Edible oil adulterations

food

1. Seminar Leader Dr. Indu Chopra Presented by Shubham Yadav Roll No – 10730 AC-691 Techniques for Adulteration Detection in Edible Oils Division of Agricultural Chemicals, ICAR-IARI, New Delhi

2. Edible oils A food substance of whatever origin, source or composition that is manufactured for human consumption wholly or in part from a fat or oil other than milk and dairy product. (Government of Ontario, Canada on 12-Oct-2017) Some properties of oil: Liquid at room temperature Mostly p lant origin Less saturated Lower melting point Less stable Examples- hazelnut oil, olive oil , mustard oil, sunflower oil etc.

3. Year Area (million hectare) Production (Million Tonnes) 2001-02 2002-03 2003-04 2004-05 2005-06 2006-07 2007-08 2008-09 2009-10 2010-11 2011-12 2012-13 2013-14 2014-15 2015-16 2016-17 22.64 21.49 23.66 27.52 27.86 26.51 26.69 27.56 25.96 27.22 26.31 26.48 28.05 25.59 26.13 - 20.66 14.84 25.19 24.35 27.98 24.29 29.76 27.72 24.88 32.48 29.8 30.94 32.75 27.51 25.3 32.09 Source: Handbook of Agriculture, 2016

4. Source: Handbook of Agriculture, 2016

5. Health Benefits of Edible Oils Decrease in Risk for Heart Disease Decrease in Risk for Breast Cancer O mega-3 Fatty Acids B etter digestion V itamin E Promote Cell Growth A ntioxidants Indelicato et.al. 2017 Improvement of Metabolism Source of energy

6. Current issues in edible oil adulteration in India A dmixing cold press oil with refined one. Mustard oil adulterated with argemone oil. High price oil adulterations with low price oil. Loose edible oil adulterations. Reputed brands adulterate with ordinary palm oil or other cheap oils. Mineral oil, karanja oil , castor oil, and artificial colours are used in edible oil adulterations. Navya et.al. 2017

7. Composition of Edible Oils Triacylglycerol's (TAGs) shares 90-95% in edible oil. Minor component in edible oils Mono and Diacylglycerols- mono and di-esters of fatty acids and glycerol. Free Fatty Acids- unattached fatty acids present in a edible oil. Phosphatides- glycerol + FA + PA + N- Containing compounds. Sterols- phyto-sterols, Sito -sterol, and stigma-sterol. Fatty Alcohols- Long chain alcohols. Tocopherols- methylated phenols, antioxidants to retard rancidity. Carotenoids and Chlorophyll Indelicato et.al. 2017

8. Glycerol Tri-hydric alcohol and popular name glycerin. It is synthesized in the body from glucose. Colourless viscous oily liquid with sweet taste.

9. Fatty Acids Fatty acids are aliphatic mono-carboxylic acids that are mostly obtained from the hydrolysis of natural fats and oils. Have the general formula R- (CH 2 )n-COOH and mostly have straight chain. In this formula "n" is mostly an even number of carbon atoms (2-34 ). According to presence or absence of double bonds they are classified into : Saturated Fatty Acids Un-Saturated Fatty Acids

10. Saturated Fatty Acids T hey contain no double bonds with 2-24 or more carbons. They are solid at room temperature except if they are short chained. They may be even or odd numbered. They have the following molecular formula, C n H 2n+1 COOH . Acetic F.A. (2C ) CH 3 -COOH . Butyric F.A. (4C ) CH 3 -(CH 2 ) 2 -COOH. Caproic F.A. (6C ) CH 3 -(CH 2 ) 4 -COOH. C aprylic (8 C ) CH 3 -(CH 2 ) 6 -COOH. Capric (10 C ) CH 3 -( CH 2 ) 8 -COOH Palmitic ( 16C) CH 3 -(CH 2 ) 14 -COOH S tearic (18 C ) CH 3 -(CH 2 ) 16 -COOH L ignoceric (24C ) CH 3 -(CH 2 ) 22 -COOH

11. Unsaturated Fatty Acids They contain double bonds. M onounsaturated T hey contain one double bonds . ( CnH 2 n-1 COOH) P olyunsaturated They contain more the one double bond (C n H 2 n-more than 1 COOH). Palmitoleic acid : It is found in all fats. It is C16:1∆9, i.e., has 16 carbons and one double bond located at carbon number 9 and involving carbon 10. CH 3 - ( CH 2 ) 5 CH = CH-(CH 2 ) 7 – COOH Oleic acid Is the most common fatty acid in natural fats. It is C18:1∆9, i.e., has 18 carbons and one double bond located at carbon number 9 and involving carbon 10 . CH 3 - (CH 2 ) 7 - CH=CH – (CH 2 ) 7 -COOH

12. Linoleic C18:2∆9, 12 It is the most important since other essential fatty acids can be synthesized from it in the body. CH 3 - (CH 2 ) 4 -CH = CH-CH 2 -CH=CH-( CH 2 ) 7 -COOH Linolenic acid C18:3∆9, 12, 15 I n corn, linseed, peanut, olive, cottonseed and soybean oils. CH 3 -CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH- ( CH 2 ) 7 -COOH Arachidonic acid C20:4∆5, 8, 11, 14 It is an important component of phospholipids in animal and in peanut oil from which prostaglandins are synthesized. CH 3 - (CH 2 ) 4 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-(CH 2 ) 3 -COOH

13. Adulteration Adulteration usually refers to mixing other matter of an inferior and sometimes harmful quality with edible oil intended to be sold. As a result of adulteration, oil becomes impure and unfit for human consumption.

14. Effects of adulterations G allbladder cancer Mishra et.al. 2011 Edible oil adulterants, argemone oil and butter yellow, as aetiological factors for gall bladder cancer. Edible oils adulterated with Mineral oil causes damage to liver and carcinogenic effects. Vegetable oil adulterated with Castor oil causes stomach problem.

15. Argemone oil mix with Edible oils causes- Epidemic dropsy & Glaucoma Loss of eyesight Das et. a l. 1997

16. Different Analytical Techniques

17. Purity Detection Techniques Limitations Saponification Value Acid Value Iodine Value Refractive Index Specific Gravity Insensitive at lower concentration Time consuming Usage of chemicals, maximal Sample preparations Insensitive in sophisticated adulterations

18. Major technologies for the detection of edible oil fraud, as reported in the literature from 2005 to 2015. Edible Oils and fat Hong et.al. 2017

19. Gas and liquid chromatography The most frequent and most popular techniques used in the analysis of edible oils are gas and liquid chromatography with different detection systems. The combined techniques are also often used, with the sample first undergoing separation with high-pressure liquid chromatography, and then the resultant fractions being analysed with the use of gas chromatography. The most popular detectors are flame ionization detectors (FID ) and UV-DAD because of their availability and cost, and also a mass spectrometer, which enables easy identification of an analysed sample’s components. These techniques enable the analysis of compounds which belong to different groups with the use of a single instrument. Table-1 lists examples of chromatographic techniques used for the analysis of individual component groups present in vegetable oils. Gromadzka et.al. 2011

20. Infrared Spectroscopy (IR ) NIR has found application in the discovery of camellia oils adulterations with soybean oil , as well as at the differentiation and classification stages of vegetable oils . Using mid-infrared ( IR) spectroscopy the detection limit of extra virgin olive oil adulteration was determined as 5 % for corn – sunflower binary mixture , cottonseed and rapeseed oils. Fourier transforms infrared spectroscopy ( FTIR) allowed for the differentiation of stages of the oxidation process and determination of the oxidation level of the analysed oil sample . Automated FTIR technique, with the use of an auto sampler, allows conducting even 90 analyses within one hour . Such an automated technique was used for the determination of free fatty acids in vegetable oils. The identification of fatty acids is done on the basis of absorption of the carbonyl group (1573 cm-1 ). Gromadzka et.al. 2011

21. Nuclear Magnetic Resonance (NMR) Analysing proton NMR data , genuine oil can be differentiated from those with even 5 % added hazelnut oil, which is the hardest adulteration to uncover. The geographic region of origin can also be determined, as well as the type and quality of the oil (extra virgin, virgin olive oil ). Similarly , the application of coupled proton NMR and phosphorous(31) NMR as well as multidimensional statistical analysis allowed for the classification of 13 types of vegetable oils, and enabled the discovery of adulterations of olive oils with these oils at a 5% level . Using differentiation analysis and carbon(13) NMR data from the olefin group, the majority of adulterations with other vegetable oils, which can be met in olive oil, can be determined. Carbon(13) NMR analysis can also be a powerful tool for evaluating the oxidative stability of oil samples. Gromadzka et.al. 2011

22. Raman Spectroscopy A dulterations of olive oils with hazelnut oil can be determined using Raman spectroscopy. Olive oil adulteration with waste cooking oil was detected and quantified by combining optical Raman scattering spectroscopy and chemo-metrics. Spectra of 96 olive oil samples with waste cooking oil (2.5%, 5%, 10%, 20%, 30% and 50%) were collected by the portable Raman spectroscopy system. The established model could make effective quantitative analysis on adulteration of waste cooking oil. It provides a quick accurate method for adulteration detection of waste cooking oil in olive oil. Olive oil is among the most commonly adulterated food products. Raman spectroscopy has the potential to be utilized in measurements of olive oil to establish purity from adulteration with other, less expensive, oils. This application note reviews some of the most compelling data present in recent scientific literature to support this claim . The adulteration of extra virgin olive oil (EVOO) is a big problem in food safety. The Raman spectra characterize different kinds of vegetable oils in the region 800–1800 cm− 1 . The method based on Bay-LS-SVM and Raman spectroscopy is also easy to operate, non-destructive and ‘lipid sensitive’, and it is considered to be suitable for online detection of adulterated olive oil. Gromadzka et.al. 2011

23. Rapid Authentication of Olive Oil Adulteration by Raman Spectrometry Normalized Raman spectra of olive oil, soybean oil, sunflower seed oil, rapeseed oil, and corn oil. Zou et.al. 2009

24. PCR A CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism) method based on PCR technology was established to distinguish olive, soybean, sunflower, peanut, sesame, maize. Sensitivity of the method detecting DNA and oil mixture at different ratio was at level as low as 10% (V/V). PCR-CE-SSCP used to authenticate edible oils . Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils . PCR-CE assay proved equally efficient as gas chromatography analysis in detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Zhang et.al. 2012

25. ELISA In MILK The enzyme-linked immune-sorbent assay technique involves the detection of the complex between target protein and specific mono- or polyclonal antibodies, thereby enabling both the quantitative and qualitative detection of that protein . the individual casein fractions and peptide fragments of caseins can be detected using ELISA . Azad et.al. 2016

26. Adulteration Detection Techniques

27. C old press oil adulteration The amount of the non-TAG varies with the oil source, extraction process, season and geographical source. Removal of non-TAG constituents from the oil with the least possible damage to the TAG and minimal loss of desirable constituents is the objective of the refining process. Refining can affect minor components present in the unsaponifiable fraction of vegetable oils. During refining processes, particularly during deodorisation and bleaching, trans fatty acids and steradienes are also formed which are generally absent in cold press oil. Virgin olive oil adulteration with refined vegetable oils can be detected using trans fatty acid or steradienes as markers. It should be noted that detection of steradienes and trans fatty acids isomer in other cold press oils are also a sign of adulteration with refined oils. T rans fatty acids are not essential and they do not promote good health. The consumption of trans fatty acids increases risk of coronary heart disease. Trans fats from partially hydrogenated oils are more harmful than naturally occurring oils . Example- GLC Damirchi et.al. 2015

28. Case study-1 A study was carried out to assess the effectiveness of Fourier transform infrared ( FTIR) spectroscopy in detecting adulteration of virgin coconut oil with palm kernel olein as a potential adulterant. Multi-bounce attenuated total reflectance measurements were made on pure and adulterated samples of virgin coconut oil. Detection of adulteration up to 1% was possible. Discriminant analysis using 10 principal components was able to classify pure and adulterated samples on the basis of their spectra. MATERIALS AND METHODS Virgin coconut oil and palm kernel olein were mixed to obtain a series of standard or trained sets of 14 pure and 18 adulterated samples containing 1–50 % palm kernel olein. The samples containing palm kernel olein were marked as adulterated, while pure virgin coconut oil samples were marked as virgin coconut oil and classified using the FTIR spectra. Manaf et.al. 2007

29. Manaf et.al. 2007

30. Manaf et.al. 2007

31. CONCLUSIONS The presence of palm kernel olein as adulterant in virgin coconut oil can be successfully detected by using FTIR spectroscopy. A multivariate classification method such as discriminant analysis was able to classify pure and adulterated virgin coconut oil samples successfully down to an adulterated level of 1%. Multivariate quantification based on PLS successfully quantified the composition of palm kernel olein in virgin coconut oil.

32. Adulteration is a common practice in the field of virgin olive oil because of its greater demand at national and international level. The physicochemical properties studied were: density, refraction index and saponification index. Methods Density measurements, Refraction index of the analysed samples was measured using an Abbe refractrometer at 20 ° C, Saponification index C 3 H 5 (OOCR) 3 + 3KOH 3RCOOK + C 3 H 5 (OH) 3 Oroian et.al. 2014 Case study-2 -

33. Physicochemical parameters of virgin olive oil Density- 0.915 g/cm3 Refraction index- 1.467 Saponification index- 187.51 Oroian et.al. 2014

34. Results The saponification index can be used for the identification of virgin olive oil adulteration with sunflower and corn oils in substitutions greater than 30%. The suitable parameter for the adulteration identification is the density, at percentages of substitution greater than 5% it can be observed that the parameter values is getting out of the domain.

35. Argemone mexicana oil detection in edible oils Mexicana seeds contain 22–36% of a pale yellow non-edible oil, called argemone oil or katkar oil, which contains the toxic alkaloids sanguinarine and di-hydro-sanguinarine. S anguinarine D ihydrosanguinarine Sanguinarine is a toxin that kills animal cells through its action on the Na + /K + -ATPase trans-membrane protein. Epidemic dropsy is a disease that results from ingesting sanguinarine . Das et. al. 1997

36. Case study-3 M ustard oils for Argemone In number of cases, adulteration of Argemone mexicana seed oil in mustard oils has been reported as cause of epidemic dropsy . Oils are extracted from the seed and samples were analysed by HPTLC. Sample preparation: An aliquot of 20μl of Argemone oil was dissolved directly in chloroform and made up to final volume of 1 ml in a certified volumetric flask. Adulterated edible oil samples (50μl, each) were also dissolved directly in chloroform and made up to 1 ml in certified volumetric flasks. Procedure: 2μl of the working standard solution was applied by means of Linomat-V sample applicator over the plates about 1cm above the edge using a bandwidth of 2mm along with 2μl edible oil and adulterated edible oil samples. The chromatogram was developed under chamber saturation condition with solvent system Hexane: Acetone: Methanol (80:15:5 ). Sheler et. al. 2011

37. HPTLC chromatogram of adulterated mustard oil Pure mustard oil sheler et. al. 2011 Hexane: Acetone: Methanol (80:15:5) 365nm

38. Result HPTLC is a effective tool for determination of argemone oil adulteration up to lower concentration 0.5% v/v.

39. Study was conducted to check the purity of vegetable oil using HPTLC fingerprinting. Adulteration of vegetable oil is usually by using mineral oils . Due to scarcity of vegetable oils , often it is adulterated with mineral oils. Mineral oils are listed as group 1 carcinogens to humans . The proposed HPTLC method was found to be simple, rapid, accurate and reproducible for the identification and estimation of mineral oil in various vegetable oils. Case study-4 Kumar et.al. 2014

40. Quantitative evaluation of the plate was performed after developing with hexane and derivatization with anisaldehyde -sulphuric acid reagent and scanning at 650 nm . Kumar et.al. 2014

41. Linear calibration curve of the mineral oil For the quantification calibration curve of mineral oil was obtained by plotting peak areas verses concentration applied . Kumar et.al. 2014

42. Recent Advances: Fatty Acids as Markers for Adulteration Detection All the techniques mentioned earlier can detect the adulteration in the product available in the market which can just be utilized to take action against the culprits. But if the mixing or adulteration can be detected before the product comes into the market it will be help to save money along with the non renewable sources of energy. Fatty acids, an important component of edible oils, can help to serve the purpose. FA can act as marker to detect the adulteration in different edible oils.

43. Why Fatty acids as markers? Triacylglycerol's (TAGs) shares 90-95% in edible oil . Fatty acids have high sensitivity to adulterations identifications. Fatty acids compositions are stable. Fatty acids can be converted in to FAME(Fatty acids methyl esters).

44. Case study-5 The detection of adulteration of high priced oils is a particular concern in food quality and safety. F atty acid profiles of five edible oils were established by gas chromatography coupled with mass spectrometry (GC/MS) in selected ion monitoring mode. 28 fatty acids were identified and employed to classify five kinds of edible oils by using unsupervised (principal component analysis and hierarchical clustering analysis ), supervised (random forests) multivariate statistical methods . Experimental Procedure of Derivatization 0.06 g of vegetable oil sample was diluted with 2 mL diethyl ether and petroleum ether (v/v 1:1), and 1mL of 0.4 M KOH−CH3OH was added. Then, it was vortex-mixed for 30 s and placed at RT for 2.5 h, and then, 2 mL of redistilled water was added. It was then vortex-mixed and centrifuged at 4500 rpm for 2 min. The organic phase (200 μL) was collected and diluted by 800 μL of petroleum ether, prior to analysis by GC-MS. Zhang et. al. 2014

45. Zhang et. al. 2014

46. From the score plot obtained from PCA , five kinds of edible oils were clearly classified into five groups, among which rapeseed and peanut oils are far from sunflower, sesame and soybean oils, respectively . Zhang et. al. 2014

47. B lend oils appear in the centre. fatty acid profiles could detect adulteration of peanut , sunflower and sesame oils with other vegetable oils about the level of 10%. For soybean and rapeseed oils, pure and adulterated oils are heavily overlapped. Zhang et. al. 2014

48. Results Fatty acid profiles of these edible oils could classify five kinds of edible vegetable oils into five groups and employed to authenticity assessment. This model could identify five kinds of edible oils and sensitively detect adulteration of edible oil with other vegetable oils about the level of 10%.

49. Case study-6 A simple spectrophotometric method for detection and quantification of adulteration of olive oil with sunflower, corn and soybean oils was developed. This was done by measuring the characteristics of the absorption bands between 200 and 400 nm. In order to quantify the adulteration; synthetic mixtures were made by 0.5%, 1%, 5%, 25%, 50% and 75% percent's for each of the sunflower, corn and soybean oil in olive oil and the absorbance of each solution was measured at 268 nm against isooctane as a blank. Amereih et. al. 2014

50. Amereih et. al. 2014

51. Calibration curves represent absorbance versus percent of adulteration (x-axis) of : A- soybean , B- sunflower and C - corn in olive oil and their rational equations enabling detection and quantification of adulteration. Amereih et. al. 2014

52. If the absorbance measured is higher than 0.2 this an indication that the sample is subjected to adulteration with other seeds and vegetables oils since olive oil at this wave length doesn't show high absorption values at 268 nm referring to the lack of poly unsaturated fatty acids . Using calibration curves of absorbance versus percent of seed oils in olive oil; one can quantify the amount of adulteration, where the minimum detectable present of the examined oils in olive oil is less than 0.5 %. Amereih et. al. 2014 Results

53. Case study-7 This fingerprinting analysis was applied to genuine samples of olive, soybean, corn, canola, sunflower, and cottonseed oil, to admixtures of these oils, and samples of aged soybean oil. Adulteration of high price olive oil with low price oil is a major fraud in edible oil. Experimental Procedures- O il samples (250.0 microliter) were homogenized in a flask with a solution containing equal parts of water and methanol, completing the final volume of 1.0 mL. The phases were allowed to separate, and the top layer was removed. Catharino et. al. 2005

54. ESI-MS fingerprints in the negative ion mode of methanol/water extracts of the following: (A) olive, (B) soybean, (C) corn, ( D) canola , (E) sunflower, and ( F) cottonseed oil . Relative abundances of the ions of m/z 279 and 281 differenced olive oil from other . olive soybean corn canola sunflower cottonseed Catharino et. al. 2005

55. PCA of ESI(-)-MS PCA treatment of the ESI(-)-MS data separates the samples in six groups . Catharino et. al. 2005

56. Catharino et.al. 2005

57. Catharino et.al. 2005

58. Results ESI-MS fingerprint clearly differentiate olive oil from the five other refined oils. The method is also shown to detect aging and adulteration of vegetable oils.

59. Conclusio n It can be concluded that there is constant development of different techniques for edible oil adulteration detection such as ESI-MS fingerprinting analysis which is applied to genuine samples of olive, soybean, corn, canola, sunflower, and cottonseed oil, to admixtures of these oils, and samples of aged soybean oil. ESI-MS fingerprints in the negative ion mode clearly differentiate olive oil from the five other oils. This method also detect aging and adulteration of vegetable oils. Now a days new techniques are appearing which use modern instruments, which are partially or fully automated. Most often their main advantage is the simplicity of performance and short times needed for individual analysis. Such methods include chromatographic techniques such as GC/MS, which is used to detect adulteration of edible oil with other vegetable oils about the level of 10% in peanut, sunflower, sesame, soybean and rapeseed oils. This model could identify five kinds of edible oils. This is very popular techniques in vegetable oil analysis. Spectrophotometric method for detection and quantification of adulteration of olive oil with sunflower, corn and soybean oils was developed. Using calibration curves of absorbance versus percent of seed oils in olive oil, amount of adulterations are quantified. HPTLC method is developed for the identification of mineral oil and argemone oil in various vegetable oils. Physico -chemical properties such as density, saponification index and refraction index are used for the identification of virgin olive oil adulteration with sunflower oil, corn oils and groundnut oil. The presence of palm kernel olein as adulterant in virgin coconut oil can be successfully detected by using FTIR spectroscopy. A multivariate classification method such as discriminant analysis was able to classify pure and adulterated virgin coconut oil samples successfully down to an adulterated level of 1%.

60. Path Ahead Edible oil adulteration detection based on fatty acid profile is not so popular so it should be considered in future research. Geographical and seasonal variations in fatty acid profile of edible oils other than olive oil should also be considered in future research. Sale of loose edible oil should be banned across the country to control the health risk of consumers.