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Edible oil adulterations
1.
2. 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
3. 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 plant origin
• Less saturated
• Lower melting point
• Less stable
Examples- hazelnut oil, olive oil , mustard oil, sunflower oil etc.
6. Health Benefits of Edible Oils
Decrease in Risk for Heart Disease
Decrease in Risk for Breast Cancer
Omega-3 Fatty Acids
Better digestion
Vitamin E
Promote Cell Growth
Antioxidants
Indelicato et.al. 2017
Improvement of Metabolism
Source of energy
7. Current issues in edible oil adulteration in India
Admixing 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
8.
9.
10.
11. 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
12. Glycerol
Tri-hydric alcohol and popular name glycerin.
It is synthesized in the body from glucose.
Colourless viscous oily liquid with sweet taste.
CH2 OH
CH
CH2 OH
HO
CHO
CH
CH2
2 H2O
Heating, KHSO4
Glycerol Acrolein
13. 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-(CH2)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
14. Saturated Fatty Acids
They 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, CnH2n+1COOH.
Acetic F.A. (2C ) CH3-COOH.
Butyric F.A. (4C ) CH3-(CH2)2-COOH.
Caproic F.A. (6C ) CH3-(CH2)4-COOH.
Caprylic (8 C ) CH3-(CH2)6-COOH.
Capric (10 C ) CH3-(CH2)8-COOH
Palmitic (16C) CH3-(CH2)14-COOH
Stearic (18 C ) CH3-(CH2)16-COOH
Lignoceric (24C ) CH3-(CH2)22-COOH
15. Unsaturated Fatty Acids
They contain double bonds.
Monounsaturated
They contain one double bonds .
(CnH2n-1 COOH)
Polyunsaturated
They contain more the one double bond (CnH2n-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.
CH3-( CH2 )5CH = CH-(CH2)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.
CH3-(CH2)7- CH=CH – (CH2)7-COOH
16. Linoleic
C18:2∆9, 12
It is the most important since other essential fatty acids can be synthesized from it in the
body.
CH3-(CH2)4-CH = CH-CH2-CH=CH-(CH2)7-COOH
Linolenic acid
C18:3∆9, 12, 15
In corn, linseed, peanut, olive, cottonseed and soybean oils.
CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)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.
CH3-(CH2)4-CH=CH-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)3-COOH
17. 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.
18. Effects of adulterations
Gallbladder 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.
19. Argemone oil mix with Edible oils
causes-
Epidemic dropsy & Glaucoma
Loss of eyesight
Das et. al. 1997
21. 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
22. 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
23. 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
24.
25.
26.
27. 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
28. 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
29. Raman Spectroscopy
Adulterations 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
30. 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
31. 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
32. 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
35. Cold 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.
Trans 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
36. 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
39. 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.
40. 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
C3H5(OOCR)3 + 3KOH 3RCOOK + C3H5(OH)3
Oroian et.al. 2014
Case study-2
-
42. 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.
43. 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.
Sanguinarine
Dihydrosanguinarine
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
44. Case study-3
Mustard 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
45. HPTLC chromatogram of adulterated mustard oil Pure mustard oil sheler et. al. 2011
Hexane: Acetone: Methanol (80:15:5)
365nm
46. Result
HPTLC is a effective tool for determination of argemone oil
adulteration up to lower concentration 0.5% v/v.
47. 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
48. 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
49. 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
50. 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.
51. 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).
52. Case study-5
The detection of adulteration of high priced oils is a particular concern in food quality and safety.
Fatty 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
54. 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
55. Blend 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
56. 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%.
57. 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
59. 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
60. 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
61. 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- Oil 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
62. 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
63. PCA of ESI(-)-MS
PCA treatment of the
ESI(-)-MS data separates
the samples in six groups.
Catharino et. al. 2005
67. Conclusion
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%.
68. 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.