RO126791A0 - Process for uv-vis and ft-ir spectroscopic assessment of quality and authenticity of vegetable oils - Google Patents

Abstract

The invention relates to a process for assessing the quality of vegetable oils. According to the invention, the process comprises complementary UV-Vis and FT-IR spectrometry techniques for determining the quality and authenticity of vegetable oils by pointing out biomarkers, such as phenolic compounds, carotenoid and chlorophylic pigments, catechols and fatty acids, as well as oxidative alteration processes.

Description

PROCEDURES FOR UV-VIS AND FTIR SPECTROSCOPIC EVALUATION (FOURIER TRANSFORMANT INFRARED SPECTROSCOPY) OF THE QUALITY AND AUTHENTICITY OF VEGETABLE OILS

DESCRIPTION OF THE INVENTION

The present invention relates to the use of molecular spectroscopic techniques, such as UV-Vis absorption spectrometry and Infrared vibrational spectrometry (with Fourier Transform) to highlight the authenticity biomarker of vegetable oils.

Vegetable oils are usually characterized by general quality indicators (water percentage, peroxide index, iodine index, refractive index) and are subject to European or national quality standards, specific to oils and related procedures.

In the last 15 years, taking into account the nutritional and biomedical implications of vegetable oils rich in antioxidants (mono- and polyunsaturated fatty acids, pigments, sterols with antagonistic action against cholesterol) interest in assessing their quality and authenticity has increased significantly. Modified gas chromatography (GC) techniques coupled with flame detection (FID) or mass spectrometry (MS) have been developed to identify and separate unsaturated fatty acids and to confirm the authenticity or detection of virgin oils with refined oils. same type or valuable oils by mixing with lower oils.

From the analysis of ISO standards published by ISO / TC 34 / SC11 Animal and vegetable fats and oils, those that indirectly refer to the analysis of authentic biomarkers are ISO 12228: 1999 - Determination of individual sterol and total sterol content by gas chromatographic method and respectively ISO 15788-1: 1999 - Determination of stigmastadienes by capillary column GC (Reference method)

European standards (CEN) for the analysis of vegetable oils are CEN / TC 307 Standardization of methods (physical and chemical) for sampling and analysis of vegetable oils, animal oils and residues, in order to harmonize European methods with international ISO and CEN 14103 - Determination of fatty acid methyl esters (FAME)

CODEX STAN 19-1981, Rev 2-1999 applied to edible oils and fats (glycerides, phosphatides, free fatty acids) include ways of discriminating between the composition of virgin and cold-pressed oils (category A) and refined ones (category B) . Thus, virgin and cold-pressed oils are obtained exclusively by physical (mechanical) methods which do not alter the original chemical composition of the oilseeds from which they are obtained, can be purified by water washing, decantation, filtration and centrifugation, without the addition of additives ( dyes). Additions of natural or identically natural flavors, natural antioxidants (ascorbic acid 500 ppm) are allowed.

Until now, the authentication of food products is done by GC type methods coupled with FID or MS, methods that require very expensive equipment and reagents, speoialteatjși personnel, laborious procedures of isolation, pre-processing and analysis. The present patent proposes the use of '^ -2010-00694'2 4 - 3b- 2010 two spectroscopic procedures which are less expensive and provide timely, rapid and non-destructive information of oil samples of any type or purity class.

The two procedures covered by the patent are:

1. Analysis by UV-Vis molecular spectrometry which allows the identification of types of molecules containing single or double bonds (in isolated or conjugated system) based on the ability of organic groups with double bonds to absorb ultraviolet (UV) radiation (180- 360 nm) or visible (360-800 nm). Molecules with single bonds or double bonds isolated from vegetable oils (saturated fatty acids or unsaturated fatty acids in free or esterified form, sterols, amino acids, esters of phenolic compounds) absorb in the UV domain, while molecules with more than 9 bonds conjugated doubles absorb Visible Field (VIS) radiation and are colored. This is the case of chlorophyll, carotenoid or quinone pigments, which are fat-soluble and are found in oils. The specific maximum absorption ranges of these molecular types are 270 -280 nm (phenolic acid derivatives), 260 and 340 nm (flavonoids), 420 - 470 nm (carotenoids and apocarotenoids), 615, 670 nm (chlorophylls).

2. Infrared Spectroscopy (FT-IR) is used to identify functional groups and specific fingerprint regions in oils, directly, without prior processing, mixtures of molecules.

These techniques are frequently used in the quantification of major components of oils, using as measurement variants the analysis of IR with absorption (diamond microcell) or with attenuated transmissions (ATR), a recent method with high sensitivity.

The literature shows that FTIR vibrational spectroscopy in combination with a multivariate statistical analysis, can successfully discriminate different types of oil and can also identify virgin or refined olive oil, or fraudulent with soybean oil, sunflower or lower oils. The generic FTIR type spectrum for oils is shown in Fig.l.

Fig. 1. The generic FTIR spectrum for highlighting the oil footprint, with © Vid ^ țieriSa functional groups _; .

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According to this generic FT-IR spectrum, the specific fingerprint area of the oils is the range 750-1250 cm ' ¹ , combined with other areas indicating the presence of cis-type double bonds (indicating virgin, non-heat-processed oils) or trans ( inferior and processed oils).

So:

• the signal from 720-722 cm ' ¹ is due to the overlap CH2 of the “rocking” vibration with the out-of-plane deformation vibration of the m-disubstituted olefmic group.

• the slightly intense band from 962-968 cm ' ¹ is associated with olefmic conjugated CH groups, with deformation vibrations, of the trans, trans- or cis, trans conjugated olefmic groups.

• bands from 1119 and 1098cm ^I st associated with elongations of the ester group CO, the first being inversely proportional to the proportion of saturated acyl groups • signal from 1377cm · 'due to deformation of CH2 groups • bands from 1238 and llbOcm' ¹ are associated with elongations of the C-0 ester group and with the deformation vibration of the CH ₂ group, both associated with the proportion of saturated acyl groups • band 1416-1418cnf ¹ due to the “rocking” vibrations of CH m-disubstituted • band from 1464 cm ' ¹ due deformation vibrations of aliphatic CH2 and CH3 • band 1650-1656 cm ' ¹ associated with the C = C extent of czs-olefmic isomers • band 1742-1744 cm' ¹ attributed to ester and carbonyl groups of triglycerides • Bands from 2923 and 2854 cm ' ¹ corresponding to asymmetric and symmetrical elongation vibrations of aliphatic CH2 • Band from 3007-3009 cm' ¹ due to the double bonds C = C cis (elongation vibration) At the same time, it is possible to highlight peroxidation processes, by bands specific for oxidized fatty acids, located between 3300-3500 cm ' ¹ .

This fast, direct and economical method does not require reagents or preliminary extractions, but is not standardized. The results obtained by FTIR were correlated with the results obtained by GC or HPLC chromatography and directly proportional, statistically significant correlations were established.

The aim of the present invention is to apply the two spectroscopic measurement procedures (UV-Vis and FTIR) in order to find, in a minimum time, with minimum costs, indications regarding the authenticity of the different types of vegetable oils, based on the specific profile.

The procedures used were applied on 6 types of edible oils with antioxidant character: olive oil (virgin or refined), sunflower oil (unrefined and refined), flaxseed oil, hemp, rapeseed and sea buckthorn.

UV-Vis molecular absorption spectra were recorded with a double-beam spectrometer, ABL Jasco 460, using the range 200-700nm.

Infrared absorption spectra (FTIR) were recorded directly using the Shimadzu IR Prestige -21 spectrophotometer with HATR (Horizontal Attenuated Total Reflectance) accessory. The spectra were recorded on the wavelength range 600-4000 cm '' ^{? L The} absorption bands characteristic of the type of connections and functional groups (expressed in cm ' ¹ ) were identified.

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Example 1. Authentication procedure for olive oils based on UV-Vis and FTIR spectra

Authentic extra virgin olive oil (PI) was used and controlled mixtures were made with refined sunflower oil (in percentages of 10 and 25%), noted as P2 and P3, respectively. the three oil samples were introduced into quartz tanks for UV-Vis analysis and the spectrum was recorded in the 350-700 nm range.

Fig.2 shows the UV-Vis spectral fingerprint.

2.2

Abs ¹

350 400

500 600

700

Wavelength [nm]

Fig. 2. UV-Vis spectral footprint (350-700 nm) specific for authentic or counterfeit extra virgin olive oil by adding refined sunflower oil.

Green - PI - genuine extra virgin olive oil. Blue- P2- extra virgin olive oil adulterated with 10% refined sunflower oil. Red - P3- extra virgin olive oil adulterated with 25% refined sunflower oil

Figure 3. shows the FTIR spectrum recorded directly for samples PI, P2 and P3, on the range 6004000 cm ' ¹ .

B

Figure 3. FTIR-ATR spectrum specific to PI, P2, P3 samples. PI - authentic virgin olive oil. P2ulei forged with 10% refined sunflower oil. P3- counterfeit PI oil with 25% refined sunflower oil. Bands 1-7 in the spectrum are characteristic of wave numbers 722 (1), 1095 (2a), 1118 (2b), 1157 (2c), 1377 (3a), 1465 (3b), 1743 (4), 2850 (5) , 2920 (6), 3008 cm ¹ (7).

¢ ^ - 2 0 1 0 - 0 0 8 9 4 -2 4 -09- 2010

Example 2. Identification of refined and unrefined sunflower oil

Unrefined, authentic sunflower oil (P4) was used and mixed with refined oil (P7). The 1: 1 mixture between P4: P7 is represented by sample P6. the three oil samples were introduced into quartz tanks for UV-Vis analysis and the spectrum was recorded in the 350700 nm range.

Fig. 4 shows the UV-Vis spectral footprint, specific for genuine, unrefined sunflower oil, compared to refined sunflower oil.

0.3 - ----------- ---------------------, 0.2

-0.2 -; --0.3 -------- ^[ ------------------- ^: ---------- -------- ----------------- ^J '

350 400 450500

Wavelength [nm]

Figure 4. The UV-Vis spectral footprint (350-500 nm) is specific for genuine, unrefined sunflower oil compared to refined sunflower oil. Blue - authentic, unrefined sunflower oil. Green - refined sunflower oil.

Fig. 5 shows the FTIR spectrum recorded directly for samples PI, P2 and P3, on the range 6004000 cm ' ¹ .

| --Ρβ |

Wavenumbercm Wiverwmbwcm Wavenumber cm ¹

A B C

Fig. 5. FTIR-ATR spectrum specific to samples P4 (A), P6 (B), P7 (C). P4- authentic, unrefined sunflower oil. P6- P 4 forged with refined oil, P7, in a 1: 1 ratio. P7- refined sunflower oil.Bands 1-7 in the spectrum are characteristic of wave numbers 722 (1), 1095 (2a), 1118 (2b), 1157 (2c), 1377 (3a), 1465 (3b), 1743 (4), 2850 (5), 2920 (6), 3008 cm ' ¹ (7) ·

6V 2 Ο 1 Ο - Ο Ο 8 9 4 2 h -09- 2010

Example 3. Identification of UV-Vis and FTIR spectroscopic fingerprints specific to authentic flax, hemp, rapeseed and sea buckthorn oils

Fig.6 shows the UV-Vis spectral imprint specific to authentic flax (A) and hemp (B) oils

¹ r:: 1 1 1 ⁰⁵ · - l · - - Abs 1 1 1 ¹ 1 1 ol-- --- - - -0.1 ^{1 1 1 1 1} 250 300 400 500

Wavelength [nm]

1.5

Abs ¹ !

0.5

600 200 300 400 500 600 700

Wavelength [nm]

A B

Figure 6. UV-Vis spectral footprint (200-700 nm) specific for genuine flaxseed oil (A) and genuine hemp oil (B)

Fig.7 shows the UV-Vis spectral imprint specific to authentic sea buckthorn (A) and rapeseed (B) oils

0.6

0.4

Abs

0.2 o ^L 200 '' \ 1.7: ^'15 ---------________ i;' _t ' ¹ '' ¹ 1 - - - - L - - _ I. . I.

II ¹ l. (Ii ί Absi l, 1,1, ° · ^{5 1} '

-L ---------- 1 ------. ------- θ ------ j— -J -----. . J— -L300 400 500 600 200 300 400500

Wavelength [nm] Wavelength [nm]

600

A B

Figure 7. UV-Vis spectral fingerprint (200-700 nm) specific for Authentic sea buckthorn oil (A) and genuine rapeseed oil (B)

Figure 8. presents the FTIR spectrum recorded directly for the samples of rapeseed, sea buckthorn, flax and hemp oils, in the range of 600-3500 cm ' ¹ .

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2.6

2.4

2.2

2.0 1.8 ώ1.6

81.4 | 1.2

8ι.θ <0.8

0.6

0.4

0.2

0.0

-A. ² I · I · | | |

3500 3000 2500 2000 1500 1000

Wavenumber (cm ¹ )

Fig. 8. FTIR spectrum specific to samples of rapeseed, sea buckthorn, flax and hemp oils, in the range 600-3500 cm ' ¹ .

Example 4. Overall analysis of vegetable oils, by UV-Vis and FTIR spectrometry with recognition markers

By recording a large number of UV-Vis spectra and overlapping the spectra, the qualitative differences and the absorption domains specific to the different groups can be highlighted. Thus fig. 9 A and B show the UV-Vis spectral fingerprint specific for 7 types of authentic oils on wavelength ranges (250-700 nm) (A) and (350-700 nm) (B), respectively.

-0.35 ^{L 1 J} ------— ¹ --- ¹ --- ¹ ----- ¹ 250 300 400 500 600

Wavelength [nm]

2.1 .: i i 1 Abs ¹ 1 - 0 i _ _ _ j ../ _ i_. £; _j -0.35 _________IT. _i.j __________, ____ 1_. _____. ..... j 700 350 400 500 600 700 Wavelength [nm]

A B

Fig. 9. UV-Vis spectral footprint for genuine oils over wavelength ranges (250-700 nm) (A) and (350-700 nm), respectively (B). Blue - extra virgin olive oil. Dark red - unrefined sunflower oil. Light green - refined sunflower oil. V ^ le -, ^ turquoise - hemp oil. Khaki - sea buckthorn oil. Purple linseed oil. Red - rapeseed oil _{? 1} '

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The spectral data highlighted the specificity of each type of oil through the functional groups that absorb in the UV and / or Vis domain. The data are presented in Table 2.

Table 2. Specific UV and / or Vis domain, authentication markers for 7 analyzed oils

Type of oil UV markers Markeri Vis Unrefined sunflower 250-310 nm Phenolic compounds 440-470 nm Carotenoids Refined sunflower 250-320 nm Phenolic compounds - olives 305, 340 nm Phenolic compounds (derived from phenolic acids and flavonoids) 418 nm- quinone 450-470 nm Carotenoid 615, 670 nm Chlorophyll Rape 243-310 nm Phenolic compounds 440-450 nm Carotenoids Into the 250-3 10 nm Phenolic compounds - Hemp 260-300 nm Phenolic compounds 408 nm Chinone 470 nm Carotenoids 615. 670 nm Chlorophyll Calina 260-280 nm Phenolic compounds 415-470 nm Carotenoid

It is found that neither the domains nor the specific markers overlap. It is thus possible to highlight the differences between the 7 oils based on the specific composition in phenolic compounds, with or without carotenoid pigments, with or without chlorophyll pigments or quinones.

FTIR spectroscopic analysis revealed not only the specific fingerprints of each oil (shown in Fig. 3, 5 and 8, but especially the bands characteristic of cis or trans double bonds that highlight the presence of unsaturated fatty acids, in native or isomerized form by Table 3 includes the characteristic absorptions at the wave numbers 720-722, 967, 1657, 3007-3008 cm · '.

Table 3. Characteristic absorptions recorded in the FTIR spectrum, at wave numbers 720-722, 967, 1657,3007-3008 cm ' ¹ .

Name of oil Nr. wave (cm ' ¹ ) 720-722 (cis deformation) 967 (deformation cis, trans; trans, trans) 1657 extent C = C cis 3007-3008 (stretch, cis) Extra virgin olive oil (PI) 0.454 0.156 0,071 0.119 Forged extra virgin olive oil with 10% sunflower oil (P2) 0.416 0,175 0.048 0.146 Falsified extra virgin olive oil with 25% sunflower oil (P3) 0.503 0.195 0.089 0.180

CL ~ 2 Ο 1 Ο - Ο ο 8 9 4 - 2 4 -09- 2010

Beef oil. Unrefined sun (P4) 0.518 0.254 0.138 0.266 Fl. Unrefined sun forged with 10% refined sunflower oil (P6) 0.617 0,293 0,140 0.296 Fl. Refined sun (P7) 0.494 0.208 0.078 0.180 Rapeseed oil 0.654 0.188 0.058 0.188 Linseed oil 0.752 .212 0,071 0.280 Cinnamon oil 0.749 .246 0.068 0,270 Seabuckthorn oil 0,701 0,233 0.056 0,219

It is identified as follows:

❖ Increases in absorption at 720-722, 967, 1657, 3007-3008 cm ' ¹ by falsification of olive oil with sunflower oil (P3 and P2 compared to PI) ❖ Refined sunflower oil has lower absorption values at 720-722, 967, 1657, 3007-3008 cm ' ¹ , compared to crude oil ❖ Functional, virgin oils, rich in unsaturated fatty acids, have high absorption values, maximum for hemp oil, followed by flaxseed oil, sea buckthorn , rapeseed. Olive oil and sunflower oil have lower values of approx. 1.2 times.

A comparative image (Fig. 10) makes it possible to correlate the concentration of saturated fatty acids (AGS), unsaturated fatty acids (AGN) with the absorbances recorded at 720-722 cm ' ¹ (3), 967 cm' ¹ (4), 1657 cm ' ¹ (5), 3007-3008 cm''(6)

Fig.10. Correlations between the concentration of saturated fatty acids (1), unsaturated fatty acids (2) and the absorbances recorded at 720-722 cm ' ¹ (3), 967 cm' ¹ (4), 1657 cm ' ¹ (5) .30073008 cm'' (6) for the analyzed oils. FSN- unrefined sunflower. FSR-refined sunflower. .. \

Claims (4)

1. Procedure for evaluating by UV-Vis spectrometry the quality and authenticity of vegetable oils, by recording the specific metabolic profile, based on specific absorption in the range of 200-700 nm, identifying identification markers: phenolic compounds, carotenoid pigments, chlorophylls and quinones. 2. Procedure for evaluation by Fourier Transform Infrared Spectrometry (FTIR-ATR) of the quality and authenticity of vegetable oils based on the specific absorption in the range of 600-4000 cm ' ¹ . Specific fingerprinting bands have been identified, with the identification of cis or trans type double bonds specific to unsaturated fatty acids. 3. Complementary procedures (UV-Vis and FTIR) for the comparative assessment of genuine, virgin oils, compared to refined oils of the same type, or of counterfeit oils by mixing unrefined oils with refined oils, superior oils with inferior oils. 4. Complementary procedures (UV-Vis and FTIR) for comparative evaluation and authentication of six types of vegetable oils, functional, sunflower, olive, flax, hemp, sea buckthorn, rape. The tested procedures are useful in assessing the quality and authenticity of these oils, from different sources, under conditions of different technological production, or storage at different temperatures and humidity. It is also possible to highlight the processes of oxidative alteration and falsification.