Monitoring the shelf life of extra virgin olive oil using Fluorescence spectroscopy

Monitoring the shelf life of extra virgin olive oil using Fluorescence spectroscopy | 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 Monitoring the shelf life of extra virgin olive oil using Fluorescence spectroscopy Rimsha Hafeez, Naveed Ahmad, Areeba Ansar, Muhammad Saleem, Hina Ali This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4741827/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 To maintain the quality of extra virgin olive oil (EVOO), one important factor is to resist its oxidation during processing and storage. It is challenging task to prevent oxidation in EVOOs since various physical and chemical factors must be under control. In the present study extra virgin olive oil was stored under normal storage conditions for three months and monitored via quality parameters. A non-destructive fluorescence spectroscopy was performed to monitor the oxidation changes in EVOO from different olive growing regions of Pakistan and Al-Jouf region of the Kingdom of Saudi Arabia (KSA). Additionally, the impacts of geographic, climatic, and environmental factors on the oxidation of EVOOs were investigated. Two major changes in the fluorescence emission spectra of EVOO samples were mainly observed: a decrease in fluorescence emission intensities in the range between 500 to 600 nm, 650 to 690 nm, as well as a rise in the fluorescence emission intensities in the 365–500 nm region. These two changes were related to a degradation of beta carotene, chlorophyll content and formation of oxidation products. EVOO samples collected from Al-Jouf region, KSA showed low rate of oxidation which might be due to environmental and geographical effect, proper irrigation system, harvesting, processing and storage conditions. In a fast and non-destructive manner, The fluorescence spectroscopy successfully monitored the oxidation changes and deterioration of antioxidants in a rapid and non-destructive manner in the EVOOs. EVOO vitamin E beta carotene chlorophyll content Fluorescence spectroscopy quality assurance rate of oxidation deterioration of antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Extra virgin olive oil (EVOO) is a well-known edible oil that is produced from the fruit of the olive tree (Garcia-Oliveira et al. 2021). It is abundant in beneficial nutrients, monounsaturated fatty acids, and antioxidants (Conte et al. 2020 ). In addition, it carry modest amounts of vitamin K, vitamin E (tocopherols), vitamin A (β-carotenoids), vitamin F (oleic and linoleic acids) (Lanza and Ninfali 2020; Jimenez-Lopez et al. 2020 ; Maszewska et al. 2018 ). EVOO contains various pigments such as chlorophyll and carotenoids (Saleem et al. 2017 ). The color of olive oil is distinct, ranging from a light gold to a deep green, and is influenced by the quantity of pigments present(Tekaya et al. 2022 ). Green olives, rich in chlorophyll, contribute to the production of green oil, while ripe olives, containing carotenoids, result in the production of yellow oil(Lazzerini, Cifelli, and Domenici 2016). Throughout the years, EVOO has been the topic of numerous scientific studies due to its hidden health benefits and its importance as a staple anchor all over the world(Korkmaz 2023 ; Astolfi et al. 2021 ; Millman et al. 2021 ). Recently, there has been considerable interests in correlating monounsaturated fatty acids (mainly oleic acid) and minor components (phenolic compounds, carotenoids and alpha-tocopherol, squalene, simple triterpenes, and volatile compounds) in EVOO with health benefits (antihypertensive activity, chemo preventive activity, tumor-inhibitory activity, and anti-inflammatory activity) and positive sensory attributes(Li and Wang 2018 ). The presence of α-tocopherol, tocotrienols, β-carotene, lutein, squalene, triterpenes, sterols, and pigments as minor components in olive oil is crucial for human health, while also contributing significantly to its distinct taste and aroma(Cheng et al. 2016 ). Some studies have proven that oxygen, light and temperature are variables responsible for increasing deteriorative processes(Escudero et al. 2016 ; Sanmartin et al. 2018 ; Escuderos et al. 2009 ; Martín-Tornero et al. 2022 ; Lobo-Prieto et al. 2020 ) in EVOO as a consequence of oxidative and hydrolytic reactions(Mousavi et al. 2021 ). EVOO quality and shelf-life is reduced by oxidation(Lopes and Courrol 2023), which represents one of the greatest EVOO quality degradation factors during storage and which can be counteracted by the antioxidant activity of polyphenolic compounds and tocopherol(Martín-Tornero et al. 2022 ). The primary change that occurs to extra virgin olive oil (EVOO) after it has been extracted, filtered, and packaged. It is then stored in appropriate containers and kept out of direct sunlight and heat. This process is also referred to as rancidity (Othón-Díaz et al. 2023 ). During the autoxidation reaction, a number of new compounds are formed, while minor components are degraded, causing off-flavors, loss of nutritional value and finally consumer rejection(Sikorska, Khmelinskii, and Sikorski 2012). Tocopherols are antioxidant compounds(Singh et al. 2016 ) essential for human nutrition. Tocopherols are the best lipid-soluble antioxidants for shielding cell membranes from peroxyl radicals and mutagenic nitrogen oxide species(Sikorska et al. 2008 ). Phenolic compounds are important for olive oil stability and flavor(Tekaya et al. 2022 ). In addition, phenolic compounds of EVOOs are often used to assess their authenticity and potential health effects(Crizel et al. 2020 ). For consumers, one of the most important characteristics in EVOO is freshness, as freshness is typically associated with high quality and ensures food safety. The term “shelf life” is commonly referred to when determining the freshness and consumer acceptability of EVOO(Li and Wang 2018 ). It is essential to avoid the contact with inadequate materials such as metal containers and plastic bottles which can initiate oxidative degradative reactions, thus affecting the shelf-life of the oil(Sanmartin et al. 2018 ). Oxidation stability is considered one of the most important quality indicators of edible vegetable oils(Maszewska et al. 2018 ). It determines their usefulness in technological processes as well as shelf life. Increasing effort has been devoted to the development of methods capable of detecting and quantifying the oil oxidation. For this purpose several analytical techniques, including gas chromatography, high-performance size-exclusion chromatography (HPSEC) and UV-vis. spectrophotometry have been used(Tarapoulouzi et al. 2022 ). While these conventional methods are known for their sensitivity and precision, they entail the use of chemicals, involve extensive labor, consume significant time, incur high costs, and require meticulous sample preparation. The oxidation, thermal stability(Malvis et al. 2019 ), chemical composition, and quality of edible oils under various conditions have been previously investigated by applying different techniques such as infrared and Raman spectroscopy, 1D and 2D NMR, differential scanning calorimetry and front face fluorescence spectroscopy (FFFS)(Patrick and Karoui 2022). Among them, Fluorescence spectroscopy is a rapid, nondestructive analytical technique with high specificity and sensitivity. The importance of this technique for food analysis has increased in recent years (Sikorska et al. 2008 ). Fluorescence spectroscopy has been advance as a preference technique to scrutinize and monitor olives and olive oil(Lobo-Prieto et al. 2020 ). It has the dominance over the other conventional methods due to its speed of analysis and no sample preparation with the desertion of solvents and reagents. Moreover, fluorescence spectroscopy has been proposed to monitor olive oil during storage in different conditions(Martín-Tornero et al. 2022 ). Fluorescence spectroscopy was studied for the characterizing of various types of oil, determination of chlorophylls in olive oil(Sikorska, Khmelinskii, and Sikorski 2012), monitoring changes in frying oil, discrimination between different kinds of edible oils and between differently processed olive oils(Sikorska et al. 2008 ). The aim of this study is to monitor the oxidation stability and the qualitative deterioration of antioxidants in EVOO, during storage using fluorescence spectroscopy. Materials and methods Five EVOO samples (A, B, C, D, and E) from the AL-Jouf region of Saudi Arabia and four local EVOO samples (F from Chaman Baluchistan, G from Morgha Biodiversity park, Rawalpindi, H from Barani Agriculture Chakwal, and J from Loralai Baluchistan) were obtained for this study from various parts of Pakistan. Samples were kept between 25 and 30˚ C at room temperature for three months. Following the first, second, and third month, the impact on the shelf life of EVOO and the degradation of vitamin E, beta carotene, and chlorophyll were assessed. After each point the samples were again placed at room temperature for next time point. Five spectra of each sample were recorded. Fluorescence spectroscopy is used to determine the variations in relative intensity of antioxidant and oxidized products. Through this analysis, we tried to analyse oxidation products of fatty acids and vitamin E, beta carotene and chlorophyll that usually happen during storage period. Acquiring fluorescence spectra A right-angle configuration fluorescence spectrometer (FluoroMax-4, Horiba scientific, Jobin Yvon, USA) was used to measure the fluorescence spectra. The excitation source is a continuous 150 W ozone-free xenon arc lamp, and the detector is a photomultiplier (R928P). To capture closely spaced emission spectra events, the excitation and emission monochromators slit sizes were fixed at 3 and 2 nm, respectively. Five spectra of each sample were obtained in order to validate the data for comparison between various measurements. The emission spectra ranged from 365 to 690 nm, where all EVOO samples exhibited the strongest spectral fingerprints, was used to record the spectra. Pre-processing of fluorescence spectra A set of independently created MATLAB (Math Works release 2014a) routines were utilized for the vector normalization and preprocessing of each fluorescence spectrum to enhance the quality of data and remove unwanted noise or artifacts. Principal Component Analysis (PCA)-based statistics were also utilized to categorize various EVOO samples according to variances in spectral properties. PCA is a very effective unsupervised technique for categorizing and interpreting spectral data. To demonstrate the classification based on spectral variations, scatter plots between the first two main components PC1 and PC2 were created. Results and Discussion The fluorescence spectra of EVOO A from Saudi Arabia is shown in Fig. 1 (a), which were obtained to monitor changes in the composition of EVOO stored at room temperature for three months in shelf for the consequent measurement of the shelf life. Relative intensity variations in emission spectra which were recorded with 350 nm excitation wavelength and 365–690 nm emission wavelength. Four prominent emission bands appeared at 442, 466, 518 and 676 nm. The emission bands at 442 and 466 nm were assigned to vitamin E contents particularly the α-tocopherol (Kyriakidis and Skarkalis 2000). The emission band in the range of 440–470 nm can also be associated to the conjugated diene and triene of oil and the oxidation of oil with the passage of time in shelf life reflected by the elevated values(H. Ali et al. 2018 ; Tarapoulouzi et al. 2022 ). EVOO contains carotenoids like beta carotene and lutein (Lazzerini, Cifelli, and Domenici 2016; Kyriakidis and Skarkalis 2000) that prevents oxidation of oil by acting as an antioxidant. The emission band at 518 nm has been associated with carotenoids especially the beta carotene (H. Ali et al. 2018 ; Ahmad and Saleem 2018 ). With storage time and different storage conditions (Sikorska et al. 2008 ) found that certain compounds like chlorophyll and polyphenol decreased and some new oxidation compounds aroused that generated fluorescence emission in the intermediate range of 405–690 nm. A relatively high intensity fluorescence emission band of chlorophyll contents which are responsible for the greenish color of the oil usually presents in EVOO samples at 600–700 nm (Saleem et al. 2017 ). Typically, the emission band of pigment related to chlorophyll group, chlorophyll a and b around 660–700 nm are reported (Kyriakidis and Skarkalis 2000; Sikorska et al. 2008 ). It is established fact that the relative concentration of a biomolecule can be attributed to the intensity of emission band, while the band's position corresponds to fluorescence from intrinsic biomolecules (Ahmad and Saleem 2019). In lipids non-conjugated double and triple bonds are converted to conjugated bonds (diene and triene) upon oxidation (Kyriakidis and Skarkalis 2000). As revealed in Fig. 1 (a), 365–500 nm has been assigned to the region of oxidized products and 500–535 nm corresponds to the region of beta carotene. It is a well-established fact that the beta carotene and chlorophyll contents are very sensitive to thermal stress and started to deteriorate even just after the extraction of the EVOO and its packaging(Martín-Tornero et al. 2022 ). Therefore, as storage time increase the relative intensity of bands from 365–500 nm evolved to higher values as compared to control EVOO samples due to production of primary oxidation products and deterioration of the highly sensitive antioxidants present in the samples. The changes in relative intensity of bands with storage time is depicted in different colors, where spectra in green color represents the result after first month, blue color shows the spectra after second month and pink color shows the spectra of EVOO in shelf life after third month as compared to the control. Overall trend of the spectral variations evidenced the fact that the region from 365–500 nm shows relative increase in intensity with the simultaneous decrease in the intensity at 518 and 676 nm as a consequence of oxidation. These observations were in agreement with a previous study regarding monitoring of EVOO samples during storage with total luminescence and synchronous scanning fluorescence spectroscopy with storage time for the three commercial EVOO samples belonging to three different cultivars(Mishra et al. 2018 ) while in this study samples from two different regions have been analyzed for the initial deterioration of the EVOO for assessing shelf life. The region between 440–470 nm is typically the region for fluorescence bands related to oxidation products (Baltazar et al. 2020). These oxidation products during storage frequently produce from the auto-oxidation and photo-oxidation of oil(Martín-Tornero et al. 2022 ). However, during auto-oxidation, the peroxides react with other low molecular weight molecules present in the food matrix to oxidize them(Garcia-Oliveira et al. 2021). The oxidation of oil can get influenced by various factors such as pigments, peroxides, antioxidants, fatty acid composition of the oil, oil refining, external energy (heat or light), and the concentration of oxygen(Yun and Surh 2012; Mishra et al. 2018 ). In olive oils, the oxidation products are formed immediately after hydro peroxides formation and the resulting decomposition products of hydro peroxides cause off-flavor in the oxidized oil (Gargouri, Zribi, and Bouaziz 2015 ). Major reasons for oil oxidation basically change in spectral properties of EVOO is due to fluorophores (tocopherols, phenolic compounds and chlorophylls). Fluorophores in olive oil are components that can participate in oxidation, thus fluorescence spectroscopy can serve as a tool for better understanding of oil oxidation(Cheikhousman et al. 2018 ). Thanks to the carotenoids which are consistently acknowledge as deterrence of photo oxidation for their ability to quench singlet oxygens (Mousavi et al. 2021 ). However, under condition of autoxidation the antioxidant effect of carotenoids in EVOO seems to be very limited or even negative, owing to their oxidation products, and accelerate oxidation by possibly reacting with lipid substrate(Mousavi et al. 2021 ). The fact is that the oxides being evolved with the passage of time at particular region from 440–470 nm depicted intense fluorescence showing formation of oxidized products. The increase and decrease in intensity of the emission bands can be understood as the increase or decrease in the relative concentration of the compounds responsible for fluorescence signal. Deterioration of antioxidant in the form of relative decrease in intensity of certain bands might be the reason and fluorescence emission of new bands due to the production of some new oxidation compounds (Mishra et al. 2018 ). The variation of any particular peak was calculated using AUC method will be discussed later with respect to control observations. A statistical analysis was made by applying a MATLAB built-in routine for PCA. Principal component Analysis is a commonly used technique that uses the correlation structure of the original variables to reduce the dimensionality of the data when it is difficult to inquire any spectral changes with naked eye. This technique operates without supervision, whereby it utilizes an orthogonal conversion to transform a collection of observations with potentially correlated variables into a collection of values representing uncorrelated variables, known as principal component (PCs). These PCs are calculated based on the variance in the data and they are orthogonal to each other, i.e., they do not exhibit any correlation and are unrelated to each other. When these PCs are plotted as scatter plot, distinct set of data can be effectively separated. A score plot between PC1 and PC2 can separate both components if they are entirely orthogonal to each other. The percentage of variance exhibited by PC1 and PC2 describe the variance found within the data, which is used to distinct PCs in PCA (Smith 2012 ). Consequently, the scatter plot in Fig. 1 (b) which enables the classification of fluorescence spectra and it is also very useful technique to see the small variations (negligible variation). Principal components are created in order of the mount of deviation. Each of them donate some information of data. The distribution of the samples in the score plot Fig. 1 (b) shows that the PC1 mainly explain variations caused by the shelf life. Evidently samples are clustered far away from each other because of spectral variations that show the effect on samples from control to shelf life. The rise in the intensity of fluorescence band from 415–470 after each time point has been observed, which accounts for the deterioration of vitamin E or oxidation products of fatty acids in extra virgin olive oil as a possible result of shelf life induced oxidation that are completely separated in PCA scatter plot. The low intensity of fluorescence band from 500–535 nm after each time point has been observed, which account for the deterioration of beta carotene in extra virgin olive oil as a possible result of storage time. Figures 1 (c, d) illustrate that the loading vector based on their classification. Figure 1 c illustrate that sample after second and third months are clustered in positive side and sample after first month and control are clustered on negative side of the PC1 axis which follows from the spectral features associated with the former are loaded positively, while with the later are loaded negatively. It is clear that fluorescence emission band of oxidized products are loaded positively in the loading vector of PC1, which implies that the variation in the intensity of these bands correlates positively with the primary source of variability in the data. It is evident from PC2 that the fluorescence emission band of oxidized and beta carotene are similar to PC1 while in case of chlorophyll PC1 is orthogonal to PC2. Similarly, the spectra of shelf life of Pakistani sample EVOO H after first, second and third month is shown in Fig. 2 (a). In this figure five prominent band appeared at 436, 467, 515 and 674 nm. The bands at 436 and 467 nm are assigned to vitamin E, particularly from α-tocopherol. Band at 515 nm originate from compound of vitamin A due to the presence of beta carotene which is a precursor of vitamin A(Lavelli and Sereikait 2022). The emission bands at 436 and 467 nm have also been correlated with oxidized products of vitamin E/fatty acids along with beta-carotenes at 515 nm. Similar to previous results bands is the region of oxidized products at 365–500 nm evolved due to higher relative intensity value as storage time increase and the band in the region from 500–535 nm (beta carotene) showed decreased relative intensity with the increase in storage time. The increase and decrease in intensity of these bands can be understood as the increase or decrease in the relative concentration of the biomolecules as a result of oxidation products. Figure 2 (b) shows PCA scatter plot of EVOO H which shows good classification of data. Due to spectral variations samples are clustered far away from each other that show the effect on samples from control to shelf life after first, second and third month. Figure 2 (c, d) illustrate that PCA classification based upon its loading vector. In principal component analysis (PCA), loading vectors are used to determine the principal components, which are linear combinations of the observed variables that capture the most significant variance in the data. The loading vectors in PCA represent the correlation between each observed variable and the corresponding principal component. Loading vectors are important in PCA as they help interpret the structure of the data and identify the variables that contribute most to each principal component. By examining the loading vectors, we can understand which variables are driving the variation in the dataset and how they are related to each principal component. According to vectors major changes in oxidized products and chlorophyll region. Loading vectors are verifying the results major occur in oxidized products and chlorophyll. Figure 3 (a) shows the spectra of Saudi EVOO A control and its first month shelf life. Firstly, focused on the spectral region of oxidation products and then on the spectral region of beta carotene. The evolution of the fluorescence of the region 365–500 nm corresponds to oxidation products which has been discussed earlier. To obtain the difference between control and one month shelf life which has been calculated from the spectral region of 400–440 nm using area under the curve method, difference in spectral signatures of oxidation products is shown in Fig. 3 (b). Similarly the difference between control and first month in shelf life has been calculated from the spectral region of 500–535 nm corresponding to beta carotene which shown in Fig. 3 (c). For the sample A the values calculated from the difference plot using area under the curve method for the first month of shelf life is 0.804 for oxidized products and 0.204 for beta carotene. These numerical values have been calculated using origin software for each set of difference plot of oxidized products and beta carotene and chlorophyll content. Similarly, numerical values of oxidized and beta carotene for the second and third month of EVOO A are shown in Table 1 . Similarly, AUC method have been used to calculate numerical values of difference plots of oxidized products, beta carotene and chlorophyll contents for all EVOO samples and shown in Table 1 . The information related to shelf life of all EVOO samples for the period of first month in shelf life is provided in supplementary figures except EVOO A and H. Figure 4 (a) shows the spectra of Pakistani EVOO H control and its first month shelf life. Figure 4 (b) shows the difference between control and first month of spectral region of oxidation products and Fig. 4 (c) shows the difference of spectral region of beta carotene of the same period of time. Results shown in Fig. 4 , prominent relative intensity based spectral variations evolving at oxidation products region from 365–500 nm mainly assigned to vitamin E tocopherols or fatty acids and 500–535 nm for beta carotene. It is evident that these fluorescent compounds, such as beta carotene and tocopherols, undergo changes during time period of one month leading to alterations in their fluorescence properties. The fluorescence emission intensities, of these compounds provide us information about the oxidation state and the degree of oxidation of the oil(Baltazar et al. 2020). During first month of shelf life, fluorescence intensity of beta carotene decrease, because beta carotene content and its structural integrity can be affected and lead to the degradation thus decrease in intensity in this case consequently changing its concentration and potentially altering its fluorescence properties(Rotondi et al. 2021 ). Under first month shelf life, minor increase in fluorescence intensity of oxidized products shown in Figs. 3 & 4 , attributed to the formation of certain fluorescent compounds during the oxidation process. As the oxidation process progresses, the concentration of oxidation products within the EVOO increases. As oxidation progresses during the first month of shelf life, the concentration of oxidized products, such as peroxides, aldehydes, and ketones, also increase(Martín-Torres et al. 2022). These compounds can exhibit fluorescence properties, thereby leading to higher fluorescence intensity. Chlorophyll band of both Saudi and Pakistani EVOOs decrease in relative intensity with storage time. This shows the deterioration of chlorophyll compound in the form of relative decrease in intensity similar to the case of beta carotene discussed earlier. Figure 5 (a) shows the Fluorescence spectra of Saudi EVOO A control and its first month in shelf life. Similarly Fluorescence spectra of Pakistani EVOO H control and its first month in shelf life can be seen in Fig. 5 (c). Figure 5 (b) is the difference plot of chlorophyll band of Saudi EVOO A with control and first month in shelf life. Similarly difference plot of chlorophyll band of Pakistani EVOO H with control and first month in shelf life can be seen in Fig. 5 (d). For the sample A the values calculated from the difference plot using area under the curve method for the first month of shelf life is 0.063 for chlorophyll. Similarly value for the sample H is 0.129. These numerical values have been calculated using origin software for each set of difference plot of chlorophyll. Similarly, numerical values of oxidized products and beta carotene for the second month and third month of EVOO A have been calculated and shown in Table 1 . The higher chlorophyll degradation in the Pakistani extra virgin olive oil (EVOO) sample compared to the Saudi sample may be due to various factors including harvesting time, processing conditions, varietal differences, geographical and environmental Factors. Nonetheless, oxygen availability might be a factor in the oxidation of chlorophylls during the storage of olive oil, which would impact the color of the oil(Díez-Betriu et al. 2023 ). The Area under the Curve (AUC) method is a mathematical approach used to evaluate the quantitative deterioration of EVOO samples over their shelf life. It allows for the analysis and comparison of datasets represented by curves. The calculated numerical values have been plotted against the time period as shelf life. Figure 6 shows the rate of oxidation of fatty acid /vitamin E of different foreign and Pakistani EVOO samples with storage time calculated by area under the curve. A prominent trend of oxidation of all EVOO samples can be witnessed from first to third time period in shelf. It can be seen that EVOO samples from different regions of Pakistan show higher rate of oxidation as compared to EVOO samples from Saudi Arabia. The oxidation trend of Saudi EVOO samples show gradual oxidation trend as compared to Pakistani ones. Among the Saudi EVOO samples EVOO A have high relative intensity from first to third month in shelf life as compared to other samples. Almost same behavior can be seen in EVOO B, D and E. EVOO C exhibits less oxidation rate from first to third month as compared to all other Saudi based samples. This smooth trend of oxidation of all Saudi EVOO samples may be attributed to the harvesting, packaging, transportation, environmental and climatic conditions. Pakistani Sample from Morgha biodiversity park Rawalpindi EVOO G shows stability during first month accelerated oxidative rate during second month and third month as compared to other Pakistani EVOO samples. EVOO J from Loralai Baluchistan shows less oxidation rate during first and second month but it shows higher trend of oxidation after third month. The much stable behavior of EVOO J and F which have been collected from Baluchistan region of Pakistan which is much diverse in terms of its weather and climatic conditions and may be attributed to elevated ambient temperatures during both the production and storage of the olive oil. Furthermore, factors such as variation in weather patterns, soil type, precipitation, elevation, and varietal origin. EVOO F sample from Chaman Baluchistan oxidized in first month and later it shows oxidative stability while EVOO H sample from Barani agriculture Chakwal shows moderate oxidative stability throughout the storage time. It is well known that olive oil quality is primarily determined by its chemical composition, oxidative stability, and sensory qualities. These parameters are affected by cultivar (variety) and climatic condition(Attia Ibrahim and Ramadan Alshathly 2017). The results discussed in Fig. 6 also depends on their botanical variety, the olive-harvesting locale and environmental factors. Figure 7 shows the variations or deterioration of beta carotene calculated by area under the curve. The unusual characteristics of carotenoids performance could be linked to factors such as the specific species, methods of extraction, transportation procedures, packaging techniques applied to the EVOO samples. Presence of carotenoids in olive oil depends on olive fruits, but also on genetic factors (olive cultivar), the stage of fruit ripening, environmental factors (Lazzerini, Cifelli, and Domenici 2016). Oxidation of beta carotene shows a higher trend in Saudi based EVOO samples as compared to Pakistani EVOO samples except EVOO B. Maximum deterioration of beta carotene can be seen in EVOO A as compared to all foreign and Pakistani samples so it shows much higher trend. EVOO E also show maximum deterioration of beta carotene in shelf life. EVOO F shows stability while EVOO G shows increase in oxidation rate during third month. There is prominent change in trend of EVOO C after first month and most stable during second and shows higher trend after third month. Degradation of beta carotene is dependent on temperature variations (Lavelli and Sereikait 2022) probably due to high temperature of Al-Jouf region. Figure 7 also shows that the degradation of beta carotene is high in these samples. The overall higher deterioration trend of Saudi EVOO samples may be attributed to the warmer climatic conditions of Al-Jouf region. Figure 8 shows the rate of oxidation of chlorophyll contents of different foreign and Pakistani EVOO samples with storage time calculated by area under the curve. The method employed successfully calculated the numerical values of deterioration of the chlorophyll contents. Results shows that all samples from Al-Jouf KSA have intermediate and slow deterioration of chlorophyll as compared to Pakistani samples. Much faster deterioration rate observed in EVOO A and much slower in EVOO C samples which are required from Al-Jouf KSA. Similarly, Pakistani samples have shown an abrupt change in trends and deterioration of chlorophyll after second month was higher. It is evident that chlorophyll contents in EVOO F underwent much faster deterioration till second month which can be associated with its storage in in the plastic bottle when purchased from the farmer. Similarly, EVOO J showed slower rate of oxidation in first two months and much faster in third month in shelf life due to increased ambient temperatures during the production as well as by weather patterns, climatic conditions, and type of soil, precipitation, height and provenance. Furthermore, the Pakistani EVOO G sample showed second faster rate of chlorophyll contents deterioration in three months shelf life time. The behavior of Saudi Arabian EVOO samples showed less degradation of chlorophyll thus they proved to be more stable as compared to Pakistani EVOO samples. In present study area under the curve method proven to be very good at making a difference between Pakistani and foreign samples of extra virgin olive oil. Table 1 Area under the curve values of oxidation products, beta carotene and chlorophyll of all EVOO samples obtained from Al-Jouf Olive Farms, Saudi Arabia and various parts of Pakistan. Country Origin details Control EVOO (Fridge) Room Temperature (27–32 o C) Area under curve values 1st Month – Control 2nd Month–Control 3rd Month –Control Oxidized products Beta Carotene chlorophyll Oxidized products Beta Carotene Chlorophyll Oxidized products Beta Carotene Chlorophyll AL-Jouf region, Kingdom of Saudi Arabia Zone 1 A 0.804 0.206 0.063 1.618 0.383 0.261 2.281 0.666 0.397 Zone 2 B 0.516 0.014 0.113 1.138 0.013 0.248 1.681 0.075 0.343 Zone 3 C 0.527 0.238 0.036 0.732 0.268 0.054 1.091 0.483 0.086 Zone 4 D 0.605 0.181 0.017 1.117 0.281 0.158 1.736 0.343 0.234 Zone 5 E 0.604 0.174 0.0015 1.271 0.277 0.136 1.794 0.451 0.225 Pakistan Chaman, Baluchistan F 0.865 0.038 0.525 1.195 0.067 0.808 1.285 0.094 0.842 Morgha Biodiversity Park, Rawalpindi G 0.733 0.007 0.171 1.730 0.049 0.438 3.340 0.195 1.006 Barani Agriculture Research Institute, Chakwal, H 0.762 0.119 0.129 1.290 0.184 0.278 1.536 0.264 0.341 Loralai Baluchistan J 0.273 0.061 0.0192 0.561 0.151 0.031 2.381 0.279 0.621 The results demonstrate that the many climatic, geographic, environmental factors, such as average yearly temperature, average rainfall, and developed irrigation systems, harvesting, storage conditions packing and transport mechanism affect the quality parameters and shelf life of EVOO. As indicated from the present study, significant differences among olive oil samples from Al-Jouf regions of KSA and from different regions of Pakistan were observed. Al-Jouf region have appropriate soil properties. Summers in Al-Jouf are long and extremely hot, while winter is short with average temperatures of 26 degree Celsius which throughout the olive oil production process is crucial to preserve the quality and freshness of the oil and consequently show more stability (Attia Ibrahim and Ramadan Alshathly 2017; Fraihat et al. 2018). (Khan et al. 2021 ) reported that the Loralai and Chaman regions, located in the northern part of Baluchistan, Pakistan, undergo a climate characterized by brief, mild summers and extended, chilly winters, with an average temperature of 21°C. This climatic variation may contribute to the relatively lower stability observed in the extra virgin olive oil (EVOO) produced in these areas when compared to EVOO samples from the Al-Jouf, KSA. The results here showed fast oxidation rate of Pakistani sample EVOO F from Chaman Baluchistan due to the fact that in the market, it was sold in plastic bottles with direct exposure to sunlight which played a role in the production and acceleration of oxidized products.. Samples from Loralai region Baluchistan (province of Pakistan) showed less stability may be due to informal plantation, inappropriate irrigation system. Nonetheless, Pakistan has 5 million recently planted olive trees spread throughout 42,000 acres (17,000 hectares). This information was revealed by Muhammad Ramzan Anser, an agronomist and the deputy project director at CEFORT, on June 7, 2023, during the third annual "Olive Value Chain Conference" of Pakistan (S. Ali et al. 2024 ). Further analysis of additional EVOO samples from the different region of Pakistan is required to uncover the potential factors contributing to its superb quality. Conclusion Area under the curve method has been employed to investigate oxidation of fatty acids/vitamin E, beta carotene and chlorophyll content of different samples of EVOO during storage. Numerical values expressing oxidation rate have been calculated and plotted against shelf life. Results have shown that EVOO samples collected from Al-Jouf, KSA are more stable than EVOO samples collected from different regions of Pakistan. Furthermore, oxidation rate of fatty acids/vitamin E have much faster in EVOO samples G and J. Beta carotene showed more accelerated oxidation rate in EVOO samples A and C, which are from Saudi Arabia. Similarly degradation of chlorophyll content in EVOO samples G, F and J has also been detected. The oxidation rate of all EVOO samples may be associated with climatic, geographic, environmental factors, such as average yearly temperature, average rainfall, and developed irrigation systems, harvesting, packing and transport mechanisms. Furthermore, the quality and the stability of the samples were influenced by variety of factors, including the state of olive ripeness, the method of extraction, the geographical provenance, the fruit's harvesting, storage, and extraction methods, as well as every circumstance that may arise during its commercial life. These results show that fluorescence spectroscopy has the potential to be a quick and affordable method for EVOO quality analysis. Declarations Acknowledgments The Authors would like to acknowledge Ms. Mha Albqmi (KSA) and Muhammad Ramzan Anser (BARI Chakwal) for providing EVOO samples. Ethical Approval Ethical approval was not required as no human or animal was involved in conducting present study. Competing interests There are no competing interests of a financial or personal nature. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection were performed by Areeba Ansar and Dr. Hina Ali. The first draft of the manuscript was written by Rimsha Hafeez and Dr. Naveed Ahmad and all authors commented on previous versions of the manuscript. Major editing and rewriting of the manuscript is done by Dr. Naveed Ahmad. Funding Not Applicable Availability of data and materials The data that support the findings of this study are available from [Dr. Naveed Ahmad]. Consent to participate and Consent to Publish All the authors who contributed in this manuscript has fully authorized the corresponding author (Dr. Naveed Ahmad) for submission and publishing of the same manuscript. References Ahmad, Naveed, and M. Saleem. 2018. “Studying Heating Effects on Desi Ghee Obtained from Buffalo Milk Using Fluorescence Spectroscopy.” PLoS ONE 13 (5): 1–17. https://doi.org/10.1371/journal.pone.0197340. 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Supplementary Files S1.jpeg Supplementary Figures S1: (a) Fluorescence spectra of Saudi EVOO B for three months in shelf life (b) Difference plot of Saudi EVOO B with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S2.jpeg S2: (a) Fluorescence spectra of Saudi EVOO C in three months shelf life (b) Difference plot of Saudi EVOO C with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S3.jpeg S3: (a) Fluorescence spectra of Saudi EVOO D in three months shelf life (b) Difference plot of Saudi EVOO D with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S4.jpeg S4: (a) Fluorescence spectra of Saudi EVOO E in three months shelf life (b) Difference plot of Saudi EVOO E with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S5.jpeg S5: (a) Fluorescence spectra of Pakistani EVOO F in three months shelf life (b) Difference plot of Pakistani EVOO B with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S6.jpeg S6: (a) Fluorescence spectra of Pakistani EVOO G in three months shelf life (b) Difference plot of Pakistani EVOO G with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. S7.jpeg S7: (a) Fluorescence spectra of Pakistani EVOO J in three months shelf life (b) Difference plot of Pakistani EVOO J with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life. 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-4741827","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":334551439,"identity":"aed328f9-6aeb-4a41-881b-5732d6602082","order_by":0,"name":"Rimsha Hafeez","email":"","orcid":"","institution":"Mirpur University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Rimsha","middleName":"","lastName":"Hafeez","suffix":""},{"id":334551441,"identity":"14ff746c-ac77-47db-9b52-82a9e1db5c62","order_by":1,"name":"Naveed 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carotene.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/bf1778035a5b0ef226eadcce.png"},{"id":62023808,"identity":"9f051e84-860b-4e9b-934c-2d7184cf31b6","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2140498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eFluorescence spectra of Saudi EVOO A with first month in shelf life \u003cstrong\u003e(b)\u003c/strong\u003e difference plot of Saudi EVOO A with control and first month for chlorophyll \u003cstrong\u003e(c)\u003c/strong\u003e Fluorescence spectra of Pakistani EVOO H with first month in shelf life \u003cstrong\u003e(d)\u003c/strong\u003e difference plot of Pakistani EVOO H with control and first month for chlorophyll.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/8a259eb57dc0df101b64e07c.png"},{"id":62023809,"identity":"aa93f834-539d-47cb-a3b0-73689a489989","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2309714,"visible":true,"origin":"","legend":"\u003cp\u003eOxidation of vitamin E/fatty acidswith storage time.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/e06bd9b62b940a5f454d4214.png"},{"id":62025135,"identity":"2848342c-4729-43d5-8635-2ac7d86d64e6","added_by":"auto","created_at":"2024-08-08 10:39:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2618495,"visible":true,"origin":"","legend":"\u003cp\u003eOxidation of beta carotene with the storage time\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/7f8060167c2080a934194ccb.png"},{"id":62024410,"identity":"34a49798-faf9-495e-be19-5ec5bd741b33","added_by":"auto","created_at":"2024-08-08 10:31:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1769540,"visible":true,"origin":"","legend":"\u003cp\u003eOxidation of chlorophyll contents with storage time.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/beb74d77833275c942b937e9.png"},{"id":62033784,"identity":"2c247250-c3bd-486d-b3a3-167223a13f84","added_by":"auto","created_at":"2024-08-08 12:50:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18527731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/a15ec81f-69a6-45a9-81f2-88d0ca27831d.pdf"},{"id":62023805,"identity":"c094bd45-2c57-450b-ab86-f6e6e07cadd5","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1422863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS1: \u003c/strong\u003e(a) Fluorescence spectra of Saudi EVOO B for three months in shelf life (b) Difference plot of Saudi EVOO B with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/ab4c6013298e5f1fee557120.jpeg"},{"id":62024405,"identity":"e9d9feb8-bb73-45f7-bced-b464f4b6cc58","added_by":"auto","created_at":"2024-08-08 10:31:17","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1421520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS2:\u003c/strong\u003e (a) Fluorescence spectra of Saudi EVOO C in three months shelf life (b) Difference plot of Saudi EVOO C with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/7295ff44b96397330ed1425f.jpeg"},{"id":62023812,"identity":"f7d1efa4-3355-43cf-81fa-f08791a793af","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1407584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS3:\u003c/strong\u003e (a) Fluorescence spectra of Saudi EVOO D in three months shelf life (b) Difference plot of Saudi EVOO D with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/28bedd1ab32d4405bd85de3b.jpeg"},{"id":62025134,"identity":"ef94e587-3b44-44d6-be85-a4f7ced56921","added_by":"auto","created_at":"2024-08-08 10:39:17","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1431458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS4:\u003c/strong\u003e (a) Fluorescence spectra of Saudi EVOO E in three months shelf life (b) Difference plot of Saudi EVOO E with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/6cf2f9dc0ebd207ed241088e.jpeg"},{"id":62024408,"identity":"6b44f373-fe67-4086-9a10-5e0122f78091","added_by":"auto","created_at":"2024-08-08 10:31:17","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1455401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS5:\u003c/strong\u003e (a) Fluorescence spectra of Pakistani EVOO F in three months shelf life (b) Difference plot of Pakistani EVOO B with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/37d07f936946d70c3e956a12.jpeg"},{"id":62023815,"identity":"450fc5b8-3437-4eb4-9931-0de04387d205","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1443163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS6:\u003c/strong\u003e (a) Fluorescence spectra of Pakistani EVOO G in three months shelf life (b) Difference plot of Pakistani EVOO G with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/082a18ef4206b563a415ec5a.jpeg"},{"id":62023817,"identity":"57e7eea9-5180-4667-8fc0-020c60d5ef8c","added_by":"auto","created_at":"2024-08-08 10:23:17","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1421693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS7:\u003c/strong\u003e (a) Fluorescence spectra of Pakistani EVOO J in three months shelf life (b) Difference plot of Pakistani EVOO J with first month for oxidation products (c) Difference plot of beta carotene with first month in shelf life (d) Difference plot of chlorophyll with first month in shelf life.\u003c/p\u003e","description":"","filename":"S7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4741827/v1/dd5ab6fedec89bc6fd46afc9.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Monitoring the shelf life of extra virgin olive oil using Fluorescence spectroscopy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtra virgin olive oil (EVOO) is a well-known edible oil that is produced from the fruit of the olive tree (Garcia-Oliveira et al. 2021). It is abundant in beneficial nutrients, monounsaturated fatty acids, and antioxidants (Conte et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, it carry modest amounts of vitamin K, vitamin E (tocopherols), vitamin A (β-carotenoids), vitamin F (oleic and linoleic acids) (Lanza and Ninfali 2020; Jimenez-Lopez et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Maszewska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). EVOO contains various pigments such as chlorophyll and carotenoids (Saleem et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The color of olive oil is distinct, ranging from a light gold to a deep green, and is influenced by the quantity of pigments present(Tekaya et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Green olives, rich in chlorophyll, contribute to the production of green oil, while ripe olives, containing carotenoids, result in the production of yellow oil(Lazzerini, Cifelli, and Domenici 2016). Throughout the years, EVOO has been the topic of numerous scientific studies due to its hidden health benefits and its importance as a staple anchor all over the world(Korkmaz \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Astolfi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Millman et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, there has been considerable interests in correlating monounsaturated fatty acids (mainly oleic acid) and minor components (phenolic compounds, carotenoids and alpha-tocopherol, squalene, simple triterpenes, and volatile compounds) in EVOO with health benefits (antihypertensive activity, chemo preventive activity, tumor-inhibitory activity, and anti-inflammatory activity) and positive sensory attributes(Li and Wang \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The presence of α-tocopherol, tocotrienols, β-carotene, lutein, squalene, triterpenes, sterols, and pigments as minor components in olive oil is crucial for human health, while also contributing significantly to its distinct taste and aroma(Cheng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome studies have proven that oxygen, light and temperature are variables responsible for increasing deteriorative processes(Escudero et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sanmartin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Escuderos et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mart\u0026iacute;n-Tornero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lobo-Prieto et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in EVOO as a consequence of oxidative and hydrolytic reactions(Mousavi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). EVOO quality and shelf-life is reduced by oxidation(Lopes and Courrol 2023), which represents one of the greatest EVOO quality degradation factors during storage and which can be counteracted by the antioxidant activity of polyphenolic compounds and tocopherol(Mart\u0026iacute;n-Tornero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The primary change that occurs to extra virgin olive oil (EVOO) after it has been extracted, filtered, and packaged. It is then stored in appropriate containers and kept out of direct sunlight and heat. This process is also referred to as rancidity (Oth\u0026oacute;n-D\u0026iacute;az et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During the autoxidation reaction, a number of new compounds are formed, while minor components are degraded, causing off-flavors, loss of nutritional value and finally consumer rejection(Sikorska, Khmelinskii, and Sikorski 2012). Tocopherols are antioxidant compounds(Singh et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) essential for human nutrition. Tocopherols are the best lipid-soluble antioxidants for shielding cell membranes from peroxyl radicals and mutagenic nitrogen oxide species(Sikorska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Phenolic compounds are important for olive oil stability and flavor(Tekaya et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, phenolic compounds of EVOOs are often used to assess their authenticity and potential health effects(Crizel et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For consumers, one of the most important characteristics in EVOO is freshness, as freshness is typically associated with high quality and ensures food safety. The term \u0026ldquo;shelf life\u0026rdquo; is commonly referred to when determining the freshness and consumer acceptability of EVOO(Li and Wang \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is essential to avoid the contact with inadequate materials such as metal containers and plastic bottles which can initiate oxidative degradative reactions, thus affecting the shelf-life of the oil(Sanmartin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Oxidation stability is considered one of the most important quality indicators of edible vegetable oils(Maszewska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It determines their usefulness in technological processes as well as shelf life. Increasing effort has been devoted to the development of methods capable of detecting and quantifying the oil oxidation. For this purpose several analytical techniques, including gas chromatography, high-performance size-exclusion chromatography (HPSEC) and UV-vis. spectrophotometry have been used(Tarapoulouzi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While these conventional methods are known for their sensitivity and precision, they entail the use of chemicals, involve extensive labor, consume significant time, incur high costs, and require meticulous sample preparation. The oxidation, thermal stability(Malvis et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), chemical composition, and quality of edible oils under various conditions have been previously investigated by applying different techniques such as infrared and Raman spectroscopy, 1D and 2D NMR, differential scanning calorimetry and front face fluorescence spectroscopy (FFFS)(Patrick and Karoui 2022). Among them, Fluorescence spectroscopy is a rapid, nondestructive analytical technique with high specificity and sensitivity. The importance of this technique for food analysis has increased in recent years (Sikorska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFluorescence spectroscopy has been advance as a preference technique to scrutinize and monitor olives and olive oil(Lobo-Prieto et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has the dominance over the other conventional methods due to its speed of analysis and no sample preparation with the desertion of solvents and reagents. Moreover, fluorescence spectroscopy has been proposed to monitor olive oil during storage in different conditions(Mart\u0026iacute;n-Tornero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fluorescence spectroscopy was studied for the characterizing of various types of oil, determination of chlorophylls in olive oil(Sikorska, Khmelinskii, and Sikorski 2012), monitoring changes in frying oil, discrimination between different kinds of edible oils and between differently processed olive oils(Sikorska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The aim of this study is to monitor the oxidation stability and the qualitative deterioration of antioxidants in EVOO, during storage using fluorescence spectroscopy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eFive EVOO samples (A, B, C, D, and E) from the AL-Jouf region of Saudi Arabia and four local EVOO samples (F from Chaman Baluchistan, G from Morgha Biodiversity park, Rawalpindi, H from Barani Agriculture Chakwal, and J from Loralai Baluchistan) were obtained for this study from various parts of Pakistan. Samples were kept between 25 and 30˚ C at room temperature for three months. Following the first, second, and third month, the impact on the shelf life of EVOO and the degradation of vitamin E, beta carotene, and chlorophyll were assessed. After each point the samples were again placed at room temperature for next time point. Five spectra of each sample were recorded. Fluorescence spectroscopy is used to determine the variations in relative intensity of antioxidant and oxidized products. Through this analysis, we tried to analyse oxidation products of fatty acids and vitamin E, beta carotene and chlorophyll that usually happen during storage period.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAcquiring fluorescence spectra\u003c/h2\u003e \u003cp\u003eA right-angle configuration fluorescence spectrometer (FluoroMax-4, Horiba scientific, Jobin Yvon, USA) was used to measure the fluorescence spectra. The excitation source is a continuous 150 W ozone-free xenon arc lamp, and the detector is a photomultiplier (R928P). To capture closely spaced emission spectra events, the excitation and emission monochromators slit sizes were fixed at 3 and 2 nm, respectively. Five spectra of each sample were obtained in order to validate the data for comparison between various measurements. The emission spectra ranged from 365 to 690 nm, where all EVOO samples exhibited the strongest spectral fingerprints, was used to record the spectra.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePre-processing of fluorescence spectra\u003c/h2\u003e \u003cp\u003eA set of independently created MATLAB (Math Works release 2014a) routines were utilized for the vector normalization and preprocessing of each fluorescence spectrum to enhance the quality of data and remove unwanted noise or artifacts. Principal Component Analysis (PCA)-based statistics were also utilized to categorize various EVOO samples according to variances in spectral properties. PCA is a very effective unsupervised technique for categorizing and interpreting spectral data. To demonstrate the classification based on spectral variations, scatter plots between the first two main components PC1 and PC2 were created.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe fluorescence spectra of EVOO A from Saudi Arabia is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), which were obtained to monitor changes in the composition of EVOO stored at room temperature for three months in shelf for the consequent measurement of the shelf life. Relative intensity variations in emission spectra which were recorded with 350 nm excitation wavelength and 365\u0026ndash;690 nm emission wavelength. Four prominent emission bands appeared at 442, 466, 518 and 676 nm. The emission bands at 442 and 466 nm were assigned to vitamin E contents particularly the α-tocopherol (Kyriakidis and Skarkalis 2000). The emission band in the range of 440\u0026ndash;470 nm can also be associated to the conjugated diene and triene of oil and the oxidation of oil with the passage of time in shelf life reflected by the elevated values(H. Ali et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tarapoulouzi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). EVOO contains carotenoids like beta carotene and lutein (Lazzerini, Cifelli, and Domenici 2016; Kyriakidis and Skarkalis 2000) that prevents oxidation of oil by acting as an antioxidant. The emission band at 518 nm has been associated with carotenoids especially the beta carotene (H. Ali et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ahmad and Saleem \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). With storage time and different storage conditions (Sikorska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that certain compounds like chlorophyll and polyphenol decreased and some new oxidation compounds aroused that generated fluorescence emission in the intermediate range of 405\u0026ndash;690 nm.\u003c/p\u003e \u003cp\u003eA relatively high intensity fluorescence emission band of chlorophyll contents which are responsible for the greenish color of the oil usually presents in EVOO samples at 600\u0026ndash;700 nm (Saleem et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Typically, the emission band of pigment related to chlorophyll group, chlorophyll a and b around 660\u0026ndash;700 nm are reported (Kyriakidis and Skarkalis 2000; Sikorska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is established fact that the relative concentration of a biomolecule can be attributed to the intensity of emission band, while the band's position corresponds to fluorescence from intrinsic biomolecules (Ahmad and Saleem 2019). In lipids non-conjugated double and triple bonds are converted to conjugated bonds (diene and triene) upon oxidation (Kyriakidis and Skarkalis 2000). As revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), 365\u0026ndash;500 nm has been assigned to the region of oxidized products and 500\u0026ndash;535 nm corresponds to the region of beta carotene. It is a well-established fact that the beta carotene and chlorophyll contents are very sensitive to thermal stress and started to deteriorate even just after the extraction of the EVOO and its packaging(Mart\u0026iacute;n-Tornero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, as storage time increase the relative intensity of bands from 365\u0026ndash;500 nm evolved to higher values as compared to control EVOO samples due to production of primary oxidation products and deterioration of the highly sensitive antioxidants present in the samples. The changes in relative intensity of bands with storage time is depicted in different colors, where spectra in green color represents the result after first month, blue color shows the spectra after second month and pink color shows the spectra of EVOO in shelf life after third month as compared to the control. Overall trend of the spectral variations evidenced the fact that the region from 365\u0026ndash;500 nm shows relative increase in intensity with the simultaneous decrease in the intensity at 518 and 676 nm as a consequence of oxidation. These observations were in agreement with a previous study regarding monitoring of EVOO samples during storage with total luminescence and synchronous scanning fluorescence spectroscopy with storage time for the three commercial EVOO samples belonging to three different cultivars(Mishra et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) while in this study samples from two different regions have been analyzed for the initial deterioration of the EVOO for assessing shelf life.\u003c/p\u003e \u003cp\u003eThe region between 440\u0026ndash;470 nm is typically the region for fluorescence bands related to oxidation products (Baltazar et al. 2020). These oxidation products during storage frequently produce from the auto-oxidation and photo-oxidation of oil(Mart\u0026iacute;n-Tornero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, during auto-oxidation, the peroxides react with other low molecular weight molecules present in the food matrix to oxidize them(Garcia-Oliveira et al. 2021). The oxidation of oil can get influenced by various factors such as pigments, peroxides, antioxidants, fatty acid composition of the oil, oil refining, external energy (heat or light), and the concentration of oxygen(Yun and Surh 2012; Mishra et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In olive oils, the oxidation products are formed immediately after hydro peroxides formation and the resulting decomposition products of hydro peroxides cause off-flavor in the oxidized oil (Gargouri, Zribi, and Bouaziz \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Major reasons for oil oxidation basically change in spectral properties of EVOO is due to fluorophores (tocopherols, phenolic compounds and chlorophylls). Fluorophores in olive oil are components that can participate in oxidation, thus fluorescence spectroscopy can serve as a tool for better understanding of oil oxidation(Cheikhousman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thanks to the carotenoids which are consistently acknowledge as deterrence of photo oxidation for their ability to quench singlet oxygens (Mousavi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, under condition of autoxidation the antioxidant effect of carotenoids in EVOO seems to be very limited or even negative, owing to their oxidation products, and accelerate oxidation by possibly reacting with lipid substrate(Mousavi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fact is that the oxides being evolved with the passage of time at particular region from 440\u0026ndash;470 nm depicted intense fluorescence showing formation of oxidized products. The increase and decrease in intensity of the emission bands can be understood as the increase or decrease in the relative concentration of the compounds responsible for fluorescence signal. Deterioration of antioxidant in the form of relative decrease in intensity of certain bands might be the reason and fluorescence emission of new bands due to the production of some new oxidation compounds (Mishra et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The variation of any particular peak was calculated using AUC method will be discussed later with respect to control observations.\u003c/p\u003e \u003cp\u003eA statistical analysis was made by applying a MATLAB built-in routine for PCA. Principal component Analysis is a commonly used technique that uses the correlation structure of the original variables to reduce the dimensionality of the data when it is difficult to inquire any spectral changes with naked eye. This technique operates without supervision, whereby it utilizes an orthogonal conversion to transform a collection of observations with potentially correlated variables into a collection of values representing uncorrelated variables, known as principal component (PCs). These PCs are calculated based on the variance in the data and they are orthogonal to each other, i.e., they do not exhibit any correlation and are unrelated to each other. When these PCs are plotted as scatter plot, distinct set of data can be effectively separated. A score plot between PC1 and PC2 can separate both components if they are entirely orthogonal to each other. The percentage of variance exhibited by PC1 and PC2 describe the variance found within the data, which is used to distinct PCs in PCA (Smith \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Consequently, the scatter plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) which enables the classification of fluorescence spectra and it is also very useful technique to see the small variations (negligible variation). Principal components are created in order of the mount of deviation. Each of them donate some information of data. The distribution of the samples in the score plot Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) shows that the PC1 mainly explain variations caused by the shelf life. Evidently samples are clustered far away from each other because of spectral variations that show the effect on samples from control to shelf life. The rise in the intensity of fluorescence band from 415\u0026ndash;470 after each time point has been observed, which accounts for the deterioration of vitamin E or oxidation products of fatty acids in extra virgin olive oil as a possible result of shelf life induced oxidation that are completely separated in PCA scatter plot. The low intensity of fluorescence band from 500\u0026ndash;535 nm after each time point has been observed, which account for the deterioration of beta carotene in extra virgin olive oil as a possible result of storage time.\u003c/p\u003e \u003cp\u003eFigures\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c, d) illustrate that the loading vector based on their classification. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec illustrate that sample after second and third months are clustered in positive side and sample after first month and control are clustered on negative side of the PC1 axis which follows from the spectral features associated with the former are loaded positively, while with the later are loaded negatively. It is clear that fluorescence emission band of oxidized products are loaded positively in the loading vector of PC1, which implies that the variation in the intensity of these bands correlates positively with the primary source of variability in the data. It is evident from PC2 that the fluorescence emission band of oxidized and beta carotene are similar to PC1 while in case of chlorophyll PC1 is orthogonal to PC2.\u003c/p\u003e \u003cp\u003eSimilarly, the spectra of shelf life of Pakistani sample EVOO H after first, second and third month is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a). In this figure five prominent band appeared at 436, 467, 515 and 674 nm. The bands at 436 and 467 nm are assigned to vitamin E, particularly from α-tocopherol. Band at 515 nm originate from compound of vitamin A due to the presence of beta carotene which is a precursor of vitamin A(Lavelli and Sereikait 2022). The emission bands at 436 and 467 nm have also been correlated with oxidized products of vitamin E/fatty acids along with beta-carotenes at 515 nm.\u003c/p\u003e \u003cp\u003eSimilar to previous results bands is the region of oxidized products at 365\u0026ndash;500 nm evolved due to higher relative intensity value as storage time increase and the band in the region from 500\u0026ndash;535 nm (beta carotene) showed decreased relative intensity with the increase in storage time. The increase and decrease in intensity of these bands can be understood as the increase or decrease in the relative concentration of the biomolecules as a result of oxidation products.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b) shows PCA scatter plot of EVOO H which shows good classification of data. Due to spectral variations samples are clustered far away from each other that show the effect on samples from control to shelf life after first, second and third month. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c, d) illustrate that PCA classification based upon its loading vector. In principal component analysis (PCA), loading vectors are used to determine the principal components, which are linear combinations of the observed variables that capture the most significant variance in the data. The loading vectors in PCA represent the correlation between each observed variable and the corresponding principal component. Loading vectors are important in PCA as they help interpret the structure of the data and identify the variables that contribute most to each principal component. By examining the loading vectors, we can understand which variables are driving the variation in the dataset and how they are related to each principal component. According to vectors major changes in oxidized products and chlorophyll region. Loading vectors are verifying the results major occur in oxidized products and chlorophyll.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) shows the spectra of Saudi EVOO A control and its first month shelf life. Firstly, focused on the spectral region of oxidation products and then on the spectral region of beta carotene. The evolution of the fluorescence of the region 365\u0026ndash;500 nm corresponds to oxidation products which has been discussed earlier.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo obtain the difference between control and one month shelf life which has been calculated from the spectral region of 400\u0026ndash;440 nm using area under the curve method, difference in spectral signatures of oxidation products is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b). Similarly the difference between control and first month in shelf life has been calculated from the spectral region of 500\u0026ndash;535 nm corresponding to beta carotene which shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c). For the sample A the values calculated from the difference plot using area under the curve method for the first month of shelf life is 0.804 for oxidized products and 0.204 for beta carotene. These numerical values have been calculated using origin software for each set of difference plot of oxidized products and beta carotene and chlorophyll content. Similarly, numerical values of oxidized and beta carotene for the second and third month of EVOO A are shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Similarly, AUC method have been used to calculate numerical values of difference plots of oxidized products, beta carotene and chlorophyll contents for all EVOO samples and shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The information related to shelf life of all EVOO samples for the period of first month in shelf life is provided in supplementary figures except EVOO A and H.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a) shows the spectra of Pakistani EVOO H control and its first month shelf life. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b) shows the difference between control and first month of spectral region of oxidation products and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c) shows the difference of spectral region of beta carotene of the same period of time. Results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, prominent relative intensity based spectral variations evolving at oxidation products region from 365\u0026ndash;500 nm mainly assigned to vitamin E tocopherols or fatty acids and 500\u0026ndash;535 nm for beta carotene. It is evident that these fluorescent compounds, such as beta carotene and tocopherols, undergo changes during time period of one month leading to alterations in their fluorescence properties. The fluorescence emission intensities, of these compounds provide us information about the oxidation state and the degree of oxidation of the oil(Baltazar et al. 2020). During first month of shelf life, fluorescence intensity of beta carotene decrease, because beta carotene content and its structural integrity can be affected and lead to the degradation thus decrease in intensity in this case consequently changing its concentration and potentially altering its fluorescence properties(Rotondi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under first month shelf life, minor increase in fluorescence intensity of oxidized products shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, attributed to the formation of certain fluorescent compounds during the oxidation process. As the oxidation process progresses, the concentration of oxidation products within the EVOO increases. As oxidation progresses during the first month of shelf life, the concentration of oxidized products, such as peroxides, aldehydes, and ketones, also increase(Mart\u0026iacute;n-Torres et al. 2022). These compounds can exhibit fluorescence properties, thereby leading to higher fluorescence intensity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChlorophyll band of both Saudi and Pakistani EVOOs decrease in relative intensity with storage time. This shows the deterioration of chlorophyll compound in the form of relative decrease in intensity similar to the case of beta carotene discussed earlier. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a) shows the Fluorescence spectra of Saudi EVOO A control and its first month in shelf life. Similarly Fluorescence spectra of Pakistani EVOO H control and its first month in shelf life can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b) is the difference plot of chlorophyll band of Saudi EVOO A with control and first month in shelf life. Similarly difference plot of chlorophyll band of Pakistani EVOO H with control and first month in shelf life can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d). For the sample A the values calculated from the difference plot using area under the curve method for the first month of shelf life is 0.063 for chlorophyll. Similarly value for the sample H is 0.129. These numerical values have been calculated using origin software for each set of difference plot of chlorophyll. Similarly, numerical values of oxidized products and beta carotene for the second month and third month of EVOO A have been calculated and shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The higher chlorophyll degradation in the Pakistani extra virgin olive oil (EVOO) sample compared to the Saudi sample may be due to various factors including harvesting time, processing conditions, varietal differences, geographical and environmental Factors. Nonetheless, oxygen availability might be a factor in the oxidation of chlorophylls during the storage of olive oil, which would impact the color of the oil(D\u0026iacute;ez-Betriu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Area under the Curve (AUC) method is a mathematical approach used to evaluate the quantitative deterioration of EVOO samples over their shelf life. It allows for the analysis and comparison of datasets represented by curves. The calculated numerical values have been plotted against the time period as shelf life.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the rate of oxidation of fatty acid /vitamin E of different foreign and Pakistani EVOO samples with storage time calculated by area under the curve. A prominent trend of oxidation of all EVOO samples can be witnessed from first to third time period in shelf. It can be seen that EVOO samples from different regions of Pakistan show higher rate of oxidation as compared to EVOO samples from Saudi Arabia. The oxidation trend of Saudi EVOO samples show gradual oxidation trend as compared to Pakistani ones. Among the Saudi EVOO samples EVOO A have high relative intensity from first to third month in shelf life as compared to other samples. Almost same behavior can be seen in EVOO B, D and E. EVOO C exhibits less oxidation rate from first to third month as compared to all other Saudi based samples. This smooth trend of oxidation of all Saudi EVOO samples may be attributed to the harvesting, packaging, transportation, environmental and climatic conditions.\u003c/p\u003e \u003cp\u003ePakistani Sample from Morgha biodiversity park Rawalpindi EVOO G shows stability during first month accelerated oxidative rate during second month and third month as compared to other Pakistani EVOO samples. EVOO J from Loralai Baluchistan shows less oxidation rate during first and second month but it shows higher trend of oxidation after third month. The much stable behavior of EVOO J and F which have been collected from Baluchistan region of Pakistan which is much diverse in terms of its weather and climatic conditions and may be attributed to elevated ambient temperatures during both the production and storage of the olive oil. Furthermore, factors such as variation in weather patterns, soil type, precipitation, elevation, and varietal origin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEVOO F sample from Chaman Baluchistan oxidized in first month and later it shows oxidative stability while EVOO H sample from Barani agriculture Chakwal shows moderate oxidative stability throughout the storage time. It is well known that olive oil quality is primarily determined by its chemical composition, oxidative stability, and sensory qualities. These parameters are affected by cultivar (variety) and climatic condition(Attia Ibrahim and Ramadan Alshathly 2017). The results discussed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e also depends on their botanical variety, the olive-harvesting locale and environmental factors.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the variations or deterioration of beta carotene calculated by area under the curve. The unusual characteristics of carotenoids performance could be linked to factors such as the specific species, methods of extraction, transportation procedures, packaging techniques applied to the EVOO samples. Presence of carotenoids in olive oil depends on olive fruits, but also on genetic factors (olive cultivar), the stage of fruit ripening, environmental factors (Lazzerini, Cifelli, and Domenici 2016). Oxidation of beta carotene shows a higher trend in Saudi based EVOO samples as compared to Pakistani EVOO samples except EVOO B. Maximum deterioration of beta carotene can be seen in EVOO A as compared to all foreign and Pakistani samples so it shows much higher trend. EVOO E also show maximum deterioration of beta carotene in shelf life. EVOO F shows stability while EVOO G shows increase in oxidation rate during third month. There is prominent change in trend of EVOO C after first month and most stable during second and shows higher trend after third month. Degradation of beta carotene is dependent on temperature variations (Lavelli and Sereikait 2022) probably due to high temperature of Al-Jouf region. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e also shows that the degradation of beta carotene is high in these samples. The overall higher deterioration trend of Saudi EVOO samples may be attributed to the warmer climatic conditions of Al-Jouf region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the rate of oxidation of chlorophyll contents of different foreign and Pakistani EVOO samples with storage time calculated by area under the curve. The method employed successfully calculated the numerical values of deterioration of the chlorophyll contents. Results shows that all samples from Al-Jouf KSA have intermediate and slow deterioration of chlorophyll as compared to Pakistani samples. Much faster deterioration rate observed in EVOO A and much slower in EVOO C samples which are required from Al-Jouf KSA. Similarly, Pakistani samples have shown an abrupt change in trends and deterioration of chlorophyll after second month was higher. It is evident that chlorophyll contents in EVOO F underwent much faster deterioration till second month which can be associated with its storage in in the plastic bottle when purchased from the farmer. Similarly, EVOO J showed slower rate of oxidation in first two months and much faster in third month in shelf life due to increased ambient temperatures during the production as well as by weather patterns, climatic conditions, and type of soil, precipitation, height and provenance. Furthermore, the Pakistani EVOO G sample showed second faster rate of chlorophyll contents deterioration in three months shelf life time. The behavior of Saudi Arabian EVOO samples showed less degradation of chlorophyll thus they proved to be more stable as compared to Pakistani EVOO samples.\u003c/p\u003e \u003cp\u003eIn present study area under the curve method proven to be very good at making a difference between Pakistani and foreign samples of extra virgin olive oil.\u003c/p\u003e \u003cp\u003e \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\u003eArea under the curve values of oxidation products, beta carotene and chlorophyll of all EVOO samples obtained from Al-Jouf Olive Farms, Saudi Arabia and various parts of Pakistan.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eCountry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eOrigin details\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eControl EVOO\u003c/p\u003e \u003cp\u003e(Fridge)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c12\" namest=\"c4\"\u003e \u003cp\u003eRoom Temperature (27\u0026ndash;32 \u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c12\" namest=\"c4\"\u003e \u003cp\u003eArea under curve values\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1st Month \u0026ndash; Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e2nd Month\u0026ndash;Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003e3rd Month \u0026ndash;Control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOxidized products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBeta\u003c/p\u003e \u003cp\u003eCarotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echlorophyll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOxidized products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBeta Carotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eChlorophyll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eOxidized products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eBeta Carotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eChlorophyll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAL-Jouf region, Kingdom of Saudi Arabia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZone 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.804\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.397\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZone 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.343\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZone 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZone 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZone 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePakistan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChaman,\u003c/p\u003e \u003cp\u003eBaluchistan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.842\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorgha\u003c/p\u003e \u003cp\u003eBiodiversity Park, Rawalpindi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.733\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarani Agriculture\u003c/p\u003e \u003cp\u003eResearch Institute,\u003c/p\u003e \u003cp\u003eChakwal,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLoralai\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eBaluchistan\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.621\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results demonstrate that the many climatic, geographic, environmental factors, such as average yearly temperature, average rainfall, and developed irrigation systems, harvesting, storage conditions packing and transport mechanism affect the quality parameters and shelf life of EVOO. As indicated from the present study, significant differences among olive oil samples from Al-Jouf regions of KSA and from different regions of Pakistan were observed. Al-Jouf region have appropriate soil properties. Summers in Al-Jouf are long and extremely hot, while winter is short with average temperatures of 26 degree Celsius which throughout the olive oil production process is crucial to preserve the quality and freshness of the oil and consequently show more stability (Attia Ibrahim and Ramadan Alshathly 2017; Fraihat et al. 2018). (Khan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that the Loralai and Chaman regions, located in the northern part of Baluchistan, Pakistan, undergo a climate characterized by brief, mild summers and extended, chilly winters, with an average temperature of 21\u0026deg;C. This climatic variation may contribute to the relatively lower stability observed in the extra virgin olive oil (EVOO) produced in these areas when compared to EVOO samples from the Al-Jouf, KSA. The results here showed fast oxidation rate of Pakistani sample EVOO F from Chaman Baluchistan due to the fact that in the market, it was sold in plastic bottles with direct exposure to sunlight which played a role in the production and acceleration of oxidized products.. Samples from Loralai region Baluchistan (province of Pakistan) showed less stability may be due to informal plantation, inappropriate irrigation system. Nonetheless, Pakistan has 5\u0026nbsp;million recently planted olive trees spread throughout 42,000 acres (17,000 hectares). This information was revealed by Muhammad Ramzan Anser, an agronomist and the deputy project director at CEFORT, on June 7, 2023, during the third annual \"Olive Value Chain Conference\" of Pakistan (S. Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Further analysis of additional EVOO samples from the different region of Pakistan is required to uncover the potential factors contributing to its superb quality.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eArea under the curve method has been employed to investigate oxidation of fatty acids/vitamin E, beta carotene and chlorophyll content of different samples of EVOO during storage. Numerical values expressing oxidation rate have been calculated and plotted against shelf life. Results have shown that EVOO samples collected from Al-Jouf, KSA are more stable than EVOO samples collected from different regions of Pakistan. Furthermore, oxidation rate of fatty acids/vitamin E have much faster in EVOO samples G and J. Beta carotene showed more accelerated oxidation rate in EVOO samples A and C, which are from Saudi Arabia. Similarly degradation of chlorophyll content in EVOO samples G, F and J has also been detected. The oxidation rate of all EVOO samples may be associated with climatic, geographic, environmental factors, such as average yearly temperature, average rainfall, and developed irrigation systems, harvesting, packing and transport mechanisms. Furthermore, the quality and the stability of the samples were influenced by variety of factors, including the state of olive ripeness, the method of extraction, the geographical provenance, the fruit's harvesting, storage, and extraction methods, as well as every circumstance that may arise during its commercial life. These results show that fluorescence spectroscopy has the potential to be a quick and affordable method for EVOO quality analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors would like to acknowledge Ms. Mha Albqmi (KSA) and\u0026nbsp;Muhammad Ramzan Anser (BARI Chakwal)\u0026nbsp;for providing EVOO samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was not required as no human or animal was involved in conducting present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;There are no competing interests of a financial or personal nature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection were performed by Areeba Ansar and Dr. Hina Ali. The first draft of the manuscript was written by Rimsha Hafeez and Dr. Naveed Ahmad and all authors commented on previous versions of the manuscript. Major editing and rewriting of the manuscript is done by Dr. Naveed Ahmad.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from [Dr. Naveed Ahmad].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and Consent to Publish\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the authors who contributed in this manuscript has fully authorized the corresponding author (Dr. Naveed Ahmad) for submission and publishing of the same manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad, Naveed, and M. Saleem. 2018. \u0026ldquo;Studying Heating Effects on Desi Ghee Obtained from Buffalo Milk Using Fluorescence Spectroscopy.\u0026rdquo; \u003cem\u003ePLoS ONE\u003c/em\u003e 13 (5): 1\u0026ndash;17. https://doi.org/10.1371/journal.pone.0197340.\u003c/li\u003e\n\u003cli\u003eAhmad, Naveed, and M Saleem. 2019. \u0026ldquo;Raman Spectroscopy Based Characterization of Desi Ghee Obtained from Buffalo and Cow Milk.\u0026rdquo; \u003cem\u003eInternational Dairy Journal\u003c/em\u003e 89: 119\u0026ndash;28. https://doi.org/10.1016/j.idairyj.2018.08.013.\u003c/li\u003e\n\u003cli\u003eAli, Hina, Muhammad Saleem, Muhammad Ramzan Anser, Saranjam Khan, Rahat Ullah, and Muhammad Bilal. 2018. \u0026ldquo;Validation of Fluorescence Spectroscopy to Detect Adulteration of Edible Oil in Extra Virgin Olive Oil (EVOO) by Applying Chemometrics.\u0026rdquo; \u003cem\u003eApplied Spectroscopy\u003c/em\u003e 72 (9): 1371\u0026ndash;79. https://doi.org/10.1177/0003702818768485.\u003c/li\u003e\n\u003cli\u003eAli, Sajjad, Abdul Mueed, Muhammad Jahangir, Shehla Sammi, Shahbaz Ahmad Zakki, Ammad Amin, Khurshid Anwar, et al. 2024. \u0026ldquo;Evolution of Olive Farming, Industry, and Usage in Pakistan: A Comprehensive Review.\u0026rdquo; \u003cem\u003eJournal of Agriculture and Food Research\u003c/em\u003e 16 (September 2023): 101091. https://doi.org/10.1016/j.jafr.2024.101091.\u003c/li\u003e\n\u003cli\u003eAstolfi, Maria Luisa, Federico Marini, Maria Agostina Frezzini, Lorenzo Massimi, Anna Laura Capriotti, Carmela Maria Montone, and Silvia Canepari. 2021. \u0026ldquo;Multielement Characterization and Antioxidant Activity of Italian Extra-Virgin Olive Oils.\u0026rdquo; \u003cem\u003eFrontiers in Chemistry\u003c/em\u003e 9 (November): 1\u0026ndash;12. https://doi.org/10.3389/fchem.2021.769620.\u003c/li\u003e\n\u003cli\u003eAttia Ibrahim, Amal, and Mona Ramadan Alshathly. 2017. \u0026ldquo;Influence of Environmental Factors on Olive Oil Production and Quality in the Northern Region of Kingdom of Saudi Arabia. INFERRING THE EFFICACY AND SAFETY OF GREEN TEA WATER EXTRACTAFTER PROLONGED CONSUMPTION AGAINST EPIRUBICIN-INDUCED HEPATOTOXICITY IN.\u0026rdquo; \u003cem\u003eJournal of American Science\u003c/em\u003e, no. January: 61\u0026ndash;66. https://www.researchgate.net/publication/312040621.\u003c/li\u003e\n\u003cli\u003eBaltazar, Paola, Natalia Hern\u0026aacute;ndez-S\u0026aacute;nchez, Bel\u0026eacute;n Diezma, and Lourdes Lle\u0026oacute;. 2020. \u0026ldquo;Development of Rapid Extra Virgin Olive Oil Quality Assessment Procedures Based on Spectroscopic Techniques.\u0026rdquo; \u003cem\u003eAgronomy\u003c/em\u003e 10 (1): 1\u0026ndash;14. https://doi.org/10.3390/agronomy10010041.\u003c/li\u003e\n\u003cli\u003eCheikhousman, R., M. Zude, D. J-R Bouveresse, D. N Rutledge, and I. Birlouez-Aragon. 2018. \u0026ldquo;Fluorescence Spectroscopy for Monitoring Extra Virgin Olive Oil Deterioration upon Heating.\u0026rdquo; \u003cem\u003eCzech Journal of Food Sciences\u003c/em\u003e 22 (SI-Chem. Reactions in Foods V): S147\u0026ndash;50. https://doi.org/10.17221/10640-cjfs.\u003c/li\u003e\n\u003cli\u003eCheng, Y.-L., C.-Y. Lee, Y.-L. Huang, Carly A. Buckner, R. M. Lafrenie, J. A. D\u0026eacute;nomm\u0026eacute;e, J. M. Caswell, et al. 2016. \u0026ldquo;We Are IntechOpen , the World \u0026rsquo; s Leading Publisher of Open Access Books Built by Scientists , for Scientists TOP 1 %.\u0026rdquo; \u003cem\u003eIntech\u003c/em\u003e 11 (tourism): 1\u0026ndash;23. https://www.intechopen.com/books/advanced-biometric-technologies/liveness-detection-in-biometrics.\u003c/li\u003e\n\u003cli\u003eConte, Lanfranco, Andrea Milani, Sonia Calligaris, Pierangela Rovellini, Paolo Lucci, and Maria Cristina Nicoli. 2020. \u0026ldquo;Temperature Dependence of Oxidation Kinetics Of.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 9: 1\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eCrizel, Rosane Lopes, Jessica Fernanda Hoffmann, Giovana Paula Zandon\u0026aacute;, Paula Mendon\u0026ccedil;a, Shild Lobo, Rogerio Oliveira Jorge, and Fabio Clasen Chaves. 2020. \u0026ldquo;Characterization of Extra Virgin Olive Oil from Southern Brazil.\u0026rdquo; \u003cem\u003eEuropean Journal of Lipid Science and Technology\u003c/em\u003e 1900347: 11\u0026ndash;14. https://doi.org/10.1002/ejlt.201900347.\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;ez-Betriu, Anna, Julen Bustamante, Agust\u0026iacute; Romero, Antonia Ninot, Alba Tres, Stefania Vichi, and Francesc Guardiola. 2023. \u0026ldquo;Effect of the Storage Conditions and Freezing Speed on the Color and Chlorophyll Profile of Premium Extra Virgin Olive Oils.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 12 (1): 1\u0026ndash;11. https://doi.org/10.3390/foods12010222.\u003c/li\u003e\n\u003cli\u003eEscudero, Alfredo, Natividad Ramos, M. Dolores La Rubia, and Rafael Pacheco. 2016. \u0026ldquo;Influence of Extreme Storage Conditions on Extra Virgin Olive Oil Parameters: Traceability Study.\u0026rdquo; \u003cem\u003eJournal of Analytical Methods in Chemistry\u003c/em\u003e 2016: 1\u0026ndash;10. https://doi.org/10.1155/2016/7506807.\u003c/li\u003e\n\u003cli\u003eEscuderos, M. E., A. Sayago, M. T. Morales, and R. 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Prieto, and Jesus Simal-Gandara. 2020. \u0026ldquo;Bioactive Compounds and Quality of Extra Virgin Olive Oil.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 9 (8): 1\u0026ndash;31. https://doi.org/10.3390/foods9081014.\u003c/li\u003e\n\u003cli\u003eKhan, Saif Ullah, Surriya Shahab, Muhammad Ishaque Fani, Abdul Wahid, Mahmood Ul Hassan, and Aslam Khan. 2021. \u0026ldquo;Climate and Weather Condition of Balochistan Province, Pakistan.\u0026rdquo; \u003cem\u003eInternational Journal of Economic and Environmental Geology\u003c/em\u003e 12 (2): 65\u0026ndash;71. https://doi.org/10.46660/ijeeg.vol12.iss2.2021.589.\u003c/li\u003e\n\u003cli\u003eKorkmaz, Aziz. 2023. \u0026ldquo;Characterization and Comparison of Extra Virgin Olive Oils of Turkish Olive Cultivars.\u0026rdquo; \u003cem\u003eMolecules\u003c/em\u003e 28 (3): 1\u0026ndash;16. https://doi.org/10.3390/molecules28031483.\u003c/li\u003e\n\u003cli\u003eKyriakidis, Nikolaos B., and Paulos Skarkalis. 2000. \u0026ldquo;Fluorescence Spectra Measurement of Olive Oil and Other Vegetable Oils.\u0026rdquo; \u003cem\u003eJournal of AOAC International\u003c/em\u003e 83 (6): 1435\u0026ndash;39. https://doi.org/10.1093/jaoac/83.6.1435.\u003c/li\u003e\n\u003cli\u003eLanza, Barbara, and Paolino Ninfali. 2020. \u0026ldquo;Antioxidants in Extra Virgin Olive Oil and Table Olives: Connections between Agriculture and Processing for Health Choices.\u0026rdquo; \u003cem\u003eAntioxidants\u003c/em\u003e 9 (1): 1\u0026ndash;17. https://doi.org/10.3390/antiox9010041.\u003c/li\u003e\n\u003cli\u003eLavelli, Vera, and Jolanta Sereikait. 2022. \u0026ldquo;Foods-11-00437-V3.Pdf,\u0026rdquo; 1\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eLazzerini, Cristina, Mario Cifelli, and Valentina Domenici. 2016. \u0026ldquo;Pigments in Extra‐Virgin Olive Oil: Authenticity and Quality.\u0026rdquo; \u003cem\u003eProducts from Olive Tree\u003c/em\u003e, no. October: 1\u0026ndash;18. https://doi.org/10.5772/64736.\u003c/li\u003e\n\u003cli\u003eLi, Xueqi, and Selina C. Wang. 2018. \u0026ldquo;Shelf Life of Extra Virgin Olive Oil and Its Prediction Models.\u0026rdquo; \u003cem\u003eJournal of Food Quality\u003c/em\u003e 2018: 1\u0026ndash;16. https://doi.org/10.1155/2018/1639260.\u003c/li\u003e\n\u003cli\u003eLobo-Prieto, Ana, Noelia Tena, Ram\u0026oacute;n Aparicio-Ruiz, Diego L. Garc\u0026iacute;a-Gonz\u0026aacute;lez, and Ewa Sikorska. 2020. \u0026ldquo;Monitoring Virgin Olive Oil Shelf-Life by Fluorescence Spectroscopy and Sensory Characteristics: A Multidimensional Study Carried out under Simulated Market Conditions.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 9 (12): 1\u0026ndash;20. https://doi.org/10.3390/foods9121846.\u003c/li\u003e\n\u003cli\u003eLopes, Carla Regina Borges, and Lilia Coronato Courrol. 2023. \u0026ldquo;Evaluation of Steady-State and Time-Resolved Fluorescence Spectroscopy as a Method for Assessing the Impact of Photo-Oxidation on Refined Soybean Oils.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 12 (9): 1\u0026ndash;16. https://doi.org/10.3390/foods12091862.\u003c/li\u003e\n\u003cli\u003eMalvis, Ana, Peter \u0026Scaron;imon, Tibor Dubaj, Alexandra Sl\u0026aacute;dkov\u0026aacute;, Ale\u0026scaron; H\u0026aacute;z, M. Jablonsky, Stanislav Sekret\u0026aacute;r, et al. 2019. \u0026ldquo;Determination of the Thermal Oxidation Stability and the Kinetic Parameters of Commercial Extra Virgin Olive Oils from Different Varieties.\u0026rdquo; \u003cem\u003eJournal of Chemistry\u003c/em\u003e 2019. https://doi.org/10.1155/2019/4567973.\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;n-Tornero, El\u0026iacute;sabet, Antonio Fern\u0026aacute;ndez, Isabel Dur\u0026aacute;n-Mer\u0026aacute;s, and Daniel Mart\u0026iacute;n-Vertedor. 2022. \u0026ldquo;Fluorescence Monitoring Oxidation of Extra Virgin Olive Oil Packed in Different Containers.\u0026rdquo; \u003cem\u003eMolecules\u003c/em\u003e 27 (21). https://doi.org/10.3390/molecules27217254.\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;n-Torres, Sandra, Juan Antonio Tello-Jim\u0026eacute;nez, Rafael L\u0026oacute;pez-Blanco, Antonio Gonz\u0026aacute;lez-Casado, and Luis Cuadros-Rodr\u0026iacute;guez. 2022. \u0026ldquo;Monitoring the Shelf Life of Refined Vegetable Oils under Market Storage Conditions\u0026mdash;A Kinetic Chemofoodmetric Approach.\u0026rdquo; \u003cem\u003eMolecules\u003c/em\u003e 27 (19). https://doi.org/10.3390/molecules27196508.\u003c/li\u003e\n\u003cli\u003eMaszewska, Magdalena, Anna Florowska, Elzbieta Dłuzewska, Małgorzata Wroniak, Katarzyna Marciniak-Lukasiak, and Anna Zbikowska. 2018. \u0026ldquo;Oxidative Stability of Selected Edible Oils.\u0026rdquo; \u003cem\u003eMolecules\u003c/em\u003e 23 (7): 15\u0026ndash;17. https://doi.org/10.3390/molecules23071746.\u003c/li\u003e\n\u003cli\u003eMillman, Jasmine F., Shiki Okamoto, Taiki Teruya, Tsugumi Uema, Shinya Ikematsu, Michio Shimabukuro, and Hiroaki Masuzaki. 2021. \u0026ldquo;Extra-Virgin Olive Oil and the Gut-Brain Axis: Influence on Gut Microbiota, Mucosal Immunity, and Cardiometabolic and Cognitive Health.\u0026rdquo; \u003cem\u003eNutrition Reviews\u003c/em\u003e 79 (12): 1362\u0026ndash;74. https://doi.org/10.1093/nutrit/nuaa148.\u003c/li\u003e\n\u003cli\u003eMishra, Puneet, Lourdes Lle\u0026oacute;, Teresa Cuadrado, Margarita Ruiz-Altisent, and Natalia Hern\u0026aacute;ndez-S\u0026aacute;nchez. 2018. \u0026ldquo;Monitoring Oxidation Changes in Commercial Extra Virgin Olive Oils with Fluorescence Spectroscopy-Based Prototype.\u0026rdquo; \u003cem\u003eEuropean Food Research and Technology\u003c/em\u003e 244 (3): 565\u0026ndash;75. https://doi.org/10.1007/s00217-017-2984-1.\u003c/li\u003e\n\u003cli\u003eMousavi, Soraya, Roberto Mariotti, Vitale Stanzione, Saverio Pandolfi, Valerio Mastio, Luciana Baldoni, and Nicol G M Cultrera. 2021. \u0026ldquo;Evolution of Extra Virgin Olive Oil Quality under Different Storage Conditions,\u0026rdquo; 1\u0026ndash;19.\u003c/li\u003e\n\u003cli\u003eOth\u0026oacute;n-D\u0026iacute;az, Elsa Daniela, Jorge O. Fimbres-Garc\u0026iacute;a, Marcela Flores-Sauceda, Brenda A. Silva-Espinoza, Leticia X. L\u0026oacute;pez-Mart\u0026iacute;nez, Ariadna T. Bernal-Mercado, and Jesus F. Ayala-Zavala. 2023. \u0026ldquo;Antioxidants in Oak (Quercus Sp.): Potential Application to Reduce Oxidative Rancidity in Foods.\u0026rdquo; \u003cem\u003eAntioxidants\u003c/em\u003e 12 (4): 1\u0026ndash;21. https://doi.org/10.3390/antiox12040861.\u003c/li\u003e\n\u003cli\u003ePatrick, Eliot, and Romdhane Karoui. 2022. \u0026ldquo;3D Front Face Fluorescence Spectroscopy as a Tool for Monitoring the Oxidation Level of Edible Vegetable Oil during Storage at 60 ◦ C.\u0026rdquo; \u003cem\u003eLWT\u003c/em\u003e 154 (July 2021): 112659. https://doi.org/10.1016/j.lwt.2021.112659.\u003c/li\u003e\n\u003cli\u003eRotondi, Annalisa, Lucia Morrone, Gianpaolo Bertazza, and Luisa Neri. 2021. \u0026ldquo;Effect of Duration of Olive Storage on Chemical and Sensory Quality of Extra Virgin Olive Oils.\u0026rdquo; \u003cem\u003eFoods\u003c/em\u003e 10 (10): 1\u0026ndash;9. https://doi.org/10.3390/foods10102296.\u003c/li\u003e\n\u003cli\u003eSaleem, M, Naveed Ahmad, H Ali, M Bilal, Saranjam Khan, Rahat Ullah, M Ahmed, and S Mahmood. 2017. \u0026ldquo;Investigating Temperature Effects on Extra Virgin Olive Oil Using Fluorescence Spectroscopy.\u0026rdquo; \u003cem\u003eLaser Physics\u003c/em\u003e 27 (12): 125602. https://doi.org/10.1088/1555-6611/aa8cd7.\u003c/li\u003e\n\u003cli\u003eSanmartin, Chiara, Francesca Venturi, Cristina Sgherri, Anita Nari, Monica Macaluso, Guido Flamini, Mike Frank Quartacci, Isabella Taglieri, Gianpaolo Andrich, and Angela Zinnai. 2018. \u0026ldquo;The Effects of Packaging and Storage Temperature on the Shelf-Life of Extra Virgin Olive Oil.\u0026rdquo; \u003cem\u003eHeliyon\u003c/em\u003e 4 (11): e00888. https://doi.org/10.1016/j.heliyon.2018.e00888.\u003c/li\u003e\n\u003cli\u003eSikorska, Ewa, Igor Khmelinskii, and Marek Sikorski. 2012. \u0026ldquo;Analysis of Olive Oils by Fluorescence Spectroscopy: Methods and Applications.\u0026rdquo; \u003cem\u003eOlive Oil - Constituents, Quality, Health Properties and Bioconversions\u003c/em\u003e, no. February: 63\u0026ndash;87. https://doi.org/10.5772/30676.\u003c/li\u003e\n\u003cli\u003eSikorska, Ewa, Igor V. Khmelinskii, Marek Sikorski, Francesco Caponio, Maria T. Bilancia, Antonella Pasqualone, and Tommaso Gomes. 2008. \u0026ldquo;Fluorescence Spectroscopy in Monitoring of Extra Virgin Olive Oil during Storage.\u0026rdquo; \u003cem\u003eInternational Journal of Food Science and Technology\u003c/em\u003e 43 (1): 52\u0026ndash;61. https://doi.org/10.1111/j.1365-2621.2006.01384.x.\u003c/li\u003e\n\u003cli\u003eSingh, JP, M Kumar, A Sharma, G Pandey, K Chae, and S Lee. 2016. \u0026ldquo;We Are IntechOpen , the World \u0026rsquo; s Leading Publisher of Open Access Books Built by Scientists , for Scientists TOP 1 %.\u0026rdquo; \u003cem\u003eIntech\u003c/em\u003e 11 (tourism): 1\u0026ndash;20. https://www.intechopen.com/books/advanced-biometric-technologies/liveness-detection-in-biometrics.\u003c/li\u003e\n\u003cli\u003eSmith, Geoffrey. 2012. \u0026ldquo;A Raman Spectroscopic Study of Paint and Dairy Samples (M.Phil Thesis),\u0026rdquo; 1\u0026ndash;115.\u003c/li\u003e\n\u003cli\u003eTarapoulouzi, Maria, Sofia Agriopoulou, Anastasios Koidis, Charalampos Proestos, Hesham Ali El Enshasy, and Theodoros Varzakas. 2022. \u0026ldquo;Recent Advances in Analytical Methods for the Detection of Olive Oil Oxidation Status during Storage along with Chemometrics, Authenticity and Fraud Studies.\u0026rdquo; \u003cem\u003eBiomolecules\u003c/em\u003e 12 (9): 1\u0026ndash;21. https://doi.org/10.3390/biom12091180.\u003c/li\u003e\n\u003cli\u003eTekaya, Meriem, Mguidich Belhaj Amel, Beligh Mechri, Mohamed Ayadi, Manel Ben Slamia Aouina, Jalel Mkada, Hajer Belkhiria, et al. 2022. \u0026ldquo;Biochemical Characterization of Olive Oil Samples Obtained from Fruit Mixtures and from Oil Blends of Four Cultivars Grown in Central Tunisia.\u0026rdquo; \u003cem\u003eOCL - Oilseeds and Fats, Crops and Lipids\u003c/em\u003e 29: 1\u0026ndash;13. https://doi.org/10.1051/ocl/2021050.\u003c/li\u003e\n\u003cli\u003eYun, Jung Mi, and Jeonghee Surh. 2012. \u0026ldquo;Fatty Acid Composition as a Predictor for the Oxidation Stability of Korean Vegetable Oils with or without Induced Oxidative Stress.\u0026rdquo; \u003cem\u003ePreventive Nutrition and Food Science\u003c/em\u003e 17 (2): 158\u0026ndash;65. https://doi.org/10.3746/pnf.2012.17.2.158.\u003c/li\u003e\n\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":"EVOO, vitamin E, beta carotene, chlorophyll content, Fluorescence spectroscopy, quality assurance, rate of oxidation, deterioration of antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-4741827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4741827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo maintain the quality of extra virgin olive oil (EVOO), one important factor is to resist its oxidation during processing and storage. It is challenging task to prevent oxidation in EVOOs since various physical and chemical factors must be under control. In the present study extra virgin olive oil was stored under normal storage conditions for three months and monitored via quality parameters. A non-destructive fluorescence spectroscopy was performed to monitor the oxidation changes in EVOO from different olive growing regions of Pakistan and Al-Jouf region of the Kingdom of Saudi Arabia (KSA). Additionally, the impacts of geographic, climatic, and environmental factors on the oxidation of EVOOs were investigated. Two major changes in the fluorescence emission spectra of EVOO samples were mainly observed: a decrease in fluorescence emission intensities in the range between 500 to 600 nm, 650 to 690 nm, as well as a rise in the fluorescence emission intensities in the 365\u0026ndash;500 nm region. These two changes were related to a degradation of beta carotene, chlorophyll content and formation of oxidation products. EVOO samples collected from Al-Jouf region, KSA showed low rate of oxidation which might be due to environmental and geographical effect, proper irrigation system, harvesting, processing and storage conditions. In a fast and non-destructive manner, The fluorescence spectroscopy successfully monitored the oxidation changes and deterioration of antioxidants in a rapid and non-destructive manner in the EVOOs.\u003c/p\u003e","manuscriptTitle":"Monitoring the shelf life of extra virgin olive oil using Fluorescence spectroscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-08 10:23:12","doi":"10.21203/rs.3.rs-4741827/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":"33c93fd3-4d56-471c-8d5a-10dfa647833e","owner":[],"postedDate":"August 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-08T12:41:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-08 10:23:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4741827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4741827","identity":"rs-4741827","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}