Application of steady-state and time-resolved fluorescence spectroscopy in identification of cold-pressed vegetal oils

Monitoring the quality of food products is very important, due to the fact that substances potentially harmful to consumers may be formed during production and storage. In addition, vegetal oils are subject to falsification, which is based mainly on mixing and adding other, usually cheaper equivalents or providing false data on the composition and origin. In this way, the path from a very healthy product to a product that has a negative impact on the human body is very short. In this study fluorescence spectra and fluorescence decay kinetics of selected cold-pressed oils were characterized and the origin of the observed fluorescence bands was discussed. The obtained fluorescence characteristics were found to be the unique fingerprints of the individual oils and showed evident differences which arise from specific qualitative and quantitative compositions of the oils. The most apparent differences in the shape of fluorescence bands were observed in the emission spectrum at excitation wavelength 350 nm and in the excitation spectrum recorded at emission wavelength 500 nm. The obtained results show the utility of the applied steady-state and time –resolved fluorescence measurements in the identification of the tested vegetal oils.


Introduction
Vegetal oils are defined as liquid fat obtained from various parts of plants.In chemical terms, vegetal oils are a mixture of unsaturated and saturated fatty acid esters, as well as other accompanying compounds, which include bioactive ingredients, i.e. dyes, vitamins, phenolic compounds and other lipids (Bałasińska et al. 2010).The proportions between the individual components determine the physicochemical and biological properties of a given vegetal oil, and thus affect its quality and directly affect the health of the consumer.In particular, the composition and the content of fatty acids in edible oils may be considered as an important factor of their quality.Qualitative and quantitative of fatty acids in edible oils may be performed with the use of Gas Chromatography and Ultra-High Performance Liquid Chromatography Coupled to Mass Spectrometry (Lechhab et al. 2022;Arena et al. 2022).Nevertheless, the problem of the quality of vegetal oils is related to the low stability of lipids.Biochemical transformations of fatty acids take place at every stage of production and distribution, from sourcing, pre-treatment of the raw material, to processing and storage, therefore it is important to monitor all these stages.The main processes that have a decisive impact on the quality of lipids include: oxidation, hydrolysis, polymerization and interactions with other components.The dynamics of the occurring changes depends on internal and external factors, i.e. temperature, access to oxygen, light, water and the composition of fatty acids.Changes that occur in the product are often undesirable.Not only the sensory properties of the oil deteriorate, but also its nutritional value (Zhao et al. 2018).One of the oil production processes is cold pressing, in which the oils are obtained as a result of mechanical treatment that preserves essential fatty acids, phospholipids, glycolipids, carotenoids, tocopherols and vitamins.However, after this process, there are also substances with undesirable pro-oxidative effects, such as chlorophylls and heavy metals (Skwarek and Dolatowski 2013).
The potential of fluorescent methods in the analysis of vegetal oils has already been highlighted by researchers in the first half of the last century.In 1929-30, thanks to the popularization of the mercury lamp with a Wood filter, ultraviolet radiation was used to detect adulteration of virgin olive oil (Glantz 1930;Sidney and Willoughby 1929).Based on the visual observation of oil fluorescence, it was shown that virgin oils (high content of carotenoids and chlorophyll) exhibit a characteristic yellow fluorescence, while refined oils with a low content of natural dyes -blue.These studies allowed to characterize the fluorescence of vegetal oils obtained by different methods, as well as to detect the adulteration of virgin olive oil with other oils at the level of 5%, giving prospects for further experiments using electromagnetic wave radiation in food analysis.Currently, spectroscopic techniques are very popular due to the high sensitivity, speed and accuracy of the equipment.The research carried out so far concerns many aspects that contribute to the quality of the product, e.g.geographical origin of the raw material, production and storage conditions, counterfeiting of good quality products with cheaper equivalents (Ahmad et al. 2017).
Vegetal fats are a mixture of many compounds with different chemical properties, between which a number of processes and interactions can occur that have a direct impact on fluorescence.Many researchers propose to combine the two methods of measurement.The first measurement concerns the fluorescence of diluted samples (concentration below 1% v/v) performed in the right angle geometry.This method allows to eliminate the unfavourable effects associated with the high absorbance of the sample and to reduce the intermolecular interactions occurring in the concentrated system.The advantage of the second method, based on the analysis of undiluted samples, is the possibility of emission of compounds present in small amounts or with low fluorescence yields.Measurements for undiluted systems are made in reflection geometry.Spectra of undiluted samples are characterized by low fluorescence intensity in the shortwave and high in the long-wave range.The spectra of vegetal oils show many similarities and have unique individual features characteristic of a given type (Sikorska et al. 2012).
Fluorescent compounds found in vegetal oils include: vitamin E, chlorophyll and phenolic compounds, oxidation products (Zandomeneghi et al. 2005;Galeano Díaz et al. 2003;Giungato et al. 2002;Dupuy et al. 2005).According to the literature, the emission of tocopherols can be observed at excitation λ exc = 290 nm and emission λ em = 325 nm.In total fluorescence spectra, vitamin E can be observed through an intense band in the excitation wavelength range of 250-320 nm and the emission wavelength of 300-350 nm (Zandomeneghi et al. 2005;Sikorska et al. 2004).When analysing the excitation spectra of short-wave fluorescence, differences in the positions of the maximum of different types of oil can be noticed, which may be the result of subtle differences in the content of individual forms of tocopherols and tocotrienols (Sikorska 2008).In the synchronous fluorescence spectra, the band with a maximum of about 301 nm is due to the emission of tocopherols (Sikorska et al. 2005;Sikorska et al. 2003).Vitamin E is one of the main fluorescent components found in all vegetal oils.The emission band from chlorophyll dyes is observed at λ exc = 430-450 nm and λ em = 640-660 nm (Zandomeneghi et al. 2005).Dyes occur mainly in unprocessed oils in the form of chlorophyll a and b, their breakdown products -pheophytins a and b and pheophorbides b (Cert et al. 2000).In the synchronous spectra of some oils, a band with a maximum of about 666 nm arising from chlorophyll is visible (Sikorska 2008).Vegetal oils also contain other fluorescent components, i.e. products of thermal oxidation and those whose structure has not yet been identified.The occurrence of many compounds depends on many factors, thanks to which fluorescent methods can be successfully used in the general characteristics of vegetal oil emissions, classification according to quality and origin, detection of adulteration, control of quality changes occurring during processing (Zandomeneghi et al. 2005;Dupuy et al. 2005;Cheikhousman et al. 2005;Poulli et al. 2005;Poulli et al. 2006).
To sum up, vegetal oils can be an excellent source of health-promoting compounds, however, their excellent quality is a prerequisite.In order for them to serve health, they must be properly produced, labeled, stored and used.There is a great need for further research on the impact of consumption of individual vegetal oils on human health and the improvement of methods enabling simple and quick analysis of their quality.Monitoring the quality of food products is very important, due to the fact that substances potentially harmful to consumers may be formed during production and storage.In this way, the path from a very healthy product to a product that has a negative impact on the human body is very short.
The aim of the study was to characterize fluorescence spectra and fluorescence decays of selected cold-pressed oils.On the basis of the obtained results, attempts were made to determine the usefulness of steady state and timeresolved fluorescence methods in the initial identification of the tested oils.

Materials
In this study the following vegetal oils were used : walnut oil, cold pressed, origin: EU (OW), corn oil, cold pressed, origin: EU (K), roasted peanut oil, unrefined, origin: EU (A), Extra Virgin Olive Oil, virgin cold pressed, origin: Spain (O) and cold-pressed sunflower oil, origin: EU (S).Cold-pressed vegetal oils were stored at 4°C in brown glass bottles.The dates of use of the oils did not exceed their use-by dates (expiry dates).

Steady State Fluorescence Measurements
Fluorescence emission and excitation spectra were recorded using Horiba Jobin Yvon Fluoromax 4 spectrofluorimeter.Measurements were made at room temperature in quartz cuvettes, in the "front-face" geometry.

Time-resolved Fluorescence Measurements
Time-resolved fluorescence measurements were made with the use of the FI900cd apparatus from Edinburgh Instruments (Scotland).For fluorescence excitation, pulse diodes were used with the possibility of selecting the pulse repetition frequency.The diodes generating light with wavelengths of 405 and 450 nm were used.The half-width of the generated pulses was 900 ps, and the spectral half-width for these diodes was approx.30 nm.The excitation light passed through the monochromator, thanks to which the spectral width of the excitation pulses was fixed at 10 nm.Similarly, in the detection channel, the fluorescence light passed through a monochromator selecting a predetermined wavelength of the emitted signal with a spectral width of 10 nm.The intensity of the fluorescence excitation light was controlled with a gray filter.Similarly, in the detection channel, the incoming fluorescence intensity to the detection channel was also regulated by a gray filter.In the measurements, the cuvettes were positioned (in the "front-face" geometry) to eliminate the reflected light excitation from the surface of the cuvette and directed to the detection channel.

Steady-state Fluorescence Spectra of Cold-pressed Vegetal Oils
The emission spectra of cold-pressed vegetal oils excited at various excitation wavelengths are presented in Fig. 1.The dependence of the emission spectra on the excitation wavelength indicates the presence of a mixture of fluorophores in the tested oils.To verify the origin of the emission bands, the excitation spectra at the maxima of the emission bands were also recorded and presented in Fig. 2.
In the emission spectra of the studied oils shown in Fig. 1a (normalized) and Fig. 1b (non-normalized), excited at 290 nm, two broad emission bands may be seen in the 300 -400 nm and in the 350 -550 nm wavelength range.The maxima of the emission spectra of sunflower oil, olive oil and peanut oil were located at 325-330 nm, whereas the maxima of walnut and corn oils are slightly red-shifted.The shape of the emission spectra are similar for all the tested oils.The excitation spectra of the tested recorded at the emission wavelength set to 320 nm (Fig. 2a) showed two bands with maxima at 260 and 300 nm, whereas the excitation spectra of the tested oils at the emission wavelength set to 380 nm (Fig. 2b) have intense structure band in the 300-360 nm range and a band at 260 nm.For all the tested oils with the exception of sunflower oil, the longer-wavelength band in the excitation spectra recorded at 380 nm is more intense as compared to the shorter-wavelength band.Most probably, the emission band at about 320 nm and 380 nm arises from the mixture of fluorescent polyphenols and/or tocopherols, which content is characteristic feature for each of the tested oil.The wide emission band in the range of 350-550 nm may be seen in the spectra of all the tested oils with the exception of sunflower oil.The evident vibronic structure (420 nm, 445 nm, 475 nm, 525 nm ) may be seen in the normalized emission spectra of corn oil which is characteristic feature of this oil.A slight vibronic structure may also be seen in the emission spectra of olive oil.
The broad band at 350-550 nm range may be seen in the emission spectra of the tested oils when excited at λ exc =320 nm (Fig. 1c).The shapes of the emission spectra of walnut and peanut oils (excited at 320 nm) are very similar with the maximum located at about 410 nm (A) and 420 nm (OW).The emission band of olive oil is structured with two peaks at about 400 nm and 440 nm.In the emission spectra of sunflower and corn oil (at excitation wavelength 320 nm) the maximum is located at 370-375 nm and a shoulder located at about 515 nm may be seen in the spectra of these oils.
The emission spectra of the tested oils with the exception of peanut oil excited at 350 nm show a broad maximum in the 375-600 nm range with evident vibronic structure, which is distinct feature of each of the studied oil.The emission spectrum of peanut oil has the maximum at 425 nm with no vibronic structure.The emission spectra of the studied oils at excitation wavelength 405 nm show two bands: one with the maximum at 475 nm (for walnut, peanut and sunflower oils) and 525 nm for corn oil and the second long-wavelength structured band in the 650-750 nm wavelength range.The long wavelength band most probably arise from the presence of chlorophylls and the products of their degradation which absorb at around 450 and 650-700 nm.In the excitation spectra at emission wavelength set to 650 nm (Fig. 2d) the long wavelength band at 500-600 nm (with vibronic structure characteristic for a given oil) may be assigned to chlorophyll dyes; whereas the structured bands seen in the 330-475 nm wavelength most probably arise from the mixture of compounds whose origin is not yet clearly explained in the literature.
The excitation spectra of the studied oils recorded at λ em = 500 nm (Fig. 2c) show structured band with vibronic structure (for example at 310 nm, 325 nm, 349 nm and 370 nm for olive oil; at 318 nm, 340 nm, 350 nm, 375 nm and 390 nm for sunflower oil); which shape is characteristic for a given oil.

Time-resolved Fluorescence Spectra of Cold-pressed Vegetal Oils
The fluorescence intensity decays of the tested vegetal oils (with noise), fitting curves and excitation pulse shapes (Instrument Response Function histograms) are presented  (e) Normalized fluorescence intensity in Fig. 3 (a-l).The values of fitting parameters: α i (λ exc, λ em ) and fluorescence lifetimes τ k τ i (λ exc, λ em ) are presented in Figs. 4 and 5, respectively.The values of fluorescence lifetimes and their corresponding amplitudes (%) are gathered in Table 1.The fluorescence decays were registered at 465, 660 and 672 nm for the excitation at 405 nm; whereas at excitation 450 nm the decays were monitored at 510, 660 and 672 nm.The best fits were obtained for tri-exponential decays.Standard deviations of the determined fluorescence lifetimes were about 3-5% (numerical data analysis errors).Surveying the results obtained from numeric analysis (Table 1) it may be assumed that the differences between these parameters for individual oils are significant compared to the numerical accuracy of their estimation.These differences are also visible in the graphical presentation of the obtained results for the amplitudes and fluorescence extinction times in the three-exponential decay model, as shown in Fig. 3 and Fig. 4. It should be emphasized that the intensity of fluorescence signal was very low for the tested oils, which resulted in a relatively long time for a single measurement.For this reason, the time for measurement of a single decay was relatively long, which forced the necessity to measure decays with a very low number of photon counts in decay maxima and to resign from measuring fluorescence decays for a larger number of emission wavelengths.The obtained results showed that using time-resolved fluorescence measurement it is possible to distinguish between the types of tested oils.Both the amplitudes and the fluorescence lifetimes are effective parameters of the fluorescence intensity decay, in other words their values reflect the photophysics of all fluorophores that contribute to the fluorescence signal at a given excitation wavelength and at a given detection (emission) wavelength.The obtained results allow to postulate the thesis that applying time-resolved fluorescence spectroscopy it is possible to distinguish all the analysed oil samples.What would be needed is just standardization which should involve repeating these tests on a large number of samples of each oil, but from different manufacturers.Thanks to such tests, it would be possible

Discussion
The main fluorophores present in cold-pressed vegetal oils involve polyphenols, tocopherols and chlorophyll dyes.
Since individual vegetal oil is characterized by the specific qualitative and quantitative content of these fluorophores, which additionally may interact with each other and with other non-fluorescent constituents, the fluorescence spectra and fluorescence decays characteristics of vegetal oils are expected to be unique fingerprint for an individual oil and show evident differences which allow for their simple identification without prior preparation of the samples.Fluorescence spectra of cold-pressed vegetal oils result from the presence of typical previously identified and described fluorophores as well as from the presence of unique constituents which depend on the source of plant and oil (g-h), sunflower oil (i-j), walnut oil (k-l).Excitation wavelength in the titles; emission wavelength in the captions the type of an oil.Morever specific interactactions between oil constituents may also affect their fluorescence spectra.One of the well-known fluorophores present in cold-pressed oils are tocopherols and polyphenols, which fluorescence bands often overlap.According to Sikorska et al. (2012), the fluorescence at about λ em = 325/330 nm (at λ exc = 290 nm), observed in the emission spectra of vegetal oils may be assigned to α-tocopherol (Zandomeneghi et al. 2005).Based on the above, the observed emission bands in the range of 300-400 nm (Fig. 1 (a and b)) with a maximum of approximately 330/335 nm at excitation wavelength 290 nm arise from fluorescence of tocopherol compounds (Zandomeneghi et al. 2005).The excitation fluorescence spectra of the tested oils spectrum at λ em = 320 nm (Fig. 2a) reveal the band located at about 298/300 nm with different position of the maximum of the band (depending on the type of vegetal oil, a difference of a few nm) which can be assigned to vitamin A. This difference in the position of maximum is explained by E. Sikorska et al. (2004) by the different tocopherol compositions (αT3, βT3, γT3, δT3).In addition to the observed differences in the shape of the spectra and the shifts of their maxima, the tested oils also exhibit differences in the fluorescence intensity of Fig. 4 Values of amplitudes for multiexponential fluorescent decays for the tested oils: olive oil (a-b), peanut oil (c-d), corn oil (e-f), rapeseed oil (g-h), sunflower oil (i-j), walnut oil (k-l).Excitation wavelength in the titles; emission wavelengths are indicated in the x-axes individual components.Relatively strong tocopherol emission is observed in the case of cold-pressed sunflower oil, moderately intense for peanut oil and olive oil, and weak for walnuts and corn oils.These differences may correlate with the variable content and proportion of individual tocopherol fractions in the respective oils.For example, in sunflower oil, the content of α-tocopherol is the highest (approximately 96%), while in corn oil, α-tocopherol accounts for 20% (Zandomeneghi et al. 2005).However, due to the complexity of the tested system, any quantitative forecasts require further research.Dupuy et al. (2005) indicated that the fluorescence band in the wavelength range of 275-350 nm also may arise from the presence of phenolic compounds (caffeic acid, p-coumaric acid).According to various authors, the maximum wavelength of excitation of phenolic compounds is 264 nm, 270 nm, 280 nm, 310 nm (Lenhardt et al. 2015;Karoui et al. 2007).Since the emission spectra of tocopherols and polyphenolic compounds overlap, without application of specific separation techniques, it is not possible to assign clearly the emission maxima at the 270-290 nm range to tocopherols or phenolic compounds.The long-wavelength band located in the 650-750 nm range may be assigned to chlorophyll dyes.This band may be observed in the emission spectra of all tested oils except peanut oil.Similar results were obtained by Zandomeneghi et al. (2005) comparing sunflower oil with oil from peanuts.The author indicates the absence of a fluorescence signal at 666 nm in the emission spectrum of peanut oil.Based on the literature data, the fluorescence bands with a maximum of 652 nm and 675 nm arise from pheophytinin a and b (Zandomeneghi et al. 2005).According to Sikorska (2008), the fluorescence band at 670 nm at excitation wavelength of 405 nm may be assigned to chlorophylls, which is confirmed by the results of studies of Kyriakidis and Skarkalis (2000) and Sayago et al. (2007).The content of chlorophyll in vegetal oils is mainly determined by the type of the oil, and additionally the observed changes in chlorophyll content depends on the storage conditions as well as production and technological processes (Kyriakidis and Skarkalis 2000;Sayago et al. 2007).
It should be emphasized that the fluorescence band arising from chlorophylls (650-700 nm ) may be seen in coldpressed unrefined vegetal oils, since the vast majority of chlorophylls are removed during the refining process.
In the excitation spectrum (λ em = 650 nm) shown in Fig. 2d, several bands in the range 330-630 nm were observed.One of the bands present in the spectra of walnut oil, sunflower oil and olive oil with a maximum of about 415 nm may be due to the presence of pyropheophytins (PPP).These oils were also characterized by a high intensity of chlorophyll bands.PPP is formed after long storage time as the result of heat-induced degradation of chlorophylls.PPP fluorescence was previously observed and described by Sayago et al. (2007) in their study of refined hazelnut oil and olive oil.The bands located at 328 nm, 333 nm, 408 nm and 414 nm (observed in the excitation spectra registered at λ em = 655) were assigned by the authors to the PPP compounds.The presence of PPP may also indicate the adulteration of good quality oil by adding their refined substitutes (Sayago et al. 2007).The emission spectrum of extra virgin olive oil shown in Fig. 1d is very similar to that obtained by Guimet et al. (2004), which may indicate the utility of fluorescence methods to control/assess the quality of oils.The short wavelength and longer wavelength bands observed in the emission spectra of cold-pressed oils are assigned to tocopherols/ polyphenols and chlorophyll dyes, respectively.However, in the emission spectra of walnut, peanut, corn, sunflower and olive oil oils a structured band located in the intermediate range 330-600 nm range is observed (Fig. 1c).The position of the maxima of individual bands is different and specific for a given type of oil (370 nm for sunflower oil, 380 nm for corn oil, 409 nm, 418 nm, 435 nm for peanut oil, walnut oil and olive oil, respectively).The origin of fluorescence band located in this intermediate range 300-600 nm range is still unclear.Some researchers suggest that this band may originate from the presence of fatty acids, the others attribute this band to the oxidation products or other unidentified compounds.
According to the study of Poulli et al. (2005) the fluorescence intensity in the region 329-545 nm may be associated with the presence of oleic acid, which fluorescence band is located at 405 nm.Peanut oil, olive oil and rapeseed oil are oils characterized by a high content of oleic acid (Poulli et al. 2005), therefore it seems reasonable that the intense fluorescence at 409 nm observed in the emission spectra of peanut, walnut and olive oils (Fig. 1c)) is attributed to oleic acid.It is worth noting, however, that, according to the literature, walnut oil contains three times less oleic acid compared to peanut oil and olive oil (Poulli et al. 2005).
The absorption and fluorescence properties of some fatty acids have also been indicated by other researchers.According to Smyk et al. (2009) fluorescence spectra of tetraenes in various oils (rape, grape seed, sunflower, evening primrose, red currant and black currant, soybean, olive oil) are very similar and exhibit a fluorescence band with maximum at 415 nm.In the emission spectra (at λ exc = 320 nm) of all the tested oils, with the exception of sunflower oil.(Fig. 1c), similar bands located at 409-418 nm were observed.However, it is difficult to assume that these band arise from only one tetraene isomer.The authors also emphasize that among fatty acids, only unsaturated acids with a conjugated double bond system exhibit fluorescence.Currently, there is insufficient information in the literature on the presence of pentaenes and tetraenes in vegetal oils (Smyk et al. 2009).Data reported by Mateo et al. (1996) suggest that pentaene with four double bonds in the trans position and one double bond in the cis configuration (c-COPA) is characterized by the presence of bands with maxima at 313 nm, 331 nm and 348 nm observed in the excitation spectrum.In the excitation spectrum recorded at λ em = 500 nm (Fig. 2c), similar bands at about 310 nm, 325 nm, 350 nm for olive oil, 315 nm 340 nm, 350 nm for sunflower oil and a single band at 350 nm for walnut and peanut oil were observed.
In the emission spectra of the tested oils a small band in the 450-550 nm range was noticed (Fig. 1c).According to the literature data, the emission band (at λ exc = 320 nm ) in the 450-550 nm range with a maximum at 475 nm may be attributed to c-COPA.The emission bands located at the 300-600 nm range (λ exc = 320 nm and 350 nm) presented in Fig. 1c and were also observed by Zandomeneghi et al. (2005).The authors indicate the presence of bands with a maximum of 385 nm, 439 nm and 470 nm in the emission spectrum of olive oil.The results obtained in this study for olive oil presented in Fig. 1c are very similar to those presented in the literature.Zandomeneghi et al. (2005) also indicated lower intensity of sunflower oil as compared to that of peanut oil.
According to Sayago et al. (2007), the bands located in the 420-460 nm and 460-490 nm emission range are correlated with the presence of conjugated dienes (K 232 ), trienes (K 270 ) and hydrolysis products, although these dienes and trienes are not fluorescent.Kyriakidis and Skarkalis (2000) indicated correlation of the observed bands at 445 nm (at λ exc = 365 nm) with the level of fatty acid oxidation products (conjugated dienes -K 232 and trienes -K 270 ).Similarly, Guimet et al. (2005), in their study of olive oil, indicated the presence of two bands around λ em = 445 nm and λ em = 475 nm, suggesting that they are correlated with products formed as the result of heating.According to the authors, conjugated hydroperoxides (primary oxidation products) have high ultraviolet (UV) absorbance at 232 nm (K 232 ) and their presence is correlated with the peroxide (PV) value of the oils.Due to their low stability, hydroperoxides decompose rapidly into aldehydes, ketones and low molecular weight acids (secondary oxidation products).These compounds have a high absorbance at 270 nm (K 270 ) (Guimet et al. 2005).
The fluorescence at the wavelength of 515/520 nm (Fig. 1d) observed in the emission spectra of corn oil, sunflower oil and olive oil may arise from vitamin B 2 or FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide).These assumptions are based on the results of studies by Zandomeneghi et al. (2005) who observed a directly proportional increase in fluorescence intensity at 524 nm after adding increasing concentrations of riboflavin to the oil sample.The low intensity of the band attributed to vitamin B 2 in vegetal oil may result from the presence of other substances, such as carotenoids, which are characterized by a high extinction coefficient (Zandomeneghi et al. 2005).The alternate explanation for the presence of the fluorescence around 515/520 nm is the relation with quenching by chlorophyll.Kyriakidis and Skarkalis (2000) emphasize that reduction in the amount of chlorophyll in the tested sample resulted in the increase of the fluorescence intensity at 525 nm.

Conclusions
The results obtained in this study showed the utility of the applied steady-state and time resolved fluorescence measurements in the identification of the tested unrefined, cold-pressed vegetal oils.The most evident differences in the shape of the fluorescence bands were obtained in the emission spectrum at λ exc = 350 nm and in the excitation spectrum at λ em = 500 nm.A similar position of the fluorescence bands indicate that the fluorescence arise from similar group of fluorophores naturally occurring in oils.However, differences between the proportions and the amount of compounds result in a unique fluorescent image of a given type of oil.Also the results of timeresolved fluorescence spectroscopy of the tested oils showed differences in the obtained parameters: lifetimes and their contributions in the fluorescence decays monitored in various wavelength, which allows to distinguish the tested vegetal oils.

Fig. 3
Fig. 3 Fluorescence intensity decays of the tested vegetal oils (with noise), fitting curves for multi-exponent decay model for the tested vegetal oils: olive oil (a-b), peanut oil (c-d), corn oil (e-f), rapeseed