Thermal Effects on the Quality Parameters of Extra Virgin Olive Oil Using Fluorescence Spectroscopy

Extra virgin olive oil is one of the superlative due to its health benefits. In this work, the Fluorescence spectra of extra virgin olive oil (EVOO) from different olive growing regions of Pakistan and Al-Jouf region from the Kingdom of Saudi Arabia (KSA) were obtained. The emission bands depicted relative intensity variations in all non-heated and heated EVOO samples. Prominent emission bands at 385, 400, 435 and 470 nm represent oxidized products of fatty acids, bands at 520 and 673 nm has been assigned to beta carotene and chlorophyll isomers respectively. All EVOO samples collected from Al-Jouf region, KSA and from Pakistan (Loralai Baluchistan, Barani Agricultural Research Institute, Chakwal and Morgha Biodiversity Park, Rawalpindi) regions showed thermal stability. Other EVOO samples from Chaman Baluchistan and one sample from wild specie (Baluchistan) bought directly from farmers showed denatured spectra even without heating. Chemical characteristics of all EVOO samples changed significantly at 200 °C. Relatively, EVOO samples from Al-Jouf showed more thermal stability which might be due to geographical distribution, environmental effects, genetic background and processing or storage conditions. These results demonstrated fluorescence spectroscopy as a quick, cost-effective and reliable approach to assess the quality and thermal stability of EVOO. These characteristics of fluorescence spectroscopy may lead to the development of portable device for the onsite monitoring of EVOO.


Introduction
Extra virgin olive oil (EVOO) is an edible oil that is extracted from fruits of the olive tree by mechanical processes (centrifugation, malaxation and crushing) [1].EVOO consumption is increasing day by day due to its significant health benefits including high phenolic and antioxidant contents as compared to other edible oils [2].The fatty acid contour of olive oil is an important factor in determining its quality and is important from a health standpoint [3].EVOO is high in monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA), tocopherol and phytosterols [4].It contains MUFA in the form of oleic acid (55-83%) that make up the majority of olive oil's fatty acid profile.Other fatty acids included palmitoleic acid (0.3-3.5%), saturated fatty acids i.e. palmitic (7.5-20%) and stearic (0.5-5%), n-6 PUFA (linoleic acid,3.5-21%)and n-3 PUFA (alpha-linolenic acid,0-1%) [5].Oleic acid reduces low-density lipoprotein (LDL) and increases high-density lipoprotein (HDL) thus helps in lowering cholesterol levels [4].It also beneficially reduces the chances of cardiovascular diseases [6,7], cancer, inflammatory and joint issues [6][7][8].
Similarly, EVOO is a source of vitamins E (tocopherol), vitamin A (β-carotene) [9], polyphenols, sterols, hydrocarbons, terpinols, terpenoids, monoacylglycerols, diacylglycerols as well as other bioactive small molecules [10].The green and yellow hues of EVOO is due to the presence of chlorophylls and carotenoids [11].The amount of antioxidants in EVOO depends on a variety of elements like geography, climate, variety, ripening stage at harvesting, oil extraction process and storage conditions.[12].
Generally each type of an oil has a particular smoke point, which is determined by the oil's quality.Oil with a lower smoke point is of higher grade whereas a high smoke point oil is ideal for cooking and other applications [13].Unsaturated fatty acids are vulnerable to oxidation due to the presence of at least one double bond, which makes it simple for chemical processes to take place.The nutritional ingredients in vegetable oils undergo a number of chemical processes like oxidation and hydrolysis during household cooking and deep frying [14].The heating effect on EVOO is essential to identify a safe range of temperatures for cooking and frying [2].After thermal processing, compositional changes in olive oil are anticipated along with Millard reactions with dietary components, triacylglycerol (TAG) hydrolysis and polymerization, fatty acid and sterol oxidation [14].Some of these degraded chemicals can damage vitamins, block enzymes and possibly even cause mutations or gastrointestinal irritations, all of which are detrimental to human health [15].EVOO shows greater thermal stability as compared to other vegetable oils due to its high oleic acid content, phenolic compounds and vitamin E [16,17].
Various experimental techniques, including Gas chromatography [18], Mass spectrometry [19], Nuclear magnetic resonance (NMR) [20] has been used for the quantitation and identification of volatile components respectively.These classical methods are tedious, costly and destructive with several limitations and unsuitable for monitoring on regular basis.On the other hand, Raman spectroscopy [21], Fourier transform Raman spectroscopy [22] and Microwave reflectometry [23] have been reported to provide quick and reliable analysis.
The main objective of the present work is to look for differences in the chemical composition and thermal stability of EVOO samples collected from KSA (Al-Jouf) and different regions of Pakistan, based on the geographical distribution using the potential of fluorescence spectroscopy.

Sample Collection and Preparation
The study comprised of five EVOO samples acquired from Al-Jouf, Kingdom of Saudi Arabia (KSA) and five samples from different regions of Pakistan (Table 1).All samples were harvested from October to November 2021 both in KSA and Pakistan and kept at 4 °C till analysis.The thermal stability of EVOO samples were conducted by heating them in a dry oven for a time period of 30 min at different temperatures of 110, 130, 150, 170 and 200 °C.Fluorescence spectroscopy was utilized to determine the intensity variations of betacarotene, chlorophyll and oxidized fatty acids.

Fluorescence Spectral Acquisition
Fluorescence spectra were measured in 1 cc cuvette in a right-angle configuration using a fluorescence spectrometer (FluoroMax-4, Horiba scientific, Jobin Yvon, USA) with an excitation wavelength of 350 nm.A continuous 150 W ozone free xenon arc lamp is used as the excitation source and the photomultiplier (R928P) as a detector.The slit size of the excitation and emission monochromators were fixed at 3 and 2 nm respectively, to record closely spaced emission spectral events.Five spectra of each sample were acquired to validate the results for comparison between different measurements.

Pre-processing of Fluorescence Spectra
For preprocessing of every fluorescence spectrum to eliminate unnecessary noise and for the vector normalization, a collection of self-written MatLab (Math Works release 2014a) routines were used.The statistical method based on Principal Component Analysis (PCA) was also used for the classification of different EVOO samples based on the variations in spectral features.

Results and Discussion
Fluorescence spectra were recorded in the emission range from 365 to 690 nm where all EVOO samples ref lected maximum spectral signatures except one EVOO sample extracted from wild olives growing in the region of Baluchistan with emission from 500 to 800 nm.In all spectra, low-intensity bands at 385 nm, a triplet at 400, 435 and 470 nm, a strong band at 520 nm and another high intensity band at 673 nm were observed.The band position represents fluorescence from intrinsic biomolecules while peak intensity can be assigned to its relative concentration in different samples [35].The low-intensity triplet in the 400-470 nm region represent conjugated hydroperoxides and is linked to fatty acid oxidation as non-conjugated double and triple bonds in lipids are converted to conjugated bonds (diene and triene) and hydrolysis products [36][37][38][39][40].Studies have shown correlation of emission bands (440, 455 and 475 nm) with oxidized products of fatty acids as their intensity increased with the oxidation state of oil but the emission bands at 440, 475 and 525 nm was also reported to have association with tocopherols or vitamin E [37,41,42].The band at 673 nm was recognized as chlorophyll [2,9,37] and is very sensitive to heat and degrades at high temperature [43].EVOO also includes carotenoids (betacarotene and lutein), which give it yellow hue and function as an antioxidant that act as singlet oxygen quenchers and radical scavengers in lipid oxidation [17,40].Fluorescence emission in the range of 520-560 nm are caused by the presence of beta-carotene [44].
The low intensity of triplet (400, 435 and 470 nm) was seen in all EVOO samples, whereas increased intensity of these bands was observed in sample 6 specifically at band position 400 nm that can be related to the oxidation of oil due to the production of substantial amounts of conjugated hydroperoxides.The formation of oxidation products during the processing of oil is linked to the presence of polyunsaturated fatty acids [40,45].EVOO6 showed different behavior as compared to other EVOO samples with increased fluorescence emission from oxidized products as mentioned above might be due to increased ambient temperatures during the production or storage or probably by weather patterns, climatic conditions, type of soil, precipitation, height and provenance.Moreover, EVOO 6 was purchased from farmer and was stored in a clear plastic bottle that can also be the reason for its degradation as compared to other samples that were stored in glass bottles.Strong emission at 520 nm was observed in EVOO1 from KSA but beta carotene with low intensity was seen in EVOO2 and EVOO5 which were acquired from Al-Jouf, KSA and EVOO6 from Chaman (Baluchistan, Pakistan) while rest of the EVOO samples showed intermediate intensity in this region.The greater shift in emission band of beta carotene was seen in EVOO6 and smaller shift in EVOO3 which was acquired from Al-Jouf KSA, EVOO8 from Loralai (Baluchistan) and EVOO9 from Barani Agriculture Research Institute (Chakwal) respectively.Blue shift in beta carotene was observed in EVOO samples with high intensity of emission bands linked with free fatty acids in non-heated samples.The emission band position at 673 nm in sample 2 and 5 from Al-Jouf region showed maximum relative intensity with negligible variation that correlates with high concentration of chlorophyll contents as compared to all other samples.EVOO6 had a lower intensity of chlorophyll reflecting the ripening stage as photosynthetic activity decreases as ripening progresses.Moreover it was reported that chlorophyll act as an antioxidant in the dark but acts as a pro-oxidant during exposure to light.This might be the reason that non-heated EVOO6 had an exceptionally high intensity of oxidation compounds that may attribute to cultivar, harvesting stage, climatic or storage conditions (Fig. 1).
PCA scatter plot of all non-heated EVOO samples (Fig. 2) represented that most of the samples were different from each other while few of them had similar chemical composition owing to their scattering position on the plot.EVOO6 positioned itself on the negative side of the scatter plot away from the rest of the EVOO samples clearly showing spectral variation due to oxidized products and decreased chlorophyll contents (Fig. 3).
Fluorescence spectra of EVOO from wild olive (EVOO10) from the region of Baluchistan showed an emission wavelength ranging from 365 to 830 nm with an excitation wavelength of 350 nm.Emission bands at 525, 550, 650, 685 and 717 nm can be seen in EVOO10..No fluorescence emission was detected in the region from 385 to 500 nm and different spectral signatures of beta carotene band can be seen in EVOO10 samples.
An additional band at 715 nm can be seen in EVOO10 spectrum extracted from wild olives as compared to EVOO samples from cultivated varieties with chlorophyll band at 673 nm only.The spectrum of EVOO10 was characterized by a strong signal in the region between 650-750 nm with an intense peak at 685 nm.Spectral region from 600-750 nm o has been associated with chlorophyll and pheophytins [37,38].

Effects of Temperature on EVOO Samples
In order to investigate the effects of temperature on EVOO samples collected from KSA and Pakistan, samples were heated at temperature range from 110 to 200 °C with the interval of 20 °C for 30 min at each temperature.Heating introduces prominent spectral changes that effects the chemical composition of EVOO samples due to thermal oxidation of fatty acids and other compounds.
The fluorescence emission bands were not very visibly affected at temperature range from 110-150 °C, but these spectral variation appeared with the increase in temperature around 170 and 200 °C (Figs. 4 and 5).It is worth mentioning that the chemical constituents of all EVOO began to transform, degrade or oxidized as the temperature reached 170 °C, a noticeable spectral variation was observed in comparison to non-heated EVOO samples.The region from 400-500 nm reflected the most prominent changes in the emission spectrum of EVOO during the oxidation process.Thermal degradation of the natural components of EVOO resulted in broad-band fluorescence emission with a shift towards shorter wavelengths.Thermal oxidation in oil generates free radicals and lipid peroxidation that degrade the quality of oil and can visibly be seen in spectral signatures of EVOO tested.The oxidation begins with the onset of primary to secondary organic products (aldehydes and ketones) and ends with ultimate tertiary oxidation products near to smoke point that affect the sensory quality of oil [9,41].The intensity of emission bands between 400 and 500 nm corresponds to the oxidation products and increased with increasing temperature.It was reported that concentration of phenolic compounds and vitamin E decreased during heating and as a result maximum fluorescence intensity shifts to longer wavelength with the generation of oxidized products of vitamin E and hydrolysis products as depicted by synchronous fluorescence spectra and quantified by standard methods [46].We have used single excitation wavelength of 350 nm that covered the emission spectra of photo-oxidized products due to oil processing, storage conditions etc. and thermo-oxidation products after heating.Betacarotene (520 nm) was affected by thermal treatment in EVOO1 and 5 at high temperature (200 °C) whereas slightly changed up to 170 °C.Decreasing trend at 520 nm band was observed in EVOO4 and an elevated one in EVOO5 as compared to EVOO1, 2 and 3.This anomalous behavior of beta-carotene may be attributed to the cultivars.Similarly, chlorophyll contents decreased with increasing temperature.
The chlorophyll band showed a slight decrease in all KSA samples except EVOO5 that showed up with lowest intensity and drastic change was observed at 200 °C.
Emission spectra of all Pakistani EVOO samples is depicted in Fig. 6 showing characteristic spectral changes Fig. 1 Fluorescence emission spectra of non-heated EVOO samples from 1 to 9 collected from KSA and Pakistan in response to thermal treatment.Spectral alteration with relative intensity and band shift started near 150 °C but more pronounced at 200 °C.Figure 6a shows emission spectra of EVOO6 (Chaman, Baluchistan) with elevated level of band from 380 -520 nm different from all other EVOO samples.EVOO6 already had denatured spectra with decreased chlorophyll contents as discussed above probably due to packaging in plastic bottle.Furthermore, minute spectral changes were seen in the fluorescence emission spectra between 110 and 130 °C in EVOO7, 8 and 9 with almost similar spectral signatures (Fig. 6b-d)but changes became obvious in EVOO7 at 150 °C.Chlorophyll band intensity in EVOO samples 6, 7, 8 and 9 reduced gradually after thermal treatment and reduction was more severe as compared to KSA samples.All EVOO samples demonstrated distorted spectral band with the formation of a big hump ranging from 400 to 500 nm with maxima around 450 nm.The spectral changes in this region were typically due to oxidized products of fatty acid contents.Emission bands from 380 to 520 nm showed an increased intensity with an increase in temperature above 170 °C.This increase in intensity is due to oxidation of unsaturated fatty acids resulting in the formation of and hydroperoxides and free fatty acids as unstable primary oxidation products evolved above 170 °C and promptly degrade to secondary oxidation products [17].As the temperature rises secondary oxidation products like aldehydes and ketones appears reflects as change in band intensity and shape [2].Pigments like beta-carotene proved to be thermally stable up to 170 °C in all EVOO samples tested except EVOO 4 and 5 from KSA that showed decreased and increased intensity respectively.Degradation of betacarotenes in virgin olive oils was previously reported due to thermal stress with subsequent decrease in their concentration [47].Chlorophyll bands intensity also affected due to pigment degradation resulting in a loss of magnesium in the chlorophyll molecule due to thermal treatment [48].
Therefore, it is suggested that appearance of prominent spectral changes owe to formation of primary and secondary oxidation products and subsequently degrading the natural ingredients of EVOO [17,37].
EVOO10 from wild type displayed an increase in the spectral range between 500 and 600 nm when heated above 110 °C as illustrated in Fig. 7.Even at 200 °C, the comparative spectra of heated and non-heated EVOO10 sample revealed no spectral change in chlorophyll at 650 nm but a slight decrease in pheophytins at 717 nm.The spectra of EVOO10 sample appeared to be thermally unstable and started to degrade as the temperature increased above 110 °C was obvious from its color change with a rancid smell.The only unaffected region was in the range of 680-750 nm, attributed to chlorophyll and pheophytins otherwise the relative band intensity at different band positions was badly affected after thermal treatment.
Sequential separation of EVOO samples collected from KSA and Pakistan is presented in PCA scatter plot (Fig. 8).PCA is an extremely powerful unsupervised method for classification of spectral data and its interpretation.Scatter plots were produced between the first two principal components PC1 and PC2 to illustrate the classification based on spectral variations.PCA scatter plot classified non-heated and heated EVOO samples when plotted between PC1 and PC2 according to spectral alterations that evolved as a result of thermal treatment.Both PCs have variance in data as 80% and 12% respectively.Several overlapping and difficult-to-see spectral fingerprints were categorized using PCA which successfully classified all heated and non-heated EVOO samples.PCA is a non-supervised statistical technique that converts a collection of observations of possibly correlated variables into a collection of values of uncorrelated variables known as principal components.Principal components are orthogonal to each other but have no correlation among them.The PCA scatter plot mainly classified all the EVOO samples in to two major groups with red and blue colors.This shows that variation exists between EVOO samples collected from Pakistan and KSA due to thermal treatments.Degradation of components was more pronounced in most of the Pakistani EVOO as compared to KSA that proved to be more thermally stable.
Pakistani EVOO8 from BARI (Chakwal, Punjab province) and EVOO9 from Loralai (Baluchistan province) comprising of non-heated and heated up to 150 °C showed a close grouping with KSA samples which may be due to Fig. 4 Fluorescence emission spectra of non-heated and heated EVOO1 from Al-Jouf, KSA at different temperatures for 30 min genetic similarity of cultivars or chemical composition of oil conferring thermal stability.Pakistani EVOO sample 6 from (Chaman, Baluchistan) and EVOO7 from Morgha Biodiversity Park (Rawalpindi) degraded more quickly around 150 °C.EVOO2 and 4 from KSA clustered towards positive side of PCA scatter plot due to thermal stability as compared to EVOO 1 and 5. Low thermal stability of EVOO 1 and 5 may be attributed to cultivar or chemical nature of oil even then proved to be more thermally stable as compared to Pakistani EVOO samples.
In PCA scatter plot, EVOO6 showed completely different behavior and it clustered away from all other EVOO samples due to deviated spectral signatures as discussed above.All the EVOO samples from KSA were stable in the temperature range of 130 -170 °C, with low level of deterioration in their natural components whereas only two EVOO samples 8 and 9 from Pakistan showed thermal stability.The stability of samples 8 and 9 might be due to cultivar or the involvement of other factors like cultivation practices, extraction and processing of oil as these two samples were collected from well-established farm at Loralai Baluchistan and Barani Agricultural Research Institute, Chakwal.
EVOO is by definition produced from the initial cold mechanical press of the olive fruit.The International Olive Council has defined the different categories of olive oils [49].However, following pressing, the oil's characteristics will change due to degradation of its certain components which will increase the accumulation of oxidation products during storage.The spectrum variance might be due to the geographical distribution and post-harvest processes or possibly by the addition of antioxidants or artificial colorants by vendors that could possibly had an impact on the deterioration of EVOO.Additionally, it is well known that oxidation process occurs during storage due to high temperature and other factors affecting the oil chemical composition and consequently affect the oil's fluorescence characteristics [50].KSA EVOO sample proved to be more thermos stable even at 170 °C whereas Pakistani EVOO samples lost most of their natural ingredients when heated at high temperature around 170 °C.All KSA and Pakistani EVOO samples turned rancid, changed color and sour odour after thermal treatment at high temperature (200 °C) due to production of aldehydes and ketones as depicted in fluorescence emission spectra and PCA plot.The fluorescence spectra of wild EVOO10 were excluded from PCA due to high spectral variation in comparison to EVOO from cultivated varieties.thermal stability of KSA EVOO samples owe to different environmental and geographical conditions like average annual temperature, average rainfall and developed irrigation system [51].The Al-Jouf region of KSA experiences long summer and short winters with average temperatures of 26° C which casts a good impact on the quality parameters of olive oil produced here and consequently towards more thermal stability.While the Loralai and Chaman areas which lie in the Northern region of Baluchistan (Pakistan) experience short mild summer and cold long winters with average temperature of 21 °C [52].Loralai and Chaman regions have almost same climatic conditions even then sample no 8 from Loralai was thermally stable than EVOO 6 from Chaman due to cultivar and influence of agronomic practices.Moreover, EVOO6 from the Chaman region was sold in a plastic bottle which is not recommended for EVOO storage.It has been studied that photo-oxidation of olive oil occurred that was stored even in clear and green glass bottles under light affecting the pigments like chlorophyll and carotenoids producing secondary oxidized products [53].Tin and dark glass containers preserves the quality of oil thus retains organoleptic and physiochemical properties of EVOO whereas clear glass and plastic containers significantly lowers the antioxidants like carotenoids, chlorophyll and phenolic contents [54] It was observed that Baluchistan region produces the large sized fruit from the same varieties as compared to the olive fruit produced in Potohar regions of Pakistan.There is a need to analyze more samples from Chaman, Baluchistan region to dig out the possible factors for its low quality for the improvement of quality oil.

Conclusion
Fluorescence spectroscopy was utilized to examine the excitation emission properties and temperature effects on the chemical composition of various KSA and Pakistani EVOO samples from different regions.The pigments that give extra virgin olive oil its greenish hue serve as a proxy for the quality index and thermal stability of extra virgin olive oil.Fluorescence spectroscopy has the potential to provide insight into the geographical variations and quality including thermal stability of extra virgin olive oils through these quality parameters that have intrinsic fluorescence emission.All EVOO samples from KSA and two samples from Pakistan were found to be more thermally stable due to the cultivation practices, harvesting, oil processing and storage conditions.The stability of olive oil is influenced by the fatty acid composition and thermal treatment contribute to the formation of oxidation products that can be easily visualized by the spectral signatures.There is a need to improve the agronomical practices and storage conditions of EVOO in Pakistan to preserve the quality of oil that will contribute in extending shelf life and thermal stability.These results demonstrate the promising potential of fluorescence spectroscopy for quality assurance of EVOO as a rapid and cost-effective tool for its implantation in laboratories for quality analysis.

Fig. 2 Fig. 3
Fig. 2 Principal Component Analysis of non-heated EVOO samples from KSA and Pakistan