groups(n = 3): (a) Control (Uncoated fresh produce)(b) GTIN-SFNPs nanocomposite coated Fresh produce, and (c) GTIN-Honey-SFNPs nanocomposite coated Fresh produce.

[071] Example (1a): Tomato coated with GTIN-Honey-SFNPs nanocomposite

Figure 7, supported by the data in Table 3, illustrates a comparative study of tomato preservation across three groups: (a) uncoated tomato (Control), (b) tomato coated with GTIN-SFNPs nanocomposite, and (c) tomato coated with GTIN-Honey-SFNPs nanocomposite. The control samples exhibited visible spoilage by Day 8, while tomatoes coated with GTIN-SFNPs nanocomposite showed signs of spoilage by Day 24. In contrast, tomatoes coated with GTIN-Honey-SFNPs nanocomposite remained firm and odour-free and spoilage for up to initiating between Day 28 and Day 36. The enhanced preservation in the GTIN-Honey-SFNPs group is attributed to reduced surface dehydration and the biological antimicrobial activity of the coating. Honey is a naturally occurring, multifunctional substance and its composition is rich in sugars, enzymes, amino acids, and polyphenols, so it also acts as a preservative and a bioactive enhancer in postharvest applications.

[072] Table 3: Comparative Evaluation of Shelf-Life Parameters in tomato preservation across three treatment groups
Sample Firm Odor-Free Spoilage
a. Control Day 6 Day 7 Day 8
b. Tomato coated with GTIN-SFNPs nanocomposite Day 14 Day 22 Day 34
c. Tomato coated with GTIN-Honey-SFNPs nanocomposite Day 14 Day 28 Day 36

[073] Example (1b): Ivy gourd coated with GTIN-Honey-SFNPs nanocomposite

Figure 8, supported by the data in Table 4, illustrates a comparative study of Ivy gourd preservation across three groups: (a) uncoated Ivy gourd (Control), (b) Ivy gourd coated with GTIN-SFNPs nanocomposite, and (c) Ivy gourd coated with GTIN-Honey-SFNPs nanocomposite. The uncoated Ivy gourd ripens after 5 days, but it cannot be consumed because, side by side, it starts decaying at the same time.

The GTIN-Honey-SFNPs nanocomposite coating delays the ripening process of Ivy gourdby 3–4 days, and after ripening, it can be used as a fruit (the color changes to pink during initial ripening). One of the primary causes of the uncoated Ivy gourd's early deterioration was fungal development, while the coated Ivy gourd did not exhibit any fungal growth, due to the presence of Gossypetin, act as a antibacterial ingredient in SFNPs. Other benefits of using Honey are it acts as a natural humectant, helping retain moisture and prevent desiccation of fruit surfaces. Its antimicrobial properties, primarily due to hydrogen peroxide and low pH, inhibit the growth of spoilage-causing bacteria and fungi.

[074] Table 4: Comparative Analysis of Ivy gourd preservation across three groups
Sample Ripen Deterioration
Control Day 6 Day 13
Ivy gourd coated with GTIN-SFNPs nanocomposite Day 17 Day 22
Ivy gourd coated with GTIN-Honey-SFNPs nanocomposite Day 23 Day 27

[075] Example (1c): Eggplant coated with GTIN-Honey-SFNPs nanocomposite:
Figure 9, supported by the data in Table 5, illustrates a comparative study of Eggplant preservation across three groups: (a) uncoated Eggplant (Control), (b) eggplant coated with GTIN-SFNPs nanocomposite, and (c) Eggplant coated with GTIN-Honey-SFNPs nanocomposite. The Eggplant was selected as a model vegetable due to its short shelf life of about two to three days. An Eggplant covered with edible GTIN-Honey-SFNPs nanocomposite had a seven-day shelf-life extension while maintaining its quality, texture, and rigidity. As seen in Fig. 9, an uncoated Eggplant selected as the control, after eight days, lost its rigidity and began to smell bad.
[076] Table 5: Comparative Analysis of Eggplant preservation across three groups
Sample Deterioration Shelf Life
Control
( Uncoated Eggplant) Day 3 Day 5
Eggplant coated with GTIN-SFNPs nanocomposite Day 7 Day 10
Eggplant coated with GTIN-Honey-SFNPs nanocomposite Day17 Day 20

[077] Example (1d): Green banana coated with GTIN-Honey-SFNPs nanocomposite:

Figure 10, supported by the data in Table 6, illustrates a comparative study of Green banana preservation across three groups: (a) uncoated Green banana (Control), (b) Green banana coated with GTIN-SFNPs nanocomposite, and (c) Green banana coated with GTIN-Honey-SFNPs nanocomposite. The immature green banana is a vegetable that undergoes a two-week delay in ripening when coated with GTIN-Honey-SFNPs nanocomposite. Once ripened, the banana can be used as a fruit. In contrast, an uncoated banana ripens after two weeks but cannot be consumed because it begins to decay simultaneously with the coated banana. As can be seen in Fig.10, the fungal growth is shown on the uncoated banana, which is one of the primary causes of its early deterioration. In contrast, coated bananas do not exhibit any fungal growth because SFNPs contain Gossypetin, an antibacterial agent. Additionally, Honey contributes to antioxidant protection, delaying oxidative browning and preserving the sensory quality of fruits. Honey can be blended with biopolymers to form composite coatings with improved mechanical strength and barrier properties.

[078] Table 6: Comparative Analysis of Green banana preservation across three groups
Sample Ripen Deterioration
Control
Uncoated Green banana Day 5 Day 8 to Day 11
Green bananacoated with GTIN-SFNPs nanocomposite Day 9 Day 9 to Day 11
Green banana coated with GTIN-Honey-SFNPs nanocomposite Day 19 Day 22

 
[079] Example.2 Antimicrobial and Antifungal activityof GTIN-Honey-SFNPs Nanocomposite:

In this experimental Embodiment, Antimicrobial and Antifungal Activity of GTIN-Honey-SFNPs Nanocomposite is analysed. In this analysis, the antimicrobial efficacy of GTIN-Honey-SFNPs nanocomposite was evaluated against Escherichia coli and Bacillus subtilis, while antifungal activity was assessed against Aspergillus flavus. The zone of inhibition was analyzed at varying concentrations of the nanocomposite to determine its inhibitory potential across microbial types. Thus the detection of spoilage-related and pathogenic microorganisms highlights the necessity for robust preservation strategies.

[080] Isolation and Characterization of Microorganisms from Spoiled Tomato Samples

Fresh tomatoes (Procured from Ooravayal, Karaikudi (Taluk), Tamil Nadu, India) were exposed to ambient conditions for 7 days to facilitate natural spoilage. Microbial isolation was conducted using serial dilutions (10⁻¹ to 10⁻⁶) of fruit suspensions prepared in sterile distilled water and plated on nutrient agar medium (NAM). Plates were incubated at 37 °C for 24 hours, and morphological assessment was performed. Selected bacterial colonies were subcultured on NAM slants and stored at 4 °C.

[081] Fungal isolation followed the pour-plate method (Barnett & Hunter, 1987). Colonies were transferred to Sabouraud Dextrose Agar (SDA) plates and incubated at 28–30 °C for 5–7 days. Purified isolates were maintained on SDA slants at 10–15 °C for further analysis.

[082] Fungal isolation followed the pour-plate method (Barnett & Hunter, 1987). Microbial identification was carried out through cultural, morphological, microscopic, and biochemical methods, including Gram staining and IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate). Reference standards (Holt et al., 1994; Goldman & Green, 2008; Shermanand& Cappuccino, 2014) were used to validate the findings.

[083] Microbial Targets for Biological Activity Assessment:

[084] Figure 11 depicts the microbiological characterization of selected bacterial and fungal strains:
(a) colony morphology of Bacillus subtilis,
(b) positive Gram staining result of Bacillus subtilis (magnification 40×),
(c) colony morphology of Escherichia coli,
(d) negative Gram staining result of Escherichia coli (magnification 40×),
(e) colony morphology of Aspergillus flavus, and
(f) microscopic analysis of Aspergillus flavuscell morphology (magnification 40×)

[085] The following species were identified from spoiled fruit samples and selected for biological evaluation:

• Escherichia coli — short rod-shaped, Gram-negative
• Bacillus subtilis — long rod-shaped, Gram-positive
• Aspergillus flavus — filamentous fungus

The presence of spoilage-associated and pathogenic microorganisms underscores the importance of effective preservation systems.

[086]Example 2(a) Antibacterial Activity of GTIN-Honey-SFNPs Nanocomposite:

[087] This example, six different concentrations of GTIN-Honey-SFNPs (50 μg/ mL, 100 μg/ mL, 200 μg/ mL, 400 μg/ mL and 500μg/mL) were prepared from a stock solution of GTIN-Honey-SFNPs.Antibacterial activity of GTIN-Honey-SFNPs nanocomposites was performed against Gram-positive and negative bacteria, namely E. coli and Bacillus subtilis, as shown in Figure 12.
[088] E. coli is more sensitive to the extract with an average zone of 12.8±1.2mm, while Bacillus subtilis is less sensitive to an average zone of inhibition of 11±0.7 mm at a maximum concentration of GTIN-Honey-SFNPs nanocomposite at 500μg/mL exhibited good antibacterial activity respectively in comparison with the control, where the zone of inhibition was 14mm as shown in Table 7.
[089] Table.7: The zone of inhibition of the GTIN-Honey-SFNPs against Gram positive bacteria (Bacillus subtilis) and Gram-negative bacteria (E. coli).
S.No Name of the strain (Zone of inhibition mm)
Concentration of GTIN-Honey-SFNPs
Control 50μg/mL 100μg/mL 200μg/mL 400μg/mL 500μg/mL
1. E. coli 14 -
9.0±1.2 10.5±0.9 11.5±0.8 12.8±1.2
2. B. subtilis
14
-

-

10±0.5
11±1.9
12±0.7

[090] Example 2(b): In vitro Antifungal activityof GTIN-Honey-SFNPs Nanocomposite:

The antifungal potential of the GTIN-Honey-SFNPs nanocomposite is assessed using the disc diffusion method, a standard qualitative technique for evaluating zone of inhibition (Yoon et al., 2015).The synthesized GTIN-Honey-SFNPs appear to be alternative antifungal agents to antibiotics and can overcome antibiotic-resistant fungi. Therefore, it is necessary to develop GTIN-Honey-SFNPs as antifungal agents.

[091] In this example, nine different concentrations of GTIN-Honey-SFNPs (3.125 μg/ mL, 6.2 μg/ mL, 12.5 μg/ mL, 25 μg/ mL, 50 μg/ mL, 100 μg/ mL, 200 μg/ mL, 400 μg/ mL and 500μg/mL) were prepared from a stock solution of GTIN-Honey-SFNPs.The antifungal potential of the GTIN-Honey-SFNPs nanocomposite against Aspergillus flavus was evaluated using the agar-disc diffusion method, as illustrated in Figure 13. The distinct formation of inhibition zones around discs impregnated with the nanocomposite confirmed its antifungal efficacy. A notable reduction in fungal colony growth is observed, with clear inhibition zones forming consistently across replicates. To assess formulation stability, the assay was performed in triplicate over a 5-day period using the same storage solution, demonstrating retained antifungal activity throughout.

[092] Table.8 The zone of inhibition of the GTIN-Honey-SFNPs against Aspergillus flavus:

[093] The average length of these inhibition zones (average, standard deviation) was calculated. Aspergillus flavus is more sensitive to the extract, with an average zone of 14.1±1.9 mm at a maximum concentration of GTIN-Honey-SFNPs nanocomposite at 500μg/mL exhibited good antifungal activity, respectively, in comparison with the control, where the zone of inhibition was 15 mm, as shown in Table 8.

[094] The results demonstrated that the GTIN-Honey-SFNPs nanocomposites that were prepared with a higher concentration of GTIN-Honey-SFNPs nanocomposites had outstanding effects against both fungi, which shows a clear region around the disc for fungi, whereas GTIN-Honey-SFNPs nanocomposites synthesized at lower concentrations demonstrate very low activity. The higher concentration of GTIN-Honey-SFNPs nanocomposite employed the stronger antifungal activity against Aspergillus flavus.

[095] Example 3: Proximate Composition Analysis of GTIN-Honey-SFNPs Coated Tomatoes:

[096] In this experimental Embodiment, a comparative study is conducted to evaluate the proximate of GTIN-Honey-SFNPs nanocomposite coated tomatoes with uncoated tomatoes (as control) and GTIN-SFNPs coated tomato. The assessment focuses on parameters including moisture content, crude protein, crude fat, ash, crude fiber, and carbohydrates to determine the impact of the coating on nutritional retention during room temperature storage.

[097] Table 9: Proximate composition of tomato samples

Contents Fresh tomato GTIN-SFNPs Coated tomato GTIN-Honey-SFNPs Coated tomato
Moisture (%) 75.12±0.15 92.79±0.12 92.12±0.78
Crude protein (%) 0.26±0.87 0.24±0.12 0.21±0.14
Crude fat (%) 0.06±0.01 0.05±0.98 0.04±0.07
Ash (%) 0.14±0.14 0.12±0.12 0.10±0.27
Carbohydrate (%) 6.78±0.04 6.53±0.01 7.14±0.02

[098] Proximate results indicate that the uncoated fresh tomatoes exhibit moisture content, crude protein, crude fat, ash, and carbohydrate levels of 75.12%, 0.26%, 0.06%, 0.14%, and 6.78%, respectively. Tomatoes coated with GTIN-SFNPs retain quality for up to 21 days, displaying corresponding values of 92.79%, 0.24%, 0.05%, 0.12%, and 6.53%.
[099] Whereas the Tomatoes coated with GTIN-Honey-SFNPs exhibit extended shelf life up to 27 days, with moisture content, crude protein, crude fat, ash, and carbohydrate levels recorded as 92.12%, 0.21%, 0.04%, 0.10%, and 7.14%, respectively, as presented in Table 9.
[100] The results demonstrate that the incorporation of Honey facilitates nutrient preservation and prolongs the shelf life of tomatoes by an additional six days compared to GTIN-SFNPs alone.
[101] Example 4: Antioxidant Potential of GTIN-Honey-SFNPs via DPPH Assay and Determination of IC50 Value:
[102] In this experimental embodiment, the antioxidant properties of the GTIN-Honey-SFNPs nanocomposite are evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, as illustrated in Figure 15. This method evaluates the material's ability to neutralize nitrogen-centred free radicals, which are visually indicated by a colour shift from deep violet to yellow upon reduction. The assay involves treating a methanolic DPPH solution with varying concentrations of the nanocomposite and monitoring the decrease in absorbance at 517 nm. As illustrated in Fig. 15, the free radical scavenging activity exhibited a dose-dependent trend. An increase in the concentration of the GTIN-Honey-SFNPs nanocomposite resulted in a corresponding enhancement in scavenging efficiency, rising from 30% to 60%.

[103] As depicted in Fig. 15, the IC50 value derived from the dose-response curve was approximately 190 μg/mL for the GTIN-Honey-SFNPs nanocomposite. In a comparable study, Pratap Singh and Packirisamy (2022) reported a dose-dependent increase in free radical scavenging activity for nanocurcumin, ranging the IC50 value at 17.64 μg/mL.

[104] Example 5: Hemocompatibility of GTIN-Honey-SFNPs Formulation for Intravenous Administration
[105] In one embodiment, the GTIN-Honey-SFNPs formulation is demonstrated to be safe for intravenous injection, as determined by its hemocompatibility profile evaluated through a hemolysis assay. This procedure confirms that the formulation induces minimal red blood cell lysis across tested concentrations, indicating favorable compatibility with systemic administration.
[106] The Figure 16 presents the comparative hemocompatibility analysis of GTIN-Honey-SFNPs-Tween and GTIN-Honey-SFNPs formulations based on an in vitro hemolysis assay.Figure 16(a) illustrates the hemolysis rate (%) of red blood cells exposed to varying concentrations (100, 200, 300, 400, and 500 μg/mL) of GTIN-Honey-SFNPs and GTIN-Honey-SFNPs-Tween formulations.Data are expressed as mean ± standard error (SE) for three independent replicates (n = 3) with statistically significant differences are indicated ***P,0.000.
[107] Figure 16(b) displays representative microscopic images of red blood cells (RBCs) following treatment with GTIN-Honey-SFNPs-Tween and GTIN-Honey-SFNPs formulations. The images depict morphological variations in RBCs indicative of differential hemolytic responses, highlighting membrane integrity preservation in samples treated with GTIN-Honey-SFNPs as compared to those treated with the Tween-containing formulation.
[108] Referring to Figure 16(a), the GTIN-Honey-SFNPs-Tween formulation exhibited elevated hemolytic activity, reaching 15.89% at a concentration of 200 μg/mLand peaking at approximately 80% at 500 μg/mL. In contrast, the GTIN-Honey-SFNPs formulation consistently maintained hemolysis rates below the widely accepted intravenous safety threshold of 5.0%, as established by Cao et al. (2011), at concentrations of 100, 200, 300, and 400 μg/mL. A marginal increase was observed at 500 μg/mL. Therefore, these results underscore the excellent hemocompatibility of the GTIN-Honey-SFNPs formulation, reinforcing its potential suitability for safe intravenous administration.
 
[109] Example 6: Biocompatibility and Toxicity Evaluation:
[110] Bioimaging-Based Assessment of GTIN-Honey-SFNPs Nanocomposite Demonstrating High Biocompatibility and Low Toxicity:
[111] In one embodiment, a comparative analysis is performed to evaluate the biocompatibility and toxicity characteristics of synthesized SFNPs, GTIN, GTIN-SFNPs, and GTIN-Honey-SFNPs formulations following their introduction into the body of Caenorhabditis elegans. The investigation assesses cellular viability, morphological integrity, and fluorescent behaviour to determine suitability for biological applications.
[112] Figure 17 presents fluorescence imaging of Caenorhabditis elegans exposed to SFNPs, GTIN, GTIN-SFNPs, and GTIN-Honey-SFNPs bioactive formulations. Upon laser excitation at 470 nm, the organisms exhibit green fluorescence, indicating high biocompatibility and minimal toxicity.
The study investigates four distinct treatments SFNPs (I), GTIN (II), GTIN-SFNPs (III), and GTIN-Honey-SFNPs (IV) across three concentration levels: 0.5 mg/mL, 1 mg/mL, and 2 mg/mL. Panel (a) represents the control group, where C. elegans were fed with E. coli OP50 and received no treatment. Panels (b), (c), and (d) illustrate the fluorescence behavior of C. elegans incubated with the respective test formulations at 0.5 mg/mL, 1 mg/mL, and 2 mg/mL concentrations. Each treatment demonstrates distinct emission profiles, confirming successful dose-dependent internalization and optical traceability. The formulations exhibit excellent biocompatibility with minimal observable toxicity, highlighting their imaging potential for bio-preservative and biomedical applications.
 
[113] Table 10: Comparison table of Fluorescence Imaging in Caenorhabditis elegan
Formulations Concentration
0.5 mg/mL 1 mg/mL 2 mg/mL
SFNPs Very low visibility of Green low visibility of Green Minimum visibility of Green
GTIN Very low visibility of Green low visibility of Green Minimum visibility of Green
GTIN-SFNPs low visibility of Green Minimum visibility of Green Medium visibility of Green
GTIN-Honey-SFNPs Minimum visibility of Green Medium visibility of Green Intense Green

[114] The bioimage analysis of the produced SFNPs, GTIN, GTIN-SFNPs, and GTIN-Honey-SFNPs revealed that, even at 0.5 mg/mL, they did not significantly lower the viability of Caenorhabditis elegans cells. Moreover, no morphological alterations were noted in the cells following their incubation with the SFNPs, GTIN, GTIN-SFNPs, and GTIN-Honey-SFNPs.

[115] It is confirmed that thebioimage analysis of synthesized GTIN-Honey-SFNPs at concentration of 2mg/ml shows high fluorescent intensity and high biocompatibility and are thus appropriate for biomedical and other biological applications because of their photo stability, water stability, and bioimaging properties when compared to SFNPs, GTIN, and GTIN-SFNPs.

[116] Example 7: In Vitro Cytotoxicity Assay:
[117] In another embodiment, the cytotoxic potential of the formulation was evaluated using two complementary assays: the MTT assay, which measures the loss of cell viability based on metabolic activity, and fluorescence-based staining methods, which detect the mode of cell death—specifically, apoptosis—through visualization of characteristic cellular changes.
[118] Culture Medium:
[119] The bacterial culture medium, Luria Bertani (LB) and Nutrient Broth (NB), used for bacterial assays, were procured from Merck (Germany) and Himedia(India), respectively. The HCT-116 colorectal cancer cell lines were received from the National Center for Cell Science (Pune, India), and the [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT ≥ 97.5%), was received from Sigma-Aldrich (Bangalore, India) Dulbecco’s Modified Eagle Medium (DMEM) (LOT0000481561) Dulbecco’s Phosphate Buffered Saline (LOT SLBZ6118). All the reagents were of analytical grade and used without further modification.
[120] The MTT assay was employed to evaluate the cytotoxic potential of the GTIN-Honey-SFNPs nanocomposite against colorectal cancer cells under in vitro conditions. Figure 14 illustrates the viability percentage of colorectal cancer cells (HCT-116) after 24-hour exposure to varying concentrations (0 to 10 μM)of GTIN-Honey-SFNPs nanocomposite, demonstrating its concentration-dependent cytotoxic effects. The half-maximal inhibitory concentration (IC50) was determined to assess the cytotoxic potency of the GTIN-Honey-SFNPs nanocomposite. It was observed that a concentration of 6 μM effectively induced dose-dependent cell death in colorectal cancer cells (HCT-116). The IC50value corresponded to apoptotic cell death, indicating the nanocomposite’s efficacy in triggering programmed cell death pathways.

[121] Example 7b: Fluorescence-Based Staining Reveals Apoptosis Induction in Colorectal Cancer Cells by GTIN-Honey-SFNPs Nanocomposite
[122] Furthermore, a dual-staining method (as described by Solairaj et al. (2016)). is implemented using acridine orange (AO) and ethidium bromide (EtBr) dyes to differentiate between normal and apoptotic cells.
[123] Figure 15 illustrates the fluorescence-based staining analysis performed to assess the apoptotic response of colorectal cancer cells following treatment with the GTIN-Honey-SFNPs nanocomposite. Panels (a–b) show dual staining using acridine orange (AO) and ethidium bromide (EtBr), which differentiate viable and apoptotic cells based on green and red fluorescence emissions, respectively. AO, a cell-permeable nucleic acid-selective dye, emits green fluorescence upon binding to DNA in intact cells, enabling visualization of nuclei and cytoplasm. EtBr, which penetrates cells with compromised membrane integrity, binds to nuclear DNA and emits bright red fluorescence, marking apoptotic or necrotic cells.
[124] Panel (a) displays control cells with uniform green fluorescence, indicating the absence of apoptotic features. In contrast, panel (b) shows cells treated with the GTIN-Honey-SFNPs nanocomposite at the half-maximal inhibitory concentration (IC50), exhibiting both green and red fluorescence—corresponding to viable and apoptotic cells, respectively. These results confirm the cytotoxic nature of the GTIN-Honey-SFNPs nanocomposite and its role in inducing apoptosis.
[125] Panels (c–d) present the propidium iodide (PI) exclusion assay for live/dead cell detection. Control cells in panel (c) exclude PI, reflecting intact membrane integrity, while treated cells in panel (d) incorporate PI, indicating membrane disruption and cell death.
[126] Panels (e–f) depict nuclear staining using Hoechst 33344 to evaluate chromatin condensation. Hoechst 33344 is a non-intercalating dye that binds to the minor groove of AT-rich regions in DNA and emits blue fluorescence upon UV illumination. This fluorescence marks pyknotic nuclei—characterized by irreversible chromatin condensation and indicative of apoptotic progression. Control cells in panel (e) show normal nuclear morphology, whereas treated cells in panel (f) exhibit prominent pyknotic nuclei. A higher frequency of such nuclei is observed in treated samples, supporting the hypothesis that the GTIN-Honey-SFNPs nanocomposite induces apoptosis via DNA damage mechanisms.
[127] Collectively, these fluorescence-based staining results substantiate the apoptotic potential of the GTIN-Honey-SFNPs nanocomposite. All panels include a scale bar of 20 μm.s
[128] Advantages of the present disclosure:
[129] The present disclosure discloses an Edible bio-preservative coating composition for enhancing the shelf life of fresh fruits and vegetablescomprising a synergistic combination of 1 mL of Silk fibroin aqueous solution (1% w/v), 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0), and 5% Honey, relative to the Gossypetin-Silk fibroin solution volume with effective antioxidant, anticarcinogenic, and antibacterial properties. Further, the disclosed synergistic nanocomposite effectively enhances the shelf life and preserves the physical integrity of various perishable vegetables, including tomatoes, ivy gourd (ripe and unripe), green banana (ripe and unripe), and eggplant.
[130] Furthermore, the disclosed GTIN-Honey-SFNPs nanocomposite demonstrates significant advantages in preserving the shelf life and quality of perishable produce. Experimental results confirm that tomatoes coated with the GTIN-Honey-SFNPs nanocomposite remain edible for up to 28 days, retaining texture and quality, compared to uncoated tomatoes which deteriorate within 5–7 days. Similarly, coated ivy gourd functions for more than seven days, and it loses much less weight than the uncoated variety, which loses its rigidity and ripens on the twenty-ninth day, while the coated variety is in fine shape. Comparable to the coated and uncoated varieties of green banana, the Coated Green bananas with the nanocomposite maintain structural integrity for over three weeks, whereas uncoated samples ripen and lose stiffness within five days. Eggplants subjected to dip-coating remain viable for up to 20 days, further validating the the coating’s efficacy across diverse produce types. Therefore, the preservative effect of the edible coating was experimentally validated on a variety of fresh produce. As shown in Table 1, the application of the coating resulted in a significant extension of shelf life when compared to untreated controls. For example, tomatoes with a natural shelf life of 6-7 days showed a shelf life of up to 28 days when coated with the composition, representing a four-fold increase. Similar results were observed with ivy gourd, green bananas, and eggplant.s
[131] Additionally, the GTIN-Honey-SFNPs nanocomposite, as disclosed herein, demonstrates that the individual components alone do not yield the same level of efficacy. Rather, it is the synergistic composition of Gossypetin, Honey, and Silk fibroin nanoparticles that confers superior advantages in preserving the shelf life and quality of perishable produce. The combined formulation exhibits enhanced bioactivity and functional performance, which are not observed when the constituents are applied independently. This synergistic effect is critical to the disclosed invention and contributes to its effectiveness in maintaining texture, reducing weight loss, and extending edibility across a range of fruits and vegetables.
[132] In addition to its proven efficacy in extending the shelf life and preserving the physical quality of fruits and vegetables, the edible coating composition disclosed herein offers a distinct advantage over existing commercial coatings. Unlike conventional edible coatings that are costly and inaccessible to regional vendors, the disclosed Silk fibroin-based nanocomposite offers a natural, affordable, and scalable solution. In addition to its preservative function, the formulation exhibits cytotoxic activity against colorectal cancer cells, suggesting its potential as a health-promoting edible biomaterial. Accordingly, the GTIN-Honey-SFNPs nanocomposite provides a dual benefit extending food shelf life and contributing to cancer prevention thereby addressing critical needs in food nanotechnology and public health.
[133] Hence, the present disclosure employs a combinatorial approach that yields a synergistic effect through the integration of Gossypetin, Honey, and Silk fibroin nanoparticles. This strategic formulation enhances the functional properties of each component, resulting in a nanocomposite with superior preservative efficacy and therapeutic potential. Overall, the composition disclosed herein is directed toward the development of a cost-effective, non-toxic, and biocompatible edible coating material. It not only extends the shelf life and maintains the quality of perishable produce but also contributes to health protection particularly in the prevention of colorectal cancer thereby addressing critical needs in food preservation, public health, and sustainable nanotechnology.
[134] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
, Claims:I/We Claim:
1. A Gossypetin-encapsulated Silk fibroin nanocomposite based edible bio-preservative coating composition comprising:
a. 1 mL of Silk Fibroin protein nanoparticles;
b. 1 mL of ethanol containing 1 mg of Gossypetin, (1% w/v aqueous solution, pH 7.0); and
c. 5% of Honey, where in the volume is relative to the combined volume of the Gossypetin-Silk fibroin solution; and

Wherein the said composition exhibits inhibitory activity against the microbial growth of Bacillus subtilis, Escherichia coli, and Aspergillus flavus thereby contributing to enhanced preservation efficacy and prolonged shelf life of fruits and vegetables.

2. The composition as claimed in claim 1, wherein the said composition exhibits high biocompatibility, excellent hemocompatibility, minimal toxicity, and enhanced free radical scavenging efficiency.

3. The composition as claimed in Claim 1, wherein the said composition exhibits antibacterial activity against Escherichia coli, with a zone of inhibition ranging from 9.0 ± 1.2 mm to 12.8 ± 1.2 mm at concentrations between 100 µg/mL and 500 µg/mL.

4. The composition as claimed in Claim 1, wherein the said composition exhibits antibacterial activity against Bacillus subtilis with a zone of inhibition ranging from 10±0.5 mm to 12±0.7 mm at concentration between 200 µg/mL and 500 µg/mL.

5. The composition as claimed in Claim 1, wherein the said composition exhibits antifungal activity against Aspergillus flavus with a zone of inhibition ranging from 8±0.9 mm to 14.1±1.9 mm at concentration between 6.2 µg/mL and 500 µg/mL.

6. The composition as claimed in Claim 1, wherein the said composition exhibits colorectal cancer-preventive properties, by selectively inducing dose-dependent cytotoxicity in colorectal cancer cells (HCT-116) and demonstrating antioxidant activity with an IC₅₀ value of approximately 190 μg/mL.

7. A method for preparing Gossypetin-encapsulated Silk fibroin nanocomposite based edible bio-preservative coating composition configured to prolong the shelf life of fresh produce, the method comprising:
• synthesizing Silk fibroin protein nanoparticles (SFNPs) via a desolvation-based protocol, wherein 1 mL of an aqueous Silk fibroin solution (1% w/v, pH 7.0) is gradually added to 1 mL of ethanol containing 1 mg of Gossypetin (GTIN), thereby inducing self-assembly of Silk fibroin molecules into uniform, structurally stable nanoparticles;
• incorporating Honey into the resulting GTIN-SFNP dispersion at a concentration of 5% relative to the total volume; and
• subjecting the blend to magnetic stirring at ambient temperature for a total duration of 50 minutes, comprising an initial 10-minute stirring phase to ensure homogeneity, followed by a 40-minute phase to achieve coating.