Biotechnological Production and Characterization of Innovative Antimicrobial and Antioxidant Edible Films Enriched with Safflower (Carthamus tinctorius L.) Extract

Biotechnological Production and Characterization of Innovative Antimicrobial and Antioxidant Edible Films Enriched with Safflower (Carthamus tinctorius L.) 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Extract ELİF AYÇA GÜLER, ELİF ÖZBEY This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7747329/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 Background Sustainable and functional alternatives to petroleum-based plastics are urgently needed in food biotechnology. Biopolymer-based edible films, particularly those enriched with bioactive plant extracts, provide innovative solutions for extending shelf life while reducing environmental impact. Safflower ( Carthamus tinctorius L.), rich in phenolics and flavonoids, offers strong antimicrobial and antioxidant potential for active packaging development. Methods Edible films were prepared using whey protein isolate with varying concentrations of safflower extract. Physicochemical, thermal, and structural properties were examined by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Antimicrobial activity was tested against Escherichia coli and Candida albicans . Moisture retention and solubility assays were performed to evaluate stability and biodegradability. Results The highest extract concentration (EF4) showed the strongest inhibition against both microorganisms. TGA confirmed biodegradability with ~ 90% total weight loss, while DSC demonstrated distinct thermal transitions indicating stability. FTIR verified successful incorporation of hydroxyl, carbonyl, and glycerol-derived groups. Higher film-forming volumes improved moisture retention, whereas high solubility indicated rapid degradability. Conclusions Safflower-enriched films present a biotechnological innovation in sustainable food packaging. The integration of bioactive plant compounds into whey protein–based matrices yields active films combining antimicrobial and antioxidant activity with environmental compatibility. These findings highlight their potential as next-generation materials in food biotechnology, with future studies needed for scale-up and industrial validation. Safflower (Carthamus tinctorius L.) Whey protein isolate (WPI) Edible films Antimicrobial activity Antioxidant activity Biodegradable packagin Food biotechnology Thermal stability (TGA DSC) FTIR spectroscopy Active food packaging Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Food biotechnology has increasingly focused on the development of bio-based packaging materials as sustainable alternatives to synthetic plastics. The incorporation of plant-derived bioactive compounds into protein- or polysaccharide-based matrices represents a biotechnological innovation that enables functionalization of edible films with antimicrobial and antioxidant activities (Campos et al., 2024 ; Zhang et al., 2023 ). Unlike conventional plastics, these films can be engineered through bioprocessing approaches that not only provide barrier properties but also contribute to active food protection and environmental sustainability (Mellinas et al., 2016 ; Versino et al., 2023 ). In this context, safflower ( Carthamus tinctorius L.), a medicinal plant rich in phenolic compounds and flavonoids, is an attractive candidate for functional biotechnological applications. The integration of safflower extract into whey protein isolate (WPI)-based matrices allows the production of edible films with enhanced bioactivity, combining food biotechnology innovation with environmental biotechnology perspectives (Kim et al., 2015 ; González et al., 2022 ). Such dual benefits position safflower-enriched edible films as next-generation materials with potential to reduce plastic waste while improving food preservation. Food packaging is a technology engineered to extend the shelf life of food products, prevent physical and chemical deterioration, and guarantee hygiene and convenience during transportation (Chauhan et al., 2022 ; Alves et al., 2022 ). While conventional packaging materials such as plastic, glass, metal, and paper have long dominated the sector, the persistence of plastic waste in the environment has heightened the importance of biodegradable and sustainable alternatives (Sastre et al., 2022 ; Versino et al., 2023 ; Foods, 2025 ). The advancement of packaging technologies has progressed beyond merely safeguarding food, leading to the emergence of smart and active packaging systems. Active packaging directly interacts with the food product to extend its shelf life and maintain quality by incorporating components such as oxygen scavengers, antimicrobial agents, and antioxidants. These systems are designed to absorb or release substances that control the internal environment of the package, thereby inhibiting Active packaging helps inhibit spoilage while maintaining the food’s sensory and nutritional qualities (Jiang et al., 2023 ; Vilela et al., 2018 ). In contrast, smart packaging consists of systems integrated with sensors and indicators that monitor environmental conditions, offering consumers real-time information about the product’s quality and safety (Ahmed et al., 2022 ; Liu et al., 2021 ). The implementation of these technologies facilitates more effective monitoring of food shelf life and contributes to the reduction of waste. In recent years, edible films thin biopolymer-based layers have gained prominence within active packaging, as they can be applied directly onto food surfaces or used as standalone packaging materials (Campos et al., 2024 ; Susmita Devi et al., 2024 ; Polymer Bulletin, 2025). These films are synthesized from natural polymers, including proteins, polysaccharides, and lipids, thereby offering a sustainable alternative for prolonging food shelf life and minimizing packaging waste (Mellinas et al., 2016 ). 1.1. Materials utilized in the preparation of edible films Edible film coatings are categorized into polysaccharides, proteins, lipids, or composites based on their constituent components. Polysaccharide-based films exhibit favorable mechanical resistance and gas barrier properties; however, their hydrophilic nature makes them susceptible to moisture, potentially compromising their integrity (Hashemi et al., 2023 ; Zhao et al., 2021 ). Protein-based films, typically formulated from solutions or dispersions, demonstrate high flexibility but are subject to limitations such as moisture susceptibility and potential allergic reactions (Chen et al., 2019 ; Mihalca et al., 2021 ). Lipid-based coatings are primarily employed to establish moisture barriers; yet, they possess certain drawbacks related to their brittle structure and waxy taste (Devi et al., 2024 ; Usman et al., 2025 ). Composite films are produced by blending different polymers with the aim of enhancing mechanical strength and barrier properties for improved food packaging performance (Liu et al., 2023 ; Zhang and Kumar, 2024 ). 1.2. Application methods of edible films Edible films are applied to the surface of food products utilizing various techniques to establish a protective barrier. These methods encompass dipping, spraying, pouring, extrusion, and brushing (Krochta and De Mulder-Johnston, 1997). Dipping consists of immersing the food product into an edible film or coating solution, allowing for uniform coverage, which is especially beneficial for products with irregular or complex surfaces (Souza et al., 2022 ; Ahmed et al., 2021 ). The spraying technique entails the application of the film solution onto the surface in a thin layer and is predominantly favored for fruits, vegetables, and meat products (Cerqueira et al., 2017 ). The pouring method involves spreading the edible film solution over a surface followed by drying, rendering it suitable for the production of independent films intended as packaging materials (Kester and Fennema, 1986). The extrusion method is executed by processing film-forming materials under conditions of heat and pressure, facilitating large-scale production within the food industry (Mellinas et al., 2016 ). The brushing technique, commonly employed in small-scale applications, involves the manual application of edible films or coatings onto food surfaces using a brush. This method allows for precise control over the application, making it suitable for laboratory-scale experiments and small production batches. Factors such as brush type, bristle material, and application pressure can influence the uniformity and effectiveness of the coating (Wang, 2023 ; Krishnan et al., 2025 ). The selection of the appropriate method is contingent upon various factors, including film composition, the specific food product targeted, and the desired barrier properties (Zhao, 2011 ). 1.3. Safflower and general properties The safflower plant ( Carthamus tinctorius L.) is an important crop cultivated for multiple industrial applications, including edible oil production, animal feed, textile dyes, and pharmaceutical products (Abbas et al., 2021 ; Li et al., 2023 ). The seeds of safflower ( Carthamus tinctorius ) contain 35–50% oil, of which approximately 90% consists of unsaturated fatty acids (Tahmasebpour et al., 2011 ; Kiprovski et al., 2021 ). The fatty acid profile is typically composed of 71–75% linoleic acid (C18:2), 16–20% oleic acid (C18:1), 6–8% palmitic acid (C16:0), and 2–3% stearic acid (C18:0) (Kurt et al., 2025 ). Linoleic acid is particularly important due to its potential role in reducing cardiovascular disease risk and improving cholesterol levels (Verywell Health, 2024 ). The flowers of safflower contain phenolic compounds and flavonoids, which give the plant strong antioxidant properties. Compounds such as gallic acid, chlorogenic acid, quercetin-3-galactoside, and epicatechin are present in safflower flowers, and these compounds may contribute to the prevention of cardiovascular diseases and cancer due to their antioxidant effects (Erbaş et al., 2023; Ren, 2025 ). Moreover, the oleic acid-rich variant of safflower oil demonstrates quality comparable to olive oil and shares similar sensory attributes, including aroma, flavor, and appearance, with sunflower oil (González et al., 2022 ; Patel and Singh, 2023 ). 2. MATERIAL AND METHOD 2.1. Biotechnological production of edible films Edible films were produced using a biopolymer-based matrix composed of whey protein isolate (WPI), glycerol as a plasticizer, and safflower ( Carthamus tinctorius L. ) flower extract as a source of bioactive compounds. Film-forming solutions were prepared by dispersing WPI in distilled water under continuous stirring and heating to ensure complete solubilization and protein denaturation. Glycerol was incorporated as a plasticizer to enhance flexibility and reduce brittleness. The bioactive extract was added at different concentrations to achieve films with antimicrobial and antioxidant functionality. The final dispersions were cast onto sterile Petri dishes and dried under controlled temperature and humidity conditions to allow solvent evaporation and film formation. This production process represents a simple, scalable, and environmentally friendly biotechnological approach for developing active edible packaging materials. Table 1 Composition of Edible Films (EF: Edible Film) Film composition Whey isolate (% w/w) Glycerol (% w/w) Guargum (% w/w) Safflower extract (% v/v) Control 10 5 0.7 - Ef1 10 5 0.7 0.5 Ef2 10 5 0.7 1 Ef3 10 5 0.7 1.5 Ef4 10 5 0.7 2 2.2. Extraction of safflower bioactive compounds Safflower petals were dried, ground, and subjected to aqueous-ethanolic extraction. The extract was filtered, concentrated under reduced pressure, and stored at 4°C until use. This extraction step provided phenolic-rich bioactive compounds, which were subsequently integrated into the film matrix to enhance its biological activity. The incorporation of plant-derived compounds into a protein-based carrier system demonstrates a biotechnological strategy for functional material development. 2.3. Bioactivity assays (antimicrobial and antioxidant evaluation) The antimicrobial properties of the films were assessed against Escherichia coli and Candida albicans using the agar diffusion method. Films containing different concentrations of safflower extract were placed on inoculated agar plates, and inhibition zones were measured after incubation. Antioxidant activity was evaluated using the DPPH radical scavenging assay to determine the ability of films to prevent oxidative deterioration. These bioactivity assays allowed for the functional validation of the biotechnological coating system. 2.4. Colony forming unite (cfu) To assess the microbial inhibition of edible film compositions against Escherichia coli and Candida albicans, 100 µL of the stock solution containing approximately 107 CFU was extracted from the cell suspensions and inoculated into conical flasks with a total volume of 100 ml, which contained 20 ml of freshly prepared liquid medium. Following the incubation period, edible film compositions with varying concentrations of Safflower flower extract (EF1, EF2, EF3, and EF4) were introduced into the incubated conical flasks along with 20 µL of the inoculum. Control group conical flasks were maintained without the addition of Safflower flower extract. After incubating E. coli at 37°C and C. albicans at 28°C, each sample underwent a serial dilution (10 − 1 to 10 − 4). For the purpose of quantifying microbial inhibition, 100 µL from each dilution was transferred to petri dishes containing solid medium to calculate colony forming units (CFU). 2.5. Biotechnological characterization of edible films The physical and structural properties of the films were analyzed to evaluate their suitability as bio-based packaging materials. Moisture content and solubility tests were performed to determine water interaction properties. Thermal stability was characterized using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), while molecular interactions and structural integrity were investigated through Fourier-transform infrared spectroscopy (FTIR). These advanced characterization techniques provided insights into the stability, hydrogen bonding, and incorporation of bioactive compounds within the protein matrix, highlighting the scientific and technological potential of the developed films. 2.6. Moisture determination Moisture determination was conducted in accordance with AOAC (1990). Edible film samples, positioned within glass drying containers, were subjected to a drying process for 2 hours at 105 ± 2 ºC until a constant weight was achieved. The samples were cut to specified dimensions, weighed using a precision scale, and arranged within the glass drying containers to ensure even distribution. Subsequently, the drying process was prolonged for 24 hours at 105 ± 2 ºC. Following this duration, the quantity of moisture lost by the sample was quantified, and the moisture content was calculated based on the initial weight. 2.7. Solubility in water The solubility (S) of edible film samples in water is determined by the ratio of the initial dry weight of the samples (Ai) to the final dry weight of the insoluble portion (Af). Initially, the samples were cut into squares measuring 2×2 cm and subjected to drying at 105°C for 24 hours. Subsequently, the dry weight of each sample was recorded (Ai). These samples were then placed in beakers containing specified volumes of pure water (20 ml and 50 ml), covered, and agitated continuously at 25°C for 24 hours. Following this agitation period, the insoluble film residue remaining in the beaker was dried again at 105°C for an additional 24 hours, and the final dry weight (Af) was determined. This procedure was conducted in triplicate, and the results were averaged. The solubility percentage was calculated using the provided formula. $$\:\%\:S=\frac{Ai-Af}{Ai}\:x\:100$$ 1 2.8. Instrumental analyses 2.7.1. Thermogravimetric analysis (Tga) Thermogravimetric analysis (TGA) serves as a technique to ascertain the mass loss or gain of materials corresponding to variations in temperature or time. This analytical method quantitatively assesses the changes in the mass of a sample as it undergoes temperature elevation. TGA analyses conducted to evaluate the thermal stability of edible film samples were executed under an air atmosphere. These analyses utilized a Shimadzu TGA-50 analyzer, employing samples of 10 mg for each test. The heating rate was established at 10°C/ min, and the scanning range spanned from 30°C to 700°C. This analytical approach yields insights into the thermal resistance and stability of the films, enabling an understanding of their stability particularly under elevated temperature conditions. 2.7.2. Differential scanning calorimetry (Dsc) In this investigation, differential scanning calorimetry (DSC) tests were conducted employing a Shimadzu DSC-60 device. The tests were administered under an air atmosphere, with a scanning speed set at 10°C /min. The temperature range was specified from 30°C to 500°C, utilizing a sample weight of 5 mg. This analytical technique effectively assesses the thermal transitions and thermal stability of materials. 2.7.3. Fourier transform ınfrared spectroscopy (Ftır) Fourier Transform Infrared Spectroscopy (FTIR) spectra of edible film dispersions were recorded at room temperature. These measurements were performed utilizing a detector within the range of 4000 − 400 cm⁻¹ at a resolution of 4 cm⁻¹. The ATI UNICAM Systems 2000 Fourier Transform Spectrometer was employed to acquire the spectra. This analysis aimed to investigate the chemical structure and functional groups present in the samples. FTIR spectra represent a widely employed technique for elucidating molecular bonds and structures, thereby providing critical insights to enhance the understanding of the properties of edible film materials. 3. RESULTS AND DISCUSSION 3.1. Film formation Edible films were successfully formed from whey protein isolate (WPI) enriched with different concentrations of safflower extract. Films obtained from the EF4 formulation (15 ml casting volume, 2% extract) exhibited the most favorable characteristics, including ~ 1 mm thickness and a uniform dark-yellow appearance (Fig. 1 ). This indicates that higher extract concentrations improve film-forming ability and visual uniformity. 3.2. Antimicrobial activity (cfu) of edible films The antimicrobial properties of the films against E. coli and C. albicans are summarized in Table 2 . A clear concentration-dependent inhibition trend was observed, with EF4 showing the highest microbial reduction. The incorporation of safflower extract into WPI-based films markedly enhanced their antimicrobial efficacy, most likely due to the phenolic and flavonoid constituents of the extract. Similar antimicrobial improvements have been reported in edible films enriched with plant-derived compounds, highlighting the role of bioactive phytochemicals in suppressing microbial growth (Singh et al., 2022 ; Zając et al., 2023 ). Table 2 Quantification of microbial inhibition by edible films using the CFU method. Microorganisms Edible Film Samples Serial Dilution 10 − 1 10 − 2 10 − 3 10 − 4 E. Coli Control ∞ ∞ ∞ 265 EF1 ∞ 235 114 76 EF2 ∞ 196 101 42 EF3 ∞ ∞ 168 55 EF4 144 102 76 23 C. Albicans Control ∞ ∞ 202 118 EF1 ∞ 182 84 55 EF2 ∞ 155 72 44 EF3 ∞ 196 115 72 EF4 205 127 95 56 3.3. Characterization analyses 3.3.1. Moisture retention capacity Moisture retention capacity increased with higher film-forming volumes (8–15 ml), reaching a maximum of 97.1% in EF4 (Table 3 ). This is consistent with the hydrophilic nature of protein-based matrices, which exhibit high water-binding capacity (Liu et al., 2023 ). Enhanced moisture retention is desirable for improving the mechanical stability of edible films during storage and handling. Table 3 Moisture retention percentage of EC4 film at various concentrations. Film composition amount (ml) Sample Moisture rate (%) 8 1 94.56 2 95.2 3 92.4 12 1 95.5 2 94.6 3 96.2 15 1 95.8 2 96.4 3 97.1 3.3.2. Solubility of edible films in water All films displayed high solubility in water, with values ranging between 92.5% and 96.1% (Table 4 ). Film 1 showed nearly complete solubility, while EF4 maintained high but slightly lower solubility. These findings indicate that safflower-enriched WPI films are rapidly degradable in aqueous environments, supporting their potential as environmentally sustainable packaging materials. The results are in agreement with recent reports highlighting the biodegradability of protein- and polysaccharide-based films (Versino et al., 2023 ). Table 4 Water Solubility of Film Samples Sample Film weight Initial weight: ai (g) Final weight: af (g) Solubility in water (%) 1 0.130 0.005 96.1 2 0.127 0.007 94.4 3 0.135 0.010 92.5 3.4. Instrumental analyses 3.4.1. Tga results Thermogravimetric analysis (TGA) was employed to investigate the thermal behavior of the material. The weight loss curve presented in Fig. 2 illustrates the physical or chemical changes occurring at various temperature ranges. Figure 2 provides a detailed account of these weight losses, including percentage values and corresponding temperature ranges. This analysis offers comprehensive insights into the decomposition mechanisms and thermal stability of the material. The TGA analysis elucidated the decomposition mechanisms of the material across different temperature ranges. The initial weight loss of % 9.985 was ascribed to the presence of moisture or volatile components, whereas the subsequent losses were linked to the degradation of the main structure, as well as organic and inorganic components. The total weight loss, approximately %90, suggests that the material is biodegradable and demonstrates typical thermal behavior. 3.4.2. Differential scanning calorimetry (DSC) DSC analysis identified three major thermal transitions (Fig. 3 ). The first minor transition (22.53 J/g) occurred at 60–105°C and was attributed to glass transition phenomena. A stronger exothermic event (105.90 J/g) was detected between 105–196°C, corresponding to partial crystallization or protein denaturation. A major thermal event (672.94 J/g) occurred between 196–375°C, associated with structural decomposition. The multi-stage transitions demonstrate that the films possess adequate thermal stability for practical food packaging applications, in agreement with previous findings for protein-based matrices (Zhang and Kumar, 2024 ). 3.4.3. Structural analysis (FTIR) FTIR spectra (Fig. 4 ) confirmed the incorporation of safflower extract into the WPI film matrix. The broad absorption band at 3283 cm⁻¹ indicated hydroxyl (-OH) groups, associated with phenolic compounds and hydrophilic moieties. The peak at 1638 cm⁻¹ corresponded to carbonyl (C = O) groups from proteins, esters, or aromatic compounds, while bands near 1013 and 950 cm⁻¹ suggested the presence of C–O and C–OH groups, linked to glycerol and carbohydrate structures. These findings confirm that safflower bioactives and glycerol acted synergistically to improve both bioactivity and flexibility of the films (González et al., 2022 ; Patel and Singh, 2023 ). 4. DISCUSSION In this study, biodegradable coating materials were developed by incorporating safflower (Carthamus tinctorius L.) flower extract into whey protein isolate (WPI)-based edible films, and their antimicrobial, physicochemical, and structural properties were evaluated. The results indicated that the EF4 formulation, containing the highest extract concentration, exhibited the strongest inhibitory effect against both Escherichia coli and Candida albicans . The presence of plant-derived phenolic compounds and flavonoids within the film matrix likely played a significant role in suppressing microbial growth. Recent studies have similarly reported that edible films enriched with plant extracts or essential oils can effectively reduce microbial load (Campos et al., 2024 ; Zhang et al., 2023 ). Physicochemical analysis revealed that an increase in the film-forming solution volume significantly enhanced moisture retention capacity, consistent with the hydrophilic nature of protein-based films (Liu et al., 2023 ). High solubility in water suggests rapid degradability and a strong biodegradability potential from an environmental perspective (Versino et al., 2023 ). Thermogravimetric analysis (TGA) results indicated approximately 90% weight loss, characteristic of biodegradable materials, while DSC analysis confirmed thermal stability across a wide temperature range with multiple phase transitions (Zhang and Kumar, 2024 ). FTIR spectra confirmed the presence of phenolic hydroxyl, carbonyl, and glycerol-derived functional groups in the film matrix, indicating successful incorporation of bioactive and plasticizing components that enhance both biological activity and mechanical flexibility. Recent literature emphasizes that the high phenolic and flavonoid content of safflower extract is a key contributor to its antioxidant and antimicrobial properties (González et al., 2022 ; Patel and Singh, 2023 ). Overall, WPI-based edible films enriched with safflower extract represent a promising, eco-friendly, and functional alternative for active food packaging applications. However, before large-scale industrial application, further research is recommended to include sensory testing in various food systems, long-term storage assessments, and production scale-up studies. 5. CONCLUSION This study successfully developed whey protein isolate (WPI)-based edible films incorporated with safflower ( Carthamus tinctorius L. ) extract and evaluated their physicochemical, antimicrobial, and structural properties. The highest concentration (EF4) demonstrated the strongest inhibitory effect against Escherichia coli and Candida albicans , confirming the antimicrobial potential of safflower-derived phenolics and flavonoids. Moisture retention capacity increased with higher film-forming volumes, while high solubility in water confirmed rapid biodegradability, supporting environmental sustainability. Thermal analyses (TGA and DSC) demonstrated multi-stage degradation and high stability across a wide temperature range. FTIR spectra verified the successful incorporation of bioactive and plasticizing components, enhancing both functional and mechanical properties. Overall, safflower-enriched WPI edible films represent a promising biotechnological alternative to petroleum-based plastics, combining antimicrobial activity with eco-friendly degradation. Future work should focus on sensory evaluation, long-term storage stability, and industrial-scale production to ensure practical applicability in the food industry. Declarations CONFLICT OF INTEREST All authors declare that they have no conflict of interest for this study. FUNDING DECLARATION This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution Idea/hypothesis, research, consultant EO; data processing, data analysis, visualisation, writing-review-editing EAG. All authors declare that they have seen/read and approved the final version of the article ready for publication. ACKNOWLEDGEMENTS I would like to thank Malatya Turgut Özal University and İnönü University for their support inconducting the research. Data Availability The authors confirm that the data supporting the findings of this study are available in the article and supplementary materials.Additional data supporting the findings of this study can be obtained from the corresponding author (E.A.G.) upon reasonable request Research Involving Plants All experimental research on plants complied with international guidelines. The safflower ( Carthamus tinctorius L .) flowers used in this study belong to the Dinçer variety, obtained from the Geçit Kuşağı Agricultural Research Institute (Turkey) and cultivated under controlled greenhouse conditions at Malatya Turgut Özal University. The study did not involve endangered or protected species; therefore, it was not subject to restrictions under the IUCN Policy Statement on Research Involving Species at Risk of Extinction or the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). No special permissions or licenses were required for this research. 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09:52:02","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133761,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/766ffc1ddce985016d11b190.html"},{"id":92934089,"identity":"bb5385f7-072d-423f-a9bc-b4b38044648c","added_by":"auto","created_at":"2025-10-07 09:43:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267841,"visible":true,"origin":"","legend":"\u003cp\u003eFilm samples obtained from EF4 film dispersion\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/f0fb85b2fa3a8ea42154a581.png"},{"id":92934084,"identity":"5273eb8e-b8e4-4b3d-8de8-9397f36773bd","added_by":"auto","created_at":"2025-10-07 09:43:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87068,"visible":true,"origin":"","legend":"\u003cp\u003eA) and B) Thermogravimetric analysis TGA) graphs of the edible film\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/f4c0ff2fa18e0c0da83771c1.png"},{"id":92934086,"identity":"465fdacc-3044-4da7-89c7-f327bf323b5e","added_by":"auto","created_at":"2025-10-07 09:43:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148890,"visible":true,"origin":"","legend":"\u003cp\u003eA) and B) Differential Scanning Calorimetry (DSC) analysis graphs of the edible film\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/e130f267d27a35de7ed9bef9.png"},{"id":92934085,"identity":"3cbc6369-24b7-4c1d-b40b-c0511a299b8e","added_by":"auto","created_at":"2025-10-07 09:43:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55798,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR analysis graph of edible film\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/a58f83fb1324a12a5f85b4f7.png"},{"id":96919944,"identity":"ee7f9370-f60c-43c0-be4b-30baf02dfbbf","added_by":"auto","created_at":"2025-11-27 14:14:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1633891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7747329/v1/0938fc81-1f27-49cb-a429-430c3b6def18.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biotechnological Production and Characterization of Innovative Antimicrobial and Antioxidant Edible Films Enriched with Safflower (Carthamus tinctorius L.) Extract","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eFood biotechnology has increasingly focused on the development of bio-based packaging materials as sustainable alternatives to synthetic plastics. The incorporation of plant-derived bioactive compounds into protein- or polysaccharide-based matrices represents a biotechnological innovation that enables functionalization of edible films with antimicrobial and antioxidant activities (Campos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Unlike conventional plastics, these films can be engineered through bioprocessing approaches that not only provide barrier properties but also contribute to active food protection and environmental sustainability (Mellinas et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Versino et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this context, safflower (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e L.), a medicinal plant rich in phenolic compounds and flavonoids, is an attractive candidate for functional biotechnological applications. The integration of safflower extract into whey protein isolate (WPI)-based matrices allows the production of edible films with enhanced bioactivity, combining food biotechnology innovation with environmental biotechnology perspectives (Kim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Such dual benefits position safflower-enriched edible films as next-generation materials with potential to reduce plastic waste while improving food preservation.\u003c/p\u003e\u003cp\u003eFood packaging is a technology engineered to extend the shelf life of food products, prevent physical and chemical deterioration, and guarantee hygiene and convenience during transportation (Chauhan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Alves et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While conventional packaging materials such as plastic, glass, metal, and paper have long dominated the sector, the persistence of plastic waste in the environment has heightened the importance of biodegradable and sustainable alternatives (Sastre et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Versino et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Foods, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe advancement of packaging technologies has progressed beyond merely safeguarding food, leading to the emergence of smart and active packaging systems. Active packaging directly interacts with the food product to extend its shelf life and maintain quality by incorporating components such as oxygen scavengers, antimicrobial agents, and antioxidants. These systems are designed to absorb or release substances that control the internal environment of the package, thereby inhibiting Active packaging helps inhibit spoilage while maintaining the food\u0026rsquo;s sensory and nutritional qualities (Jiang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Vilela et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, smart packaging consists of systems integrated with sensors and indicators that monitor environmental conditions, offering consumers real-time information about the product\u0026rsquo;s quality and safety (Ahmed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The implementation of these technologies facilitates more effective monitoring of food shelf life and contributes to the reduction of waste.\u003c/p\u003e\u003cp\u003eIn recent years, edible films thin biopolymer-based layers have gained prominence within active packaging, as they can be applied directly onto food surfaces or used as standalone packaging materials (Campos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Susmita Devi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Polymer Bulletin, 2025). These films are synthesized from natural polymers, including proteins, polysaccharides, and lipids, thereby offering a sustainable alternative for prolonging food shelf life and minimizing packaging waste (Mellinas et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1. Materials utilized in the preparation of edible films\u003c/h2\u003e\u003cp\u003eEdible film coatings are categorized into polysaccharides, proteins, lipids, or composites based on their constituent components. Polysaccharide-based films exhibit favorable mechanical resistance and gas barrier properties; however, their hydrophilic nature makes them susceptible to moisture, potentially compromising their integrity (Hashemi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Protein-based films, typically formulated from solutions or dispersions, demonstrate high flexibility but are subject to limitations such as moisture susceptibility and potential allergic reactions (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mihalca et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lipid-based coatings are primarily employed to establish moisture barriers; yet, they possess certain drawbacks related to their brittle structure and waxy taste (Devi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Usman et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Composite films are produced by blending different polymers with the aim of enhancing mechanical strength and barrier properties for improved food packaging performance (Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang and Kumar, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.2. Application methods of edible films\u003c/h2\u003e\u003cp\u003eEdible films are applied to the surface of food products utilizing various techniques to establish a protective barrier. These methods encompass dipping, spraying, pouring, extrusion, and brushing (Krochta and De Mulder-Johnston, 1997). Dipping consists of immersing the food product into an edible film or coating solution, allowing for uniform coverage, which is especially beneficial for products with irregular or complex surfaces (Souza et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ahmed et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The spraying technique entails the application of the film solution onto the surface in a thin layer and is predominantly favored for fruits, vegetables, and meat products (Cerqueira et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The pouring method involves spreading the edible film solution over a surface followed by drying, rendering it suitable for the production of independent films intended as packaging materials (Kester and Fennema, 1986). The extrusion method is executed by processing film-forming materials under conditions of heat and pressure, facilitating large-scale production within the food industry (Mellinas et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The brushing technique, commonly employed in small-scale applications, involves the manual application of edible films or coatings onto food surfaces using a brush. This method allows for precise control over the application, making it suitable for laboratory-scale experiments and small production batches. Factors such as brush type, bristle material, and application pressure can influence the uniformity and effectiveness of the coating (Wang, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Krishnan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The selection of the appropriate method is contingent upon various factors, including film composition, the specific food product targeted, and the desired barrier properties (Zhao, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.3. Safflower and general properties\u003c/h2\u003e\u003cp\u003eThe safflower plant (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e L.) is an important crop cultivated for multiple industrial applications, including edible oil production, animal feed, textile dyes, and pharmaceutical products (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The seeds of safflower (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e) contain 35\u0026ndash;50% oil, of which approximately 90% consists of unsaturated fatty acids (Tahmasebpour et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kiprovski et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The fatty acid profile is typically composed of 71\u0026ndash;75% linoleic acid (C18:2), 16\u0026ndash;20% oleic acid (C18:1), 6\u0026ndash;8% palmitic acid (C16:0), and 2\u0026ndash;3% stearic acid (C18:0) (Kurt et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Linoleic acid is particularly important due to its potential role in reducing cardiovascular disease risk and improving cholesterol levels (Verywell Health, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe flowers of safflower contain phenolic compounds and flavonoids, which give the plant strong antioxidant properties. Compounds such as gallic acid, chlorogenic acid, quercetin-3-galactoside, and epicatechin are present in safflower flowers, and these compounds may contribute to the prevention of cardiovascular diseases and cancer due to their antioxidant effects (Erbaş et al., 2023; Ren, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, the oleic acid-rich variant of safflower oil demonstrates quality comparable to olive oil and shares similar sensory attributes, including aroma, flavor, and appearance, with sunflower oil (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patel and Singh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"2. MATERIAL AND METHOD","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Biotechnological production of edible films\u003c/h2\u003e\u003cp\u003eEdible films were produced using a biopolymer-based matrix composed of whey protein isolate (WPI), glycerol as a plasticizer, and safflower (\u003cem\u003eCarthamus tinctorius L.\u003c/em\u003e) flower extract as a source of bioactive compounds. Film-forming solutions were prepared by dispersing WPI in distilled water under continuous stirring and heating to ensure complete solubilization and protein denaturation. Glycerol was incorporated as a plasticizer to enhance flexibility and reduce brittleness. The bioactive extract was added at different concentrations to achieve films with antimicrobial and antioxidant functionality. The final dispersions were cast onto sterile Petri dishes and dried under controlled temperature and humidity conditions to allow solvent evaporation and film formation. This production process represents a simple, scalable, and environmentally friendly biotechnological approach for developing active edible packaging materials.\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\u003eComposition of Edible Films (EF: Edible 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=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFilm composition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhey isolate (% w/w)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycerol (% w/w)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGuargum (% w/w)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSafflower extract (% v/v)\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=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEf1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEf2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEf3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEf4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Extraction of safflower bioactive compounds\u003c/h2\u003e\u003cp\u003eSafflower petals were dried, ground, and subjected to aqueous-ethanolic extraction. The extract was filtered, concentrated under reduced pressure, and stored at 4\u0026deg;C until use. This extraction step provided phenolic-rich bioactive compounds, which were subsequently integrated into the film matrix to enhance its biological activity. The incorporation of plant-derived compounds into a protein-based carrier system demonstrates a biotechnological strategy for functional material development.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Bioactivity assays (antimicrobial and antioxidant evaluation)\u003c/h2\u003e\u003cp\u003eThe antimicrobial properties of the films were assessed against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e using the agar diffusion method. Films containing different concentrations of safflower extract were placed on inoculated agar plates, and inhibition zones were measured after incubation. Antioxidant activity was evaluated using the DPPH radical scavenging assay to determine the ability of films to prevent oxidative deterioration. These bioactivity assays allowed for the functional validation of the biotechnological coating system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Colony forming unite (cfu)\u003c/h2\u003e\u003cp\u003eTo assess the microbial inhibition of edible film compositions against Escherichia coli and Candida albicans, 100 \u0026micro;L of the stock solution containing approximately 107 CFU was extracted from the cell suspensions and inoculated into conical flasks with a total volume of 100 ml, which contained 20 ml of freshly prepared liquid medium. Following the incubation period, edible film compositions with varying concentrations of Safflower flower extract (EF1, EF2, EF3, and EF4) were introduced into the incubated conical flasks along with 20 \u0026micro;L of the inoculum. Control group conical flasks were maintained without the addition of Safflower flower extract. After incubating \u003cem\u003eE. coli\u003c/em\u003e at 37\u0026deg;C and \u003cem\u003eC. albicans\u003c/em\u003e at 28\u0026deg;C, each sample underwent a serial dilution (10\u0026thinsp;\u0026minus;\u0026thinsp;1 to 10\u0026thinsp;\u0026minus;\u0026thinsp;4). For the purpose of quantifying microbial inhibition, 100 \u0026micro;L from each dilution was transferred to petri dishes containing solid medium to calculate colony forming units (CFU).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Biotechnological characterization of edible films\u003c/h2\u003e\u003cp\u003eThe physical and structural properties of the films were analyzed to evaluate their suitability as bio-based packaging materials. Moisture content and solubility tests were performed to determine water interaction properties. Thermal stability was characterized using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), while molecular interactions and structural integrity were investigated through Fourier-transform infrared spectroscopy (FTIR). These advanced characterization techniques provided insights into the stability, hydrogen bonding, and incorporation of bioactive compounds within the protein matrix, highlighting the scientific and technological potential of the developed films.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Moisture determination\u003c/h2\u003e\u003cp\u003eMoisture determination was conducted in accordance with AOAC (1990). Edible film samples, positioned within glass drying containers, were subjected to a drying process for 2 hours at 105\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C until a constant weight was achieved. The samples were cut to specified dimensions, weighed using a precision scale, and arranged within the glass drying containers to ensure even distribution. Subsequently, the drying process was prolonged for 24 hours at 105\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C. Following this duration, the quantity of moisture lost by the sample was quantified, and the moisture content was calculated based on the initial weight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Solubility in water\u003c/h2\u003e\u003cp\u003eThe solubility (S) of edible film samples in water is determined by the ratio of the initial dry weight of the samples (Ai) to the final dry weight of the insoluble portion (Af). Initially, the samples were cut into squares measuring 2\u0026times;2 cm and subjected to drying at 105\u0026deg;C for 24 hours. Subsequently, the dry weight of each sample was recorded (Ai). These samples were then placed in beakers containing specified volumes of pure water (20 ml and 50 ml), covered, and agitated continuously at 25\u0026deg;C for 24 hours. Following this agitation period, the insoluble film residue remaining in the beaker was dried again at 105\u0026deg;C for an additional 24 hours, and the final dry weight (Af) was determined. This procedure was conducted in triplicate, and the results were averaged.\u003c/p\u003e\u003cp\u003eThe solubility percentage was calculated using the provided formula.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:S=\\frac{Ai-Af}{Ai}\\:x\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Instrumental analyses\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1. Thermogravimetric analysis (Tga)\u003c/h2\u003e\u003cp\u003eThermogravimetric analysis (TGA) serves as a technique to ascertain the mass loss or gain of materials corresponding to variations in temperature or time. This analytical method quantitatively assesses the changes in the mass of a sample as it undergoes temperature elevation. TGA analyses conducted to evaluate the thermal stability of edible film samples were executed under an air atmosphere. These analyses utilized a Shimadzu TGA-50 analyzer, employing samples of 10 mg for each test. The heating rate was established at 10\u0026deg;C/ min, and the scanning range spanned from 30\u0026deg;C to 700\u0026deg;C. This analytical approach yields insights into the thermal resistance and stability of the films, enabling an understanding of their stability particularly under elevated temperature conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2. Differential scanning calorimetry (Dsc)\u003c/h2\u003e\u003cp\u003eIn this investigation, differential scanning calorimetry (DSC) tests were conducted employing a Shimadzu DSC-60 device. The tests were administered under an air atmosphere, with a scanning speed set at 10\u0026deg;C /min. The temperature range was specified from 30\u0026deg;C to 500\u0026deg;C, utilizing a sample weight of 5 mg. This analytical technique effectively assesses the thermal transitions and thermal stability of materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.7.3. Fourier transform ınfrared spectroscopy (Ftır)\u003c/h2\u003e\u003cp\u003eFourier Transform Infrared Spectroscopy (FTIR) spectra of edible film dispersions were recorded at room temperature. These measurements were performed utilizing a detector within the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1;. The ATI UNICAM Systems 2000 Fourier Transform Spectrometer was employed to acquire the spectra. This analysis aimed to investigate the chemical structure and functional groups present in the samples. FTIR spectra represent a widely employed technique for elucidating molecular bonds and structures, thereby providing critical insights to enhance the understanding of the properties of edible film materials.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Film formation\u003c/h2\u003e\n \u003cp\u003eEdible films were successfully formed from whey protein isolate (WPI) enriched with different concentrations of safflower extract. Films obtained from the EF4 formulation (15 ml casting volume, 2% extract) exhibited the most favorable characteristics, including\u0026thinsp;~\u0026thinsp;1 mm thickness and a uniform dark-yellow appearance (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This indicates that higher extract concentrations improve film-forming ability and visual uniformity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Antimicrobial activity (cfu) of edible films\u003c/h2\u003e\n \u003cp\u003eThe antimicrobial properties of the films against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e are summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. A clear concentration-dependent inhibition trend was observed, with EF4 showing the highest microbial reduction. The incorporation of safflower extract into WPI-based films markedly enhanced their antimicrobial efficacy, most likely due to the phenolic and flavonoid constituents of the extract. Similar antimicrobial improvements have been reported in edible films enriched with plant-derived compounds, highlighting the role of bioactive phytochemicals in suppressing microbial growth (Singh et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zając et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eQuantification of microbial inhibition by edible films using the CFU method.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMicroorganisms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eEdible Film Samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eSerial Dilution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eE. Coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cem\u003eC. Albicans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026infin;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Characterization analyses\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Moisture retention capacity\u003c/h2\u003e\n \u003cp\u003eMoisture retention capacity increased with higher film-forming volumes (8\u0026ndash;15 ml), reaching a maximum of 97.1% in EF4 (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This is consistent with the hydrophilic nature of protein-based matrices, which exhibit high water-binding capacity (Liu et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Enhanced moisture retention is desirable for improving the mechanical stability of edible films during storage and handling.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMoisture retention percentage of EC4 film at various concentrations.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFilm composition amount (ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMoisture rate (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. Solubility of edible films in water\u003c/h2\u003e\n \u003cp\u003eAll films displayed high solubility in water, with values ranging between 92.5% and 96.1% (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Film 1 showed nearly complete solubility, while EF4 maintained high but slightly lower solubility. These findings indicate that safflower-enriched WPI films are rapidly degradable in aqueous environments, supporting their potential as environmentally sustainable packaging materials. The results are in agreement with recent reports highlighting the biodegradability of protein- and polysaccharide-based films (Versino et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eWater Solubility of Film Samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eFilm weight\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInitial weight: ai (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFinal weight: af (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSolubility in water (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Instrumental analyses\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1. Tga results\u003c/h2\u003e\n \u003cp\u003eThermogravimetric analysis (TGA) was employed to investigate the thermal behavior of the material. The weight loss curve presented in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the physical or chemical changes occurring at various temperature ranges. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e provides a detailed account of these weight losses, including percentage values and corresponding temperature ranges. This analysis offers comprehensive insights into the decomposition mechanisms and thermal stability of the material.\u003c/p\u003e\n \u003cp\u003eThe TGA analysis elucidated the decomposition mechanisms of the material across different temperature ranges. The initial weight loss of % 9.985 was ascribed to the presence of moisture or volatile components, whereas the subsequent losses were linked to the degradation of the main structure, as well as organic and inorganic components. The total weight loss, approximately %90, suggests that the material is biodegradable and demonstrates typical thermal behavior.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2. Differential scanning calorimetry (DSC)\u003c/h2\u003e\n \u003cp\u003eDSC analysis identified three major thermal transitions (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The first minor transition (22.53 J/g) occurred at 60\u0026ndash;105\u0026deg;C and was attributed to glass transition phenomena. A stronger exothermic event (105.90 J/g) was detected between 105\u0026ndash;196\u0026deg;C, corresponding to partial crystallization or protein denaturation. A major thermal event (672.94 J/g) occurred between 196\u0026ndash;375\u0026deg;C, associated with structural decomposition. The multi-stage transitions demonstrate that the films possess adequate thermal stability for practical food packaging applications, in agreement with previous findings for protein-based matrices (Zhang and Kumar, \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.3. Structural analysis (FTIR)\u003c/h2\u003e\n \u003cp\u003eFTIR spectra (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) confirmed the incorporation of safflower extract into the WPI film matrix. The broad absorption band at 3283 cm⁻\u0026sup1; indicated hydroxyl (-OH) groups, associated with phenolic compounds and hydrophilic moieties. The peak at 1638 cm⁻\u0026sup1; corresponded to carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups from proteins, esters, or aromatic compounds, while bands near 1013 and 950 cm⁻\u0026sup1; suggested the presence of C\u0026ndash;O and C\u0026ndash;OH groups, linked to glycerol and carbohydrate structures. These findings confirm that safflower bioactives and glycerol acted synergistically to improve both bioactivity and flexibility of the films (Gonz\u0026aacute;lez et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patel and Singh, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eIn this study, biodegradable coating materials were developed by incorporating safflower \u003cem\u003e(Carthamus tinctorius L.)\u003c/em\u003e flower extract into whey protein isolate (WPI)-based edible films, and their antimicrobial, physicochemical, and structural properties were evaluated. The results indicated that the EF4 formulation, containing the highest extract concentration, exhibited the strongest inhibitory effect against both \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e. The presence of plant-derived phenolic compounds and flavonoids within the film matrix likely played a significant role in suppressing microbial growth. Recent studies have similarly reported that edible films enriched with plant extracts or essential oils can effectively reduce microbial load (Campos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhysicochemical analysis revealed that an increase in the film-forming solution volume significantly enhanced moisture retention capacity, consistent with the hydrophilic nature of protein-based films (Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). High solubility in water suggests rapid degradability and a strong biodegradability potential from an environmental perspective (Versino et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thermogravimetric analysis (TGA) results indicated approximately 90% weight loss, characteristic of biodegradable materials, while DSC analysis confirmed thermal stability across a wide temperature range with multiple phase transitions (Zhang and Kumar, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFTIR spectra confirmed the presence of phenolic hydroxyl, carbonyl, and glycerol-derived functional groups in the film matrix, indicating successful incorporation of bioactive and plasticizing components that enhance both biological activity and mechanical flexibility. Recent literature emphasizes that the high phenolic and flavonoid content of safflower extract is a key contributor to its antioxidant and antimicrobial properties (Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patel and Singh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, WPI-based edible films enriched with safflower extract represent a promising, eco-friendly, and functional alternative for active food packaging applications. However, before large-scale industrial application, further research is recommended to include sensory testing in various food systems, long-term storage assessments, and production scale-up studies.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThis study successfully developed whey protein isolate (WPI)-based edible films incorporated with safflower (\u003cem\u003eCarthamus tinctorius L.\u003c/em\u003e) extract and evaluated their physicochemical, antimicrobial, and structural properties. The highest concentration (EF4) demonstrated the strongest inhibitory effect against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e, confirming the antimicrobial potential of safflower-derived phenolics and flavonoids. Moisture retention capacity increased with higher film-forming volumes, while high solubility in water confirmed rapid biodegradability, supporting environmental sustainability. Thermal analyses (TGA and DSC) demonstrated multi-stage degradation and high stability across a wide temperature range. FTIR spectra verified the successful incorporation of bioactive and plasticizing components, enhancing both functional and mechanical properties.\u003c/p\u003e\u003cp\u003eOverall, safflower-enriched WPI edible films represent a promising biotechnological alternative to petroleum-based plastics, combining antimicrobial activity with eco-friendly degradation. Future work should focus on sensory evaluation, long-term storage stability, and industrial-scale production to ensure practical applicability in the food industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eAll authors declare that they have no conflict of interest for this study.\u003c/p\u003e\u003ch2\u003eFUNDING DECLARATION\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eIdea/hypothesis, research, consultant EO; data processing, data analysis, visualisation, writing-review-editing EAG. All authors declare that they have seen/read and approved the final version of the article ready for publication.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eI would like to thank Malatya Turgut \u0026Ouml;zal University and İn\u0026ouml;n\u0026uuml; University for their support inconducting the research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available in the article and supplementary materials.Additional data supporting the findings of this study can be obtained from the corresponding author (E.A.G.) upon reasonable request\u003c/p\u003e\u003cp\u003e\u003cb\u003eResearch Involving Plants\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All experimental research on plants complied with international guidelines. The safflower (\u003cem\u003eCarthamus tinctorius L\u003c/em\u003e.) flowers used in this study belong to the Din\u0026ccedil;er variety, obtained from the Ge\u0026ccedil;it Kuşağı Agricultural Research Institute (Turkey) and cultivated under controlled greenhouse conditions at Malatya Turgut \u0026Ouml;zal University. The study did not involve endangered or protected species; therefore, it was not subject to restrictions under the IUCN Policy Statement on Research Involving Species at Risk of Extinction or the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). No special permissions or licenses were required for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas S, Khan MA, Farooq M. Safflower (Carthamus tinctorius L.): Agronomic, nutritional, and industrial perspectives. 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Foods. 2021;10(8):1845\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10081845\u003c/span\u003e\u003cspan address=\"10.3390/foods10081845\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"Safflower (Carthamus tinctorius L.), Whey protein isolate (WPI), Edible films, Antimicrobial activity, Antioxidant activity, Biodegradable packagin, Food biotechnology, Thermal stability (TGA, DSC), FTIR spectroscopy, Active food packaging","lastPublishedDoi":"10.21203/rs.3.rs-7747329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7747329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSustainable and functional alternatives to petroleum-based plastics are urgently needed in food biotechnology. Biopolymer-based edible films, particularly those enriched with bioactive plant extracts, provide innovative solutions for extending shelf life while reducing environmental impact. Safflower (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e L.), rich in phenolics and flavonoids, offers strong antimicrobial and antioxidant potential for active packaging development.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eEdible films were prepared using whey protein isolate with varying concentrations of safflower extract. Physicochemical, thermal, and structural properties were examined by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Antimicrobial activity was tested against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e. Moisture retention and solubility assays were performed to evaluate stability and biodegradability.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe highest extract concentration (EF4) showed the strongest inhibition against both microorganisms. TGA confirmed biodegradability with ~\u0026thinsp;90% total weight loss, while DSC demonstrated distinct thermal transitions indicating stability. FTIR verified successful incorporation of hydroxyl, carbonyl, and glycerol-derived groups. Higher film-forming volumes improved moisture retention, whereas high solubility indicated rapid degradability.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eSafflower-enriched films present a biotechnological innovation in sustainable food packaging. The integration of bioactive plant compounds into whey protein\u0026ndash;based matrices yields active films combining antimicrobial and antioxidant activity with environmental compatibility. These findings highlight their potential as next-generation materials in food biotechnology, with future studies needed for scale-up and industrial validation.\u003c/p\u003e","manuscriptTitle":"Biotechnological Production and Characterization of Innovative Antimicrobial and Antioxidant Edible Films Enriched with Safflower (Carthamus tinctorius L.) Extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 09:43:54","doi":"10.21203/rs.3.rs-7747329/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":"36952e07-f632-4c81-b86b-cf601709fa64","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-27T05:53:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-07 09:43:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7747329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7747329","identity":"rs-7747329","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}