applied sciences

Article Raman Analysis of Free in

Jin Qiu 1,2, Hua-Yi Hou 1, In-Sang Yang 2,* and Xiang-Bai Chen 1,*

1 Hubei Key Laboratory of Optical Information and Pattern Recognition, Wuhan Institute of Technology, Wuhan 430205, China; [email protected] (J.Q.); [email protected] (H.-Y.H.) 2 Department of Physics, Ewha Womans University, Seoul 03760, Korea * Correspondence: [email protected] (I.-S.Y.); [email protected] (X.-B.C.)

 Received: 18 September 2019; Accepted: 18 October 2019; Published: 24 October 2019 

Featured Application: Reliable and rapid analysis of free fatty acid (FFA) in olive oil is possible from the relative intensity ratio of characteristic vibrational modes observed by Raman spectroscopy.

Abstract: Free fatty acid (FFA) is one of the most critical parameters for evaluating the quality of olive oil. In this paper, we present a simple and rapid Raman spectroscopy method for analyzing free fatty acid in olive oil. First, FFA degradation of carotenoids in olive oil is confirmed by analyzing the relative intensity of characteristic vibrational modes and introducing an intensity decrease factor. Second, it is demonstrated that the relative intensity ratio of the two characteristic vibrational modes 1 1 at 1525 cm− and 1655 cm− presents a good and rapid analysis of FFA content in olive oil; the relative intensity ratio decreases linearly with FFA content. In addition, resonance Raman scattering of carotenoid is discussed, showing that a green laser should be utilized to study FFA in olive oil.

Keywords: free fatty acid; olive oil; Raman spectroscopy; relative intensity analysis; resonance Raman

1. Introduction Olive oil is extracted from olive fruit mechanically without any thermal or chemical treatment. It is highly prized for its health benefits, delicious taste and fine aroma [1–5]. Free fatty acid (FFA) content is one of the most critical parameters to evaluate the quality of olive oil, since FFA causes flavor deterioration and shelf life decrease of olive oil [6–8]. According to the International Olive Oil Council, the FFA content in extra virgin olive oil should be lower or equal to 0.8%, while low-quality olive oil with FFA content above 3.3% is considered unsuitable for human consumption and must be refined prior to consumption [9]. Therefore, rapid analysis of FFA content in olive oil deserves research attention. Various methods have been applied for the analyses of FFA content in oil, such as method [10–12]; pH-metric, chromatographic, colorimetric techniques [13,14]; electrical impedance spectroscopy [15]; Fourier transform infrared absorption [16–19]; Fourier transform Raman scattering with infrared excitation at 1064 nm [1,20]; and Raman scattering with visible excitation [6,7]; and so forth. Practically, the titration method of the American Oil Chemists’ Society (AOCS) with NaOH is commonly applied for the determination of FFA content in olive oil. The Raman method has the advantages of short measurement time, nondestructive analyses, high sensitivity, no sample preparation, no consumption of chemicals, and so forth. Also, a visible excitation source can help to resonantly excite vibrational bands of many important constituents of oils [6,21]. In a previous visible Raman study of FFA content in 1 extra virgin olive oil, it was reported that the spectral window of 945–1600 cm− is a useful fingerprint region for prediction of the FFA% [6]. In the study, Raman scattering signal obtained directly from

Appl. Sci. 2019, 9, 4510; doi:10.3390/app9214510 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 4510 2 of 9 experiment (i.e., absolute Raman intensity) were analyzed. For practical applications, the analysis of absolute Raman intensity would be difficult, since comparison of absolute intensity would require same experimental conditions. In this paper, relative Raman intensity analysis — the study of relative intensity ratios of characteristic vibrational modes, is applied to investigate FFA in olive oils. Relative intensity ratios are more reliable parameters than absolute Raman intensities, thus would make practical application of Raman method possible. Our relative intensity analysis shows that in the spectral window 1 1 1 of 945–1600 cm− , the two characteristic vibrational modes at 1525 cm− and 1155 cm− are correlated 1 with FFA content in olive oil. In addition, the relative intensity ratio of vibrational modes at 1525 cm− 1 and 1655 cm− presents a good and rapid analysis of FFA content in extra virgin olive oil; the intensity ratio decreases linearly with FFA content. Portable Raman system operating at 785 nm had been applied for prediction of fatty acid composition in vegetable oils and identification of waste cooking oils [22,23]. The relative intensity analysis is not dependent on experimental conditions, thus applying it in portable Raman system operating with a visible laser would be very helpful for quick on-site quality evaluation of olive oils.

2. Experiment Five extra virgin olive oil samples, one pomace olive oil sample, and their mixtures have been investigated. Sample A: Pago Baldios San Carlos Extra Virgin Olive Oil (originally from Spain) and sample B: Lorenzo No. 1 Extra Virgin Olive Oil (originally from Italy) were purchased from a Shinsegae Department Store, in Seoul, Korea. Sample C is Huilerie Loued Extra Virgin Olive Oil (originally from Tunisia), which was purchased from a duty-free store in Wuhan, China. Sample D: Luhua Extra Virgin Olive Oil (originally from Spain) and sample E: Olivoila Extra Virgin Olive Oil (originally from Italy) were purchased from a Carrefour store in Wuhan, China. Sample F is Costa d’Oro Pomace Olive Oil (originally from Italy), which was purchased from Taobao in China. The FFA contents of Sample A, B, C, D, E, and F were 0.1%, 0.15%, 0.4%, 0.6%, 0.8%, and 3%, respectively; which were declared by the producers of these samples. Also, the mixtures of C and F, C and D, D and E, and C and E were prepared by mixing two samples with a desired volume ratio under stirring, then waiting one day for equilibrium. Raman spectra of the samples were obtained in backscattering configuration with two different micro-Raman systems, XperRam200 system from NANOBASE company and DXR system from ThermoFisher company (Waltham, MA, USA). For the XperRam200 system, the excitation source was a 532 nm green laser. For the DXR system, two excitation sources were used: a 633 nm red laser and a 785 nm near infrared laser. The scattered signal was detected by air-cooled charge-coupled device (CCD) detector. The samples were placed on glass flasks. The laser power on sample was varied up to 5 mW to check the laser power influences on the Raman spectral features of the samples, which indicated that laser heating or damage of the samples was negligible in our experiment. All the spectra were recorded at room temperature about 25 ◦C with 15 s acquisition time and 2-scan average. All the spectra were baseline corrected with Origin software, and wavenumber calibrated 1 using the 521 cm− vibrational mode of a Si substrate. Also, the Si spectrum was measured after experiments to ensure the calibration was not changed during the measurements. For each sample, 5 random spectra were measured and the average spectrum was used for the analyses of relative intensity ratios.

3. Results and Discussion Figure1 presents the Raman scattering spectra of the six olive oil samples of A, B, C, D, E, and F with 532 nm green laser excitation. All these samples show characteristic vibrational modes 1 1 1 1 1 1 1 at 1080 cm− , 1265 cm− , 1300 cm− , 1440 cm− , 1655 cm− , and 1750 cm− . The mode at 1080 cm− 1 can be assigned to (C–C) stretching of the (CH2)n group; the mode at 1265 cm− can be assigned to 1 (=C–H) deformation of cis (R–HC=CH–R); the mode at 1300 cm− can be assigned to (C–H) bending Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 9

RC=OOR [24–27]. These are the common characteristic vibrational modes of almost all edible vegetable oils. The major difference between low-quality pomace olive oil and extra virgin olive oil samples is the appearance of the two vibrational modes at 1155 cm−1 and 1525 cm−1. In the extra virgin olive oil Appl. Sci. 2019, 9, 4510 3 of 9 samples, these two modes can be observed, while in the low-quality pomace olive oil sample, these two modes are not detectable. Also, the intensity of these two modes varies with the FFA content in 1 −1 extratwist virgin of the olive CH2 group;oil samples. the mode The mode at 1440 at cm1155− cmcanbe can assigned be assigned to (C–H) to C– scissoringC stretching of of CH carotenoids,2; the mode 1 −1 1 andat 1655 the cm mode− can at be 1525 assigned cm can to (C be= C) assigned of cis (RHC to C=C=CHR); stretch theing mode of carotenoids at 1750 cm− [26,can 28 be, 29 assigned]. The carotenoidsto (C=O) stretching play an important of RC=OOR role [24 as– 27natural]. These antioxidants are the common in olive characteristic oils. The observation vibrational of these modes two of modesalmost would all edible be very vegetable helpful oils. for the analyses of different quality olive oils.

1440 1655 1300 1525 1080 1155 1265 1750 A

B

C

D Raman Intensity Raman E

F

1000 1200 1400 1600 1800 Wavenumber (cm-1)

Figure 1. Raman spectra of olive oil samples A, B, C, D, E, and F of different origin. Each spectrum is Figurey-shifted 1. Raman for clarity. spectra of olive oil samples A, B, C, D, E, and F of different origin. Each spectrum is y-shifted for clarity. The major difference between low-quality pomace olive oil and extra virgin olive oil samples is 1 1 −1 −1 the appearanceAs can be seen of the in twoFigure vibrational 1, the intensity modes of at the 1155 two cm vibrational− and 1525 modes cm− . at In 1155 the extra cm virginand 1525 olive cm oil decreasessamples, thesesystematically two modes with can increasing be observed, FFA while content in thein olive low-quality oil, which pomace is consistent olive oil with sample, reported these resultstwo modes [6]. This are notintensity detectable. decrease Also, indicates the intensity that FFA of these would two degrade modes variescarotenoids with the in FFAolive content oil, thus in 1 higherextra virgin FFA content olive oil is samples. associated The with mode lower at 1155 intensity cm− can of becarotenoids assigned tovibrational C–C stretching modes. of carotenoids,Due to the 1 FFAand thedegradation mode at 1525 of carotenoids, cm− can be when assigned an extra to C= virginC stretching olive oil of is carotenoids mixed with [26 a, lower28,29].-quality The carotenoids pomace oliveplay anoil, important the intensity role decrease as natural of antioxidantscarotenoids inRaman olive oils.modes The due observation to volume of increase these two would modes be wouldmore significantbe very helpful than for that the of analyses other modes of different in extra quality virgin olive olive oils. oil. Below, we present a simple Raman 1 1 methodAs can to confirm be seen in the Figure FFA1 , degradation the intensity of of the carotenoids two vibrational by analyzing modes at the1155 relative cm − and intensity 1525 cm of− characteristicdecreases systematically vibrational modes with increasing and introducing FFA content an intensity in olive decrease oil, which factor. is consistent This method with would reported be helpfulresults also [6]. to This study intensity possible decrease degradation indicates process that in other FFA wouldmaterials. degrade carotenoids in olive oil, thusFigure higher 2 FFA presents content the is Raman associated spectra with of lower the mixtures intensity of of sample carotenoids C and vibrational F with volume modes. ratios Due of to 2:1,the 3:2, FFA 1:1, degradation 2:3, and 1:2. of For carotenoids, comparison, when the anRaman extra spectra virgin oliveof sample oil is C mixed and F withare also a lower-quality included in thepomace figure, olive and oil, the the intensities intensity of decrease all the spectra of carotenoids are normalized Raman modesusing the due intensity to volume of increasethe vibrational would modebe more at 1655 significant cm−1 as thana reference. that of In other Figure modes 2, the in spectra extra virginwere normalized olive oil. Below, only for we better present observation a simple ofRaman the difference method toof confirm relative theintensity FFA degradation ratios. The ofmode carotenoids at 1655 bycm−1 analyzing mode has the relatively relative intensitygood line of- shapecharacteristic and is not vibrational much affected modes by and the introducingneighboring an modes, intensity thus decrease was chosen factor. for Thisnormalization. method would As can be behelpful seen in also Figure to study 2, the possible intensity degradation decrease of process the two in vibrational other materials. modes at 1155 cm−1 and 1525 cm−1 is muchFigure faster 2th presentsan other themodes Raman of extra spectra virgin of theolive mixtures oil. Also, of the sample intensity C and decrease F with of volume the two ratios modes of 2:1, 3:2, 1:1, 2:3, and 1:2. For comparison, the Raman spectra of sample C and F are also included in the figure, and the intensities of all the spectra are normalized using the intensity of the vibrational 1 mode at 1655 cm− as a reference. In Figure2, the spectra were normalized only for better observation 1 of the difference of relative intensity ratios. The mode at 1655 cm− mode has relatively good line-shape Appl. Sci. 2019, 9, 4510 4 of 9 and is not much affected by the neighboring modes, thus was chosen for normalization. As can be seen Appl. Sci. 2019, 9, x FOR PEER REVIEW 1 1 4 of 9 in Figure2, the intensity decrease of the two vibrational modes at 1155 cm − and 1525 cm− is much faster than other modes of extra virgin olive oil. Also, the intensity decrease of the two modes is faster is faster than the volume increase when sample F is added to sample C. Furthermore, the intensity of than the volume increase when sample F is added to sample C. Furthermore, the intensity of these these two modes has more significant intensity decrease when the volume ratio of sample F increases two modes has more significant intensity decrease when the volume ratio of sample F increases in in the mixture. These observations indicate that when FFA content increases in the mixture, FFA the mixture. These observations indicate that when FFA content increases in the mixture, FFA would would have more significant degradation of carotenoid, whose characteristic vibrational modes are1 have more−1 significant degradation−1 of carotenoid, whose characteristic vibrational modes are 1155 cm− 1155 cm and 11525 cm . and 1525 cm− .

1440

1655

1525 1155 C C:F(2:1)

C:F(3:2)

C:F(1:1) Raman Intensity C:F(2:3)

C:F(1:2) F

1000 1200 1400 1600 1800 Wavenumber (cm-1)

Figure 2. Raman spectra of the mixtures of sample C and F with volume ratios of 2:1, 3:2, 1:1, 2:3, Figureand 1:2. 2. ForRaman comparison, spectra of the the spectra mixtures of of sample sample C andC and F areF with included volume in ra thetios figure. of 2:1, The 3:2,intensities 1:1, 2:3, and of 1 1:2.all theFor spectracomparison, are normalized the spectra using of sample the intensity C and F ofare the included vibrational in the mode figure. at 1655The intensities cm− as a reference.of all the spectraEach spectrum are normalized is y-shifted using for the clarity. intensity of the vibrational mode at 1655 cm−1 as a reference. Each spectrum is y-shifted for clarity. 1 For a clear representation of the intensity decrease of the two vibrational modes at 1155 cm− 1 and 1525For a cmclear− inrepresentation the mixture, anof the intensity intensity decrease decrease factor of thex is two introduced vibrational as follows. modes at When 1155 samples cm−1 and C 1 1525and Fcm are−1 in mixed, the mixture, for example an inte withnsity a decrease volume ratio factor of x 1:1, is introduced we assume as that follows. the intensities When sample of 1155s C cm and− 1 Fand are 1525mixed, cm for− modes example in thewith mixture a volume would ratio be ofx 1:1,/2 ( xweshould assume be smallerthat the thanintensities 1) of their of 1155 intensities cm−1 and in 1525sample cm C,−1 modes and the in intensities the mixture of would all the otherbe x/2 modes (x should in samples be smaller C and than F would 1) of their be half intensities of their in intensities. sample C,Then and in the the intensities mixture (M), of theall relativethe other intensity modes ratioin sample of IM,1440s C /IandM,1525 F wouldand IM,1655 be half/IM,1525 of theircan be intensities. estimated Thenby (IC,1440 in the/x ImixtureC,1525 + I F,1440(M), /thexIF,1525 relative) and intensity (IC,1655/x IratioC,1525 of+ IIF,1655M,1440//IxM,1525IF,1525 and), respectively. IM,1655/IM,1525 Therefore, can be estimated through 1 bythe (I analysisC,1440/xIC,1525 of + relative IF,1440/xIF,1525 intensity) and ratios,(IC,1655/x theIC,1525 intensity + IF,1655/xI decreaseF,1525), respectively. factor x of Therefore, 1525 cm − throughmode thecan analysisbe calculated of relative with intensity the ratio ratios, of {(I C,1440the intensity/IC,1525) decrease(IC,1655 factor/IC,1525 x) of(IF,1440 1525/ IcmF,1655−1 mode)} to {(IcanM,1440 be calculated/IM,1525) − · 1 − with(IM,1655 the/I ratioM,1525 of) (I {(IF,1440C,1440/I/IF,1655C,1525)}.) − The(IC,1655 calculated/IC,1525)·(IF,1440 values/IF,1655 for)} intensityto {(IM,1440 decrease/IM,1525) − factor(IM,1655/Ix M,1525of 1525)·(IF,1440 cm−/IF,1655mode)}. · 1 Theare presented calculated in values Table1 for. Similarly, intensity the dec intensityrease factor decrease x of factor 1525 x cmof−1 1155 mode cm are− mode presented can be in calculated; Table 1. 1 1 Similarly,the values the are intensity also included decrease in Table factor1. x The of 1155 intensity cm−1 decreasemode can factor be calculated for 1155 cm; the− valuesand 1525 are cmalso− includedmodes should in Table be same,1. The theintensity small didecreasefference factor between for the1155 two cmx−1values and 1525 would cm−1 be m mainlyodes should due to be analysis same, 1 theerror small from difference measuring bet theween intensities the two ofx values the vibrational would be modes. mainly Thedue modeto analys at 1525is error cm −fromis less measuring affected 1 1 theby itsintensities surrounding of the peaks vibrational than that modes. of 1155 The cm− mode, thus at the 1525 intensity cm−1 decreaseis less affected factor xbyof its 1525 surrounding cm− mode 1 peakswould than be more that accurateof 1155 cm than−1, thatthus ofthe 1155 intensity cm− . Asdecrease can be factor seen in x Tableof 15251, thecm−1 intensity mode would decrease be factormore accurate than that of 1155 cm−1. As can be seen in Table 1, the intensity decrease factor x is indeed smaller than 1 and it decreases systematically with increasing FFA content in the mixture. This further confirms the FFA degradation of carotenoid in olive oil.

Appl. Sci. 2019, 9, 4510 5 of 9 x is indeed smaller than 1 and it decreases systematically with increasing FFA content in the mixture. This further confirms the FFA degradation of carotenoid in olive oil.

Table 1. The experimental relative intensity ratios: I1440/I1525,I1440/I1655,I1655/I1525; and the calculated intensity decrease factors: x1525, x1155.

Sample C C:F (2:1) C:F (3:2) C:F (1:1) C:F (2:3) C:F (1:2) F

I1440/I1525 4.58 8.30 9.35 10.97 15.83 24.91 - I1440/I1655 1.47 1.22 1.22 1.12 1.11 1.08 0.92 I1655/I1525 3.11 6.78 7.64 9.33 14.29 23.08 - x1525 - 0.84 0.75 0.73 0.65 0.48 - x1155 - 0.89 0.74 0.70 0.72 0.44 -

The above results show that relative Raman intensity analysis is very helpful for investigating 1 FFA content in extra virgin olive oil. The analysis confirmed that the two vibrational modes at 1155 cm− 1 and 1525 cm− are characteristic Raman modes for analyzing FFA content in extra virgin olive oil, and their intensities decrease systematically with increasing FFA content in extra virgin olive oil. 1 This is consistent with the reported result that the spectral region of 945–1600 cm− , which includes the carotenoid Raman modes, is important for predicting the FFA content in olive oil [6]. In the study, absolute Raman intensities of various FFA content extra virgin olive oil samples were analyzed, 1 and it was found that the absolute Raman intensity of the spectral window 945–1600 cm− increases systematically with decreasing FFA content in extra virgin olive oils. In the Raman study, the analysis of relative intensity ratios would be easier and more accurate than absolute intensity, since absolute Raman intensity depends on various factors such as alignment of the Raman system, laser power stability, focus of the laser, absorption coefficient of the sample, and so forth. We show below that 1 1 the study of relative intensity ratio of vibrational modes at 1525 cm− and 1655 cm− presents a good and rapid analysis of FFA content in extra virgin olive oil, which would be helpful for quick quality evaluation of extra virgin olive oil. Figure3 presents the relative intensity ratios of I 1525/I1655 and I1155/I1655 as a function of FFA content in extra virgin olive oil samples; for comparison, the relative intensity ratio of I1440/I1655 is also presented in the figure. Seven different FFA contents from 0.1% to 0.8% are included in Figure3. Samples A, B, C, D, and E had FFA content of 0.1%, 0.15%, 0.4%, 0.6%, and 0.8%, respectively. Mixtures of C and D, and D and E with volume ratio 1:1 were prepared to obtain FFA content of 0.5% and 0.7%, respectively. Also, a mixture of C and E with volume ratio 1:1 was prepared to obtain FFA content of 0.6%, for comparison with sample D. As can be seen in Figure3, the relative intensity ratios of I 1525/I1655 and I1155/I1655 decrease linearly with increasing FFA contentin extra virgin olive oils. The intensity ratio of I1525/I1655 shows a better linear correlation with FFA content in extra virgin olive oil than 1 that of I1155/I1655. This would be due to the vibrational mode at 1525 cm− being less affected by its 1 surrounding peaks than that of 1155 cm− , consistent with the result in Table1. Therefore, the relative intensity ratio of I1525/I1655 would be a good parameter for rapid analyses of FFA content in extra virgin olive oil. 1 In the above analysis, the mode at 1655 cm− was chosen as a reference for calculation of relative intensity ratios. This would be valid for the Raman study of same type of vegetable oils. For comparison 1 of different types of vegetable oils, the mode at 1440 cm− could be chosen as a reference [30]. In our 1 1 recent study, the relative intensity ratio of 1655 cm− to 1440 cm− has been systematically investigated in various types of vegetable oils and three different types of pure unsaturated fatty acids (oleic acid, 1 linoleic acid, and α-linolenic acid) [31]. This study showed that the mode at 1440 cm− can be chosen as a reference for first order differentiation of different types of vegetable oils; and for more accurate 1 analysis of same type of vegetable oils, the mode at 1655 cm− can be chosen as a reference. In addition, a systematic density functional theory (DFT) calculation of the Raman spectra of unsaturated fatty Appl. Sci. 2019, 9, 4510 6 of 9 acids up to six carbon–carbon double bonds, is currently underway, which also supports the above method for choosing the reference mode. The DFT result will be presented elsewhere. The extra virgin olive oil samples were purchased from standard supermarkets in China and Korea; theAppl. samples Sci. 2019, 9 and, x FOR their PEER mixtures REVIEW were investigated without other treatment. Through relative intensity6 of 9 1 analysis, we first confirmed that the intensities of characteristic vibrational modes at 1155 cm− cm−1 and 15251 cm−1 of extra virgin olive oil decrease systematically with increasing FFA content; then and 1525 cm− of extra virgin olive oil decrease systematically with increasing FFA content; then showed showed that the intensity ratio of I1525/I1655 decreases linearly with FFA content. This linear correlation that the intensity ratio of I1525/I1655 decreases linearly with FFA content. This linear correlation presents apresents good and a good rapid and analysis rapid of analysis FFA content of FFA in extra content virgin in oliveextra oil.virgin In recent olive years,oil. In portablerecent years, and handheld portable Ramanand handheld systems Raman have beensystems developed. have been One developed. advantage One of advantage our method of our is that method it is notis that depending it is not ondepending experimental on experimental conditions. conditions. Thus, when Thus, our when method our is method combined is combinedwith portable with and portable handheld and Ramanhandheld systems, Raman itsystems, would beit would powerful be powerful for quick for on-site quick quality on-site evaluation quality evaluation of extra virginof extra olive virgin oil inolive supermarkets. oil in supermarkets.

1.8

1.6

1.4

0.6 A B

0.5 C

0.4 C:D(1:1) D

0.3 RelativeIntensity Ratio C:E(1:1) D:E(1:1) 0.2 E

0.1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 FFA content (%)

Figure 3. Relative intensity ratios of I1525/I1655 (bullet), I1155/I1655 (triangle), and I1440/I1655 (square) asFigure a function 3. Relative of free intensity fatty acid ratios (FFA) of content I1525/I1655 in ( extrabullet virgin), I1155 olive/I1655 (triangle) oil. The straight, and I1440 line/I1655 is a(square) linear fitting as a function of free fatty acid (FFA) content in extra virgin olive oil. The straight line is a linear fitting of of I1525/I1655. I1525/I1655. The main purpose of this paper is to propose that relative Raman intensity analysis is very usefulThe for main quick purpose quality of evaluation this paper of is extrato propose virgin that olive relative oils. For Raman practical intensity application analysis of is our very method, useful afor standard quick quality correlation evaluation between of FFA extra content virgin and olive relative oils. intensity For practical ratio I 1525 application/I1655 should of our be established, method, a 1525 1655 whichstandard would correlation require between accurate FFA measurements content and ofrelative FFA% int andensity I1525 /ratioI1655 .I With/I 532 should nm laser be established, excitation, 1 1525 1655 thewhich intensity would of require 1525 cm accurate− mode measurements is quite weak; of a FFA% significant and I enhancement/I . With of532 this nm mode laser wouldexcitation, be very the −1 helpfulintensity to of improve 1525 cm themode accuracy is quite of I 1525weak/I1655; a .significant Thus, a resonance enhancement investigation of this mode would would be necessary, be very ashelpful discussed to improve below. the accuracy of I1525/I1655. Thus, a resonance investigation would be necessary, as 1 discussedTo achieve below. significant enhancement of 1525 cm− mode, the wavelength of excitation laser should be closeTo achieve to the absorptionsignificant enhancement edge of carotenoids. of 1525 cm The−1 mode, absorption the wavelength study showed of excitation that the laser absorption should 1 edgebe close of carotenoidsto the absorption is about edge 510 of nmcarotenoids. [32]. Therefore, The absorption the intensity study of showed the 1525 that cm the− absorptionmode would edge be stronglyof carotenoids enhanced is about with 510 excitation nm [32]. aboutTherefore, 510 nm,the intensity and decrease of the with 1525 highercm−1 mode excitation would wavelength. be strongly Thisenhanced is consistent with excitation with a reported about 510 study nm, with and 514.5 decrease nm laser with [6 higher]; and our excitation study with wavelength. 532 nm, 633 This nm, is andconsistent 785 nm with lasers, a reported which isstudy presented with 514.5 in Figure nm laser4. It [6]; was and shown our study that with with 514.5 532 nm, nm laser633 nm, excitation, and 785 1 1 −1 1525nm lasers, cm− whichmode is have presented very strong in Figure intensity, 4. It was which shown is even that higherwith 514.5 than nm that laser of 1655excitation, cm− 1525mode cm [6]. mode have very strong intensity, which is even higher than that of 1655 cm−1 mode [6]. While 1525 cm−1 mode is much weaker than 1655 cm−1 mode with 532 nm laser excitation, as can be seen in Figure 4, 1525 cm−1 mode is too weak to be observed with 785 nm laser excitation, and with 633 nm laser excitation, a strong luminescence peak at about 670 nm is observed, and no vibrational modes can be observed due to the strong luminescence background. Therefore, a green laser of 514.5 nm would be ideal for practical application of relative intensity analysis of FFA in olive oils. Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 of 9

Our above results indicate that the study of relative intensity ratio of the two characteristic vibrational modes at 1525 cm−1 and 1655 cm−1 with 514.5 nm green laser excitation would be of great interest for quick on-site quality evaluation of olive oils. This simple relative intensity analysis of 1525 cm−1 and 1655 cm−1 modes would be powerful only for quick evaluation of similar types of high- quality olive oils, such as extra virgin olive oils. For quick on-site detection of olive oil adulteration, further systematic studies would be needed. For instance, if olive oils are adulterated with other types of vegetables oils, then the mode at 1655 cm−1 would not be a good reference, thus the intensity ratio Appl. Sci. 2019, 9, 4510 7 of 9 of I1525/I1655 cannot be simply applied for detecting olive oil adulteration. In a recent study, Li et al. [33] showed that Raman spectroscopy combined with iPLS and SiPLS is helpful for detection of olive oil adulteration with1 waste cooking oil. In our recent study,1 it was shown that Raman spectroscopy While 1525 cm− mode is much weaker than 1655 cm− mode with 532 nm laser excitation, as can be combined with two-dimensional1 correlation spectroscopy (2DCOS) is helpful for differentiating seen in Figure4, 1525 cm − mode is too weak to be observed with 785 nm laser excitation, and with different633 nm lasertypes excitation, of vegetable a strong oils [31]. luminescence These studies peak suggest at about that 670 Raman nm is spectroscopy observed, and combined no vibrational with 2DCOSmodes canand be chemometrics observed due method to the wouldstrong luminescencebe helpful for background. practical application Therefore, of a on green-site qualitylaser of evaluation514.5 nm would of olive be oil ideal adulteration. for practical application of relative intensity analysis of FFA in olive oils.

633 nm

1155 1525 Raman Intensity Raman

532 nm

785 nm

800 1000 1200 1400 1600 1800 Wavenumber (cm-1)

Figure 4. Raman spectra of olive oil sample C by three different wavelength lasers of 532 nm, 633 nm, Figure 4. Raman spectra of olive oil sample C by three different wavelength lasers of 532 nm, 633 nm, and 785 nm. Each spectrum is y-shifted for clarity. and 785 nm. Each spectrum is y-shifted for clarity. Our above results indicate that the study of relative intensity ratio of the two characteristic 4. Conclusions 1 1 vibrational modes at 1525 cm− and 1655 cm− with 514.5 nm green laser excitation would be of greatIn interest conclusion, for quick Raman on-site spectroscopy quality evaluation has been ofapplied olive oils.for rapid This simpleanalysis relative of FFA intensity content in analysis olive 1 1 oil.of 1525Our results cm− and show 1655 that cm the− intensitiesmodes would of the be two powerful characteristic only for vibration quick evaluational modes at of 1525 similar cm−1 types and 1155of high-quality cm−1 decrease olive systematically oils, such as extrawith increasingvirgin olive FFA oils. content For quick in olive on-site oil, detection due to carotenoid of olive oil degradationadulteration, caused further by systematic FFA. In addition, studies wouldthe relative be needed. intensity For ratio instance, of the ifvibrational olive oils aremodes adulterated at 1525 1 cmwith−1 and other 1655 types cm−1 of decreases vegetables linearly oils, thenwith FFA themode content, at 1655thus it cm can− wouldbe applied not for be arapid good analysis reference, of thethus quality the intensity of olive oil. ratio Furthermore, of I1525/I1655 itcannot has been be discussed simply applied that a 514.5 for detecting nm green olive laser oilwould adulteration. be ideal forIn aRaman recent study study, of Li FFA et al.in [olive33] showed oil. that Raman spectroscopy combined with iPLS and SiPLS is helpful for detection of olive oil adulteration with waste cooking oil. In our recent study, it was shown Author Contributions: J.Q. performed the experiments, analyzed the data, and wrote the original draft; H.-Y.H. that Raman spectroscopy combined with two-dimensional correlation spectroscopy (2DCOS) is helpful analyzed the data and participated in discussions of the research; I.-S.Y. and X.-B.C. supervised the project and revisedfor diff theerentiating manuscript. diff erent types of vegetable oils [31]. These studies suggest that Raman spectroscopy combined with 2DCOS and chemometrics method would be helpful for practical application of on-site quality evaluation of olive oil adulteration.

4. Conclusions In conclusion, Raman spectroscopy has been applied for rapid analysis of FFA content in olive 1 oil. Our results show that the intensities of the two characteristic vibrational modes at 1525 cm− 1 and 1155 cm− decrease systematically with increasing FFA content in olive oil, due to carotenoid 1 degradation caused by FFA. In addition, the relative intensity ratio of the vibrational modes at 1525 cm− 1 and 1655 cm− decreases linearly with FFA content, thus it can be applied for rapid analysis of the quality Appl. Sci. 2019, 9, 4510 8 of 9 of olive oil. Furthermore, it has been discussed that a 514.5 nm green laser would be ideal for Raman study of FFA in olive oil.

Author Contributions: J.Q. performed the experiments, analyzed the data, and wrote the original draft; H.-Y.H. analyzed the data and participated in discussions of the research; I.-S.Y. and X.-B.C. supervised the project and revised the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 11574241) and the National Research Foundation of Korea (NRF) grant funded by the Korean Ministry of Science and ICT (No. 2017R1A2B2009309). Conflicts of Interest: The authors declare no conflict of interest.

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