Development of Biodegradable Smart Packaging for Virgin Olive Oil Utilizing Cellulose Nanofiber, Methylene Blue, and Vitamin C

Development of Biodegradable Smart Packaging for Virgin Olive Oil Utilizing Cellulose Nanofiber, Methylene Blue, and Vitamin C | 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 Development of Biodegradable Smart Packaging for Virgin Olive Oil Utilizing Cellulose Nanofiber, Methylene Blue, and Vitamin C Sina Sadeghi, Sajad Pirsa, Narmela Asefi, Mehdi Gharekhani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6429267/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 In this study, four types of cellulose (Cel) nanofiber films modified with methylene blue (MB) and vitamin C (VC) were prepared and examined using scanning electron microscopy (SEM). The effects of the films and storage time on the oil's chemical, sensory, and color properties was studied. A mathematical relationship was obtained between the storage time of the oil and the color changes of the films (from white to blue) and it was used to determine the shelf life and expiration date of the oil. The results indicated a significant increase in acidity and peroxide values in control samples over time, reflecting a decline in oil quality. In contrast, packages containing antioxidants like methylene blue and vitamin C effectively maintained lower acidity and peroxide values, while total phenol content remained stable, showcasing their role in preserving oil freshness and antioxidant capacity. Color changes were also less pronounced in antioxidant packages, which maintained better color stability. Sensory evaluations revealed that the combined package received the highest ratings in taste, odor, and overall acceptability, while the control group rated the lowest. This study underscores the importance of selecting appropriate packaging and using antioxidant agents to enhance the quality and shelf life of virgin olive oil. Also, the cellulose/methylene blue/vitamin C kit in olive oil packaging with antioxidant properties can estimate the shelf life of the oil by changing its color from white to blue due to oxidation. The color changes of the sensor are easily visible and using color software, the color factor b can be recorded and the exact shelf life of the oil can be calculated. Kit Sensor Smart packaging Quality control Olive oil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Extra virgin olive oil is obtained by crushing and pulping washed olives. The olive pulp is kneaded and after the oil is extracted from the olive fruit pulp, the best quality virgin oil is separated from the fruit pulp and water using a centrifuge. The taste, color and aroma of olive oil depend on the geographical area where it is grown and the type of olive tree variety. Of course, the time when the olives are harvested for use in oil pressing can also make a difference in its taste and aroma. This oil is an excellent source of antioxidants and healthy fats. Studies have consistently linked a diet rich in monounsaturated fat with favorable effects on markers of heart disease and stroke, including reduced markers of chronic inflammation, blood pressure, cholesterol levels and blood glucose levels. A large review study that included data from over 840,000 people found that those who consumed the most olive oil were 9% less likely to develop heart disease and 11% less likely to die prematurely, compared to those who consumed the least [1 and 2]. Despite the fact that olive oil has many beneficial properties, it is vulnerable to oxidation and loses its desirable properties. Therefore, it is essential to protect this oil against oxidative phenomena. Despite the complexity and extent of the oxidation process in edible oils and fats, the most important and fundamental reactions and factors involved in autoxidation, photo-oxidation and enzymatic oxidation have been identified and defined. In addition, temperature and oxygen can also cause the oxidation of olive oil. Although it is not possible to completely distinguish the separate effects of temperature and oxygen on the oxidation of oils, the general principles and differences in the stability of oils at high and low temperatures can be summarized in a few cases. As temperature increases, the solubility of oxygen decreases. In addition, the rate of oxidation reactions accelerates with increasing temperature. As oxygen pressure decreases, the importance of the initial oxidation reactions becomes more evident [3 and 4]. Active packaging is designed in such a way that components are intentionally incorporated into the structure of the packaging film that release or absorb substances into the package or from the packaged food or from the environment around the food. Active packaging materials are used to increase the shelf life or preserve or improve the condition of packaged food [ 5 – 7 ]. Smart food packaging is the latest technology in the field of food packaging. For this reason, significant advances have been made in the field of smart packaging in the world. Researchers have also made achievements in the field of smart packaging production, which include labels sensitive to carbon dioxide concentration, labels sensitive to oxygen levels, time-temperature labels, and labels sensitive to changes in the pH of the food. Due to the presence of special identifiers, this type of packaging can detect environmental conditions and changes in the food and provide information about the quality and health or unhealthiness of the food to the consumer through these indicators. In recent years, packaging has been introduced that is both active and smart. These systems both actively extend the shelf life of food and can intelligently display the processes inside the food [ 8 – 11 ]. Cellulose is an organic compound that is known as the most abundant biopolymer on Earth. It is a complex carbohydrate or polysaccharide that consists of hundreds to thousands of glucose molecules linked together to form chains. Cellulose is the main structural component of cell walls in plants and algae and has important derivatives that are generally biodegradable and renewable resources. These compounds are usually non-toxic and non-allergenic. Among its derivatives are cellulose nanofibers, celluloid, cellulose acetate, methylcellulose, hydroxyl propyl methylcellulose, and carboxymethylcellulose, all of which are used in the production of biodegradable films [ 12 – 14 ]. Methylene blue is a dye with special properties that acts as a redox indicator and has different colors (blue/colorless) in the oxidized and reduced states. One of the important tests for assessing milk quality is the methylene blue reduction test, which helps estimate the number of bacteria present in raw milk. It is known as an organic chloride salt and a thiazine dye that has oxazine or antioxidant properties and can be used as an effective indicator to detect the amount of oxygen present in solutions [ 15 ]. Vitamin C is an antioxidant (or reducing agent) that can prevent damage caused by free radicals and harmful chemicals. This vitamin also helps protect the skin from the harmful effects of ultraviolet radiation, strengthens the immune system, and helps strengthen gums and teeth. Vitamin C also helps produce collagen (the most important part of connective tissue) and helps prevent high blood cholesterol and blood clots in the arteries [16 and 17]. Due to the problems of oxidation of virgin olive oil, the preservation of this food and its long-term use are facing many problems. Therefore, in this research, an active and intelligent biodegradable film was designed that, in addition to being able to delay the oxidation of olive oil and increase its shelf life, can also intelligently monitor the oxidation process of olive oil and estimate its shelf life and expiration date. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C was used to package virgin olive oil. Methylene blue is naturally blue in color, which becomes colorless when composited with vitamin C (as a reducing agent). Therefore, cellulose film modified with methylene blue and vitamin C is a white film. This film inside the olive oil packaging increases the shelf life of olive oil because both methylene blue and vitamin C have antioxidant properties and prevent the oxidation of olive oil. In addition, as olive oil is stored for a long time, the cellulose nanofiber film modified with methylene blue and vitamin C changes color (from white to blue), which can be considered as a factor indicating the storage time of olive oil. 2. Materials and Methods 2.1. Chemicals Virgin olive oil was purchased from Daneh Khak Salamat brand in Urmia, Iran. Also, cellulose film with nanofiber characteristics, whose thickness is between 30 and 100 nm and its porosity is between 5 and 10 µm (with a molecular weight of 100,000 to 200,000 Daltons), was provided from Zardab Company in Tabriz. In addition, chemicals such as methylene blue, vitamin C, sodium hydroxide, silica gel and other compounds were also provided from reputable companies such as Merck (Germany) and Aldrich (USA) and were used without further purification steps. 2.2. Preparation of Nanofiber Cellulose/Methylene Blue/Vitamin C Kit To prepare the standard methylene blue solution, 500 mg of commercial methylene blue powder was dissolved in one liter of distilled water. Dissolution of this substance was continued at room temperature and with stirring for 10 minutes. To make the films, first cellulose films measuring 10×10 cm were prepared. Then, 50 ml of distilled water was added to a 100 ml beaker and the cellulose film was placed inside it (solution 1). To prepare the cellulose film containing methylene blue, 100 µl of methylene blue solution was added to solution 1 and dissolved well so that methylene blue was absorbed by the film. To prepare the cellulose film containing vitamin C, 5% vitamin C powder was added to solution 1 and dissolved completely so that vitamin C was fixed on the film. To prepare cellulose film containing methylene blue and vitamin C, the film containing methylene blue was removed from the solution and dried in an oven at 50°C. After drying, the colored film was immersed in 50 mL of 5% NaOH solution for 10 minutes. The film was then removed from the solution and dried again in an oven at 50°C. In the next step, the dried film was immersed in a solution containing vitamin C at a concentration of (5%), which resulted in its color changing to white. Finally, the film was removed from the solution and dried again in an oven at 50°C. 2.2.1. Microstructure of Kits Microstructural analysis of the kits was performed to examine their microscopic features and details using a Sigma VP scanning electron microscope (SEM) from ZEISS (Germany). Initially, in order to improve image quality and increase contrast, the samples were coated with a thin layer of gold. This coating step helps to prevent electrical charging on the sample surface, which in turn leads to improved image characteristics and analysis accuracy. After the coating steps, a scanning electron microscope was used for analysis. This device is capable of providing very high-resolution images of the sample surface and accurately shows microscopic structures. Finally, by analyzing the images obtained from SEM, useful results can be obtained regarding the structure and physical properties of the films, which can help optimize processes and improve material performance. 2.3. Oil packaging with kit To package the oil, plastic containers with a capacity of 15 ml and plastic caps were used. In this process, the prepared films were cut into 1×2 cm dimensions and placed in the bottom of the bottles. After placing the films, 10 ml of olive oil was slowly added into the bottle. After filling the bottle, the cap was placed tightly on it and special sealing methods were used to prevent any air from entering the bottle. This step of the packaging process is very crucial, because preventing air from entering can help maintain the quality and freshness of the oil and prevent oxidation (Fig. 1 ). Figure 1 2.4. Oil tests 2.4.1. Acidity To measure the acidity of the oil, method number Cd 3–63 (AOCS, 1993) was used. In this process, first 5 g of the desired oil was mixed with 20 to 30 ml of ethanol or another neutral alcohol. Then, by adding a few drops of phenolphthalein, the solution was titrated so that a pink color was determined [ 18 ]. The acidity number of the oil was calculated using the following formula: $$\:\text{A}=\frac{28.2\times\:\text{N}\times\:100\times\:\text{V}}{1000\times\:\text{W}}\times\:100$$ 1 In this relation: N: Normality of the used oil V: Volume of used oil in mL W: Weight of the sample in g A: Amount of free fatty acids in terms of oleic acid in 100 g of sample This method allows for an accurate assessment of the quality of the oil in terms of acidity, which indicates the oxidation and storage status of the oil. 2.4.2. Refractive index To measure the refractive index of oil samples, these samples were placed in a refractometer and tested at a temperature of 25°C. The results obtained from these measurements were recorded and analyzed according to the standard method Cc 7–25 (AOCS, 1993) [ 19 ]. This test is important for determining the optical properties of oils and can provide useful information about the purity and quality of oils. The refractive index, especially due to its relationship with chemical and physical properties, is one of the key parameters in assessing the quality of food. 2.4.3. Peroxide value Measurement of the peroxide value of olive oil samples was performed using the AOCS method Cd 8–53. In this test, 5 g of olive oil was added to a 250 mL Erlenmeyer flask. Then, 30 mL of acetic acid/chloroform solution in a ratio of 3:2 was added to the flask and the mixture was shaken until the oil was completely dissolved. After that, 0.5 mL of saturated potassium iodide solution was added to the flask and the mixture was kept in the dark for one minute. This step was performed to prevent oxidation and the effect of light on the results. Then, 30 ml of distilled water was added to the flask and 0.5 mL of 1% starch indicator solution was added to it. The final solution was then titrated with 0.1 N sodium thiosulfate until the blue color of the solution was completely eliminated. For the control titration, all the above steps were also performed without adding the oil sample to determine the effect of the volume of indicator and sodium thiosulfate on the accurate results. Finally, the peroxide value was calculated using the following formula [ 20 ]. \(\:PV=\left(\frac{S-B}{W}\right)\) ×N×1000 (2) PV: Peroxide value in milliequivalents of oxygen per kilogram of oil sample S: Amount of tetrazole used in the sample B: Amount of tetrazole used in the control N: Normality of tetrazole used in equivalents per liter W: Weight of oil sample in g 2.4.2. Total phenol content To determine the amount of total phenolic compounds, a colorimetric method using Folin-Ciocalteu reagent was used. In this process, one gram of each sample was mixed with three mL of methanol and water solution in a ratio of 90 to 10 and stirred well for 4 minutes. Then, the solution was centrifuged for 5 minutes at a speed of 3000 rpm to separate the upper liquid phase of the solution. After centrifugation, 20 microliters of the upper phase of methanol extraction was mixed with 2.8 mL of water and 0.5 mL of Folin reagent. After mixing, one mL of 10% sodium carbonate solution was added to this mixture. This mixture was kept at room temperature and in the dark for one hour to complete the chemical reactions. After one hour, the absorbance of the samples was read at room temperature using an ultraviolet spectrophotometer at a wavelength of 765 nm. Gallic acid was used at different concentrations (0 to 1000 µg/mL) to draw a standard curve. Finally, the total phenolic content was reported as mg of gallic acid per kilogram of sample. This method, due to its high accuracy and sensitivity, provides a suitable opportunity to evaluate the phenolic content in the samples and its relationship with antioxidant properties. 2.4.5. Sensory properties The sensory properties of the oil samples, including color, aroma, and flavor, as well as the total acceptance, were evaluated by 20 evaluators in the form of a 5-point descriptive hedonic test. In this evaluation, each characteristic was given a score using a scale of 1 to 5: 1: Very low 2: Low 3: Medium 4: High 5: Very high This sensory evaluation method allows evaluators to express their opinions on various characteristics of the oil in a qualitative and quantitative manner. This type of evaluation is of great importance, especially in the food industry, in order to measure and improve the quality of products. The results of this evaluation can be used as a guide for future research and the development of new products. 2.4.6. Color properties of oil and kit To record the color properties of oil samples and kits used in packaging, a designed system was used (Fig. 1 ). The first step involved calibrating the device using a standard white plate. After this step, the oil was placed evenly on a white paper and their color was measured using the Color Grab software available on a Samsung mobile phone (A21-S). The kits were also placed in a special sample position in the designed system and their color characteristics were recorded. The results of these measurements included three color dimensions with the following quantitative indices: Lightness (from 0 = L* for black to 100 L* = for white), green to red (from − 60 a* = for green to + 60 a* = for red) and blue to yellow (from − 60 b* = blue to + 60 b* = for yellow) This method can accurately analyze the color of oils and provide useful information about their visual characteristics and quality. The use of software along with appropriate calibration increases the accuracy and reliability of the results. 2.5. Color changes of the kit to estimate the shelf life of the oil In this stage, the color factors of the cellulose kit containing methylene blue and vitamin C used for packaging were analyzed. Given that the film changes from white to blue, the b* factor was further investigated. In this factor, negative numbers indicate the intensity of the blue color, and as the number moves towards more positive numbers, it indicates a decrease in the blue color. Accordingly, with the increase in the shelf life of olive oil and the effect of oxidizing agents on the film, the color of the film moved towards blueness. At this stage, a mathematical relationship was established between the intensity of the blueness of the film and the shelf life of the film, and this relationship was used to estimate the storage time or expiration date of the oil. 2.6. Statistical analysis To study the effect of packaging type and storage time on the chemical, sensory and color properties of virgin olive oil, a statistical design (One Factor) was used according to Table 1 . For this purpose, 4 types of packaging were used, including 1- regular packaging, 2- packaging with cellulose film modified with methylene blue, 3- packaging with cellulose film modified with vitamin C, and 4- packaging with cellulose film modified with methylene blue and vitamin C. The storage time was also considered to be 30 days, and the tests were examined on days 1, 15 and 30. The response surface method was used to examine the relationship between independent variables (packaging type and storage time) and dependent variables and modeling. Data analysis was performed at the 95% probability level and mathematical curves and models were drawn using Design Expert-10 software. Table 1 List of tests performed for olive oil packaging Run Factor A: Storage time (Day) Factor B: Packaging type 1 1.00 Control 2 1.00 Cel/MB 3 1.00 Control 4 30.00 Cel/MB 5 15.00 Cel/MB/VC 6 30.00 Control 7 30.00 Cel/VC 8 15.00 Cel/MB/VC 9 1.00 Cel/VC 10 30.00 Control 11 15.00 Cel/MB 12 30.00 Cel/MB/VC 13 1.00 Cel/MB 14 30.00 Cel/MB 15 1.00 Cel/MB/VC 16 30.00 Cel/MB 17 30.00 Cel/MB/VC 18 30.00 Cel/VC 19 1.00 Cel/MB/VC 20 1.00 Cel/VC * Cellulose (Cel); Methylene blue (MB); Vitamin C (VC) Table 1 3. Results and Discussion 3.1. Surface Morphology of the kits Figure 2 shows SEM images of cellulose kit and its various composites. According to SEM images, pure cellulose film has a fibrous structure with dimensions of 20 to 100 nm and significant pores on the film surface. By adding methylene blue to the film, the pores on the film surface are partially filled and a uniform surface is created. In the case of cellulose film containing vitamin C, it is observed that this vitamin has filled most of the surface of the fibers, but the pores on the surface of the cellulose film are still visible. In the cellulose kit containing both methylene blue and vitamin C, the polymer surface is largely saturated with additives and the penetration paths of various gases through the polymer surface are significantly reduced. This study can be a guide to the design and production of films with improved properties for various applications in the packaging or medical industry. Figure 2 3.2. Response Surface Procedure and Modeling Linear interaction curves are one of the important tools in data analysis that are used as a graphical representation of the relationships between two types of variables (numerical and non-numerical) in various scientific and industrial fields. These curves help us to gain a better understanding of how variables affect each other and to identify patterns in the data. In this particular study, we have examined virgin olive oil. In this regard, the storage time of the oil is considered as a numerical variable and the type of packaging as a non-numerical variable. The storage time of the oil is one of the key factors in maintaining its quality and flavor; so that the physicochemical and sensory properties of the oil may change over time. On the other hand, the type of packaging can also have a significant impact on these properties, because different packaging can protect the oil from light, oxygen, and moisture in order to maintain its quality. Table 1 presents the mathematical model data. It includes regression coefficients and modified regression coefficients and depicts the relationship between physicochemical properties such as oxidative stability, sensory properties (including odor, taste, and texture), and color of olive oil with the independent variables of storage time and packaging type. These relationships allow us to analyze the interactions of these variables and reach scientific conclusions about the best practices for storing and packaging virgin olive oil. The ultimate goal is to ensure that this high-quality product reaches consumers and that all its nutritional and sensory benefits are preserved. Thus, this study is not only scientifically important, but can also provide practical guidance for olive oil producers to improve product quality and attract consumers. Table 2 Table 2 Mathematical models and relationships between independent variables and dependent variables Response Equations R² R² Adj Acidity (mg KOH/g oil) =+0.37 + 0.14*A + 0.096*B[ 1 ]-0.036*B[ 2 ]-0.052*B[ 3 ] + 0.10*AB[ 1 ]-0.034*AB[ 2 ]-0.056*AB[ 3 ]-0.023*A2 0.994 0.991 Refractive index =+1.90 + 0.21*A + 0.16*B[ 1 ]-0.018*B[ 2 ]-0.11*B[ 3 ] + 0.22*AB[ 1 ]-6.817E-003*AB[ 2 ]-0.095*AB[ 3 ] 0.776 0.646 PV(mEq O2/Kg oil) =+4.86 + 1.65*A + 1.03*B[ 1 ]-0.38*B[ 2 ]-0.62*B[ 3 ] + 1.03*AB[ 1 ]-0.19*AB[ 2 ]-0.62*AB[ 3 ]-2.02*A2 0.976 0.960 Total Phenol (mg GAE/kg) Total Phenol (mg GAE/kg) = + 147.56–38.43*A- 34.70*B[ 1 ] + 13.87*B[ 2 ] + 19.73*B[ 3 ]-35.31 *AB[ 1 ] + 7.68*AB[ 2 ] + 18.43*AB[ 3 ] + 41.21*A2 0.946 0.904 Flavor =+4.46 − 0.36*A-0.23*B[ 1 ] + 0.074*B[ 2 ] + 0.19*B[ 3 ]-0.17*AB[ 1 ] + 0.051*AB[ 2 ] + 0.16*AB[ 3 ] 0.976 0.963 Color =+4.71 − 0.10*A-0.024*B[ 1 ] + 0.037*B[ 2 ]-0.024*B[ 3 ]-0.049*AB[ 1 ] + 0.048*AB[ 2 ] + 0.051*AB[ 3 ] + 0.12*A2 0.901 0.821 Odor =+4.37–0.37*A-0.17*B[ 1 ] + 0.068*B[ 2 ] + 0.11*B[ 3 ]-0.13*AB[ 1 ] + 0.042*AB[ 2 ] + 0.14*AB[ 3 ] + 0.20*A2 0.980 0.964 Total acceptance =+4.53 − 0.34*A-0.21*B[ 1 ] + 0.064*B[ 2 ] + 0.19*B[ 3 ]-0.19*AB[ 1 ] + 0.060*B[ 2 ] + 0.11*AB[ 3 ] 0.938 0.902 L* =+87.48–3.57*A-2.31*B[ 1 ] + 1.02*B[ 2 ] + 1.69*B[ 3 ]-2.43*AB[ 1 ] + 0.80*AB[ 2 ] + 1.57*AB[ 3 ] + 2.33*A2 0.983 0.972 a* =+0.35 + 0.68*A + 0.31*B[ 1 ]-0.16*B[ 2 ]-0.25*B[ 3 ] + 0.37*AB[ 1 ]-0.19*AB[ 2 ]-0.23*AB[ 3 ] 0.940 0.905 b* =+0.70 + 0.53*A + 0.77*B[ 1 ]-0.32*B[ 2 ]-0.44*B[ 3 ] + 0.76*AB[ 1 ]-0.27*AB[ 2 ]-0.44*AB[ 3 ] 0.986 0.978 **A = Stogareg time (Day); B1 = Control packaging; B2 = Cel/MB; B3 = Cel/VC and B4 = Cel/MB/VC 3.3. Acidity and Refractive Index The acid index indicates the number of milligrams of potassium required to neutralize the free fatty acids in one gram of fatty substance. This measurement refers to the acidic strength of the substance, regardless of the type of fatty acid. In contrast, acidity determines the amount of free fatty acids in 100 grams of the substance and is usually expressed in grams of oleic acid. Increased acidity in an oil or fat is an indication of hydrolysis of triglycerides by the enzyme lipase and can indicate adverse conditions in production or storage, such as high temperatures and humidity. The refractive index of light is one of the well-known methods for identifying organic compounds. The speed of light varies in different media, and this difference causes the light to bend when it crosses the boundary between the two media. The angle of refraction of light depends on factors such as density, type of molecules, temperature, and wavelength of light. Using these angles, it is possible to identify specific properties of different materials, which provides important information about the composition of fats and oils. Figure 3 shows the interaction curve of the effect of storage time and packaging type on the acidity and refractive index for virgin olive oil. In this packaging, four types of conventional packaging (control) (B1), packaging with cellulose modified with methylene blue (B2), packaging with cellulose modified with vitamin C (B3) and packaging with cellulose modified with methylene blue and vitamin C (B4) were used. The single-factor curve of the effect of packaging type on the 30th day is also shown. In general, an increase in acidity and refractive index with storage time is observed in all types of packaging. This means that with time, olive oil is affected by environmental and biochemical factors that lead to an increase in free fatty acids. Also, in all curves, the refractive index tends to increase with time. This increase could indicate chemical changes or deterioration of the oil quality. The control packaging curve (B1) shows the fastest increase in acidity and light scattering coefficient, which indicates its weakness in protecting the oil. Packaging B2, B3 and B4 all have a smaller increase in acidity and light scattering coefficient than B1 and it seems that these packaging have better protective properties. In particular, packaging B4 has the lowest increase in acidity and light scattering coefficient, which indicates the synergistic effect of the combination of methylene blue and vitamin C in maintaining the quality of the oil. The single-factor curve includes the four types of packaging mentioned earlier and the average acidity and light scattering coefficient have been calculated for each. Here again, it is observed that the control packaging has the highest acidity and light scattering coefficient, indicating the damage caused by oxidation and degradation of the oil components. Both the (B2) cellulose/methylene blue) and (B3) cellulose/vitamin C packaging offer a significant reduction in acidity. This could be due to the antioxidant and protective properties of these components. The (B4) cellulose/methylene blue/vitamin C packaging shows the lowest acidity. This combination effectively protects the oil from adverse quality changes. The caveat that these factors are involved in an interaction suggests that the effects of form and time dependence may be more complex than a simple assessment. These two figures demonstrate the importance of packaging in protecting the quality of virgin olive oil. Packaging containing modified agents, such as methylene blue and vitamin C, have been able to effectively control the properties of olive oil. Rotondi et al. (2021) conducted a similar study to investigate the effect of storage conditions on the quality of virgin olive oil, including acidity and peroxide value. The results showed that in olive oil samples stored under incorrect conditions, acidity increased significantly, indicating a decrease in oil quality. While samples stored under optimal conditions showed lower acidity [ 21 ]. Figure 3 3.4. Peroxide value and total phenol content Peroxide is the primary product of lipid oxidation, and increasing the degree of unsaturation in oils can lead to the production of volatile substances such as aldehydes and short-chain fatty acids, which are effective in the development of undesirable odors and flavors. The higher the peroxide value, the more advanced the oxidation and the lower the quality of the oil. Polyphenols, which are part of flavonoid compounds, are present as antioxidants in oils and other plant compounds and are known as anticancer substances. A high total phenol content in the oil indicates antioxidant properties and higher quality of the oil. Figure 4 shows the interaction curve of the effect of storage time and packaging type on peroxide value and total phenol content for virgin olive oil. Also, the single-factor curve of the effect of packaging type on the 30th day is shown. According to the peroxide value and total phenol curves, an increase in peroxide value and a decrease in total phenol content in all curves with time are clearly visible, indicating a type of oxidation in the oil. The curve corresponding to the control packaging (B1) has a steeper slope, indicating that this type of packaging rapidly deteriorates in quality. Packaging with celluloses modified with methylene blue and vitamin C with a slope indicates the lowest oxidation rate and the lowest loss in total phenol content, indicating the highest stability in oil quality. The control packaging has the highest peroxide value and the lowest phenol content, indicating its weakness in protecting the oil against oxidation. The modified packaging has a significant decrease in peroxide value and preservation of total phenol content, indicating the protective effect of methylene blue and vitamin C. These compounds effectively slow down oxidation. The B4 packaging has the best performance, showing the lowest peroxide value and the highest total phenol content. For producers, this result can serve as a recommendation for the use of antioxidant compounds in order to maintain the quality of the oil. The total phenol content refers to milligrams of gallic acid per kilogram of oil (mg GAE/kg). This measurement can be an indicator of the antioxidant properties of the oil. With increasing storage time, the total phenol content decreases continuously. This decrease indicates the oxidation and destruction of antioxidants in the oil. Caipo et al. (2021) have investigated the effect of packaging type and storage conditions on phenolic compounds and other volatiles. The results of their study confirm the results of the present study in terms of the effect of active packaging on maintaining the quality and total phenol content of virgin olive oil during storage [ 22 ]. Figure 4 3.5. Color Properties Colors are important characteristics in foods that have a great influence on consumer acceptance. They are not only effective in stimulating appetite and associating with taste, but can also indicate the quality and safety of foods. Color changes may be related to health risks and, therefore, color can be an indicator of the freshness or staleness of a food. Finally, consumers often evaluate food quality based on its color. Figure 5 shows the interaction curve of the effect of storage time and packaging type on color characteristics for virgin olive oil. Also, the single-factor curve of the effect of packaging type on the 30th day is shown. According to the results, all curves show a decrease in L* with increasing storage time, which could be due to oxidation and chemical changes. B4 packaging has the best protection and maintains a brighter color over time. The parameter a* indicates the amount of red to green color. Positive values ​​indicate red color and negative values ​​indicate green. As storage time increases, a* tends to increase in red. This change can be related to oxidation and degradation of the color compounds of the oil. The B4 packaging package with the least changes in a* indicates better color stability. b* indicates the amount of yellow to blue color. Positive values ​​indicate yellow color and negative values ​​indicate blue color. An increase in b* means the effect of oxidation and changes in the color of the oil. Better quality oils should practically show less color and less yellowness during storage. Here again, B4 packaging is the best for maintaining the desired color over time. The color results clearly indicate the effects of storage time and type of packaging on the quality of the olive oil. Packaging containing modified materials, especially (B4), offers the best protection of the oil against color changes and oxidation. This information can help producers make better decisions about packaging and storage methods for olive oil and maintain product quality over time. Di Stefano and Melilli (2020) investigated the impact of packaging conditions on the physicochemical and color properties of extra virgin olive oil. The results of Di Stefano and Melilli all point to the importance of proper management of storage time and packaging type in maintaining the quality of virgin olive oil and report that with increasing storage time, the color quality of olive oil decreases, which is due to oxidation. The results of the present study indicate a better performance of B4 packaging in maintaining the color and quality of the oil, which is consistent with the paper’s emphasis on the importance of packaging type in reducing oxidation and maintaining quality. Both studies show that oxidation affects color and quality changes in olive oil and that appropriate packaging can prevent these changes. This consistency in results indicates the importance of managing storage time and choosing appropriate packaging to maintain the quality of virgin olive oil [ 23 ]. Figure 5 3.6. Sensory Properties One of the tools that humans naturally have is the five senses. Using the sense of touch, the quality of a substance can be recognized, and by tasting the taste of food, one can ensure that it has not spoiled. In the case of identifying the quality of olive oil, the five senses play an important role. In particular, the taste and smell of this oil can be good criteria for evaluating its quality. In this study, flavor, taste, color, and total acceptance are considered as sensory characteristics for identifying the quality of olive oil. Figure 6 shows the interaction curve of the effect of storage time and packaging type on sensory characteristics for virgin olive oil. The single-factor curve of the effect of packaging type on the 30th day is also shown. According to the results, with increasing storage time, the taste of the oil clearly decreases. Packaging (B4) is identified as the best option with the least loss in taste. Over time, the color and odor of the oil generally decrease, which can be due to oxidation and chemical changes. Packaging (B4) best protects the color of the oil. The odor of the oil decreases significantly with increasing storage time. Packaging B4 performs best in preserving the odor and can be identified as an ideal option. The control packaging has the lowest acceptance rate, indicating a negative effect of storage time on its quality. Modified packaging shows better results compared to the controls, especially B4, which has the best average acceptance. In all these sensory characteristics, packaging containing modified ingredients, especially the combination of methylene blue and vitamin C, clearly protects the olive oil against oxidation and quality loss. The total acceptance of olive oil during storage is clearly affected by the type of packaging. Packaging containing antioxidant ingredients significantly prevents deterioration of quality, taste and odor, making it easier to maintain the overall quality of the oil. According to the results, the use of this type of packaging can help improve acceptance, customer satisfaction and higher sales. Figure 6 3.7. Smart packaging with cellulose/methylene blue/vitamin C kit In this section, the color characteristics of the cellulose/methylene blue/vitamin C kit used in oil packaging were recorded during 30 days of storage, and the b* factor was considered as the basis of color changes. Despite having antioxidant properties, the cellulose/methylene blue/vitamin C kit changed color from white to blue over time under the influence of the oxidation phenomenon (Fig. 1 ), and the b* factor was driven towards more negative numbers. By calibrating the b* factor and the storage time, the following mathematical relationship was obtained. By recording the color of the kit on each day of storage and examining this mathematical relationship, the shelf life of the oil can be estimated (Fig. 1 ). It is worth noting that the color changes of the sensor are visually visible and the spoilage of the oil can be estimated without the need for special tools. Also, to accurately determine the shelf life of oil, you can use the Color Grab software on a smartphone to record the color factor b* for the kit at a desired time and calculate the shelf life from the resulting mathematical equation. b*=-0.1367 Storage time(Day) + 0.5506 R² = 0.997 (3) Mignani et al. (2007) designed a smart cap that uses optochemical sensors to detect olive oil spoilage. Given the importance of maintaining olive oil quality and preventing oxidation, this technology is able to detect chemical changes and product spoilage in real time. They used optochemical sensors based on optical and chemical changes to identify compounds caused by oil spoilage. The smart cap is designed to be easily installed on olive oil containers and continuously monitor the quality of the oil. Their experiments showed that these sensors are able to detect changes caused by oxidation and spoilage of olive oil with high accuracy. This technology not only helps to detect the time of spoilage but can also provide producers and consumers with valuable information about the quality of olive oil. Using this smart shelf, it is possible to ensure the optimal quality of olive oil until consumption. The results of the study by Mignani et al. confirm the results of the present study in terms of the ability to detect and measure oil spoilage during storage [ 24 ]. 4. Conclusion The results of this study show that the type of packaging and storage conditions have a great impact on the quality of virgin olive oil. With increasing storage time, the acidity of the oil increases and its quality decreases. Packaging containing antioxidants, especially methylene blue and vitamin C, reduce this negative trend. The refractive index of the oil is also related to quality changes and modified packaging indicates the stability of oil quality over time. The peroxide value, as an indicator of oxidation, increased in all samples, but antioxidant packaging reduced the peroxide value and increased the quality retention time. The phenol content in the modified packaging was more stable and, given the sensitivity of the oil to oxygen and light, these compounds help maintain quality, taste and aroma. The B4 packaging provided the best results in terms of color and sensory quality. Also, the cellulose/methylene blue/vitamin C kit in the antioxidant olive oil packaging can estimate the shelf life of the oil by changing its color from white to blue due to oxidation. The color changes of the sensor are easily visible and using the Color Grab software, the color factor b can be recorded and the exact shelf life of the oil can be calculated. Considering all these findings, it can be concluded that the use of packaging containing antioxidants and modifiers can significantly help maintain the quality of virgin olive oil during storage periods and has the ability to detect shelf life and expiration date without the need for physicochemical tests. These results can help producers design optimal packaging to extend the shelf life of olive oil and maintain its quality. Ethics statement of sensory evaluation This sensory evaluation study was conducted in accordance with the guidelines approved by Ethics Committee of Urmia University. The authors confirm that all procedures involving human participants adhered to the ethical standards of the responsible committee and complied with relevant guidelines. All participants provided informed consent, and their rights and privacy were protected throughout the study, including measures to ensure voluntary participation, full disclosure of study details, and the ability to withdraw at any time. No participant data was released without their prior knowledge or consent. Declarations Ethics statement of sensory evaluation: This sensory evaluation study was conducted in accordance with the guidelines approved by Ethics Committee of Urmia University. The authors confirm that all procedures involving human participants adhered to the ethical standards of the responsible committee and complied with relevant guidelines. All participants provided informed consent, and their rights and privacy were protected throughout the study, including measures to ensure voluntary participation, full disclosure of study details, and the ability to withdraw at any time. No participant data was released without their prior knowledge or consent. Consent to participate: Informed consent was obtained from all participants involved in the study. The participants were informed of the purpose, potential risks, and benefits of the study, and they voluntarily agreed to participate. Clinical trial: This study has no clinical trial. Author Contribution: Sajad Pirsa conceived of the presented idea, Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it. Funding: No funding was received for this study. Data availability : The data that support the findings of this study are available on request from the corresponding author. Competing interests: Author A (Sina Sadeghi) declares that he has no conflict of interest. Author B (Sajad Pirsa) declares that he has no conflict of interest. Author C (Narmela Asefi) declares that he has no conflict of interest. Author C (Mehdi Gharekhani) declares that he has no conflict of interest. Author Contribution Sajad Pirsa conceived of the presented idea, Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it. References Yubero-Serrano, E.M., Lopez-Moreno, J., Gomez-Delgado, F. and Lopez-Miranda, J., 2019. Extra virgin olive oil: More than a healthy fat. European journal of clinical nutrition, 72(Suppl 1), pp.8–17. Jimenez-Lopez, C., Carpena, M., Lourenço-Lopes, C., Gallardo-Gomez, M., Lorenzo, J.M., Barba, F.J., Prieto, M.A. and Simal-Gandara, J., 2020. Bioactive compounds and quality of extra virgin olive oil. Foods, 9(8), p.1014. Gargouri, B., Zribi, A. and Bouaziz, M., 2015. Effect of containers on the quality of Chemlali olive oil during storage. Journal of Food Science and Technology, 52, pp.1948–1959. Reboredo-Rodríguez, P., Figueiredo-González, M., González-Barreiro, C., Simal-Gándara, J., Salvador, M.D., Cancho-Grande, B. and Fregapane, G., 2017. State of the art on functional virgin olive oils enriched with bioactive compounds and their properties. International Journal of Molecular Sciences, 18(3), p.668. Wyrwa, J. and Barska, A., 2017. Innovations in the food packaging market: Active packaging. European Food Research and Technology, 243, pp.1681–1692. Abdolsattari, P., Pirsa, S., Peighambardoust, S.J., Fasihnia, S.H. and Peighambardoust, S.H., 2020. Investigating microbial properties of traditional Iranian white cheese packed in active LDPE films incorporating metallic and organoclay nanoparticles. Chemical Review and Letters, 3(4), pp.168–174. Pirsa, S., Mahmudi, M. and Ehsani, A., 2023. Biodegradable film based on cress seed mucilage, modified with lutein, maltodextrin and alumina nanoparticles: Physicochemical properties and lutein controlled release. International Journal of Biological Macromolecules, 224, pp.1588–1599. Schaefer, D. and Cheung, W.M., 2018. Smart packaging: opportunities and challenges. Procedia Cirp, 72, pp.1022–1027. Salgado, P.R., Di Giorgio, L., Musso, Y.S. and Mauri, A.N., 2021. Recent developments in smart food packaging focused on biobased and biodegradable polymers. Frontiers in Sustainable Food Systems, 5, p.630393. Pirsa, S., 2017. Fast Determination of Water Content of Some Organic Solvents by Smart Sensor Based on PPy-Ag Nanocomposite. Nanoscience and Nanotechnology Asia, 6 (2), 119–127. Pirsa, S. and Nejad, F.M., 2017. Simultaneous analysis of some volatile compounds in food samples by array gas sensors based on polypyrrole nano-composites. Sensor Review, 37(2), pp.155–164. Zanchetta, E., Damergi, E., Patel, B., Borgmeyer, T., Pick, H., Pulgarin, A. and Ludwig, C., 2021. Algal cellulose, production and potential use in plastics: Challenges and opportunities. Algal Research, 56, p.102288. Pirsa, S., Bener, M. and Şen, F.B., 2024. Biodegradable film of carboxymethyl cellulose modified with red onion peel powder waste and boron nitride nanoparticles: Investigation of physicochemical properties and release of active substances. Food Chemistry, 445, p.138721. Pirsa, S., 2024. Cellulose-based cartons: production methods, modification, and smart/active packaging. Cellulose, 31(6), pp.3421–3445. Stoean, B., Lehene, M., Zagrean-Tuza, C., Silaghi-Dumitrescu, R., Cristea, C. and Gaina, L., 2024. Transient radical species and oxygen colorimetric indicators grounded on phenothiazinium dyes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 320, p.124602. Pehlivan, F.E., 2017. Vitamin C: An antioxidant agent. Vitamin C, 2, pp.23–35. Chiarappa, G., De’Nobili, M.D., Rojas, A.M., Abrami, M., Lapasin, R., Grassi, G., Ferreira, J.A., Gudiño, E., de Oliveira, P. and Grassi, M., 2018. Mathematical modeling of L-(+)-ascorbic acid delivery from pectin films (packaging) to agar hydrogels (food). Journal of Food Engineering, 234, pp.73–81. Joseph, K.S., Bolla, S., Joshi, K., Bhat, M., Naik, K., Patil, S., Bendre, S., Gangappa, B., Haibatti, V., Payamalle, S. and Shinde, S., 2017. Determination of chemical composition and nutritive value with fatty acid compositions of African mangosteen (Garcinia livingstonei). Erwerbs-Obstbau, 59(3), pp.195–202. Petrauskaite, V., De Greyt, W., Kellens, M. and Huyghebaert, A., 1998. Physical and chemical properties of trans-free fats produced by chemical interesterification of vegetable oil blends. Journal of the American Oil Chemists' Society, 75(4), pp.489–493. AOCS, 1998, 1998 & 2009. Official methods and recommended practices of the American oil chemistry society. Sampling and analysis of commercial fats and oils, Cd 8–53. Peroxide value, Ca 5a-40. Free fatty acids & Cd 19–90. 2-Thiobarbituric acid value. Rotondi, A., Morrone, L., Bertazza, G. and Neri, L., 2021. Effect of duration of olive storage on chemical and sensory quality of extra virgin olive oils. Foods, 10(10), p.2296. Caipo, L., Sandoval, A., Sepúlveda, B., Fuentes, E., Valenzuela, R., Metherel, A.H. and Romero, N., 2021. Effect of storage conditions on the quality of arbequina extra virgin olive oil and the impact on the composition of flavor-related compounds (phenols and volatiles). Foods, 10(9), p.2161. Di Stefano, V. and Melilli, M.G., 2020. Effect of storage on quality parameters and phenolic content of Italian extra-virgin olive oils. Natural Product Research, 34(1), pp.78–86. Mignani, A.G., Ciaccheri, L., Mencaglia, A.A., Paolesse, R., Mastroianni, M., Monti, D., Buonocore, G., Del Nobile, A., Mentana, A. and Grimaldi, M.F., 2007, September. A smart cap for olive oil rancidity detection using optochemical sensors. In Advanced Environmental, Chemical, and Biological Sensing Technologies V (Vol. 6755, pp. 127–132). SPIE. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6429267","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465378739,"identity":"5a8868b6-19e1-44f1-aff8-77c93141f3cb","order_by":0,"name":"Sina Sadeghi","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Sina","middleName":"","lastName":"Sadeghi","suffix":""},{"id":465378740,"identity":"2e9cc0a8-566b-41d9-a4a3-6793f959e38e","order_by":1,"name":"Sajad Pirsa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBAC+2YgwfjPBkQ2HiBKi8FhEMmWBtLSQKQWsDI2sEYGIrUc53384QfPebu17YeBttTYRBPUYt/MbibZI3E7eduZRKCWY2m5DQRtYWZjY+AxuJ1sdgCohbHhMFFamD/+STiXbHb+IfFaGKR5DhywM7tBgi1s0rINyQlmN4C2JBDlF/5jzB/fNtjZm51Pf/jgQ40NYS0wkAhWmUCschCwJ0XxKBgFo2AUjDAAAAiaQthfyvZVAAAAAElFTkSuQmCC","orcid":"","institution":"Urmia University","correspondingAuthor":true,"prefix":"","firstName":"Sajad","middleName":"","lastName":"Pirsa","suffix":""},{"id":465378741,"identity":"d7afaa92-b0ad-43b4-a5fe-cfafae9c4105","order_by":2,"name":"Narmela Asefi","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Narmela","middleName":"","lastName":"Asefi","suffix":""},{"id":465378742,"identity":"e4fc8e5e-d0df-45bc-8444-c8224c6ce9f9","order_by":3,"name":"Mehdi Gharekhani","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Gharekhani","suffix":""}],"badges":[],"createdAt":"2025-04-11 14:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6429267/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6429267/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83912362,"identity":"309b41aa-9856-47be-be5f-131b1cee3473","added_by":"auto","created_at":"2025-06-04 12:07:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156101,"visible":true,"origin":"","legend":"\u003cp\u003ePlacement of kit inside the oil packaging and the calibration curve of the kit's color changes during storage time\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/5cf1163daeb40afa67e183e1.png"},{"id":83912367,"identity":"b7cfc620-866e-4d69-a679-cc2991a9754e","added_by":"auto","created_at":"2025-06-04 12:07:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398494,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of cellulose kit and its various composites\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/716e7ae17a82713d00883a68.png"},{"id":83912360,"identity":"4ec10781-9ef8-4886-acc6-5407fa9596d0","added_by":"auto","created_at":"2025-06-04 12:07:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40993,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction curve of the effect of storage time and packaging type and single-factor curve of the effect of packaging type on the 30th day on the acidity and refractive index of virgin olive oil\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/15901321400d331d2de57264.png"},{"id":83912364,"identity":"80bcef65-593c-4e84-b438-0353635c9f52","added_by":"auto","created_at":"2025-06-04 12:07:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42104,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction curve of the effect of storage time and packaging type and single-factor curve of the effect of packaging type on the 30th day on the peroxide value and phenol content of virgin olive oil\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/8d32850e6f3732483103f901.png"},{"id":83913092,"identity":"fd8f9ea8-5cdc-4c85-ad13-dcdb9ead8616","added_by":"auto","created_at":"2025-06-04 12:15:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55724,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction curve of the effect of storage time and packaging type and single-factor curve of the effect of packaging type on the 30th day on the color characteristics of virgin olive oil\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/cee418a2f2d958922ade04a7.png"},{"id":83912366,"identity":"60614f0d-eccf-49ab-87f9-092ae1c13c43","added_by":"auto","created_at":"2025-06-04 12:07:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64378,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction curve of the effect of storage time and packaging type and single-factor curve of the effect of packaging type on the 30th day on the sensory characteristics of virgin olive oil\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/ce91adb9f6c240512a09856f.png"},{"id":88866725,"identity":"51ff81b8-5aa1-47cc-aa4d-b637ecf756f7","added_by":"auto","created_at":"2025-08-12 08:38:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1680484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6429267/v1/d44df01c-21a6-4f65-8503-657914c13efe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Biodegradable Smart Packaging for Virgin Olive Oil Utilizing Cellulose Nanofiber, Methylene Blue, and Vitamin C","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExtra virgin olive oil is obtained by crushing and pulping washed olives. The olive pulp is kneaded and after the oil is extracted from the olive fruit pulp, the best quality virgin oil is separated from the fruit pulp and water using a centrifuge. The taste, color and aroma of olive oil depend on the geographical area where it is grown and the type of olive tree variety. Of course, the time when the olives are harvested for use in oil pressing can also make a difference in its taste and aroma. This oil is an excellent source of antioxidants and healthy fats. Studies have consistently linked a diet rich in monounsaturated fat with favorable effects on markers of heart disease and stroke, including reduced markers of chronic inflammation, blood pressure, cholesterol levels and blood glucose levels. A large review study that included data from over 840,000 people found that those who consumed the most olive oil were 9% less likely to develop heart disease and 11% less likely to die prematurely, compared to those who consumed the least [1 and 2].\u003c/p\u003e \u003cp\u003eDespite the fact that olive oil has many beneficial properties, it is vulnerable to oxidation and loses its desirable properties. Therefore, it is essential to protect this oil against oxidative phenomena. Despite the complexity and extent of the oxidation process in edible oils and fats, the most important and fundamental reactions and factors involved in autoxidation, photo-oxidation and enzymatic oxidation have been identified and defined. In addition, temperature and oxygen can also cause the oxidation of olive oil. Although it is not possible to completely distinguish the separate effects of temperature and oxygen on the oxidation of oils, the general principles and differences in the stability of oils at high and low temperatures can be summarized in a few cases. As temperature increases, the solubility of oxygen decreases. In addition, the rate of oxidation reactions accelerates with increasing temperature. As oxygen pressure decreases, the importance of the initial oxidation reactions becomes more evident [3 and 4].\u003c/p\u003e \u003cp\u003eActive packaging is designed in such a way that components are intentionally incorporated into the structure of the packaging film that release or absorb substances into the package or from the packaged food or from the environment around the food. Active packaging materials are used to increase the shelf life or preserve or improve the condition of packaged food [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Smart food packaging is the latest technology in the field of food packaging. For this reason, significant advances have been made in the field of smart packaging in the world. Researchers have also made achievements in the field of smart packaging production, which include labels sensitive to carbon dioxide concentration, labels sensitive to oxygen levels, time-temperature labels, and labels sensitive to changes in the pH of the food. Due to the presence of special identifiers, this type of packaging can detect environmental conditions and changes in the food and provide information about the quality and health or unhealthiness of the food to the consumer through these indicators. In recent years, packaging has been introduced that is both active and smart. These systems both actively extend the shelf life of food and can intelligently display the processes inside the food [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCellulose is an organic compound that is known as the most abundant biopolymer on Earth. It is a complex carbohydrate or polysaccharide that consists of hundreds to thousands of glucose molecules linked together to form chains. Cellulose is the main structural component of cell walls in plants and algae and has important derivatives that are generally biodegradable and renewable resources. These compounds are usually non-toxic and non-allergenic. Among its derivatives are cellulose nanofibers, celluloid, cellulose acetate, methylcellulose, hydroxyl propyl methylcellulose, and carboxymethylcellulose, all of which are used in the production of biodegradable films [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMethylene blue is a dye with special properties that acts as a redox indicator and has different colors (blue/colorless) in the oxidized and reduced states. One of the important tests for assessing milk quality is the methylene blue reduction test, which helps estimate the number of bacteria present in raw milk. It is known as an organic chloride salt and a thiazine dye that has oxazine or antioxidant properties and can be used as an effective indicator to detect the amount of oxygen present in solutions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVitamin C is an antioxidant (or reducing agent) that can prevent damage caused by free radicals and harmful chemicals. This vitamin also helps protect the skin from the harmful effects of ultraviolet radiation, strengthens the immune system, and helps strengthen gums and teeth. Vitamin C also helps produce collagen (the most important part of connective tissue) and helps prevent high blood cholesterol and blood clots in the arteries [16 and 17].\u003c/p\u003e \u003cp\u003eDue to the problems of oxidation of virgin olive oil, the preservation of this food and its long-term use are facing many problems. Therefore, in this research, an active and intelligent biodegradable film was designed that, in addition to being able to delay the oxidation of olive oil and increase its shelf life, can also intelligently monitor the oxidation process of olive oil and estimate its shelf life and expiration date. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C was used to package virgin olive oil. Methylene blue is naturally blue in color, which becomes colorless when composited with vitamin C (as a reducing agent). Therefore, cellulose film modified with methylene blue and vitamin C is a white film. This film inside the olive oil packaging increases the shelf life of olive oil because both methylene blue and vitamin C have antioxidant properties and prevent the oxidation of olive oil. In addition, as olive oil is stored for a long time, the cellulose nanofiber film modified with methylene blue and vitamin C changes color (from white to blue), which can be considered as a factor indicating the storage time of olive oil.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eVirgin olive oil was purchased from Daneh Khak Salamat brand in Urmia, Iran. Also, cellulose film with nanofiber characteristics, whose thickness is between 30 and 100 nm and its porosity is between 5 and 10 \u0026micro;m (with a molecular weight of 100,000 to 200,000 Daltons), was provided from Zardab Company in Tabriz. In addition, chemicals such as methylene blue, vitamin C, sodium hydroxide, silica gel and other compounds were also provided from reputable companies such as Merck (Germany) and Aldrich (USA) and were used without further purification steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Nanofiber Cellulose/Methylene Blue/Vitamin C Kit\u003c/h2\u003e \u003cp\u003eTo prepare the standard methylene blue solution, 500 mg of commercial methylene blue powder was dissolved in one liter of distilled water. Dissolution of this substance was continued at room temperature and with stirring for 10 minutes. To make the films, first cellulose films measuring 10\u0026times;10 cm were prepared. Then, 50 ml of distilled water was added to a 100 ml beaker and the cellulose film was placed inside it (solution 1). To prepare the cellulose film containing methylene blue, 100 \u0026micro;l of methylene blue solution was added to solution 1 and dissolved well so that methylene blue was absorbed by the film. To prepare the cellulose film containing vitamin C, 5% vitamin C powder was added to solution 1 and dissolved completely so that vitamin C was fixed on the film. To prepare cellulose film containing methylene blue and vitamin C, the film containing methylene blue was removed from the solution and dried in an oven at 50\u0026deg;C. After drying, the colored film was immersed in 50 mL of 5% NaOH solution for 10 minutes. The film was then removed from the solution and dried again in an oven at 50\u0026deg;C. In the next step, the dried film was immersed in a solution containing vitamin C at a concentration of (5%), which resulted in its color changing to white. Finally, the film was removed from the solution and dried again in an oven at 50\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Microstructure of Kits\u003c/h2\u003e \u003cp\u003eMicrostructural analysis of the kits was performed to examine their microscopic features and details using a Sigma VP scanning electron microscope (SEM) from ZEISS (Germany). Initially, in order to improve image quality and increase contrast, the samples were coated with a thin layer of gold. This coating step helps to prevent electrical charging on the sample surface, which in turn leads to improved image characteristics and analysis accuracy. After the coating steps, a scanning electron microscope was used for analysis. This device is capable of providing very high-resolution images of the sample surface and accurately shows microscopic structures. Finally, by analyzing the images obtained from SEM, useful results can be obtained regarding the structure and physical properties of the films, which can help optimize processes and improve material performance.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Oil packaging with kit\u003c/h2\u003e \u003cp\u003eTo package the oil, plastic containers with a capacity of 15 ml and plastic caps were used. In this process, the prepared films were cut into 1\u0026times;2 cm dimensions and placed in the bottom of the bottles. After placing the films, 10 ml of olive oil was slowly added into the bottle. After filling the bottle, the cap was placed tightly on it and special sealing methods were used to prevent any air from entering the bottle. This step of the packaging process is very crucial, because preventing air from entering can help maintain the quality and freshness of the oil and prevent oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Oil tests\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Acidity\u003c/h2\u003e \u003cp\u003eTo measure the acidity of the oil, method number Cd 3\u0026ndash;63 (AOCS, 1993) was used. In this process, first 5 g of the desired oil was mixed with 20 to 30 ml of ethanol or another neutral alcohol. Then, by adding a few drops of phenolphthalein, the solution was titrated so that a pink color was determined [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe acidity number of the oil was calculated using the following formula:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{A}=\\frac{28.2\\times\\:\\text{N}\\times\\:100\\times\\:\\text{V}}{1000\\times\\:\\text{W}}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn this relation:\u003c/p\u003e \u003cp\u003eN: Normality of the used oil\u003c/p\u003e \u003cp\u003eV: Volume of used oil in mL\u003c/p\u003e \u003cp\u003eW: Weight of the sample in g\u003c/p\u003e \u003cp\u003eA: Amount of free fatty acids in terms of oleic acid in 100 g of sample\u003c/p\u003e \u003cp\u003eThis method allows for an accurate assessment of the quality of the oil in terms of acidity, which indicates the oxidation and storage status of the oil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Refractive index\u003c/h2\u003e \u003cp\u003eTo measure the refractive index of oil samples, these samples were placed in a refractometer and tested at a temperature of 25\u0026deg;C. The results obtained from these measurements were recorded and analyzed according to the standard method Cc 7\u0026ndash;25 (AOCS, 1993) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This test is important for determining the optical properties of oils and can provide useful information about the purity and quality of oils. The refractive index, especially due to its relationship with chemical and physical properties, is one of the key parameters in assessing the quality of food.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Peroxide value\u003c/h2\u003e \u003cp\u003eMeasurement of the peroxide value of olive oil samples was performed using the AOCS method Cd 8\u0026ndash;53. In this test, 5 g of olive oil was added to a 250 mL Erlenmeyer flask. Then, 30 mL of acetic acid/chloroform solution in a ratio of 3:2 was added to the flask and the mixture was shaken until the oil was completely dissolved. After that, 0.5 mL of saturated potassium iodide solution was added to the flask and the mixture was kept in the dark for one minute. This step was performed to prevent oxidation and the effect of light on the results. Then, 30 ml of distilled water was added to the flask and 0.5 mL of 1% starch indicator solution was added to it. The final solution was then titrated with 0.1 N sodium thiosulfate until the blue color of the solution was completely eliminated. For the control titration, all the above steps were also performed without adding the oil sample to determine the effect of the volume of indicator and sodium thiosulfate on the accurate results. Finally, the peroxide value was calculated using the following formula [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:PV=\\left(\\frac{S-B}{W}\\right)\\)\u003c/span\u003e \u003c/span\u003e\u0026times;N\u0026times;1000 (2)\u003c/p\u003e \u003cp\u003ePV: Peroxide value in milliequivalents of oxygen per kilogram of oil sample\u003c/p\u003e \u003cp\u003eS: Amount of tetrazole used in the sample\u003c/p\u003e \u003cp\u003eB: Amount of tetrazole used in the control\u003c/p\u003e \u003cp\u003eN: Normality of tetrazole used in equivalents per liter\u003c/p\u003e \u003cp\u003eW: Weight of oil sample in g\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Total phenol content\u003c/h2\u003e \u003cp\u003eTo determine the amount of total phenolic compounds, a colorimetric method using Folin-Ciocalteu reagent was used. In this process, one gram of each sample was mixed with three mL of methanol and water solution in a ratio of 90 to 10 and stirred well for 4 minutes. Then, the solution was centrifuged for 5 minutes at a speed of 3000 rpm to separate the upper liquid phase of the solution. After centrifugation, 20 microliters of the upper phase of methanol extraction was mixed with 2.8 mL of water and 0.5 mL of Folin reagent. After mixing, one mL of 10% sodium carbonate solution was added to this mixture. This mixture was kept at room temperature and in the dark for one hour to complete the chemical reactions. After one hour, the absorbance of the samples was read at room temperature using an ultraviolet spectrophotometer at a wavelength of 765 nm. Gallic acid was used at different concentrations (0 to 1000 \u0026micro;g/mL) to draw a standard curve. Finally, the total phenolic content was reported as mg of gallic acid per kilogram of sample.\u003c/p\u003e \u003cp\u003eThis method, due to its high accuracy and sensitivity, provides a suitable opportunity to evaluate the phenolic content in the samples and its relationship with antioxidant properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5. Sensory properties\u003c/h2\u003e \u003cp\u003eThe sensory properties of the oil samples, including color, aroma, and flavor, as well as the total acceptance, were evaluated by 20 evaluators in the form of a 5-point descriptive hedonic test. In this evaluation, each characteristic was given a score using a scale of 1 to 5:\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e1: Very low\u003c/h3\u003e\n\n\u003ch3\u003e2: Low\u003c/h3\u003e\n\n\u003ch3\u003e3: Medium\u003c/h3\u003e\n\n\u003ch3\u003e4: High\u003c/h3\u003e\n\n\u003ch3\u003e5: Very high\u003c/h3\u003e\n\u003cp\u003eThis sensory evaluation method allows evaluators to express their opinions on various characteristics of the oil in a qualitative and quantitative manner. This type of evaluation is of great importance, especially in the food industry, in order to measure and improve the quality of products. The results of this evaluation can be used as a guide for future research and the development of new products.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.4.6. Color properties of oil and kit\u003c/h2\u003e \u003cp\u003eTo record the color properties of oil samples and kits used in packaging, a designed system was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The first step involved calibrating the device using a standard white plate. After this step, the oil was placed evenly on a white paper and their color was measured using the Color Grab software available on a Samsung mobile phone (A21-S). The kits were also placed in a special sample position in the designed system and their color characteristics were recorded. The results of these measurements included three color dimensions with the following quantitative indices:\u003c/p\u003e \u003cp\u003eLightness (from 0\u0026thinsp;=\u0026thinsp;L* for black to 100 L* = for white), green to red (from \u0026minus;\u0026thinsp;60 a* = for green to +\u0026thinsp;60 a* = for red) and blue to yellow (from \u0026minus;\u0026thinsp;60 b* = blue to +\u0026thinsp;60 b* = for yellow)\u003c/p\u003e \u003cp\u003eThis method can accurately analyze the color of oils and provide useful information about their visual characteristics and quality. The use of software along with appropriate calibration increases the accuracy and reliability of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Color changes of the kit to estimate the shelf life of the oil\u003c/h2\u003e \u003cp\u003eIn this stage, the color factors of the cellulose kit containing methylene blue and vitamin C used for packaging were analyzed. Given that the film changes from white to blue, the b* factor was further investigated. In this factor, negative numbers indicate the intensity of the blue color, and as the number moves towards more positive numbers, it indicates a decrease in the blue color. Accordingly, with the increase in the shelf life of olive oil and the effect of oxidizing agents on the film, the color of the film moved towards blueness. At this stage, a mathematical relationship was established between the intensity of the blueness of the film and the shelf life of the film, and this relationship was used to estimate the storage time or expiration date of the oil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eTo study the effect of packaging type and storage time on the chemical, sensory and color properties of virgin olive oil, a statistical design (One Factor) was used according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For this purpose, 4 types of packaging were used, including 1- regular packaging, 2- packaging with cellulose film modified with methylene blue, 3- packaging with cellulose film modified with vitamin C, and 4- packaging with cellulose film modified with methylene blue and vitamin C. The storage time was also considered to be 30 days, and the tests were examined on days 1, 15 and 30. The response surface method was used to examine the relationship between independent variables (packaging type and storage time) and dependent variables and modeling. Data analysis was performed at the 95% probability level and mathematical curves and models were drawn using Design Expert-10 software.\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\u003eList of tests performed for olive oil packaging\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFactor A: Storage time (Day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFactor B:\u003c/p\u003e \u003cp\u003ePackaging type\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\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=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\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=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/VC\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=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCel/VC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Cellulose (Cel); Methylene blue (MB); Vitamin C (VC)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Surface Morphology of the kits\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows SEM images of cellulose kit and its various composites. According to SEM images, pure cellulose film has a fibrous structure with dimensions of 20 to 100 nm and significant pores on the film surface. By adding methylene blue to the film, the pores on the film surface are partially filled and a uniform surface is created. In the case of cellulose film containing vitamin C, it is observed that this vitamin has filled most of the surface of the fibers, but the pores on the surface of the cellulose film are still visible. In the cellulose kit containing both methylene blue and vitamin C, the polymer surface is largely saturated with additives and the penetration paths of various gases through the polymer surface are significantly reduced. This study can be a guide to the design and production of films with improved properties for various applications in the packaging or medical industry.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Response Surface Procedure and Modeling\u003c/h2\u003e \u003cp\u003eLinear interaction curves are one of the important tools in data analysis that are used as a graphical representation of the relationships between two types of variables (numerical and non-numerical) in various scientific and industrial fields. These curves help us to gain a better understanding of how variables affect each other and to identify patterns in the data. In this particular study, we have examined virgin olive oil. In this regard, the storage time of the oil is considered as a numerical variable and the type of packaging as a non-numerical variable. The storage time of the oil is one of the key factors in maintaining its quality and flavor; so that the physicochemical and sensory properties of the oil may change over time. On the other hand, the type of packaging can also have a significant impact on these properties, because different packaging can protect the oil from light, oxygen, and moisture in order to maintain its quality. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the mathematical model data. It includes regression coefficients and modified regression coefficients and depicts the relationship between physicochemical properties such as oxidative stability, sensory properties (including odor, taste, and texture), and color of olive oil with the independent variables of storage time and packaging type. These relationships allow us to analyze the interactions of these variables and reach scientific conclusions about the best practices for storing and packaging virgin olive oil. The ultimate goal is to ensure that this high-quality product reaches consumers and that all its nutritional and sensory benefits are preserved. Thus, this study is not only scientifically important, but can also provide practical guidance for olive oil producers to improve product quality and attract consumers.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eMathematical models and relationships between independent variables and dependent variables\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEquations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u0026sup2;\u003csub\u003eAdj\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcidity (mg KOH/g oil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+0.37\u0026thinsp;+\u0026thinsp;0.14*A\u0026thinsp;+\u0026thinsp;0.096*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.036*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.052*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.10*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.034*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.056*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-0.023*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.991\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRefractive index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+1.90\u0026thinsp;+\u0026thinsp;0.21*A\u0026thinsp;+\u0026thinsp;0.16*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.018*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.11*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.22*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-6.817E-003*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.095*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.646\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV(mEq O2/Kg oil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+4.86\u0026thinsp;+\u0026thinsp;1.65*A\u0026thinsp;+\u0026thinsp;1.03*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.38*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.62*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;1.03*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.19*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.62*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-2.02*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.960\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Phenol (mg GAE/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal Phenol (mg GAE/kg)\u0026thinsp;=\u0026thinsp;+\u0026thinsp;147.56\u0026ndash;38.43*A- 34.70*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;13.87*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;19.73*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-35.31 *AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;7.68*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;18.43*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;41.21*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.904\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+4.46\u0026thinsp;\u0026minus;\u0026thinsp;0.36*A-0.23*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.074*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.19*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-0.17*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.051*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.16*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.963\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+4.71\u0026thinsp;\u0026minus;\u0026thinsp;0.10*A-0.024*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.037*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.024*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-0.049*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.048*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.051*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.12*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.901\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.821\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+4.37\u0026ndash;0.37*A-0.17*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.068*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.11*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-0.13*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.042*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.14*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.20*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.964\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal acceptance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+4.53\u0026thinsp;\u0026minus;\u0026thinsp;0.34*A-0.21*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.064*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.19*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-0.19*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.060*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.11*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.902\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+87.48\u0026ndash;3.57*A-2.31*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;1.02*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;1.69*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]-2.43*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.80*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;1.57*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;2.33*A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ea*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+0.35\u0026thinsp;+\u0026thinsp;0.68*A\u0026thinsp;+\u0026thinsp;0.31*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.16*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.25*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.37*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.19*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.23*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+0.70\u0026thinsp;+\u0026thinsp;0.53*A\u0026thinsp;+\u0026thinsp;0.77*B[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.32*B[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.44*B[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026thinsp;+\u0026thinsp;0.76*AB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]-0.27*AB[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]-0.44*AB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.978\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e**A\u0026thinsp;=\u0026thinsp;Stogareg time (Day); B1\u0026thinsp;=\u0026thinsp;Control packaging; B2\u0026thinsp;=\u0026thinsp;Cel/MB; B3\u0026thinsp;=\u0026thinsp;Cel/VC and B4\u0026thinsp;=\u0026thinsp;Cel/MB/VC\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Acidity and Refractive Index\u003c/h2\u003e \u003cp\u003eThe acid index indicates the number of milligrams of potassium required to neutralize the free fatty acids in one gram of fatty substance. This measurement refers to the acidic strength of the substance, regardless of the type of fatty acid. In contrast, acidity determines the amount of free fatty acids in 100 grams of the substance and is usually expressed in grams of oleic acid. Increased acidity in an oil or fat is an indication of hydrolysis of triglycerides by the enzyme lipase and can indicate adverse conditions in production or storage, such as high temperatures and humidity.\u003c/p\u003e \u003cp\u003eThe refractive index of light is one of the well-known methods for identifying organic compounds. The speed of light varies in different media, and this difference causes the light to bend when it crosses the boundary between the two media. The angle of refraction of light depends on factors such as density, type of molecules, temperature, and wavelength of light. Using these angles, it is possible to identify specific properties of different materials, which provides important information about the composition of fats and oils.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the interaction curve of the effect of storage time and packaging type on the acidity and refractive index for virgin olive oil. In this packaging, four types of conventional packaging (control) (B1), packaging with cellulose modified with methylene blue (B2), packaging with cellulose modified with vitamin C (B3) and packaging with cellulose modified with methylene blue and vitamin C (B4) were used. The single-factor curve of the effect of packaging type on the 30th day is also shown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn general, an increase in acidity and refractive index with storage time is observed in all types of packaging. This means that with time, olive oil is affected by environmental and biochemical factors that lead to an increase in free fatty acids. Also, in all curves, the refractive index tends to increase with time. This increase could indicate chemical changes or deterioration of the oil quality. The control packaging curve (B1) shows the fastest increase in acidity and light scattering coefficient, which indicates its weakness in protecting the oil. Packaging B2, B3 and B4 all have a smaller increase in acidity and light scattering coefficient than B1 and it seems that these packaging have better protective properties. In particular, packaging B4 has the lowest increase in acidity and light scattering coefficient, which indicates the synergistic effect of the combination of methylene blue and vitamin C in maintaining the quality of the oil. The single-factor curve includes the four types of packaging mentioned earlier and the average acidity and light scattering coefficient have been calculated for each. Here again, it is observed that the control packaging has the highest acidity and light scattering coefficient, indicating the damage caused by oxidation and degradation of the oil components. Both the (B2) cellulose/methylene blue) and (B3) cellulose/vitamin C packaging offer a significant reduction in acidity. This could be due to the antioxidant and protective properties of these components. The (B4) cellulose/methylene blue/vitamin C packaging shows the lowest acidity. This combination effectively protects the oil from adverse quality changes. The caveat that these factors are involved in an interaction suggests that the effects of form and time dependence may be more complex than a simple assessment. These two figures demonstrate the importance of packaging in protecting the quality of virgin olive oil. Packaging containing modified agents, such as methylene blue and vitamin C, have been able to effectively control the properties of olive oil.\u003c/p\u003e \u003cp\u003eRotondi et al. (2021) conducted a similar study to investigate the effect of storage conditions on the quality of virgin olive oil, including acidity and peroxide value. The results showed that in olive oil samples stored under incorrect conditions, acidity increased significantly, indicating a decrease in oil quality. While samples stored under optimal conditions showed lower acidity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Peroxide value and total phenol content\u003c/h2\u003e \u003cp\u003ePeroxide is the primary product of lipid oxidation, and increasing the degree of unsaturation in oils can lead to the production of volatile substances such as aldehydes and short-chain fatty acids, which are effective in the development of undesirable odors and flavors. The higher the peroxide value, the more advanced the oxidation and the lower the quality of the oil. Polyphenols, which are part of flavonoid compounds, are present as antioxidants in oils and other plant compounds and are known as anticancer substances. A high total phenol content in the oil indicates antioxidant properties and higher quality of the oil.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the interaction curve of the effect of storage time and packaging type on peroxide value and total phenol content for virgin olive oil. Also, the single-factor curve of the effect of packaging type on the 30th day is shown. According to the peroxide value and total phenol curves, an increase in peroxide value and a decrease in total phenol content in all curves with time are clearly visible, indicating a type of oxidation in the oil. The curve corresponding to the control packaging (B1) has a steeper slope, indicating that this type of packaging rapidly deteriorates in quality. Packaging with celluloses modified with methylene blue and vitamin C with a slope indicates the lowest oxidation rate and the lowest loss in total phenol content, indicating the highest stability in oil quality. The control packaging has the highest peroxide value and the lowest phenol content, indicating its weakness in protecting the oil against oxidation. The modified packaging has a significant decrease in peroxide value and preservation of total phenol content, indicating the protective effect of methylene blue and vitamin C. These compounds effectively slow down oxidation. The B4 packaging has the best performance, showing the lowest peroxide value and the highest total phenol content. For producers, this result can serve as a recommendation for the use of antioxidant compounds in order to maintain the quality of the oil. The total phenol content refers to milligrams of gallic acid per kilogram of oil (mg GAE/kg). This measurement can be an indicator of the antioxidant properties of the oil. With increasing storage time, the total phenol content decreases continuously. This decrease indicates the oxidation and destruction of antioxidants in the oil.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCaipo et al. (2021) have investigated the effect of packaging type and storage conditions on phenolic compounds and other volatiles. The results of their study confirm the results of the present study in terms of the effect of active packaging on maintaining the quality and total phenol content of virgin olive oil during storage [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Color Properties\u003c/h2\u003e \u003cp\u003eColors are important characteristics in foods that have a great influence on consumer acceptance. They are not only effective in stimulating appetite and associating with taste, but can also indicate the quality and safety of foods. Color changes may be related to health risks and, therefore, color can be an indicator of the freshness or staleness of a food. Finally, consumers often evaluate food quality based on its color.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the interaction curve of the effect of storage time and packaging type on color characteristics for virgin olive oil. Also, the single-factor curve of the effect of packaging type on the 30th day is shown. According to the results, all curves show a decrease in L* with increasing storage time, which could be due to oxidation and chemical changes. B4 packaging has the best protection and maintains a brighter color over time. The parameter a* indicates the amount of red to green color. Positive values ​​indicate red color and negative values ​​indicate green. As storage time increases, a* tends to increase in red. This change can be related to oxidation and degradation of the color compounds of the oil. The B4 packaging package with the least changes in a* indicates better color stability. b* indicates the amount of yellow to blue color. Positive values ​​indicate yellow color and negative values ​​indicate blue color. An increase in b* means the effect of oxidation and changes in the color of the oil. Better quality oils should practically show less color and less yellowness during storage. Here again, B4 packaging is the best for maintaining the desired color over time. The color results clearly indicate the effects of storage time and type of packaging on the quality of the olive oil. Packaging containing modified materials, especially (B4), offers the best protection of the oil against color changes and oxidation. This information can help producers make better decisions about packaging and storage methods for olive oil and maintain product quality over time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDi Stefano and Melilli (2020) investigated the impact of packaging conditions on the physicochemical and color properties of extra virgin olive oil. The results of Di Stefano and Melilli all point to the importance of proper management of storage time and packaging type in maintaining the quality of virgin olive oil and report that with increasing storage time, the color quality of olive oil decreases, which is due to oxidation. The results of the present study indicate a better performance of B4 packaging in maintaining the color and quality of the oil, which is consistent with the paper\u0026rsquo;s emphasis on the importance of packaging type in reducing oxidation and maintaining quality. Both studies show that oxidation affects color and quality changes in olive oil and that appropriate packaging can prevent these changes. This consistency in results indicates the importance of managing storage time and choosing appropriate packaging to maintain the quality of virgin olive oil [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Sensory Properties\u003c/h2\u003e \u003cp\u003eOne of the tools that humans naturally have is the five senses. Using the sense of touch, the quality of a substance can be recognized, and by tasting the taste of food, one can ensure that it has not spoiled. In the case of identifying the quality of olive oil, the five senses play an important role. In particular, the taste and smell of this oil can be good criteria for evaluating its quality. In this study, flavor, taste, color, and total acceptance are considered as sensory characteristics for identifying the quality of olive oil.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the interaction curve of the effect of storage time and packaging type on sensory characteristics for virgin olive oil. The single-factor curve of the effect of packaging type on the 30th day is also shown. According to the results, with increasing storage time, the taste of the oil clearly decreases. Packaging (B4) is identified as the best option with the least loss in taste. Over time, the color and odor of the oil generally decrease, which can be due to oxidation and chemical changes. Packaging (B4) best protects the color of the oil. The odor of the oil decreases significantly with increasing storage time. Packaging B4 performs best in preserving the odor and can be identified as an ideal option. The control packaging has the lowest acceptance rate, indicating a negative effect of storage time on its quality. Modified packaging shows better results compared to the controls, especially B4, which has the best average acceptance. In all these sensory characteristics, packaging containing modified ingredients, especially the combination of methylene blue and vitamin C, clearly protects the olive oil against oxidation and quality loss. The total acceptance of olive oil during storage is clearly affected by the type of packaging. Packaging containing antioxidant ingredients significantly prevents deterioration of quality, taste and odor, making it easier to maintain the overall quality of the oil. According to the results, the use of this type of packaging can help improve acceptance, customer satisfaction and higher sales.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Smart packaging with cellulose/methylene blue/vitamin C kit\u003c/h2\u003e \u003cp\u003eIn this section, the color characteristics of the cellulose/methylene blue/vitamin C kit used in oil packaging were recorded during 30 days of storage, and the b* factor was considered as the basis of color changes. Despite having antioxidant properties, the cellulose/methylene blue/vitamin C kit changed color from white to blue over time under the influence of the oxidation phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the b* factor was driven towards more negative numbers. By calibrating the b* factor and the storage time, the following mathematical relationship was obtained. By recording the color of the kit on each day of storage and examining this mathematical relationship, the shelf life of the oil can be estimated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is worth noting that the color changes of the sensor are visually visible and the spoilage of the oil can be estimated without the need for special tools. Also, to accurately determine the shelf life of oil, you can use the Color Grab software on a smartphone to record the color factor b* for the kit at a desired time and calculate the shelf life from the resulting mathematical equation.\u003c/p\u003e \u003cp\u003eb*=-0.1367 Storage time(Day)\u0026thinsp;+\u0026thinsp;0.5506 R\u0026sup2; = 0.997 (3)\u003c/p\u003e \u003cp\u003eMignani et al. (2007) designed a smart cap that uses optochemical sensors to detect olive oil spoilage. Given the importance of maintaining olive oil quality and preventing oxidation, this technology is able to detect chemical changes and product spoilage in real time. They used optochemical sensors based on optical and chemical changes to identify compounds caused by oil spoilage. The smart cap is designed to be easily installed on olive oil containers and continuously monitor the quality of the oil. Their experiments showed that these sensors are able to detect changes caused by oxidation and spoilage of olive oil with high accuracy. This technology not only helps to detect the time of spoilage but can also provide producers and consumers with valuable information about the quality of olive oil. Using this smart shelf, it is possible to ensure the optimal quality of olive oil until consumption. The results of the study by Mignani et al. confirm the results of the present study in terms of the ability to detect and measure oil spoilage during storage [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe results of this study show that the type of packaging and storage conditions have a great impact on the quality of virgin olive oil. With increasing storage time, the acidity of the oil increases and its quality decreases. Packaging containing antioxidants, especially methylene blue and vitamin C, reduce this negative trend. The refractive index of the oil is also related to quality changes and modified packaging indicates the stability of oil quality over time. The peroxide value, as an indicator of oxidation, increased in all samples, but antioxidant packaging reduced the peroxide value and increased the quality retention time. The phenol content in the modified packaging was more stable and, given the sensitivity of the oil to oxygen and light, these compounds help maintain quality, taste and aroma. The B4 packaging provided the best results in terms of color and sensory quality. Also, the cellulose/methylene blue/vitamin C kit in the antioxidant olive oil packaging can estimate the shelf life of the oil by changing its color from white to blue due to oxidation. The color changes of the sensor are easily visible and using the Color Grab software, the color factor b can be recorded and the exact shelf life of the oil can be calculated. Considering all these findings, it can be concluded that the use of packaging containing antioxidants and modifiers can significantly help maintain the quality of virgin olive oil during storage periods and has the ability to detect shelf life and expiration date without the need for physicochemical tests. These results can help producers design optimal packaging to extend the shelf life of olive oil and maintain its quality.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics statement of sensory evaluation\u003c/strong\u003e \u003cp\u003eThis sensory evaluation study was conducted in accordance with the guidelines approved by Ethics Committee of Urmia University. The authors confirm that all procedures involving human participants adhered to the ethical standards of the responsible committee and complied with relevant guidelines. All participants provided informed consent, and their rights and privacy were protected throughout the study, including measures to ensure voluntary participation, full disclosure of study details, and the ability to withdraw at any time. No participant data was released without their prior knowledge or consent.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement of sensory evaluation:\u0026nbsp;\u003c/strong\u003eThis sensory evaluation study was conducted in accordance with the guidelines approved by Ethics Committee of Urmia University. \u0026nbsp;The authors confirm that all procedures involving human participants adhered to the ethical standards of the responsible committee and complied with relevant guidelines. All participants provided informed consent, and their rights and privacy were protected throughout the study, including measures to ensure voluntary participation, full disclosure of study details, and the ability to withdraw at any time. No participant data was released without their prior knowledge or consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all participants involved in the study. The participants were informed of the purpose, potential risks, and benefits of the study, and they voluntarily agreed to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial:\u0026nbsp;\u003c/strong\u003eThis study has no clinical trial.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u0026nbsp;\u003c/strong\u003eSajad Pirsa conceived of the presented idea,\u0026nbsp;Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNo funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: The data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e Author A (Sina Sadeghi) declares that he has no conflict of interest. Author B (Sajad Pirsa) declares that he has no conflict of interest. Author C (Narmela Asefi) declares that he has no conflict of interest. Author C (Mehdi Gharekhani) declares that he has no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSajad Pirsa conceived of the presented idea, Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYubero-Serrano, E.M., Lopez-Moreno, J., Gomez-Delgado, F. and Lopez-Miranda, J., 2019. Extra virgin olive oil: More than a healthy fat. European journal of clinical nutrition, 72(Suppl 1), pp.8\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJimenez-Lopez, C., Carpena, M., Louren\u0026ccedil;o-Lopes, C., Gallardo-Gomez, M., Lorenzo, J.M., Barba, F.J., Prieto, M.A. and Simal-Gandara, J., 2020. Bioactive compounds and quality of extra virgin olive oil. Foods, 9(8), p.1014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGargouri, B., Zribi, A. and Bouaziz, M., 2015. Effect of containers on the quality of Chemlali olive oil during storage. Journal of Food Science and Technology, 52, pp.1948\u0026ndash;1959.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReboredo-Rodr\u0026iacute;guez, P., Figueiredo-Gonz\u0026aacute;lez, M., Gonz\u0026aacute;lez-Barreiro, C., Simal-G\u0026aacute;ndara, J., Salvador, M.D., Cancho-Grande, B. and Fregapane, G., 2017. State of the art on functional virgin olive oils enriched with bioactive compounds and their properties. International Journal of Molecular Sciences, 18(3), p.668.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWyrwa, J. and Barska, A., 2017. Innovations in the food packaging market: Active packaging. European Food Research and Technology, 243, pp.1681\u0026ndash;1692.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdolsattari, P., Pirsa, S., Peighambardoust, S.J., Fasihnia, S.H. and Peighambardoust, S.H., 2020. Investigating microbial properties of traditional Iranian white cheese packed in active LDPE films incorporating metallic and organoclay nanoparticles. Chemical Review and Letters, 3(4), pp.168\u0026ndash;174.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePirsa, S., Mahmudi, M. and Ehsani, A., 2023. Biodegradable film based on cress seed mucilage, modified with lutein, maltodextrin and alumina nanoparticles: Physicochemical properties and lutein controlled release. International Journal of Biological Macromolecules, 224, pp.1588\u0026ndash;1599.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaefer, D. and Cheung, W.M., 2018. Smart packaging: opportunities and challenges. Procedia Cirp, 72, pp.1022\u0026ndash;1027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalgado, P.R., Di Giorgio, L., Musso, Y.S. and Mauri, A.N., 2021. Recent developments in smart food packaging focused on biobased and biodegradable polymers. 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SPIE.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Kit, Sensor, Smart packaging, Quality control, Olive oil","lastPublishedDoi":"10.21203/rs.3.rs-6429267/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6429267/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, four types of cellulose (Cel) nanofiber films modified with methylene blue (MB) and vitamin C (VC) were prepared and examined using scanning electron microscopy (SEM). The effects of the films and storage time on the oil's chemical, sensory, and color properties was studied. A mathematical relationship was obtained between the storage time of the oil and the color changes of the films (from white to blue) and it was used to determine the shelf life and expiration date of the oil. The results indicated a significant increase in acidity and peroxide values in control samples over time, reflecting a decline in oil quality. In contrast, packages containing antioxidants like methylene blue and vitamin C effectively maintained lower acidity and peroxide values, while total phenol content remained stable, showcasing their role in preserving oil freshness and antioxidant capacity. Color changes were also less pronounced in antioxidant packages, which maintained better color stability. Sensory evaluations revealed that the combined package received the highest ratings in taste, odor, and overall acceptability, while the control group rated the lowest. This study underscores the importance of selecting appropriate packaging and using antioxidant agents to enhance the quality and shelf life of virgin olive oil. Also, the cellulose/methylene blue/vitamin C kit in olive oil packaging with antioxidant properties can estimate the shelf life of the oil by changing its color from white to blue due to oxidation. The color changes of the sensor are easily visible and using color software, the color factor b can be recorded and the exact shelf life of the oil can be calculated.\u003c/p\u003e","manuscriptTitle":"Development of Biodegradable Smart Packaging for Virgin Olive Oil Utilizing Cellulose Nanofiber, Methylene Blue, and Vitamin C","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 12:07:44","doi":"10.21203/rs.3.rs-6429267/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":"63528841-c999-4c38-b33e-a4d6e3811c07","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T08:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 12:07:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6429267","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6429267","identity":"rs-6429267","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}