Utilization of Watery Egg Yolk Fraction for development of Edible Film

Utilization of Watery Egg Yolk Fraction for development of Edible Film | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Utilization of Watery Egg Yolk Fraction for development of Edible Film Komal Shinde, Rahul Chudaman Ranveer, Nikheel Bhojraj Rathod, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7703353/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The watery yolk fraction is generated as waste during preparation of different egg yolk products such as granules and lipid paste. The aqueous component is primarily composed of protein and water. The current study is centered on the creation of an edible film using the aqueous protein fraction. The present research was conducted to investigate the potential of a 3% watery protein fraction, at varying concentrations of 1.5%, 3%, and 4.5%, in combination with 3% gelatin and 3% plasticizer, for the development of film. In order to enhance the film properties, the film mixture underwent ultrasonication treatment at 40 Khz for varying time intervals of 5, 7.5, and 10 minutes prior to casting. The protein and fat content of the watery protein fraction powder were analyzed and found to be 2.28 ± 0.33% and 1.40 ± 0.19%, respectively. The study found that the properties of the edible film were superior when prepared using a 1.5% concentration of watery protein fraction compared to other concentrations tested. The ultrasonication treatment was found to enhance the mechanical strength and color value of the film. The utilization of the watery protein fraction has been investigated for the preparation of edible film. Egg yolk Egg yolk fraction Edible film ultrasonication Figures Figure 1 Figure 2 1. Introduction The field of edible packaging is currently experiencing significant progress through the use of edible compounds, including proteins, polysaccharides, lipids, and resins, as well as other edible components sourced from a variety of renewable sources. Edible packaging materials are designed to be an essential component of food products and are consumed along with the products. Consequently, they possess inherent biodegradability in composting and other biological recycling process (Krochta 2002 ). Edible packaging is a type of packaging that encompasses various forms such as edible films, sheets, coatings, and pouches (Ribeiro, A. M et al. 2021). According to Krochta et al., (Krochta et al. 1997), edible films are independent structures that are created separately from the food and subsequently applied onto or between food components or enclosed within edible pouches. On the other hand, edible coatings are slim layers of edible substances that are directly formed onto the surface of food items. The motivation behind the growing interest in edible coatings is attributed to the rising consumer demand for food products that are safe, stable, and convenient, as well as the recognition of the adverse environmental impact of non-biodegradable packaging. Edible and renewable resources are utilized in the production of biofilms, which have the potential to degrade more easily than polymeric materials in many instances. It is suggested that films may have a positive impact on reducing environmental pollution, regardless of whether or not they are consumed (Bourtoom 2008 ). The categorization of components utilized in the production of edible films can be divided into four distinct groups, namely proteins, polysaccharides, lipids (including fatty acids, acylglycerol, waxes), and composites (Donhowe and Fennema 1993 ). The sources of film-forming proteins, which include animal-derived proteins such as casein, whey protein concentrates and isolate, collagen, gelatin, and egg albumin, as well as plant-derived proteins such as corn, soybean, wheat, cottonseed, peanut, and rice. The primary process involved in the creation of protein films is the denaturation of the protein, which can be triggered by heat, solvents, or a shift in pH (de Azeredo 2012 ). This is then followed by the bonding of peptide chains through novel intermolecular interactions. Protein-based films are highly desirable due to their nutritional value (Galus and Kadzińska 2015 ). The denaturation of proteins is commonly triggered by various factors such as exposure to high temperature, a solvent, or a shift in pH levels. Protein films are formed through the association of peptide chains via new intermolecular interactions, including covalent (peptide and disulfide) bonds and noncovalent (ionic, hydrogen, and van der Waals) interactions, after denaturation (de Azeredo 2012 ). On the other hand, polysaccharides only have the hydroxyl group as their reactive group. Hydrophobic interactions have been observed to occur with proteins. Proteins exhibit amphiphilic properties, which enable them to possess both positive and negative charges across a broad pH spectrum. The charge density of proteins varies with pH, allowing for a diverse range of charge densities. It has been observed that each protein exhibits a unique hydrophilic-hydrophobic balance. Proteins exhibit favourable film-forming characteristics and exhibit strong adhesion to hydrophilic surfaces (de Azeredo 2012 ). The process of forming protein films is a crucial mechanism that involves denaturation of proteins through various means such as heat, solvents, or pH changes. This denaturation leads to the formation of new intermolecular interactions, which in turn facilitate the association of peptide chains (Janjarasskul and Krochta 2010 ). It is important to highlight that, as far as current research indicates, there has been no prior documentation of the development of consumable films utilizing egg yolk watery fractions (protein). The objective of this study was to create films from egg yolk watery fractions (protein) by incorporating gelatine and utilizing glycerol as a plasticizer, as it has been shown to have favourable outcomes in other protein films. 2. Materials and Methods 2.1. Preparation of different egg yolk fractions High-grade eggs were selected and subjected to a thorough cleansing process using drinkable water. The egg was manually broken and its yolk and albumen components were subsequently separated. The procedure for extracting egg yolk fractions was followed as described by Laca et al. 2010 . 2.2. Development of edible film from egg yolk watery protein fraction The preparation of edible film was conducted following the methodology outlined by Fuertes et al. 2017 , with minor adjustments. Distilled water was used to dissolve a powder containing 3% yolk watery fraction. A mixture of gelatine (3%) and plasticizer (3%) was subjected to filtration using Whatman No.1 filter paper. The pH of the solution was adjusted to 7 by the addition of 2N NaOH. The study involved casting the film onto a Teflon pan and subsequently drying it at a controlled temperature of 35 ± 2 ºC for duration of 24 hours. Upon completion of the drying period, the film was peeled (Fuertes et al. 2017 ). 2.3. Ultrasonication Treatment In this study, an edible film mixture was subjected to ultrasound treatment at a frequency of 40 kHz at 30ºC for varying durations of 5, 7.5, and 10 minutes. Following the ultrasonication treatment, the film was cast and subsequently dried (Marcet et al. 2018 ). 2.4. Physico-chemical analysis of edible film 2.4.1. Thickness Film thickness was measured with a digital micrometre that had an accuracy of ± 0.1 µm (Marcet et al. 2018 ). The study involved the collection of three readings at various random locations of each film, followed by the computation of the average values. 2.4.2. Film Solubility The method used to determine the solubility of the films was investigated described by Pérez-Mateos et al., (Pérez-Mateos et al. 2009 ). The edible film, weighing 0.2 g, was subjected to immersion in distilled water for duration of 24 hours. The mixture was subjected to filtration using a Whatman no.1 paper filter, with the initial weight of the filter being recorded. The resulting filtrate was then subjected to drying at a temperature of 105°C for duration of 12 hours. The dry matter of films was determined by directly drying pieces of films that had not been exposed to water. The quantification of water-soluble matter was determined by measuring the weight difference pre- and post-water solubilization. Dry matter values were measured through the utilization of a halogen moisture analyser. 2.4.3. Water Vapor Transmission Rate (WVTR) The measurement of water vapour transmission rate (WVTR) was conducted in accordance with the ASTM standard Method E96-95 (1995), Deionized water was added to a polyvinyl chloride-based cup with a diameter of 6 cm and a depth of 8 cm. A gap of 1.5 cm was observed between the surface of the water and the film under surface. The measurement of film sample thickness was conducted at nine distinct points. The study involved placing mounted cups within an environmental chamber set at a temperature of 25°C and relative humidity of 50 ± 2%. The weight loss of the cups was monitored hourly for the first 10 hours and then again after 24 hours. The evaluation process involved four replicates for each film. The study involved plotting the weight loss against time and estimating the water vapour transmission rate (WVTR g/hr.m2) by dividing the slope in the linear region (R 2 > 0.998) by the film surface. The calculation of WVPR was performed using the subsequent equation. $$\:\mathbf{W}\mathbf{V}\mathbf{T}\mathbf{R}=\frac{\mathbf{G}}{\mathbf{t}\mathbf{A}}=\left(\frac{\mathbf{G}}{\mathbf{t}}\right)/\mathbf{A}$$ Where, G = weight change (from the straight line) (g) t = time (hrs) G/t = slope of the straight line (g/hr) A = test area (cup mouth area) (m 2 ) WVT = rate of water vapor transmission (g/hr.m 2 ) 2.5. Optical properties 2.5.1. Film Transparency Rectangular pieces of film were analysed for their light transmission at 600 nm using a spectrophotometer to determine their transparency. The films were subjected to spectrophotometer testing by placing them directly in the test cell. A blank test cell was utilized as the control in the experiment. Transparency was quantified as a percentage in accordance with established methodology. Specifically, the transmittance of the blank at 600 nm was designated as 100% transparency, as previously described by Marcet et al. 2018 . The transparency of the films was quantified using the following equation. $$\:\mathbf{T}\mathbf{r}\mathbf{a}\mathbf{n}\mathbf{s}\mathbf{p}\mathbf{a}\mathbf{r}\mathbf{e}\mathbf{n}\mathbf{c}\mathbf{y}={\mathbf{A}}_{600}÷\mathbf{X}$$ Where, A 600 is the absorbance of the film sample at 600 nm and x is film thickness (mm). 2.5.2. Color Value The Konica Minolta colour Reader (Make: Minolta Camera Co. Ltd.) was utilized to measure the colour value. The model designated as R-10 was utilized in the study. The study utilized a machine system to express the colour readings. The L*, a*, and b* values were utilized to indicate the darkness/whiteness, greenness/redness, and yellowness/blueness, respectively. According to research, the highest possible value for L* is 100, indicating the color white. The minimum value of L* is zero, indicating the absence of lightness or blackness. The numerical limits of a* and b* axes are not specified. The color red is associated with positive values of a* while green is associated with negative values of a*. The color yellow represents the positive values of b*, while the color blue represents the negative values of b*. The assessment of the hue of the samples was conducted following a 10-minute period of cooling at ambient temperature (Marcet et al. 2018 ). 2.6. Tensile strength The tensile strength (TS) of the films that were prepared was evaluated through the utilization of a Model 5566 Instron Universal Testing Machine. Specimens of film measuring 2.54 cm in width and 15 cm in length were prepared for analysis. In order to determine the thickness of the specimens, ten measurements were taken along each using a micrometer. The mean value of these ten measurements was then utilized in the calculation of the TS. The experimental conditions involved setting the initial grip separation to 10 cm and the cross-head speed to 5 cm/min. The calculation of TS involved the division of the peak load by the initial cross-sectional area of the specimen. The film specimens were subjected to a conditioning process for a period of 3 days in an environmental chamber at 50% RH and 25°C, in accordance with the guidelines set forth in ASTM Standard Method D 882 − 88 (1989), prior to conducting the tensile testing. The specimens were placed on Teflon-coated glass plates during the testing process. The study aimed to investigate the tensile properties of film samples under ambient conditions, which deviated slightly from the ASTM Standard Method D 882 − 88. The experiment was conducted with individually prepared and cast films as the replicated experimental units, and TS values were determined in triplicate for each type of film. It is worth noting that the recommended standard laboratory atmosphere of 23 ± 2°C was not used in this study. The mean of three sampling units (specimens) taken from the same film was used to represent each TS replicate. 2.7. FTIR (Fourier-transform infrared spectroscopy) The Varian 670-IR spectrometer was utilized to conduct FTIR analysis on film samples. The sample compartment was equipped with an attenuated total reflectance (ATR) accessory. Mid-infrared spectra ranging from 4000 to 600 cm- 1 were obtained. The automatic signals were gathered through 16 scans with a resolution of 4 cm-1(Fuertes et al. 2017 ). These signals were then compared to a background spectrum that was obtained from an empty and uncontaminated cell at a temperature of 25° C. 3. Results and Discussion 3.1. Proximate composition of watery protein faction powder Table 1 presents the results of an analysis of the proximate composition, including moisture, protein, fat, and ash, of both yolk granules and yolk watery protein fractions. The moisture, protein, fat, and ash contents of yolk granules and water fraction powder were analysed. Yolk granules were found to contain 4.52% moisture, 24.30% protein, 27.15% fat, and 1.23% ash. Meanwhile, water fraction powder was found to contain 6.36% moisture, 2.28% protein, 1.40% fat, and 1.30% ash. The yolk powder had a protein content of 26.20%, fat content of 27.62%, moisture content of 3.88%, and ash content of 0.60% (Mohammadi Nafchi et al. 2017 ). The protein and fat content of watery fractions were reported to be 1.9% and 0.27% (Laca et al. 2010 ) (Data Not Shown). Table 1 Effect of watery protein fraction concentration on properties of film Fraction concentration Thickness (µm) Moisture (%) Film solubility (%) WVTR (g/ hr.m 2 ) 1.5% 126 ± 11.401 10.26 ± 0.07 89.24 ± 0.51 24.45 ± 0.154 3% 176 ± 15.165 10.38 ± 0.56 88.67 ± 0.56 29.658 ± 0.576 4.5% 231 ± 19.235 12.31 ± 0.12 88.62 ± 0.42 34.246 ± 0.163 Values are mean ± SD of three determinations 3.2. Effect of watery protein faction concentration on film properties The present study investigated the impact of varying concentrations of watery protein faction on physical parameters such as thickness, moisture content, water solubility, and water vapor transmission rate (WVTR). The results of the study are summarized in Table 1 . The study investigated the impact of varying concentrations of watery protein fraction (1.5%, 3%, and 4.5%) on the thickness of films. Results showed that the thickness of the films increased with increasing concentration of the protein fraction. Specifically, the thickness of films prepared with 1.5%, 3%, and 4.5% protein fractions were measured to be 126 ± 11.40 µm, 176 ± 15.16 µm, and 231 ± 19.23 µm, respectively. The results of the study indicate that an increase in the concentration of watery protein fraction, specifically at 4.5%, resulted in a higher thickness in the film. Conversely, a lower concentration of watery protein fraction, specifically at 1.5%, resulted in a lower thickness in the film. To produce protein films through casting with increased thickness, it is necessary to utilize either a greater quantity of the same solution or a more concentrated film-forming solution (Sobral 2000 ). Thickness of films made from soy protein isolate (SPI) increased from 100–150 µm as the concentration of soy protein isolate was increased from 3–8% (Nandane and Jain 2015 ). The assessment of moisture content is a crucial parameter that signifies the overall void volume that water molecules occupy in the microstructure network of the film. The study found that the moisture content of the film varied depending on the concentration of the watery protein fraction used in its preparation. The film with the highest moisture content (12.31 ± 0.12%) was observed in the sample prepared with 4.5% watery protein fraction concentration, while the film with the lowest moisture content (10.26 ± 0.07%) was observed in the sample prepared with 1.5% watery protein fraction concentration. The study recorded the moisture content of a film made with whey protein isolate in the range of 12–16% (Gounga et al. 2007 ). The investigation of water solubility is a crucial aspect in the study of biodegradable films. The dual significance of this characteristic lies in its ability to determine both the degradation process of developed films and the effectiveness of food protection. The rate of degradation of films is directly proportional to their solubility. The significance of this film property is heightened in the preservation of high-moisture food products. The study investigated the water solubility of edible film derived from various concentrations of watery protein fractions, specifically 1.5%, 3%, and 4.5%. Results showed that the water solubility percentages were 89.24 ± 0.51%, 88.67 ± 0.56%, and 88.62 ± 0.42%, respectively. The water solubility of the film prepared with Whey Protein Isolate (WPI) was observed to be 90% (Gounga et al. 2007 ). The Water Vapour Transmission Rate (WVTR) (g/m2.hr) is a crucial characteristic of packaging materials. It indicates the ability of films to allow moisture transfer between food and atmosphere, which is a determining factor in the degradation of food products. The study found that the film casted with 4.5% watery protein fraction concentration exhibited the highest water vapor transmission rate (WVTR), while the film casted with 1.5% watery protein fraction concentration exhibited the lowest WVTR. The observed rise in the Water Vapor Transmission Rate (WVTR) could potentially be attributed due to possibility of bubble formation during the process of film casting. An elevated level of the aqueous protein fraction results in a rise in the viscosity of the solution and an increase in the formation of bubbles during the mixing process. The outcome led to an increased water vapor transmission rate (WVTR) of the film. The findings indicate that there is a positive correlation between the concentration of protein and the water vapor transmission rate (WVTR) of the edible film ( Shroti and Saini 2022 ). The water vapor permeability decreased with increase in soy protein isolate concentration up to 0% to 0.25% followed by increasing with increasing concentration from 0.25% to 0.45% (Jia et al. 2009 ). [Table 1 near here] 3.3. Effect of watery protein fraction concentration on Optical Properties Research suggests that consumers generally prefer higher levels of transparency, as it allows for easier evaluation of product quality and external appearance. Table 2 displays the transparency and color value of edible films that were produced using varying concentrations of watery protein fractions. An inverse relationship was observed between transparency and concentration of the watery protein fraction. The transparency of edible films was investigated in this study. The films were prepared using watery protein fractions at concentrations of 1.5%, 3%, and 4.5%. The transparency values for the films were measured and recorded as 5.40 ± 0.29, 6.31 ± 0.04, and 7.38 ± 0.10, respectively, as shown in Table 2 . Mihalca et al. 2021 reported comparable outcomes for the film produced from egg white. An increase in protein concentration in milk protein-based edible film resulted in a decrease in transparency (Fematt-Flores et al. 2022 ). Table 2 Effect of watery protein fraction concentration on optical properties Fraction concentration Transparency (A 600 /x) Colour values L* a* b* 1.5% 5.40 ± 0.29 84.17 ± 0.54 0.18 ± 0.008 15.65 ± 0.035 3% 6.31 ± 0.04 83.20 ± 0.41 -1.03 ± 0.008 20.26 ± 0.180 4.5% 7.38 ± 0.10 83.02 ± 0.62 -1.76 ± 0.008 26.05 ± 0.011 Values are mean ± SD of three determinations The results of color value testing on films made using a watery protein fraction are shown in Table 3 . The study investigated the effect of different concentrations of watery protein fractions on the properties of an edible film. Results showed that the film prepared from 1.5% concentration had the highest L value of 84.16 ± 0.54, followed by 3% with 83.20 ± 0.41, and 4.5% with 83.02 ± 0.62. An elevation in protein concentration led to a decrease in L value and a darker film color (Shroti and Saini 2022 ). Lee and Min 2013 reported a similar L* value (85.42 ± 1.2) for an edible film made from soybean meal. The study investigated a* value of edible films made from varying concentrations of watery protein fraction, specifically 1.5%, 3%, and 4.5%. The results showed that a* value decreased as the concentration of watery protein fraction increased. The values obtained were − 0.17 ± 0.08, -1.03 ± 0.08, and − 1.76 ± 0.08, respectively, as presented in Table 3 . The observation of a negative value for parameter a* suggests the presence of a green hue in the film. The study found that the film prepared with 4.5% watery protein fraction had the highest b* value (26.05 ± 0.011). Table 3 Influence of ultra-sonication on physical properties Sonication Time (min) Thickness (µm) Moisture (%) Film solubility (%) WVTR (g/ hr.m 2 ) Control 126 ± 11.401 10.26 ± 0.07 89.24 ± 0.51 34.246 ± 0.163 5 114 ± 13.02 9.70 ± 0.24 83.87 ± 0.54 24.29 ± 0.13 7.5 102 ± 8.24 8.90 ± 0.20 82.65 ± 0.60 23.28 ± 0.21 10 93 ± 5.16 8.17 ± 0.11 81.51 ± 0.35 23.27 ± 0.10 Values are mean ± SD of three determinations In the process of fractionation of the whole egg yolk, the majority of lipids are typically retained in the plasmatic fraction. However, even in the granule fraction, small amounts of these compounds can be detected. These trace amounts are responsible for the yellowish hue observed in films and other products that utilize this fraction, which is known for its high protein content (Laca et al. 2010 ).. [Table 2 near here] The study examined the impact of varying concentrations of watery protein fractions on the physical properties and characterization of edible films. Results indicated that the film produced from 1.5% fractions concentration exhibited reduced thickness and moisture levels in comparison to the films produced from 3% and 4.5% watery protein fractions concentration. The present study examines the color values of the recorded content of this film and compares it to previous recordings to determine if there has been an improvement in the quality of the recording. The results indicate that the film derived from the fraction exhibits higher solubility and transparency in water. Therefore, the film derived from the 1.5% aqueous protein fraction was selected for subsequent investigation. 3.4. Effect of Ultra-sonication on physical properties of film The present research involved the production of a film utilizing 1.5% aqueous protein fractions. The study involved subjecting the film to different ultrasound treatments to investigate their effects on its properties. Table 3 presents the outcomes of the administered treatments. An inverse relationship between sonication time and film thickness was observed. The thickness of the film was measured after subjecting it to a 10-minute sonication treatment, which resulted in the greatest reduction. Previous studies have suggested that the application of ultrasound to films results in a reduction in thickness, which may be due to the formation of a more compact structure (Cruz-Diaz et al. 2019 ). The study observed a correlation between sonication time and moisture content of the film, indicating that an increase in sonication time resulted in a decrease in moisture content. According to the experimental results, the utilization of sonication for a period of 10 minutes led to the minimum observed level of moisture content (8.17 ± 0.11%) in the film. The potential cause of the observed decrease in moisture content may be linked to an increase in the hydrophobicity of the protein structure. The modification could have potentially led to a decrease in the protein network's ability to retain water molecules while forming the film. The observed decrease in moisture content resulting from ultrasound treatment can be attributed to the augmented formation of protein cross-linking. This process results in the production of films that are denser and more compact, thereby exhibiting reduced moisture retention (Cheng and Cui 2021 ). [Table 3 near here] The relationship between water solubility and water resistance has been studied in order to determine the effectiveness of protecting food products with high humidity. The results of the study indicate that ultrasonication of the film led to a decrease in solubility. The reduced solubility observed in the dried proteins and lipoproteins may be attributed to their aggregation during the drying process (Marcet et al. 2018 ). The study found that the water vapor transmission rate (WVTR) of the film treated with ultrasonic waves was marginally reduced in comparison to the film sample that was not treated. The application of ultra-sonication treatment to the film mixture led to the elimination of a significant amount of entrapped air, potentially resulting in decreased WVTR (Banerjee et al. 1996 ). 3.5. Effect of Ultra-sonication on optical properties of film The transparency of the film is significantly impacted by sonication, according to the research findings (Table 4 ). The transparency of film is observed to increase with an increase in sonication time. The transparency of the film solution was measured after being treated with sonication for 10 minutes, and it was found to be the highest. The observed increase in transparency could potentially be attributed to the elimination of entrapped air through the application of ultrasonication treatment. Cheng and Cui 2021 reported comparable outcomes for edible film made from pea protein isolate. The results of the study indicate that ultrasonication did not have a statistically significant impact on the color value. Previous study have reported no significant effects of ultrasonication on the peanut protein film and whey protein concentrate (Cruz-Diaz et al. 2019 ). Table 4 Influence of ultra-sonication on optical properties Sonication Time (min) Transparency (A 600 /x) Colour values L* a* b* Control 5.40 ± 0.29 84.17 ± 0.26 0.18 ± 0.03 15.65 ± 0.20 5 5.25 ± 0.10 84.84 ± 0.32 -1.81 ± 0.07 15.41 ± 0.38 7.5 4.21 ± 0.08 85.36 ± 0.27 -2.64 ± 0.12 15.33 ± 0.27 10 3.23 ± 0.11 86.02 ± 0.24 -2.86 ± 0.16 15.46 ± 0.33 Values are mean ± SD of three determinations [Table 4 near here] 3.6. Effect of ultrasonication on the Tensile strength of film In order to ensure the integrity of the film during transportation, handling, and storage, it is necessary to have mechanical properties that are sufficiently high. Tensile strength is a crucial parameter that determines the ability of films to withstand breakage. Figure 1 displays the tensile strength of edible films that were produced using ultrasound-treated solution. The study investigated the impact of ultrasound treatment duration on the tensile strength of films. Four different treatment durations were tested, including 0, 5, 7.5, and 10 minutes. The results showed that the tensile strength of the films increased with longer ultrasound treatment duration. Specifically, the tensile strength values for the four treatment durations were 162.07 ± 11.96 g/cm2, 662.35 ± 15.58 g/cm2, 864.80 ± 38.23 g/cm2, and 1195.54 ± 113.28 g/cm2, respectively. The study investigated the impact of sonication time (5, 7.5, 10 min) on the tensile strength of film prepared from ultrasound treated solution. Results indicate that the film's tensile strength was highest after 10 minutes of sonication compared to the other films. The study's findings suggest that there was a statistically significant difference (p < 0.05) in the tensile strength of films produced through the utilization of ultrasound-treated solution. The mechanical properties of the films were modified by the ultrasonic pre-treatment, resulting in increased resistance to mechanical stress and improved flexibility. The hypothesis is that the application of energy input can result in heightened molecular interactions, which may subsequently cause a rise in molecular order and ultimately enhance the strength of the film. The application of ultrasounds for a duration of 10 minutes on samples resulted in the formation of aggregates, which may have the potential to hinder the formation of interactions between proteins. [Figure 1 near here] The confirmation of stronger and more flexible films resulting from increased protein-protein interactions following sonication-induced breakdown of aggregates has been documented (Marcet et al. 2018 ). Shiku et al., (Shiku et al. 2004 ) reported a range of 248 to 465 g/cm2 for the tensile strength of an edible film made from Alaska pollack. 3.7. FTIR spectra In this study, FTIR spectroscopy was utilized to analyse the functional groups present in edible films that underwent ultrasound treatment at a frequency of 40 kHz for varying durations of 0, 5, 7.5, and 10 minutes. Figure 2 presents the FT-IR spectra of both the control and ultrasonically treated samples. The absorption peaks of the film were identified through analysis. The main peaks were found to be the C = O stretching at 1640 cm_1 (amide I), N–H bending at 1545 cm-1 (amide - II), and C–H deformation at 1452 cm-1. These peaks were compared to reference absorption peaks for ultrasonic treated films, which were found to be in the ranges of 1630–1640 cm-1 (C = O stretching, amide I), 1541–1548 cm-1 (N–H bending, amide-II), 1400–1454 cm-1 (C–H deformation), and 2980–2880 cm-1 (C–H stretching of CH2 and CH3 groups of saturated structures)(Schmidt et al. 2005 ). The study involved the observation of spectra to compare the C-H stretching of CH2 and CH3 groups in saturated structures between ultra-sonication samples and control samples. Results showed that the C-H stretching in the range of 2980 − 2880 cm-1 was higher in the ultra-sonication samples at different time intervals. The disappearance of the functional group (C = O) at 1743 cm-1 was observed as a result of the application of ultra-sonication. The investigation suggests that an elevation in C-H stretching of CH2 and CH3 groups in saturated structures may enhance mechanical properties, specifically tensile strength. [Figure 2 near here] 4. Conclusion Edible films have the potential to serve as a substitute for certain synthetic packaging materials that are traditionally used to safeguard and maintain the quality of food products. In conclusion, the study found that edible films made from egg components, specifically 1.5% fractions, exhibited the best physical properties and characterization. Further treatment with ultrasound for 10 minutes resulted in good mechanical resistance. In conclusion, the use of watery fractions based edible films is a viable option for wrapping foods inside a secondary synthetic package during food distribution and storage. In conclusion, using biodegradable materials that are even edible can significantly reduce waste disposal costs while being environmentally friendly. Declarations Conflict of Interest The authors declare no conflict of interests Funding: No funding was obtained for this study Author Contribution •Komal Shinde: Formal analysis and Investigation •Rahul Chudaman Ranveer: Conceptualization and Methodology, Project administration •Nikheel Bhojraj Rathod: Draft Preparation •S. B. Patange: Conceptualization and Methodology•U. Pagarkar: Data Validation•S. B. Swami: Recourses Data Availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References ASTM. Standard test methods for water vapor transmission of materials, ASTM. PA: ASTME; 1995. pp. 96–95. ASTM. Annual Book of ASTM Standards. Philadelphia, PA: American Society for Testing and Materials; 1989. Banerjee R, Chen H, Wu J. Milk protein-based edible film mechanical strength changes due to ultrasound process. J Food Sci. 1996;61:824–8. Bourtoom T. 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Sobral P. do A (2000) Influência da espessura sobre certas propriedades de biofilmes à base de proteínas miofibrilares. Pesqui Agropecuária Bras 35:1251–1259. Zisu B, Lee J, Chandrapala J, et al. Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. J Dairy Res. 2011;78:226–32. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7703353","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":520029120,"identity":"d615a516-4f29-47cc-a510-12b38f29bdfb","order_by":0,"name":"Komal Shinde","email":"","orcid":"","institution":"PG Institute of Post Harvest Management (Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth)","correspondingAuthor":false,"prefix":"","firstName":"Komal","middleName":"","lastName":"Shinde","suffix":""},{"id":520029121,"identity":"47d0f9cd-0d2f-4f44-96c2-2d0ded7aa323","order_by":1,"name":"Rahul Chudaman Ranveer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFACHgaGB3BOBRAzMzcQ1pIA55wBaWEkRQtjG5jEr8W8/ewxiQSGe/LmEskHH1fOq43mbwdq+VGxDacWmTN5aUAtxYY7Z6QlG57ddjx3xmHGBsaeM7dxapFgyDEDaklg3HDmjJlk47ZjuQ1ALcyMbXi08L8Ba7HfcOb895+Nc47lzieoRQJiS+KG4z1sjI0NNbkbCGt5Y2yRYJCQvOF4m7Fkw7EDuRuBWg7i9Qt/juGNDxUJthsOMz/82FBTlzvv/OGDD35U4NYCAQZw1mEweYCAehRQR4riUTAKRsEoGCEAAGtXWTGSwdn5AAAAAElFTkSuQmCC","orcid":"","institution":"PG Institute of Post Harvest Management (Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth)","correspondingAuthor":true,"prefix":"","firstName":"Rahul","middleName":"Chudaman","lastName":"Ranveer","suffix":""},{"id":520029122,"identity":"b01d2703-5d98-4295-8fa2-a56105f8ae8f","order_by":2,"name":"Nikheel Bhojraj Rathod","email":"","orcid":"","institution":"PG Institute of Post Harvest Management (Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth)","correspondingAuthor":false,"prefix":"","firstName":"Nikheel","middleName":"Bhojraj","lastName":"Rathod","suffix":""},{"id":520029123,"identity":"6929f0aa-5900-44de-90f6-490671f1a251","order_by":3,"name":"S. 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06:44:08","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91963,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7703353/v1/8831c285d1b57c19ff755adc.html"},{"id":92232428,"identity":"ce458611-aac2-4a19-b54d-d99333a951f4","added_by":"auto","created_at":"2025-09-26 06:44:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23812,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ultra-sonication on tensile strength of film\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7703353/v1/7bd5fa5a9a11f8207deef262.png"},{"id":92232423,"identity":"6792359d-8c25-41fd-a9b8-b04f41d9fc1d","added_by":"auto","created_at":"2025-09-26 06:44:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155795,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR Analysis of Ultra- sonicated film\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7703353/v1/385ec057768e4d78bf7c74e0.png"},{"id":92579315,"identity":"f256e63a-0c10-40ac-8d95-faeb6f4c49d1","added_by":"auto","created_at":"2025-10-01 09:01:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1084511,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7703353/v1/3ff7370c-4f07-4757-8e11-e5078614cdaa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Utilization of Watery Egg Yolk Fraction for development of Edible Film","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe field of edible packaging is currently experiencing significant progress through the use of edible compounds, including proteins, polysaccharides, lipids, and resins, as well as other edible components sourced from a variety of renewable sources. Edible packaging materials are designed to be an essential component of food products and are consumed along with the products. Consequently, they possess inherent biodegradability in composting and other biological recycling process (Krochta \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Edible packaging is a type of packaging that encompasses various forms such as edible films, sheets, coatings, and pouches (Ribeiro, A. M et al. 2021). According to Krochta et al., (Krochta et al. 1997), edible films are independent structures that are created separately from the food and subsequently applied onto or between food components or enclosed within edible pouches. On the other hand, edible coatings are slim layers of edible substances that are directly formed onto the surface of food items. The motivation behind the growing interest in edible coatings is attributed to the rising consumer demand for food products that are safe, stable, and convenient, as well as the recognition of the adverse environmental impact of non-biodegradable packaging. Edible and renewable resources are utilized in the production of biofilms, which have the potential to degrade more easily than polymeric materials in many instances. It is suggested that films may have a positive impact on reducing environmental pollution, regardless of whether or not they are consumed (Bourtoom \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe categorization of components utilized in the production of edible films can be divided into four distinct groups, namely proteins, polysaccharides, lipids (including fatty acids, acylglycerol, waxes), and composites (Donhowe and Fennema \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The sources of film-forming proteins, which include animal-derived proteins such as casein, whey protein concentrates and isolate, collagen, gelatin, and egg albumin, as well as plant-derived proteins such as corn, soybean, wheat, cottonseed, peanut, and rice. The primary process involved in the creation of protein films is the denaturation of the protein, which can be triggered by heat, solvents, or a shift in pH (de Azeredo \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This is then followed by the bonding of peptide chains through novel intermolecular interactions. Protein-based films are highly desirable due to their nutritional value (Galus and Kadzińska \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe denaturation of proteins is commonly triggered by various factors such as exposure to high temperature, a solvent, or a shift in pH levels. Protein films are formed through the association of peptide chains via new intermolecular interactions, including covalent (peptide and disulfide) bonds and noncovalent (ionic, hydrogen, and van der Waals) interactions, after denaturation (de Azeredo \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, polysaccharides only have the hydroxyl group as their reactive group. Hydrophobic interactions have been observed to occur with proteins. Proteins exhibit amphiphilic properties, which enable them to possess both positive and negative charges across a broad pH spectrum. The charge density of proteins varies with pH, allowing for a diverse range of charge densities. It has been observed that each protein exhibits a unique hydrophilic-hydrophobic balance. Proteins exhibit favourable film-forming characteristics and exhibit strong adhesion to hydrophilic surfaces (de Azeredo \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The process of forming protein films is a crucial mechanism that involves denaturation of proteins through various means such as heat, solvents, or pH changes. This denaturation leads to the formation of new intermolecular interactions, which in turn facilitate the association of peptide chains (Janjarasskul and Krochta \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is important to highlight that, as far as current research indicates, there has been no prior documentation of the development of consumable films utilizing egg yolk watery fractions (protein). The objective of this study was to create films from egg yolk watery fractions (protein) by incorporating gelatine and utilizing glycerol as a plasticizer, as it has been shown to have favourable outcomes in other protein films.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Preparation of different egg yolk fractions\u003c/h2\u003e\u003cp\u003eHigh-grade eggs were selected and subjected to a thorough cleansing process using drinkable water. The egg was manually broken and its yolk and albumen components were subsequently separated. The procedure for extracting egg yolk fractions was followed as described by Laca et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Development of edible film from egg yolk watery protein fraction\u003c/h2\u003e\u003cp\u003eThe preparation of edible film was conducted following the methodology outlined by Fuertes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, with minor adjustments. Distilled water was used to dissolve a powder containing 3% yolk watery fraction. A mixture of gelatine (3%) and plasticizer (3%) was subjected to filtration using Whatman No.1 filter paper. The pH of the solution was adjusted to 7 by the addition of 2N NaOH. The study involved casting the film onto a Teflon pan and subsequently drying it at a controlled temperature of 35\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C for duration of 24 hours. Upon completion of the drying period, the film was peeled (Fuertes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Ultrasonication Treatment\u003c/h2\u003e\u003cp\u003eIn this study, an edible film mixture was subjected to ultrasound treatment at a frequency of 40 kHz at 30\u0026ordm;C for varying durations of 5, 7.5, and 10 minutes. Following the ultrasonication treatment, the film was cast and subsequently dried (Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Physico-chemical analysis of edible film\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Thickness\u003c/h2\u003e\u003cp\u003eFilm thickness was measured with a digital micrometre that had an accuracy of \u0026plusmn;\u0026thinsp;0.1 \u0026micro;m (Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The study involved the collection of three readings at various random locations of each film, followed by the computation of the average values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Film Solubility\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe method used to determine the solubility of the films was investigated described by P\u0026eacute;rez-Mateos et al., (P\u0026eacute;rez-Mateos et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The edible film, weighing 0.2 g, was subjected to immersion in distilled water for duration of 24 hours. The mixture was subjected to filtration using a Whatman no.1 paper filter, with the initial weight of the filter being recorded. The resulting filtrate was then subjected to drying at a temperature of 105\u0026deg;C for duration of 12 hours. The dry matter of films was determined by directly drying pieces of films that had not been exposed to water. The quantification of water-soluble matter was determined by measuring the weight difference pre- and post-water solubilization. Dry matter values were measured through the utilization of a halogen moisture analyser.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Water Vapor Transmission Rate (WVTR)\u003c/h2\u003e\u003cp\u003eThe measurement of water vapour transmission rate (WVTR) was conducted in accordance with the ASTM standard Method E96-95 (1995), Deionized water was added to a polyvinyl chloride-based cup with a diameter of 6 cm and a depth of 8 cm. A gap of 1.5 cm was observed between the surface of the water and the film under surface. The measurement of film sample thickness was conducted at nine distinct points. The study involved placing mounted cups within an environmental chamber set at a temperature of 25\u0026deg;C and relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;2%. The weight loss of the cups was monitored hourly for the first 10 hours and then again after 24 hours. The evaluation process involved four replicates for each film. The study involved plotting the weight loss against time and estimating the water vapour transmission rate (WVTR g/hr.m2) by dividing the slope in the linear region (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.998) by the film surface. The calculation of WVPR was performed using the subsequent equation.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{W}\\mathbf{V}\\mathbf{T}\\mathbf{R}=\\frac{\\mathbf{G}}{\\mathbf{t}\\mathbf{A}}=\\left(\\frac{\\mathbf{G}}{\\mathbf{t}}\\right)/\\mathbf{A}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere,\u003c/p\u003e\u003cp\u003e\u003cem\u003eG\u003c/em\u003e\u0026thinsp;=\u0026thinsp;weight change (from the straight line) (g)\u003c/p\u003e\u003cp\u003e\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;time (hrs)\u003c/p\u003e\u003cp\u003e\u003cem\u003eG/t\u003c/em\u003e\u0026thinsp;=\u0026thinsp;slope of the straight line (g/hr)\u003c/p\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u0026thinsp;=\u0026thinsp;test area (cup mouth area) (m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eWVT\u0026thinsp;=\u0026thinsp;rate of water vapor transmission (g/hr.m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Optical properties\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Film Transparency\u003c/h2\u003e\u003cp\u003eRectangular pieces of film were analysed for their light transmission at 600 nm using a spectrophotometer to determine their transparency. The films were subjected to spectrophotometer testing by placing them directly in the test cell. A blank test cell was utilized as the control in the experiment. Transparency was quantified as a percentage in accordance with established methodology. Specifically, the transmittance of the blank at 600 nm was designated as 100% transparency, as previously described by Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e. The transparency of the films was quantified using the following equation.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{T}\\mathbf{r}\\mathbf{a}\\mathbf{n}\\mathbf{s}\\mathbf{p}\\mathbf{a}\\mathbf{r}\\mathbf{e}\\mathbf{n}\\mathbf{c}\\mathbf{y}={\\mathbf{A}}_{600}\u0026divide;\\mathbf{X}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere, A\u003csub\u003e600\u003c/sub\u003e is the absorbance of the film sample at 600 nm and x is film thickness (mm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Color Value\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe Konica Minolta colour Reader (Make: Minolta Camera Co. Ltd.) was utilized to measure the colour value. The model designated as R-10 was utilized in the study. The study utilized a machine system to express the colour readings. The L*, a*, and b* values were utilized to indicate the darkness/whiteness, greenness/redness, and yellowness/blueness, respectively. According to research, the highest possible value for L* is 100, indicating the color white. The minimum value of L* is zero, indicating the absence of lightness or blackness. The numerical limits of a* and b* axes are not specified. The color red is associated with positive values of a* while green is associated with negative values of a*. The color yellow represents the positive values of b*, while the color blue represents the negative values of b*. The assessment of the hue of the samples was conducted following a 10-minute period of cooling at ambient temperature (Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Tensile strength\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe tensile strength (TS) of the films that were prepared was evaluated through the utilization of a Model 5566 Instron Universal Testing Machine. Specimens of film measuring 2.54 cm in width and 15 cm in length were prepared for analysis. In order to determine the thickness of the specimens, ten measurements were taken along each using a micrometer. The mean value of these ten measurements was then utilized in the calculation of the TS. The experimental conditions involved setting the initial grip separation to 10 cm and the cross-head speed to 5 cm/min. The calculation of TS involved the division of the peak load by the initial cross-sectional area of the specimen. The film specimens were subjected to a conditioning process for a period of 3 days in an environmental chamber at 50% RH and 25\u0026deg;C, in accordance with the guidelines set forth in ASTM Standard Method D 882\u0026thinsp;\u0026minus;\u0026thinsp;88 (1989), prior to conducting the tensile testing. The specimens were placed on Teflon-coated glass plates during the testing process. The study aimed to investigate the tensile properties of film samples under ambient conditions, which deviated slightly from the ASTM Standard Method D 882\u0026thinsp;\u0026minus;\u0026thinsp;88. The experiment was conducted with individually prepared and cast films as the replicated experimental units, and TS values were determined in triplicate for each type of film. It is worth noting that the recommended standard laboratory atmosphere of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C was not used in this study. The mean of three sampling units (specimens) taken from the same film was used to represent each TS replicate.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7. FTIR (Fourier-transform infrared spectroscopy)\u003c/h2\u003e\u003cp\u003eThe Varian 670-IR spectrometer was utilized to conduct FTIR analysis on film samples. The sample compartment was equipped with an attenuated total reflectance (ATR) accessory. Mid-infrared spectra ranging from 4000 to 600 cm-\u003csup\u003e1\u003c/sup\u003e were obtained. The automatic signals were gathered through 16 scans with a resolution of 4 cm-1(Fuertes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These signals were then compared to a background spectrum that was obtained from an empty and uncontaminated cell at a temperature of 25\u0026deg; C.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Proximate composition of watery protein faction powder\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the results of an analysis of the proximate composition, including moisture, protein, fat, and ash, of both yolk granules and yolk watery protein fractions. The moisture, protein, fat, and ash contents of yolk granules and water fraction powder were analysed. Yolk granules were found to contain 4.52% moisture, 24.30% protein, 27.15% fat, and 1.23% ash. Meanwhile, water fraction powder was found to contain 6.36% moisture, 2.28% protein, 1.40% fat, and 1.30% ash. The yolk powder had a protein content of 26.20%, fat content of 27.62%, moisture content of 3.88%, and ash content of 0.60% (Mohammadi Nafchi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The protein and fat content of watery fractions were reported to be 1.9% and 0.27% (Laca et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Data Not Shown).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of watery protein fraction concentration on properties of film\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThickness\u003c/p\u003e\u003cp\u003e(\u0026micro;m)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFilm solubility\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWVTR\u003c/p\u003e\u003cp\u003e(g/ hr.m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;11.401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e89.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e24.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.154\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e176\u0026thinsp;\u0026plusmn;\u0026thinsp;15.165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e88.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e29.658\u0026thinsp;\u0026plusmn;\u0026thinsp;0.576\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e231\u0026thinsp;\u0026plusmn;\u0026thinsp;19.235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e12.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e88.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e34.246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.163\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three determinations\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effect of watery protein faction concentration on film properties\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe present study investigated the impact of varying concentrations of watery protein faction on physical parameters such as thickness, moisture content, water solubility, and water vapor transmission rate (WVTR). The results of the study are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The study investigated the impact of varying concentrations of watery protein fraction (1.5%, 3%, and 4.5%) on the thickness of films. Results showed that the thickness of the films increased with increasing concentration of the protein fraction. Specifically, the thickness of films prepared with 1.5%, 3%, and 4.5% protein fractions were measured to be 126\u0026thinsp;\u0026plusmn;\u0026thinsp;11.40 \u0026micro;m, 176\u0026thinsp;\u0026plusmn;\u0026thinsp;15.16 \u0026micro;m, and 231\u0026thinsp;\u0026plusmn;\u0026thinsp;19.23 \u0026micro;m, respectively. The results of the study indicate that an increase in the concentration of watery protein fraction, specifically at 4.5%, resulted in a higher thickness in the film. Conversely, a lower concentration of watery protein fraction, specifically at 1.5%, resulted in a lower thickness in the film. To produce protein films through casting with increased thickness, it is necessary to utilize either a greater quantity of the same solution or a more concentrated film-forming solution (Sobral \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Thickness of films made from soy protein isolate (SPI) increased from 100\u0026ndash;150 \u0026micro;m as the concentration of soy protein isolate was increased from 3\u0026ndash;8% (Nandane and Jain \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe assessment of moisture content is a crucial parameter that signifies the overall void volume that water molecules occupy in the microstructure network of the film. The study found that the moisture content of the film varied depending on the concentration of the watery protein fraction used in its preparation. The film with the highest moisture content (12.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12%) was observed in the sample prepared with 4.5% watery protein fraction concentration, while the film with the lowest moisture content (10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07%) was observed in the sample prepared with 1.5% watery protein fraction concentration. The study recorded the moisture content of a film made with whey protein isolate in the range of 12\u0026ndash;16% (Gounga et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe investigation of water solubility is a crucial aspect in the study of biodegradable films. The dual significance of this characteristic lies in its ability to determine both the degradation process of developed films and the effectiveness of food protection. The rate of degradation of films is directly proportional to their solubility. The significance of this film property is heightened in the preservation of high-moisture food products. The study investigated the water solubility of edible film derived from various concentrations of watery protein fractions, specifically 1.5%, 3%, and 4.5%. Results showed that the water solubility percentages were 89.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51%, 88.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56%, and 88.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42%, respectively. The water solubility of the film prepared with Whey Protein Isolate (WPI) was observed to be 90% (Gounga et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Water Vapour Transmission Rate (WVTR) (g/m2.hr) is a crucial characteristic of packaging materials. It indicates the ability of films to allow moisture transfer between food and atmosphere, which is a determining factor in the degradation of food products. The study found that the film casted with 4.5% watery protein fraction concentration exhibited the highest water vapor transmission rate (WVTR), while the film casted with 1.5% watery protein fraction concentration exhibited the lowest WVTR. The observed rise in the Water Vapor Transmission Rate (WVTR) could potentially be attributed due to possibility of bubble formation during the process of film casting. An elevated level of the aqueous protein fraction results in a rise in the viscosity of the solution and an increase in the formation of bubbles during the mixing process. The outcome led to an increased water vapor transmission rate (WVTR) of the film. The findings indicate that there is a positive correlation between the concentration of protein and the water vapor transmission rate (WVTR) of the edible film ( Shroti and Saini \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The water vapor permeability decreased with increase in soy protein isolate concentration up to 0% to 0.25% followed by increasing with increasing concentration from 0.25% to 0.45% (Jia et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e near here]\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effect of watery protein fraction concentration on Optical Properties\u003c/h2\u003e\u003cp\u003eResearch suggests that consumers generally prefer higher levels of transparency, as it allows for easier evaluation of product quality and external appearance. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the transparency and color value of edible films that were produced using varying concentrations of watery protein fractions. An inverse relationship was observed between transparency and concentration of the watery protein fraction. The transparency of edible films was investigated in this study. The films were prepared using watery protein fractions at concentrations of 1.5%, 3%, and 4.5%. The transparency values for the films were measured and recorded as 5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, 6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, and 7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, respectively, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Mihalca et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e reported comparable outcomes for the film produced from egg white. An increase in protein concentration in milk protein-based edible film resulted in a decrease in transparency (Fematt-Flores et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of watery protein fraction concentration on optical properties\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFraction concentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTransparency (A\u003csub\u003e600\u003c/sub\u003e/x)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eColour values\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eL*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eb*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e84.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e83.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e20.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.180\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e83.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e26.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three determinations\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe results of color value testing on films made using a watery protein fraction are shown in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The study investigated the effect of different concentrations of watery protein fractions on the properties of an edible film. Results showed that the film prepared from 1.5% concentration had the highest L value of 84.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54, followed by 3% with 83.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41, and 4.5% with 83.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62. An elevation in protein concentration led to a decrease in L value and a darker film color (Shroti and Saini \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lee and Min \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e reported a similar L* value (85.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2) for an edible film made from soybean meal. The study investigated a* value of edible films made from varying concentrations of watery protein fraction, specifically 1.5%, 3%, and 4.5%. The results showed that a* value decreased as the concentration of watery protein fraction increased. The values obtained were \u0026minus;\u0026thinsp;0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, -1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, and \u0026minus;\u0026thinsp;1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, respectively, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The observation of a negative value for parameter a* suggests the presence of a green hue in the film. The study found that the film prepared with 4.5% watery protein fraction had the highest b* value (26.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInfluence of ultra-sonication on physical properties\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSonication Time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThickness\u003c/p\u003e\u003cp\u003e(\u0026micro;m)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFilm solubility\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWVTR\u003c/p\u003e\u003cp\u003e(g/ hr.m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;11.401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e89.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e34.246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.163\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e114\u0026thinsp;\u0026plusmn;\u0026thinsp;13.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e83.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e24.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e102\u0026thinsp;\u0026plusmn;\u0026thinsp;8.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e82.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e23.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e8.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e81.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e23.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three determinations\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the process of fractionation of the whole egg yolk, the majority of lipids are typically retained in the plasmatic fraction. However, even in the granule fraction, small amounts of these compounds can be detected. These trace amounts are responsible for the yellowish hue observed in films and other products that utilize this fraction, which is known for its high protein content (Laca et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)..\u003c/p\u003e\u003cp\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe study examined the impact of varying concentrations of watery protein fractions on the physical properties and characterization of edible films. Results indicated that the film produced from 1.5% fractions concentration exhibited reduced thickness and moisture levels in comparison to the films produced from 3% and 4.5% watery protein fractions concentration. The present study examines the color values of the recorded content of this film and compares it to previous recordings to determine if there has been an improvement in the quality of the recording. The results indicate that the film derived from the fraction exhibits higher solubility and transparency in water. Therefore, the film derived from the 1.5% aqueous protein fraction was selected for subsequent investigation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of Ultra-sonication on physical properties of film\u003c/h2\u003e\u003cp\u003eThe present research involved the production of a film utilizing 1.5% aqueous protein fractions. The study involved subjecting the film to different ultrasound treatments to investigate their effects on its properties. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the outcomes of the administered treatments. An inverse relationship between sonication time and film thickness was observed. The thickness of the film was measured after subjecting it to a 10-minute sonication treatment, which resulted in the greatest reduction. Previous studies have suggested that the application of ultrasound to films results in a reduction in thickness, which may be due to the formation of a more compact structure (Cruz-Diaz et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The study observed a correlation between sonication time and moisture content of the film, indicating that an increase in sonication time resulted in a decrease in moisture content. According to the experimental results, the utilization of sonication for a period of 10 minutes led to the minimum observed level of moisture content (8.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%) in the film. The potential cause of the observed decrease in moisture content may be linked to an increase in the hydrophobicity of the protein structure. The modification could have potentially led to a decrease in the protein network's ability to retain water molecules while forming the film. The observed decrease in moisture content resulting from ultrasound treatment can be attributed to the augmented formation of protein cross-linking. This process results in the production of films that are denser and more compact, thereby exhibiting reduced moisture retention (Cheng and Cui \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe relationship between water solubility and water resistance has been studied in order to determine the effectiveness of protecting food products with high humidity. The results of the study indicate that ultrasonication of the film led to a decrease in solubility. The reduced solubility observed in the dried proteins and lipoproteins may be attributed to their aggregation during the drying process (Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The study found that the water vapor transmission rate (WVTR) of the film treated with ultrasonic waves was marginally reduced in comparison to the film sample that was not treated. The application of ultra-sonication treatment to the film mixture led to the elimination of a significant amount of entrapped air, potentially resulting in decreased WVTR (Banerjee et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effect of Ultra-sonication on optical properties of film\u003c/h2\u003e\u003cp\u003eThe transparency of the film is significantly impacted by sonication, according to the research findings (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The transparency of film is observed to increase with an increase in sonication time. The transparency of the film solution was measured after being treated with sonication for 10 minutes, and it was found to be the highest. The observed increase in transparency could potentially be attributed to the elimination of entrapped air through the application of ultrasonication treatment. Cheng and Cui \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e reported comparable outcomes for edible film made from pea protein isolate. The results of the study indicate that ultrasonication did not have a statistically significant impact on the color value. Previous study have reported no significant effects of ultrasonication on the peanut protein film and whey protein concentrate (Cruz-Diaz et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInfluence of ultra-sonication on optical properties\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSonication Time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTransparency (A\u003csub\u003e600\u003c/sub\u003e/x)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eColour values\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eL*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eb*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e84.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e84.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e85.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e86.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three determinations\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e near here]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Effect of ultrasonication on the Tensile strength of film\u003c/h2\u003e\u003cp\u003eIn order to ensure the integrity of the film during transportation, handling, and storage, it is necessary to have mechanical properties that are sufficiently high. Tensile strength is a crucial parameter that determines the ability of films to withstand breakage. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the tensile strength of edible films that were produced using ultrasound-treated solution. The study investigated the impact of ultrasound treatment duration on the tensile strength of films. Four different treatment durations were tested, including 0, 5, 7.5, and 10 minutes. The results showed that the tensile strength of the films increased with longer ultrasound treatment duration. Specifically, the tensile strength values for the four treatment durations were 162.07\u0026thinsp;\u0026plusmn;\u0026thinsp;11.96 g/cm2, 662.35\u0026thinsp;\u0026plusmn;\u0026thinsp;15.58 g/cm2, 864.80\u0026thinsp;\u0026plusmn;\u0026thinsp;38.23 g/cm2, and 1195.54\u0026thinsp;\u0026plusmn;\u0026thinsp;113.28 g/cm2, respectively. The study investigated the impact of sonication time (5, 7.5, 10 min) on the tensile strength of film prepared from ultrasound treated solution. Results indicate that the film's tensile strength was highest after 10 minutes of sonication compared to the other films. The study's findings suggest that there was a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the tensile strength of films produced through the utilization of ultrasound-treated solution. The mechanical properties of the films were modified by the ultrasonic pre-treatment, resulting in increased resistance to mechanical stress and improved flexibility. The hypothesis is that the application of energy input can result in heightened molecular interactions, which may subsequently cause a rise in molecular order and ultimately enhance the strength of the film. The application of ultrasounds for a duration of 10 minutes on samples resulted in the formation of aggregates, which may have the potential to hinder the formation of interactions between proteins.\u003c/p\u003e\u003cp\u003e[Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e near here]\u003c/p\u003e\u003cp\u003eThe confirmation of stronger and more flexible films resulting from increased protein-protein interactions following sonication-induced breakdown of aggregates has been documented (Marcet et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Shiku et al., (Shiku et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) reported a range of 248 to 465 g/cm2 for the tensile strength of an edible film made from Alaska pollack.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.7. FTIR spectra\u003c/h2\u003e\u003cp\u003eIn this study, FTIR spectroscopy was utilized to analyse the functional groups present in edible films that underwent ultrasound treatment at a frequency of 40 kHz for varying durations of 0, 5, 7.5, and 10 minutes. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the FT-IR spectra of both the control and ultrasonically treated samples. The absorption peaks of the film were identified through analysis. The main peaks were found to be the C\u0026thinsp;=\u0026thinsp;O stretching at 1640 cm_1 (amide I), N\u0026ndash;H bending at 1545 cm-1 (amide - II), and C\u0026ndash;H deformation at 1452 cm-1. These peaks were compared to reference absorption peaks for ultrasonic treated films, which were found to be in the ranges of 1630\u0026ndash;1640 cm-1 (C\u0026thinsp;=\u0026thinsp;O stretching, amide I), 1541\u0026ndash;1548 cm-1 (N\u0026ndash;H bending, amide-II), 1400\u0026ndash;1454 cm-1 (C\u0026ndash;H deformation), and 2980\u0026ndash;2880 cm-1 (C\u0026ndash;H stretching of CH2 and CH3 groups of saturated structures)(Schmidt et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The study involved the observation of spectra to compare the C-H stretching of CH2 and CH3 groups in saturated structures between ultra-sonication samples and control samples. Results showed that the C-H stretching in the range of 2980\u0026thinsp;\u0026minus;\u0026thinsp;2880 cm-1 was higher in the ultra-sonication samples at different time intervals. The disappearance of the functional group (C\u0026thinsp;=\u0026thinsp;O) at 1743 cm-1 was observed as a result of the application of ultra-sonication. The investigation suggests that an elevation in C-H stretching of CH2 and CH3 groups in saturated structures may enhance mechanical properties, specifically tensile strength.\u003c/p\u003e\u003cp\u003e[Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e near here]\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eEdible films have the potential to serve as a substitute for certain synthetic packaging materials that are traditionally used to safeguard and maintain the quality of food products. In conclusion, the study found that edible films made from egg components, specifically 1.5% fractions, exhibited the best physical properties and characterization. Further treatment with ultrasound for 10 minutes resulted in good mechanical resistance. In conclusion, the use of watery fractions based edible films is a viable option for wrapping foods inside a secondary synthetic package during food distribution and storage. In conclusion, using biodegradable materials that are even edible can significantly reduce waste disposal costs while being environmentally friendly.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interests\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNo funding was obtained for this study\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\u0026bull;Komal Shinde: Formal analysis and Investigation \u0026bull;Rahul Chudaman Ranveer: Conceptualization and Methodology, Project administration \u0026bull;Nikheel Bhojraj Rathod: Draft Preparation \u0026bull;S. B. Patange: Conceptualization and Methodology\u0026bull;U. Pagarkar: Data Validation\u0026bull;S. B. Swami: Recourses\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eASTM. Standard test methods for water vapor transmission of materials, ASTM. PA: ASTME; 1995. pp. 96\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eASTM. Annual Book of ASTM Standards. Philadelphia, PA: American Society for Testing and Materials; 1989.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBanerjee R, Chen H, Wu J. Milk protein-based edible film mechanical strength changes due to ultrasound process. J Food Sci. 1996;61:824\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBourtoom T. Edible films and coatings: characteristics and properties. Int Food Res J. 2008;15:237\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng J, Cui L. Effects of high-intensity ultrasound on the structural, optical, mechanical and physicochemical properties of pea protein isolate-based edible film. Ultrason Sonochem. 2021;80:105809.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCruz-Diaz K, Cobos \u0026Aacute;, Fern\u0026aacute;ndez-Valle ME, et al. Characterization of edible films from whey proteins treated with heat, ultrasounds and/or transglutaminase. Application in cheese slices packaging. Food Packag Shelf Life. 2019;22:100397.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Azeredo HMC. (2012) 14 Edible Coatings. Adv Fruit Process Technol 345.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonhowe IG, Fennema O. The effects of plasticizers on crystallinity, permeability, and mechanical properties of methylcellulose films. J Food Process Preserv. 1993;17:247\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFematt-Flores GE, Aguil\u0026oacute;-Aguayo I, Marcos B, et al. Milk protein-based edible films: Influence on mechanical, hydrodynamic, optical and antioxidant properties. Coatings. 2022;12:196.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuertes S, Laca A, Oulego P, et al. Development and characterization of egg yolk and egg yolk fractions edible films. Food Hydrocoll. 2017;70:229\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalus S, Kadzińska J. Food applications of emulsion-based edible films and coatings. Trends Food Sci Technol. 2015;45:273\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGounga ME, Xu S-Y, Wang Z. Whey protein isolate-based edible films as affected by protein concentration, glycerol ratio and pullulan addition in film formation. J Food Eng. 2007;83:521\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJanjarasskul T, Krochta JM. Edible packaging materials. Annu Rev Food Sci Technol. 2010;1:415\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia D, Fang Y, Yao K. Water vapor barrier and mechanical properties of konjac glucomannan\u0026ndash;chitosan\u0026ndash;soy protein isolate edible films. Food Bioprod Process. 2009;87:7\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrochta JM. Proteins as raw materials for films and coatings: definitions, current status, and opportunities. Protein-Based Films Coat. 2002;1:1\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrochta JM, Mulder-Johnston D. others (1997) Edible and biodegradable polymer films: challenges and opportunities. Food Technol USA.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaca A, Paredes B, D\u0026iacute;az M. A method of egg yolk fractionation. Characterization of fractions. Food Hydrocoll. 2010;24:434\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee H, Min SC. Antimicrobial edible defatted soybean meal-based films incorporating the lactoperoxidase system. LWT - Food Sci Technol. 2013;54:42\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2013.05.012\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2013.05.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarcet I, \u0026Aacute;lvarez C, Paredes B, et al. Transparent and edible films from ultrasound-treated egg yolk granules. Food Bioprocess Technol. 2018;11:735\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMihalca V, Kerezsi AD, Weber A, et al. Protein-based films and coatings for food industry applications. Polymers. 2021;13:769.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohammadi Nafchi A, Olfat A, Bagheri M, et al. Preparation and characterization of a novel edible film based on Alyssum homolocarpum seed gum. J Food Sci Technol. 2017;54:1703\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNandane AS, Jain R. Study of mechanical properties of soy protein based edible film as affected by its composition and process parameters by using RSM. J Food Sci Technol. 2015;52:3645\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Mateos M, Montero P, G\u0026oacute;mez-Guill\u0026eacute;n M. Formulation and stability of biodegradable films made from cod gelatin and sunflower oil blends. Food Hydrocoll. 2009;23:53\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRibeiro AM, Estevinho BN, Rocha F. Preparation and incorporation of functional ingredients in edible films and coatings. Food Bioprocess Technol. 2021;14:209\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11947-020-02528-4\u003c/span\u003e\u003cspan address=\"10.1007/s11947-020-02528-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchmidt V, Giacomelli C, Soldi V. Thermal stability of films formed by soy protein isolate\u0026ndash;sodium dodecyl sulfate. Polym Degrad Stab. 2005;87:25\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShiku Y, Hamaguchi PY, Benjakul S, et al. Effect of surimi quality on properties of edible films based on Alaska pollack. Food Chem. 2004;86:493\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShroti GK, Saini CS. Development of edible films from protein of brewer\u0026rsquo;s spent grain: Effect of pH and protein concentration on physical, mechanical and barrier properties of films. Appl Food Res. 2022;2:100043.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSobral P. do A (2000) Influ\u0026ecirc;ncia da espessura sobre certas propriedades de biofilmes \u0026agrave; base de prote\u0026iacute;nas miofibrilares. Pesqui Agropecu\u0026aacute;ria Bras 35:1251\u0026ndash;1259.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZisu B, Lee J, Chandrapala J, et al. Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. J Dairy Res. 2011;78:226\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Egg yolk, Egg yolk fraction, Edible film, ultrasonication","lastPublishedDoi":"10.21203/rs.3.rs-7703353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7703353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe watery yolk fraction is generated as waste during preparation of different egg yolk products such as granules and lipid paste. The aqueous component is primarily composed of protein and water. The current study is centered on the creation of an edible film using the aqueous protein fraction. The present research was conducted to investigate the potential of a 3% watery protein fraction, at varying concentrations of 1.5%, 3%, and 4.5%, in combination with 3% gelatin and 3% plasticizer, for the development of film. In order to enhance the film properties, the film mixture underwent ultrasonication treatment at 40 Khz for varying time intervals of 5, 7.5, and 10 minutes prior to casting. The protein and fat content of the watery protein fraction powder were analyzed and found to be 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33% and 1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19%, respectively. The study found that the properties of the edible film were superior when prepared using a 1.5% concentration of watery protein fraction compared to other concentrations tested. The ultrasonication treatment was found to enhance the mechanical strength and color value of the film. The utilization of the watery protein fraction has been investigated for the preparation of edible film.\u003c/p\u003e","manuscriptTitle":"Utilization of Watery Egg Yolk Fraction for development of Edible Film","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 06:44:03","doi":"10.21203/rs.3.rs-7703353/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ea4f803-4d3c-4dc6-a5f6-821fc754bbfb","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T08:53:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 06:44:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7703353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7703353","identity":"rs-7703353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}