# Development and characterization of edible film containing keratin nanocarriers loaded with N. nucifera seedpod extract

Hina Fayaz Bhat, Zarka Nasir, Sheza Farooq, Zuhaib F Bhat, Syed Mudasir Andrabi, Abderrahmane Aït-Kaddour

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Hina Fayaz Bhat, Zarka Nasir, Sheza Farooq, Zuhaib F Bhat, Syed Mudasir Andrabi, et al.. Development and characterization of edible film containing keratin nanocarriers loaded with N. nucifera seedpod extract. Food Hydrocolloids for Health, 2025, 7, pp.100216. 10.1016/j.fhfh.2025.100216. hal-05201280

HAL Id: hal-05201280

https://hal.inrae.fr/hal-05201280v1

Submitted on 6 Aug 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Check for
updates

# Development and characterization of edible film containing keratin nanocarriers loaded with *N. nucifera* seedpod extract☆

Hina Fayaz Bhat<sup>a,**</sup>, Zarka Nasir<sup>a,1</sup>, Sheza Farooq<sup>a,1</sup>, Zuhaib F. Bhat<sup>b</sup>, Syed Mudasir Andrabi<sup>a</sup>, Abderrahmane Aït-Kaddour<sup>c,*</sup>

## ARTICLE INFO

**Keywords:**

Edible film

Keratin

Nelumbinis nucifera

Seedpod extract

Nanoparticles

Film characteristics

## ABSTRACT

Edible films prepared from organic macromolecules have evolved as sustainable and biodegradable alternatives to standard petroleum-based packaging. The present study aimed to develop a novel carrageenan-based bioactive film using keratin nanocarriers loaded with *N. nucifera* seedpod extract (KNP-Ex). An ultrasonication-based method was used to prepare the nanocarriers using the keratin derived from goat hairs and the extract from *N. nucifera* seedpod extract, a waste-to-wealth concept. The films containing keratin nanocarriers (KNP) and extract-loaded keratin nanocarriers (KNP-Ex) were compared with the control films without any nanocarriers. The films were characterized for various physicochemical (such as SDS-PAGE, LC/MS, SEM, TEM, FTIR, and TGA analysis), physicomechanical (thickness, tensile strength, surface analysis, biodegradability, transparency, and solubility) and bioactive properties [antioxidant (DPPH-radical scavenging assay) and antimicrobial (disk diffusion assay)]. The results of our study indicate the potential of keratin nanocarriers as payloads to infuse bioactive properties and modify the mechanical and functional characteristics of the film. This film can provide fundamental protective functions and can be applied to improve the storage quality of foods.

## 1. Introduction

Packaging is indispensable to the food industry; however, the use of non-biodegradable and non-recyclable materials has led to a major detrimental impact on the environment, raising the need for alternative environment-friendly packaging materials. Given the growing concerns about consumer health, food safety, and the adverse effects of single-use non-biodegradable packaging, it is crucial to create new packaging materials from natural sources that are recyclable and/or biodegradable to reduce environmental harm (Ruiz-Real et al., 2018; Vila-Lopez et al., 2021). Biopolymers, such as polysaccharides, proteins, and their derivatives, have been widely explored as sustainable alternatives to traditional packaging, providing potential solutions with little to no negative environmental impact (Shrivastava, 2018; Satchanska et al., 2024)

The development of edible films derived from natural biopolymers

has gained significant scientific interest due to their potential to serve as protective layers that are safe for consumption without altering the nutritional or sensory properties of foods (Marcio et al., 2022; Liyana-pathiranage et al., 2023). Edible films formulated from organic macromolecules, such as proteins and polysaccharides, offer a sustainable, biodegradable, and non-toxic alternative to conventional petroleum-based packaging materials (Gupta et al., 2022).

Recent advancements in food packaging technologies have emphasized the development of active and intelligent films, incorporating nanomaterials, bioactive agents, and biodegradable polymers to enhance mechanical, barrier, and antimicrobial properties (d'Agostino et al., 2025). These next-generation edible films can extend the shelf life and enhance the storage quality of foods, making them an innovative solution in food preservation. The incorporation of functional edible ingredients, including bioactive compounds and plant-derived extracts, can further enhance the physicochemical, mechanical, barrier, and

<footnote><sup>a</sup> Division of Animal Biotechnology, SKUAST-K, Kashmir, India</footnote>

<footnote><sup>b</sup> Division of Livestock Products Technology, SKUAST-J, Jammu, India</footnote>

<footnote><sup>c</sup> Université Clermont Auvergne, INRAE, VetAgro Sup, UMRF, 15000 Aurillac, France</footnote>

<footnote>☆ Keratin nanocarriers and bioactive edible film.</footnote>

<footnote>* Corresponding author at: Université Clermont Auvergne, INRAE, VetAgro Sup, UMRF, 15000 Aurillac, France.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: bhat.hina@skuastkashmir.ac.in (H.F. Bhat), abderrahmane.aitkaddour@vetagro-sup.fr (A. Aït-Kaddour).</footnote>

<footnote n="1">¹ These authors have equally contributed.</footnote>
<figure><img src="image_5.png" /><figcaption>Fig. 1. a) Cleaning, de-lipidation and keratin extraction. b)Goat hair SEM analysis before and after delipidation process.</figcaption></figure>

functional properties of these films. These bioactive edible films not only improve food preservation by providing antimicrobial and antioxidant properties but also contribute to the nutritional value and functional benefits of packaged foods (Nieto et al., 2023; Pei et al., 2024; Gupta et al., 2024). Additionally, such films can be tailored to meet consumer demands by integrating health-promoting bioactive substances, making them attractive for functional food applications and sustainable packaging solutions.

Among the naturally occurring macromolecules, keratin is abundantly present in nature as a structural protein that makes up most of the hard and filamentous structures considered municipal waste such as animal hair, hooves, and nails (Bragulla et al., 2009; Wang et al., 2016). Keratins are key structural proteins with a high degree of sequence similarity across humans and animals and have been extensively explored in the production of various scaffolds (such as gels, hydrogels, and models) with active principles for different biomedical applications (McLellan et al., 2019). Owing to their intrinsic properties, such as nontoxicity, biodegradability, low immunogenicity, high biocompatibility and cell proliferation, as well as appropriate strength and flexibility, researchers have turned their attention to the use of keratin-based products for varied fields (Lazarus et al., 2021). Keratin-based systems perform exceptionally well under diverse mechanical demands even compared to some of the most advanced engineered materials (Lee et al., 2011).

Recently, a large number of bioactive edible films have been developed using plant extracts as bioactive agents for applications in foods (Yadav et al., 2025). The use of plant-derived extracts and bioactive compounds with therapeutic potential has been of significant interest. Lotus (*Nelumbinis nucifera*) is a well-known medicinal plant and one of its byproducts i.e. seedpods (*Receptaculum Nelumbinis*) is often discarded as waste (Wang et al., 2022; Ling et al., 2005). However, several studies

have indicated the presence of substantial amounts of phenolic and other bioactive compounds in *R. Nelumbinis* (Sharma et al., 2017). Several phytochemicals including pro-anthocyanidins, oligomeric pro-cyanidins, polyphenols, and flavonoids (Wang et al., 2021; Wu et al., 2021; Shen et al., 2019; Lee et al., 2020) have been identified from lotus seedpods and have been proven to be effective against several chronic and lifestyle diseases such as diabetes, cancer, and cardiovascular disorders (Burton-Freeman, 2010; Rai et al., 2018; Irondi et al., 2018). Further, studies on its biological activities including ameliorating cognitive impairment, and its antioxidant, antibacterial and immunomodulatory effects have provided evidence of its significance and futuristic uses (Del Rio et al., 2013; Zhang et al., 2015).

In the current work, we propose the conversion of animal hair-derived keratin into a nanoparticle delivery system for modulating the mechanical and biological characteristics of packaging film for food preservation. Carrageenan was used as the matrix for our films due to its unique combination of characteristics such as its exceptional film-forming properties, biodegradability, easy availability and general non-toxicity (Cheng et al., 2022). Infusing carrageenan-based film with *R. Nelumbinis* extract-encapsulated keratin nanoparticles (KNP-Ex) can provide bioactive properties, such as antioxidant and antimicrobial, and potentially reduce the health risks associated with perishable food products during storage.

## 2. Materials and methods

### 2.1. Materials, reagents and chemicals

Animal fibres were collected from male Bhakarwal goats maintained at the mountain and agricultural research and extension station (MRSS&G) of SKUAST-Kashmir, J&K, India. The iota-carrageenan
<figure><img src="image_6.png" /><figcaption>Fig. 2. a) 1-D SDS-PAGE and iBRAQ distribution of extracted samples. B) Morphology (SEM) analysis and c)) DLS, Zeta Potential analysis.</figcaption></figure>

(derived from red seaweed) was purchased from Sigma Aldrich (India). All other chemicals used during the experiments were analytical or cell culture grade and supplied by Sigma Aldrich, Invitrogen, or Hi-Media (India) (unless mentioned otherwise). Cell culture reagents and chemicals, such as FBS, DMEM, trypsin-EDTA and an antimycotic/antibiotic solution, were purchased from Invitrogen and Sigma Aldrich (India). Dialysis (MWCO 6–8000 Da) membranes were obtained from Spectrum (US, Rancho Dominguez). The plasticware and cell culture plates were purchased from Corning (USA).

## 2.2. Keratin extraction, purification and characterization

Animal fibre samples were washed, cleaned, and delipidized using a 1:2 (v/v) solution of methanol and chloroform for 24 h and then washed thoroughly with SDS (0.5 %) containing water. This was followed by rinsing the fibres with water and drying them overnight under laminar air flow conditions, thereafter, the fibres were subject to brief UV exposure for sterilization. Keratin was extracted from goat wool according to a previously reported method with slight modifications

(Reichl et al., 2011; Borrelli et al., 2015). Briefly, cleaned fibres (5 g) were cut into snippets and dispersed in 100 ml of aqueous solution containing urea (8 M), sodium metabisulphite (0.5 M) and sodium dodecyl sulfate (SDS, 0.1 M), under mechanical shaking at 65 °C overnight. The mixture was filtered (2.5 µm pore size) and exhaustively dialyzed against deionized water for 24 h using a cellulose membrane (molecular weight cut-off of 6–8000 Da) and this procedure was done 4–6 times followed by 30 min of centrifugation (14,000 rpm). Bradford's method (Bradford Protein Assay, Bio-Rad, India) was used to estimate the total protein concentration according to the manufacturer's protocol using a multi-plate-reader (Biotek Instruments, Vermont, United States). The resulting aqueous solution was freeze-dried to obtain pristine keratin powder. Extracted keratin samples were analyzed by SDS–PAGE on 12–14 % SDS-polyacrylamide gels using the BioRad Criterion Cell (Bio-Rad Laboratories, Inc., Canada). The samples were diluted in a loading buffer (Bio-Rad Laboratories, Inc., Canada) before the electrophoresis run. Coomassie Brilliant Blue G-250 was used for staining protein bands. After the in-solution digestion, representative samples were sent for LC-MS/MS (reversed-phase high-pressure liquid
<figure><img src="image_7.png" /><figcaption>Fig. 3. FTIR Analysis for characterization of a) Keratin extract (KE), b) Keratin Nanoparticles (KNP) d) Keratin nanoparticles loaded with Nelumbinus extract (KNP-Ex) and e) carrageenan films.</figcaption></figure>

chromatography-electrospray ionization tandem mass spectrometry, RP-HPLC-ESI-MS/MS) analysis. AB SCIEX, Protein Pilot software (v. 5.0.1) with the Paragon algorithm for the species: *Capra hircus* and a quality: 0.05 was used. Only those peptides that showed a confidence score >0.05 were analyzed.

## 2.3. Preparation of R. Nelumbinis extract

The lotus seed pods (*R. Nelumbinis*), an underutilized local plant, were used to prepare the extract. Fresh lotus seed pods (*R. Nelumbinis*) were harvested from Dal Lake in Srinagar city, Jammu and Kashmir, India, spanning from August 2023 to September 2023. The seed pods were sundried, ground, and soaked in methanol at room temperature (25 °C) for 24 h. Subsequently, the methanolic extract underwent filtration and was concentrated to dryness under reduced pressure utilizing a rotary evaporator operating at 40 °C following methods outlined by *Etsassala et al. (2019)* and *Arooj et al. (2021)*.

## 2.4. Minimum inhibitory concentration (MIC)

The minimum level of incorporation of *R. Nelumbinis* extract to be loaded on keratin nanoparticles was decided on the results of the MIC test performed by microdilution technique using Muller Hinton broth in a 96-well plate. The plate was inoculated with a culture of *Escherichia coli* and incubated at 37 °C for 24 h. The MIC that inhibited the growth of *E. coli* was determined by observing the last well in the plate where no visible growth was noticed.

## 2.5. Preparation of keratin nanoparticles loaded with R. Nelumbinis extract (KNP-Ex)

The keratin nanoparticles were synthesized following the method outlined by Luo *et al.* (2016) with some modifications. Keratin was dissolved in phosphate-buffered saline (PBS) (5.0 % w/v). Subsequently, the keratin solutions were injected into a diluted HCl solution (pH 3.0)

under sonication using a needle and a syringe pump operating at speeds between 1.00 and 2.00 ml/min. Ultrasonication (20 kHz, 400 W for 15 min) was facilitated by an ultrasonic cell disruption system (QSonica, LLC, USA). Following the formation of the keratin nanoparticle suspension, centrifugation was conducted at 10,000 RPM for 20 min. Solvents were removed from the suspension using a rotary evaporator, and the resulting nanoparticles were lyophilized overnight using a freeze dryer (Mini Lyotrap, USA) and stored appropriately. The extract-loaded keratin nanoparticles (KNP-Ex) were prepared by a similar method except that *R. Nelumbinis* extract dissolved in ethanol (10 mg/ml) was added to the keratin nanoparticles solution while vigorously stirring at 700 rpm. After 65 min of incorporation, *R. Nelumbinis* extract-loaded nanoparticles (KNP-Ex) were formed and subsequently freeze-dried to obtain a pure powder.

## 2.6. Characterizations (particle size, zeta potential, and SEM)

The surface morphologies of the samples were analyzed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) performed using a Hitachi 3600 N Scanning Electron Microscope equipped with a 5-axis motorized stage and an ultra-dry Compact EDS Detector (Thermo Scientific™). Before imaging, the samples were coated with a thin layer of gold. The size and hydrodynamic diameter of the nanoparticles (KNP) in aqueous solutions were determined using dynamic light scattering (DLS). The DLS measurements were carried out using a 90° scattering angle with a wavelength of 633 nm for the laser. For the data analysis, we employed the Contin method for the distribution analysis, which was applied to obtain the hydrodynamic diameter distribution. The electrophoretic mobility (zeta-potential) was measured at 25 °C using a Litesizer™ 500. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used for the analysis of the film samples. The spectra were collected over the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹, and each sample was scanned 32 times to ensure reliable data.
<figure><img src="image_8.png" /><figcaption>Fig. 4. a) In-vitro cell culture, MTT and b) KNP-Ex coated dishes for Live-Dead assays using goat fibroblast cells.</figcaption></figure>

## 2.7. Preparation of edible film and its characterization

The carrageenan-based edible film (1.5 %, w/v) with glycerol as a plasticizer was used as a model film to study the modulating effects of keratin nanoparticles loaded with bioactive compounds on their functional and bioactive properties. The aqueous solution of carrageenan was continuously stirred at 80 °C and 450 rpm on a magnetic stirrer to obtain homogenous dispersion, cooled to 60 °C, glycerol (10 %) was added and stirred for an additional 30 min and the solution was cast on glass petri plates and dried at 55 °C for 5 h. KNP-infused edible films were prepared by the same method but with the addition of KNP or KNP-Ex (5.0 % w/v in dispersion) to the final mixture and stirred for an additional 60 min.

Film thickness was determined using a portable thickness gauge (SDL International Limited; Model 258) and surface properties were examined using an SEM (Hitachi 3600 N SEM, Thermo Scientific™). The transparency or opaqueness of the film was assessed using a UV-visible spectrophotometer (Jenway 7305, UK) at 560 nm. The moisture content of the film was determined by subjecting it to drying in a forced-air oven at 100 °C until a constant weight was achieved. Moisture content was then calculated using an equation.

$$ Moisturecontent(\%) = \frac{W_1 - W_2}{W_1} \times 100 $$

W₁ is the initial weight of the film and W₂ is the weight of the film after drying.

## 2.8. Determination of antimicrobial activity

The film's antimicrobial efficacy was assessed using the agar disk diffusion method. 15 mm diameter disks were cut from the films and positioned on Mueller-Hinton (M-H) agar plates with E. coli and S. aureus (10⁵ to 10⁶ CFU/ml bacteria). Each test plate comprised three discs. One positive control (control), which is a standard commercial antibiotic disc and two treated discs i.e. KNP and KNP-Ex were used. The plates were then incubated at 37 °C for 24 h. Following incubation, the diameter of the inhibition zone was measured using a precise calliper. The test was repeated three times to ensure reliability.

## 2.9. Antioxidant activity (DPPH radical scavenging assay)

A standardized method was used to evaluate the DPPH free radical scavenging activity. An aliquot of 0.5 ml of sample solutions was mixed with 2.5 ml of 0.5 mM DPPH. The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm using a UV spectrophotometer. Ascorbic acid was used as positive control. The experiments were performed in three replications and the results (mg VCEAC (Vit C equivalent antioxidant capacity)/100 g) were averaged.

## 2.10. Invitro cell culture and biocompatibility assays

### 2.10.1. Seeding efficiency

To evaluate the seeding efficiency, primary goat fibroblasts obtained from goat ear tissue were cultured in T-75 flasks. These cells were then
<figure><img src="image_9.png" /><figcaption>Fig. 5. a) Representative pictures for carrageenan based film transparency without infused keratin nanoparticles and KNP-Ex respectively with their respective SEM analysis b) TGA analysis.</figcaption></figure>

used to seed culture plates coated with control dishes and treated dishes (KNP and KNP-Ex). Cells were seeded at a density of 5 × 10⁴ cells per well and incubated for 10 h at 37 °C in a 5 % CO₂ atmosphere. After incubation, the cells were rinsed with PBS, detached using trypsin-EDTA, and subsequently counted to determine the seeding efficiency (ratio of attached cells to seeded cells). Each experiment was performed in triplicate (n = 3).

### 2.10.2. Biocompatibility assays

Goat primary fibroblast cells were cultured as previously described to perform the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and live-dead assays according to the manufacturer's procedure to evaluate the biocompatibility of the extracted materials (López-García et al., 2024). In a 96-well plate with control, KNP, and KNP-Ex, the cells were seeded in triplicate under 5 % CO₂ in air at 37 °C. Uncoated culture surfaces were used to compare the cell viability whereas the wells treated with keratin coating served as a control. After washing with 1 × PBS, the cells were incubated at 37 °C for 4 h in MTT solution (0.05 %, w/v) within the wells which was followed with the addition of DMSO. The absorbance was measured at 570 nm using a spectrophotometer (Amersham Life Sciences, USA).

### 2.11. Statistical analysis

The experimental set-up was carried out in triplicate and the results were expressed as mean±SD. The results were statistically analyzed using a two-way analysis of variance (ANOVA) followed by the Turkey multiple range test. Charts were drawn using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, California, USA). A significant level was set at *P* < 0.05.

## 3. Results and discussion

### 3.1. Keratin extraction, purification and characterization

The high availability of keratins from livestock waste, combined with their biocompatibility, non-toxicity, tunable mechanical properties, non-immunogenicity, and intrinsic tissue regeneration potential prompted our research into their extraction for nanoparticle development for their varied prospective applications. In the current work, we report the preparation of keratin nanoparticles (KNP) using low-quality animal fibres (goat hair) as a carrier system for the regulated release of payloads such as bioactive compounds and extracts. Following cleaning and de-lipidation (Fig. 1), we extracted keratins from goat hair under reducing conditions (urea and thiourea) to achieve maximum high-quality yield, followed by dialysis and lyophilization to convert it into pure powder form. Our SDS-PAGE (Fig. 2, Upper panel) based protein fractionation results show the visibility of two thick bands at 65–45kDa corresponding to the high molecular weight keratin proteins (keratin type I and type II), representing the acidic and basic keratin families along with many faint bands throughout the gel lanes. Also, matrix protein bands (10–20 kDa) represent the low molecular weight keratin-associated proteins (KAP’s). The LC/MS results of our extracted protein samples revealed a total of 138 proteins (with a higher confidence level) by searching the MS spectra against the UniProt Capra hircus database using Maxquant software, which is considered to be more stringent in identifying the proteins. Intensity-based absolute quantity (iBAQ) results (Fig. 2) in consensus with our 1-D SDS-PAGE revealed that goat hair fibre proteome mainly included two clustered protein groups viz. KRT I and II proteins in the molecular weight range of 40–65 kDa with keratin type I ranging from 40–50 kDa and type II ranging from 50–65 kDa and keratin-associated proteins (KAPs) in the molecular weight range of 10 to 25 kDa along with other non-keratin proteins present in very low number and distributed over the entire
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 6.** a) Representative pictures for anti-microbial activity of caraggenan films infused with Keratin nanoparticle (KNP, KNP-Ex) and b) their antioxidant properties analysis.</figcaption></figure>

<table><caption>**Table 1**

Mechanical properties of the film.</caption><thead><tr><th>S. No.</th><th>Film composition</th><th>Tensile strength (MPa) ± S.D</th><th>Elongation at break (%) ± S.D</th></tr></thead><tbody><tr><td>1.</td><td>Carrageenan (1.5 %)</td><td>14.74 ± 0.85<sup>b</sup></td><td>8.16 ± 0.62<sup>c</sup></td></tr><tr><td>2.</td><td>Carrageenan infused with keratin nanoparticles (KNP)</td><td>21.17 ± 1.02<sup>a</sup></td><td>11.12 ± 0.74<sup>a</sup></td></tr><tr><td>3.</td><td>Carrageenan infused with keratin nanoparticles loaded with R. Nelumbinus extract (KNP-Ex)</td><td>19.21 ± 0.91<sup>c</sup></td><td>9.86 ± 0.68<sup>b</sup></td></tr></tbody></table>

molecular weight range lanes (Kamarudin et al., 2017, He et al., 2019).

## 3.2. Nanoparticle synthesis and characterization

Keratin nanoparticles (KNP) were prepared using an ultrasonication-based method as discussed in the material and methods section. To confirm the synthesis of nanoparticles, several characterization studies were carried out. The morphology, particle size and distribution of KNP were studied using dynamic light scattering (DLS), transmission electron microscope (TEM) and scanning electron microscope (SEM) and the surface charge of the nanoparticles was determined through zeta potential measurements. The SEM images showed typical porous architectures with ellipsoidal or spheroid morphology. This highly porous structure of the keratin particles indicates high biological utility due to high surface area and better inter-connected active sites ideal for acting as carriers for payloads and encapsulation. The derived keratin nanoparticles were distinct, spherical in shape, and uniformly distributed

without any agglomeration (Fig. 2, Lower panel). The mean zeta potential and polydispersity index (PDI) of the nanoparticles obtained in our DLS results indicated a relatively stable particle size of 150 to 400 nm.

The dynamic light scattering (DLS) studies of the resultant particles revealed that the average hydrodynamic diameter of the keratin nanoparticles (540 nm) was greater than the size measured by TEM. This difference arises because DLS measures the hydrodynamic diameter, which includes not only the core particle size but also the surrounding solvent layer and any potential aggregation in the solution. In contrast, TEM provides the core particle size in a dry state, where the hydration shell and dispersed aggregates are absent (Hossain et al., 2022). Furthermore, the hydrodynamic diameter of keratin nanoparticles loaded with *Nelumbinus* extract exhibited a significant increase, ranging from 200 to 500 nm. This increase may be attributed to the interaction of the extract with the nanoparticle surface, leading to slight aggregation or changes in hydration dynamics.

The surface morphology of keratin nanoparticles, as observed under SEM, revealed a high surface-to-volume ratio, which could enhance their capacity as carriers for bioactive agents or drug molecules (Grace & Pandian, 2007). This observation supports the functionalization of the material for biomedical applications. Regarding the zeta potential, keratin nanoparticles exhibited an average value within the ideal +20 mV to -20 mV range, ensuring sufficient electrostatic repulsion for colloidal stability. The presence of polar amino acids, carboxyl (-COOH) groups, and primary amines in keratin contributed to this charge distribution. These findings align with existing literature on keratin-based nanomaterials and further support their potential therapeutic applications.

The FTIR-spectra characteristics of extracted keratin and keratin-
based nanoparticles loaded with *R. Nelumbinis* extract were analyzed in the region 400–4000 cm⁻¹ (Fig. 3). Our results confirmed the chemical structure of extracted keratin and its nanoparticles with some significant differences in the spectra of KNP loaded with the extract (KNP-Ex). The FTIR spectra showed characteristic vibration peaks corresponding to the amide I, II and III of keratins at around 1655 cm⁻¹, 1545 cm⁻¹ and 1260 cm⁻¹. Also, the carboxyl group (C—O) bond absorption was observed at around 1450 to 1600 cm⁻¹. The loaded keratin nanoparticles showed a shift towards lower wave number for these characteristic peaks on account of the presence of several functional groups in the *Nelumbinus* extract particularly the phenolic ‘OH’ groups (wang et al., 2022) present at around 3000 to 3400 cm⁻¹.

### 3.3. Biocompatibility of keratin nanoparticle formulations

To evaluate the biocompatibility and non-cytotoxic nature of the synthesized keratin nano-carriers (both with and without *R. Nelumbinus* extracts), invitro cell culture assays, viz MTT and Live-Dead assays were performed using fibroblast cells (Fig. 4). A 6-well culture dish was coated with keratin nanoparticles and seeded with goat fibroblast cells (5 × 10⁴ cells per well). After 24 h of incubation, a significant number of cells exhibiting round morphology were observed adhering to the coated dishes as well as the control group, with only a few cells remaining suspended in the media. Notably, fibroblast cells on keratin-coated surfaces displayed morphological changes to a spindle shape after 24 h and reached confluence by 96 h. In fact, seeding efficiency was seen to increase using keratin-coated dishes after 12 h as compared to control uncoated dishes. Recent studies have demonstrated that keratin-based biomaterials enhance cell adhesion, proliferation, and differentiation due to their intrinsic bioactivity and structural similarity to the extracellular matrix (Anand et al., 2024; Li et al., 2020). Furthermore, the seeding efficiency of fibroblast cells was found to be significantly higher on keratin-coated dishes after 12 h compared to uncoated control dishes, in alignment with findings that keratin scaffolds promote rapid cellular attachment (Peng et al., 2023).

The MTT assay results confirmed excellent biocompatibility of both keratin nanoparticles (KNP) and extract-loaded keratin nanoparticles (KNP-Ex), as indicated by high cell viability percentages. Additionally, the Live/Dead assay performed on dishes coated with keratin nanoparticles loaded with *R. Nelumbinus* extract (KNP-Ex) revealed robust cytocompatibility, supporting the attachment and proliferation of mammalian cells in vitro (Fig. 4). These findings are consistent with recent advancements in keratin-based biomaterials, which have been reported to facilitate cell proliferation and tissue regeneration in various biomedical applications (Jillian et al., 2010).

### 3.4. Characterization of the film infused with nanocarriers

Encapsulation technology has helped to increase the bioavailability of active molecules and substances and also serves to protect them against hydrolysis, enzymatic action, and inactivation within the packaging films. *R. Nelumbinis* extracts were loaded on keratin nanocarriers and incorporated into carrageenan (1.5 %) film to enhance its functional characteristics on account of its constituent bioactive components (Huang et al., 2019). The average thickness of the carrageenan film without any additive was 0.162±0.83 mm. The addition of KNP-Ex increased the average thickness of the film to 0.19±0.87 mm which may be attributed to the rheological changes in the solution due to the cross-linking of functional groups especially polyphenol-rich compounds in *R. Nelumbinis* extract (Akcan et al., 2017). FTIR analysis of these films (Fig. 3) showed characteristic peaks of carrageenan at 1230 cm⁻¹ (ester sulfate groups), 923 cm⁻¹ (3,6-anhydrogalactose group), 842 cm⁻¹ (galactose-4-sulphate) and 733 cm⁻¹ (3, 6-anhydro-d-galactose) and a wide absorption at wavenumber 3423.4 cm⁻¹ for the hydroxyl group (OH) and group NH (amine). In films containing keratin nanoparticles, slight shifts and variations in the intensity of these

characteristic peaks were observed, suggesting potential interactions—such as hydrogen bonding or electrostatic interactions—between the nanoparticles and the carrageenan matrix. These spectral changes support the structural integration of keratin nanoparticles within the polymer network.

Carrageenan inherently exhibits poor water resistance, which limits its applicability in food packaging and other biopolymer-based applications (Cheng et al., 2022). However, the incorporation of *Nelumbinus* extract-loaded keratin nanocarriers (KNP-Ex) resulted in a reduction in moisture content, likely due to the hydrophobic nature of keratin molecules and structural modifications induced by the bioactive compounds present within the *Nelumbinus* extract. Our results demonstrated a significant decrease (*P* < 0.05) in the transparency of films infused with keratin nanoparticles (Fig. 5a), which can be attributed to increased film density and thickness following the incorporation of keratin and bioactive compounds. Additionally, a significant (*P* < 0.05) reduction in film transmittance was observed upon the incorporation of KNP-Ex, which may be associated with alterations in hydrogen bonding, rheological properties, and viscosity.

A significant (*P* < 0.05) decrease in the water vapour transmission rate (WVTR) was also recorded with the incorporation of KNP and KNP-Ex. The incorporation of KNP and KNP-Ex alters the film's polymeric network by reducing free volume, enhancing hydrogen bonding, and increasing inter-chain interactions, which together create a more tortuous path for water vapour diffusion (Fig. 6b). This structural modification ultimately slows down the overall permeability of water molecules through the film, leading to a reduction in WVTR. The net effect of their incorporation is the formation of a denser and less permeable film matrix, which reduces water vapour transmission across the film. This aligns with previous studies that have reported a decrease in WVTR due to improved barrier properties resulting from biopolymer-nanoparticle interactions upon the incorporation of plant extracts (Darmawati & Fadhila, 2020)

FTIR analysis of these films (Fig. 3d) showed characteristic peaks of carrageenan at 1230 cm⁻¹ (ester sulfate groups), 923 cm⁻¹ (3,6-anhydrogalactose group), 842 cm⁻¹ (galactose-4-sulphate) and 733 cm⁻¹ (3, 6-anhydro-d-galactose) and a wide absorption at wavenumber 3423.4 cm⁻¹ for the hydroxyl group (OH) and group NH (amine), a characteristic of the carrageenan compound.

The mechanical properties of the films were significantly influenced by the incorporation of keratin nanoparticles. The carrageenan film exhibited a tensile strength of 14.74 ± 0.85 MPa and an elongation at break of 8.16 ± 0.62 %. The addition of keratin nanoparticles (KNP) significantly enhanced these properties, increasing the tensile strength to 21.17 ± 1.02 MPa and elongation to 11.12 ± 0.74 %, indicating a strong reinforcing effect. However, when the nanoparticles were loaded with *R. nelumbinus* extract (KNP-Ex), a slight reduction in mechanical performance was observed, with the tensile strength and elongation decreasing to 19.21 ± 0.91 MPa and 9.86 ± 0.68 %, respectively. This reduction could be attributed to possible interactions between the extract and the nanoparticle-polymer matrix, which may slightly disrupt the network structure while still maintaining improved mechanical performance compared to the control film (Table 1). The thermal stability of carrageenan films, both with and without keratin nanoparticles, was analyzed using TGA (Fig. 5b). The TGA curve exhibited three major weight loss events. The first weight loss occurred around 100 °C, corresponding to the evaporation of moisture and bound water in the film. The second weight-loss stage, observed at 171.18 °C, 215.44 °C, and 152.22 °C, with weight losses of 8.05 %, 8.78 %, and 3.61 %, respectively, is associated with the degradation of low-molecular-weight fractions and partial breakdown of the polymer matrix. The variation in degradation temperatures may be due to the polydispersity of carrageenan, leading to multiple degradation events. The final major weight loss, occurring at higher temperatures (~250 °C and above), corresponds to the decomposition of the carrageenan polysaccharide backbone and thermal degradation of keratin-based
components.

The results indicate that keratin nanoparticle incorporation influences the thermal degradation behaviour of the films, likely due to interactions between the biopolymer matrix and the nanoparticles. These findings provide insight into the thermal stability and potential application of keratin-reinforced carrageenan films in food packaging and biomedical fields.

### 3.5. Antimicrobial and antioxidant properties of edible film

In vitro studies have revealed that edible films based on biopolymers incorporated with bioactive compounds may have antimicrobial activity. The antimicrobial activity of *R. Nelumbinis* extracts against *E. coli* was evaluated by observing inhibition zones and determining MIC values. It was found that incorporating *R. nelumbinis*-loaded keratin particles enhanced the antimicrobial properties of these carrageenan-based edible films. The composite films with keratin nanoparticles significantly reduced the bacterial count, whereas the carrageenan films without these additives (control) showed a lower reduction in bacterial counts (Fig. 6a). The DPPH scavenging assay revealed higher antioxidant activity for the films incorporated with keratin nanoparticles compared to the control carrageenan film (10.3 mg VCEAC/100 g). When combined with positive control (PC), the activity increased significantly to 42.3 mg VCEAC/100 g. The inclusion of keratin nanoparticles (KNP) resulted in a moderate activity of 15.0 ± 0.6. Interestingly, the combination of carrageenan and keratin nanoparticles loaded with *R. Nelumbinis* (RN) extract showed a scavenging activity of 27.8 mg VCEAC/100 g (Fig. 6c). These findings suggest the potential benefits of combining *R. Nelumbinis* extract for enhanced antioxidant properties and the use of keratin nanocarriers as payloads to enhance the bioactivity of packaging systems.

## 4. Conclusions

The incorporation of keratin nanoparticles (KNP) and keratin nanoparticles loaded with *Nelumbo nucifera* seedpod extract (KNP-Ex) significantly enhanced the functional, mechanical, and bioactive properties of carrageenan-based edible films. The keratin nanoparticles, derived from goat hair, acted as effective carriers for our extract with bioactive compounds, improving the film's structural integrity and extending its potential applications. The results demonstrate the feasibility of utilizing keratin-based nanocarriers (KNP-Ex) as delivery systems to impart bioactive properties to edible films, offering a promising approach for improving food preservation and extending shelf life through the controlled release of bioactive compounds. Our work paves the way for optimizing the encapsulation efficiency and release kinetics of bioactive compounds from keratin nanocarriers to maximize their functional benefits. Additionally, investigating the antimicrobial and antioxidant properties of the developed films under real food storage conditions would provide further validation of their effectiveness.

### Ethical statement

The study was not performed in animals or humans.

### CRediT authorship contribution statement

**Hina Fayaz Bhat:** Writing – original draft, Visualization, Supervision, Methodology, Data curation, Conceptualization. **Zarka Nasir:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Sheza Farooq:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Zuhaib F. Bhat:** Writing – original draft, Validation, Methodology, Data curation, Conceptualization. **Syed Mudasir Andrabi:** Writing – review & editing, Visualization, Resources, Methodology, Data curation. **Abderrahmane Ait-Kaddour:** Writing – review & editing, Writing – original draft, Visualization, Validation,

Resources, Methodology, Data curation.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### References

Akcan, T., Estevez, M., & Serdaroglu, M. (2017). Antioxidant protection of cooked meatballs during frozen storage by whey protein edible films with phytochemicals from *laurus nobilis* L. and *salvia officinalis*. LWT Food Science and Technology, 77, 323–331.

Anand, S., Gupta, S., & Rani, M. (2024). Recent advances in preparation and biomedical applications of keratin-based biomaterials. *Biotechnology for Sustainable Materials, 1* (16).

Arooj, M., Imran, S., Inam-Ur-Raheem, M., Rajoka, M. S. R., Sameen, A., Siddique, R., & Sahar, A. (2021). Lotus seeds (*Nelumbinis* semen) as an emerging therapeutic seed: A comprehensive review. *Food Science and Nutrition, 9*(7), 3971–3987.

Borrelli, M., Joepen, N., Reichl, S., Finis, D., Schoppe, M., Geerling, G., & Schrader, S. (2015). Keratin films for ocular surface reconstruction: Evaluation of biocompatibility in an in-vivo model. *Biomaterials, 42*, 112–120.

Bragulla, H. H., & Homberger, D. G. (2009). Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia. *Journal of Anatomy, 214*(4), 516–559. https://doi.org/10.1111/j.1469-7580.2009.01066.x

Burton-Freeman, B. (2010). Postprandial metabolic events and fruit-derived phenolics: A review of the science. *British Journal of Nutrition, 104*, 1–14.

Cheng, C., Chen, S., Su, J., Zhu, M., Zhou, M., Chen, T., & Han, Y. (2022). Recent advances in carrageenan-based films for food packaging applications. *Frontiers in Nutrition, 9*, Article 1004588. https://doi.org/10.3389/fnut.2022.1004588

d'Agostino, D., Sportelli, M. C., Gentile, L., Desopo, M., Izzi, M., Incerti, O., ... Cioffi, N. (2025). Green Cu particles for functional and biodegradable food packaging solutions. *Food Chemistry, 464*(3), Article 141823. https://doi.org/10.1016/j.foodchem.2024.141823

Darmawati, E., & Fadhila, A. N. (2020). *Aloe vera* L. powder processing using spray drying method for coating material of agriculture products. *IOP Conference Series: Earth and Environmental Science, 542*(1), Article 012003. https://doi.org/10.1088/1755-1315/542/1/012003

Del Rio, D., Rodriguez-Mateos, A., Spencer, J. P., Tognolini, M., Borges, G., & Crozier, A. (2013). Dietary (poly) phenolics in human health: Structures, bioavailability, and evidence of protective effects against chronic diseases. *Antioxidants and Redox Signaling, 18*, 1818–1892.

Etsassala, N. G. E. R., Badmus, J. A., Waryo, T. T., Marnewick, J. L., Cupido, C. N., Hussein, A. A., & Iwuoha, E. I (2019). Alpha-glucosidase and alpha-amylase inhibitory activities of novel abietane diterpenes from *Salvia africana* Lutea. *Antioxidants, 8*(10), 421.

Grace, A., & Pandian, N. V. (2007). Antibacterial efficacy of aminoglycosidic antibiotics protected gold nanoparticles-A brief study. *Colloids and Surfaces A: Physicochemical and Engineering Aspects, 297*, 63–70.

Gupta, D., Lall, A., Kumar, S., Patil, T. D., & Gaikwad, K. K. (2024). Plant-based edible films and coatings for food packaging applications: Recent advances, applications, and trends. *Sustainable Food Technology, 2*, 1428–1455.

Gupta, V., Biswas, D., & Roy, S. (2022). A comprehensive review of biodegradable polymer-based films and coatings and their food packaging applications. *Materials, 15*(17), 5899. https://doi.org/10.3390/ma15175899

He, J., Xu, D., Li, J., Li, L., Li, W., Cui, W., & Liu, K. (2019). Highly efficient extraction of large molecular-weight keratin from wool in a water/ethanol co-solvent. *Textile Research Journal, 90*, 9–10.

Hossain, S. I., Sportelli, M. C., Picca, R. A., Gentile, L., Palazzo, G., Ditaranto, N., & Cioffi, N. (2022). Green synthesis and characterization of antimicrobial synergistic AgCl/BAC nanocolloids. *ACS Applied Bio Materials, 5*(7), 3230–3240. https://doi.org/10.1021/acsabm.2c00207

Huang, H., Belwal, T., Jiang, L., Hu, J., Limwachiranon, J., Li, L., Ren, G., Zhang, X., & Luo, Z. (2019). Valorization of lotus byproduct (*Receptaculum Nelumbinis*) under green extraction condition. *Food and Bioproducts Processing, 115*, 110–117.

Irondi, E. A., Adebara, O. O., Olateju, A., & Boligon, A. A. (2018). Phenolic constituents, anti-radicals, and enzymes inhibitory potentials of *brachystegiaeurycoma* seeds: Effects of processing methods. *International Journal of Food Properties, 20*, 1–11.

Jillian, G. R., & Mark, E. V. D. (2010). A review of keratin-based biomaterials for biomedical applications. *Materials, 3*(2), 999–1014.

Kamarudin, N. B., Sharma, S., Gupta, A., Kamarudin, N. B., Sharma, S., Gupta, A., Kee, C. G., Chik, S. M. S. B. T., & Gupta, R. (2017). Statistical investigation of extraction parameters of keratin from chicken feathers using Design-Expert. 3 *Biotech, 7*, 127. https://doi.org/10.1007/s13205-017-0767-9

Lazarus, B. S., Chadha, C., Velasco-Hogan, A., Barbosa, J. D. V., Jasiuk, I., & Meyers, M. A. (2021). Engineering with keratin: A functional material and a source
of bioinspiration. *iScience*, **24**(8), Article 102798. https://doi.org/10.1016/j.isci.2021.102798

Lee, M. S., Chyau, C. C., Wang, C. P., Wang, T. H., Chen, J. H., & Lin, H. H. (2020). Flavonoids identification and pancreatic beta-cell protective effect of lotus seedpod. *Antioxidants* (*Basel*), **9**, 658. https://doi.org/10.3390/antiox9080658

Lee, S., Novitskaya, E. E., Reynante, B., Vasquez, J., Urbaniak, R., Takahashi, T., Woolley, E., Tombolato, L., Chen, P. Y., & McKittrick, J. (2011). Impact testing of structural biological materials. *Material Science and Engineering: C*, **31**, 730–739.
https://doi.org/10.1016/j.msec.2010.10.017

Li, P., Wang, Y., Jin, X., Dou, J., Han, X., Wan, X., Yuan, J., & Shen, J. (2020). Catalytic generation of nitric oxide from poly(e-caprolactone)/phosphobetainized keratin mats for a vascular tissue engineering scaffold. *Langmuir: The ACS Journal of Surfaces and Colloids*, **36**(16), 4396–4404. https://doi.org/10.1021/acs.langmuir.0c00579

Ling, Z. Q., Xie, B. J., & Yang, E. L. (2005). Isolation, characterization, and determination of antioxidative activity of oligomeric procyanidins from the seedpod of *Nelumbo nucifera* Gaertn. *Journal of Agricultural and Food Chemistry*, **53**, 2441–2445. https://doi.org/10.1021/jf040325p. PMid:15796576.

Liyanapathiranage, A., Dassanayake, R. S., Gamage, A., Karri, R. R., Manamperi, A., Evon, P., Jayakodi, Y., Madhujith, T., & Merah, O. (2023). Recent developments in edible films and coatings for fruits and vegetables. *Coatings*, **13**, 1177.

López-García, S., Sanz, J. L., Oñate-Sánchez, R. E., Forner, L., García-Bernal, D., Murcia, L., ... Llena, C. (2024). In vitro biocompatibility of ammonia-free silver fluoride products on human dental pulp stem cells. *Tissue and Cell*, **86**, 102283.
https://doi.org/10.1016/j.tice.2023.102283

Luo, T., Hao, S., Chen, X., Wang, J., Yang, Q., Wang, Y., Weng, Y., Wei, H., Zhou, J., & Wang, B. (2016). Development and assessment of kerateine nanoparticles for use as a hemostatic agent. *Material Science and Engineering: C*, **63**, 352–358.

Marcio, S. A., Ishikawa, O. H., Costa, R. S., Seixas, M. V. S., Rodrigues, R. C. L. B., & Moura, E. A. B. (2022). Development of sustainable food packaging material based on biodegradable polymer reinforced with cellulose nanocrystals. *Food Packaging and Shelf Life*, **31**, Article 100807.

McLellan, J., Thornhill, S.G., Shelton, S., & Kumar, M. (2019). Keratin-based biofilms, hydrogels, and biofibers. In: Keratin as a protein biopolymer (Sharma, S., & Kumar, A., Editors). Springer, pp. 187–200, DOI: https://doi.org/10.1007/978-3-030-02901-2.

Nieto, G., Martínez-Zamora, L., Peñalver, R., Marín-Iniesta, F., Taboada-Rodríguez, A., López-Gómez, A., & Martínez-Hernández, G. B. (2023). Applications of plant bioactive compounds as replacers of synthetic additives in the food industry. *Foods (Basel, Switzerland)* (*Basel, Switzerland*), **13**(1), 47. https://doi.org/10.3390/foods13010047

Pei, J., Palanisamy, C. P., Srinivasan, G. P., Panagal, M., Kumar, S. S. D., & Mironescu, M. (2024). A comprehensive review on starch-based sustainable edible films loaded with bioactive components for food packaging. *International Journal of Biological Macromolecules*, **274**, https://doi.org/10.1016/j.ijbiomac.2024.133332. Article 133332.

Peng, M., Zhao, Q., Wang, M., & Du, X. (2023). Reconfigurable scaffolds for adaptive tissue regeneration. *Nanoscale*, **15**(10), 4567–4580.

Rai, S., Kureel, A. K., Dutta, P. K., & Mehrotra, G. K. (2018). Phenolic compounds based conjugates from dextran aldehyde and BSA: Preparation, characterization and

evaluation of their anti-cancer efficacy for therapeutic applications. *International Journal of Biological Macromolecules*, **110**, 425–436.

Reichl, S., Borrelli, M., & Geerling, G. (2011). Keratin films for ocular surface reconstruction. *Biomaterials*, **32**(13), 3375–3386.

Ruiz-Real, J. L., Uribe-Toril, J., Valenciano, J. D. P., & Gázquez-Abad, J. C. (2018). Worldwide research on circular economy and environment: A bibliometric analysis. *International Journal of Environmental Research and Public Health*, **15**, 2699. https://doi.org/10.3390/ijerph15122699

Satchanska, G., Davidova, S., & Petrov, P. D. (2024). Natural and synthetic polymers for biomedical and environmental applications. *Polymers*, **16**(8), 1159. https://doi.org/10.3390/poly16081159

Sharma, B. R., Gautam, L. N. S., Adhikari, D., & Karki, R. (2017). A comprehensive review on chemical profiling of *Nelumbo nucifera*: Potential for drug development. *Phytotherapy Research*, **31**, 3–26.

Shen, Y. B., Guan, Y. F., Song, X., He, J. L., Xie, Z. X., Zhang, Y. W., et al. (2019). Polyphenols extract from lotus seedpod (*Nelumbo nucifera* Gaertn.): Phenolic compositions, antioxidant, and antiproliferative activities. *Nutrition and Food Science*, **7**, 3062–3070. https://doi.org/10.1002/fsn3.1165

Shrivastava A. Introduction to plastics engineering. William Andrew Publishing; Boston, MA, USA: 2018. [(accessed on 15 March 2024)]. pp. 1–16.

Vila-Lopez, N., & Küster-Boluda, I. (2021). A bibliometric analysis on packaging research: Towards sustainable and healthy packages. *British Food Journal*, **123**, 684–701. https://doi.org/10.1108/BFJ-03-2020-0245

Wang, B., Yang, W., McKittrick, J., & Meyers, M. A. (2016). Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration. *Progress in Material Science*, **76**, 229–318.

Wang, J. M., Yu, T., Sheng, L. Q., Zhang, H., Chen, F., Zhu, J., et al. (2021). Lotus seedpod proanthocyanidins protect against light-induced retinal damage via antioxidative stress, anti-apoptosis, and neuroprotective effects. *Medical Science Monitor*, **27**, Article e935000. https://doi.org/10.12659/MSM.935000

Wang, Y.-F., Shen, Z.-C., Li, J., Liang, T., Lin, X.-F., Li, Y.-P., Zeng, W., Zou, Q., Shen, J.-L., & Wang, X.-Y. (2022). Phytochemicals, biological activity, and industrial application of lotus seedpod (*R. Nelumbinis*): A review. *Frontiers in Nutrition*, **9**, Article 1022794. https://doi.org/10.3389/fnut.2022.1022794

Wu, Q., Feng, Y. N., Ouyang, Y., Liang, Y. G., Zhao, K. Q., Wang, Y., et al. (2021). Inhibition of advanced glycation end productS formation by lotus seedpod oligomeric procyanidins through RAGE-MAPK signaling and NF-κB activation in high AGEs-diet mice. *Food and Chemical Toxicology*, **156**, Article 112481. https://doi.org/10.1016/j.fct.2021.112481

Yadav, P., Mahapatra, U., Sharma, R., Rustagi, S., Katyal, M., Sharma, M., Nayak, P. K., Nath, P. C., & Sridhar, K. (2025). Recent advances in plant protein-based sustainable edible film and coatings for applications in the food-pharma industry - opportunities and challenges: A review. *International Journal of Biological Macromolecules*, **296**, Article 139698. https://doi.org/10.1016/j.ijbiomac.2025.139698

Zhang, Y., Lu, X., Zeng, S., Huang, X., Guo, Z., Zheng, Y., Tian, Y., & Zheng, B. (2015). Nutritional composition, physiological functions and processing of lotus (*Nelumbo nucifera* Gaertn.) seeds: A review. *Phytochemistry Reviews*, **14**, 321–334.