Oilseeds methods and tests used to measure quality

Oilseeds quality tests carried out by the Grain Research Laboratory are based on or calibrated against, methods developed by internationally recognized standards writing agencies such as the International Organization for Standardization (ISO) or the American Oil Chemists' Society (AOCS).

These methods have been accepted by many countries as the reference procedures to be used when settling trade disputes and have well-defined limits of repeatability and reproducibility established by collaborative testing.

Methods and tests

Chlorophyll content
Chlorophyll content is determined by International Organization for Standardization method reference number ISO 10519:1997(E), Rapeseed-Determination of chlorophyll content—Spectrometric method. Results are expressed as milligrams per kilogram (mg/kg), seed basis.
For analysis by the reference ISO 10519 method, chlorophyll is extracted from the seed with suitable solvents and determined by spectroscopy. Our laboratoy also uses near infrared spectrometers (NIR), calibrated with the ISO 10519 method and modified to include visible wavelengths to allow rapid prediction of the chlorophyll content of grain samples.
Chlorophyll is the green pigment found in all green plants and is essential for photosynthesis. In oilseeds, immature seeds contain large amounts of chlorophyll as they develop. This chlorophyll is metabolized as seeds mature, fully ripe seeds contain very little chlorophyll. Because of the indeterminate nature of the flowering and ripening process in B. napus canola, canola seed can contain large amounts of chlorophyll. This is exacerbated by Canadian canola growing conditions, a short growing season with seeding in May–June and harvesting in August–October. This causes problems during processing as the chlorophyll is extracted into the oil giving it a green or brown coloration which is hard and expensive to remove. Besides being of undesirable color, oils with high levels of chlorophyll are subject to become rancid and are difficult to make into margarine or shortening (hydrogenate).
For canola, chlorophyll is estimated in grading by visual assessment of "percent distinctly green seeds". It has been shown that a level of about 24 mg/kg (ppm) chlorophyll corresponds to about 2% distinctly green seeds when Canadian canola was made of B. rapa and B. napus. Trading specifications for canola oils have limits of 25 mg/kg to 30 mg/kg which corresponds to 22 mg/kg to 24 mg/kg in the seed.
Fatty acid composition
Fatty acid composition is determined according to ISO 12966-1:2014 (Animal and vegetable fats and oils—Gas chromatography of fatty acid methyl esters—Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters) and ISO 12966-2:2011 (Animal and vegetable fats and oils—Gas chromatography of fatty acid methyl esters—Part 2: Preparation of methyl esters of fatty acids) using a Supelcowax 10 (15m by 0.32mm column with a 0.25 µm). Major and important fatty acids are reported although samples may also contain as much as 1% of other minor fatty acids which are included in the calculations.
Fatty acid composition and iodine values (calculated from the fatty acid composition) are determined by a process which starts with the extraction of a portion of the oil followed by the chemical conversion of the fatty acids of the triacylglycerols into individual fatty acid methyl esters via alkaline derivatization. The fatty acid methyl esters are then analyzed by gas chromatography where they are separated and according to their carbon chain length and their number of unsaturation. The method is rapid, accurate and reliable and can be used to analyze large number of samples.
The functional and nutritional values of different vegetable oils are dependent on the nature of the different fatty acids which are incorporated like building blocks into the oil as triacylglycerols. For example, erucic acid makes up about 50% of the fatty acids of traditional rapeseed oil and is the desired product for most industrial uses of this oil. Canola oil, on the other hand, was designed to contain "zero" erucic acid (C22:1) for nutritional reasons. By definition, erucic acid must make up less than 2% of all fatty acids in canola oil. The term "canola oil" is used to describe an oil derived from the seed of the genus Brassica (Brassica napus, Brassica rapa and Brassica juncea), with less than 2% of all fatty acids as erucic acid. Canola oil also contains low levels (less than 7.0% )of saturated fatty acids, mostly palmitic and stearic acid, high levels of mono-unsaturated fatty acids (mostly oleic acid an omega-9), and has a good ratio of polyunsaturated fatty acids (linolenic acid an omega-6 versus α-linoleic an omega-3, close to 2).
Fatty acid composition is mostly effected by variety and growing environment. It is important to know the effect of environment on the fatty acid composition of canola in order to determine where seed can be harvested to make oils meeting the specifications for saturated fatty acids required by U.S. processors (less than 7%).
Iodine value
Iodine value is a measure of unsaturation calculated from the fatty acid composition according to AOCS Recommended Practice Cd 1c-85, revised 1995 and re-approved 1997, Calculated Iodine Value.
Iodine value, or iodine number, is a measure of the total amount of unsaturated fatty acids in an oil. By definition, it is the measure of the number of grams of iodine which will combine with 100 grams of the oil. While the reference method originally involved actually reacting samples of oil with iodine, iodine value can be calculated from the fatty acid composition of the oil as it is done using AOCS Cd 1c-85.
In routine surveys, iodine values for flaxseed or canola may be predicted using an NIR spectrophotometer calibrated with AOCS Cd 1c-85 method. In flaxseed, high iodine values (189 or greater) are preferred for the manufacture of paints and inks whereas somewhat lower values (ca. 182) are needed for linoleum manufacture.
Free fatty acids content
Free fatty acid content is determined by a method adapted from the procedure of Ke et al, Analytica Chemica Acta 99:387–391 (1978), and is expressed as a percentage by weight of oleic acid in the oil. Oleic acid with a molecular weight of 282 is used as the fatty acid for the expression of the results. The indicator and reagent options are also listed in ISO 660 Animal and vegetable fats and oils-Determination of acid value and acidity.
Free fatty acids are measured by an acid-base titration of the oil extracted from the seed during an oil content determination.
This test measures the portion of the oil (triacylglycerols) which has broken down (hydrolyzed) due to chemical or microbiological activity. Free fatty acids must be removed during processing as they reduce the smoke point in frying fats and rapidly oxidize to give rancid flavors. The test gives a direct measure of the ability of the oil to be processed and is actually used to estimate the amount of lye required to refine oils. As this test is a reflection of seed quality, it reflects seed damage and grade. Top grade canola seed usually has less than 0.7% free fatty acids although in certain years in Eastern Canada and in 1989 in Western Canada this level was exceeded for as yet undetermined reasons. International specifications for top grade oil usually are set at 2% free fatty acids.
Glucosinolate content
Glucosinolate content is determined by International Organization for Standardization method reference number ISO 9167–3: 2007 (E) Rapeseed—Determination of glucosinolate content—Part 3: Spectrometric method for total glucosinolates by glucose release. Results are total seed glucosinolates expressed as micromoles per gram (µmol/g), calculated to an 8.5% moisture basis for canola or on a dry matter basis for all mustard seeds.
Glucosinolates are ionic, that is they are charged molecules. They are determined by first extracting them from the seed into boiling water, then isolating them from interfering components by ion-exchange chromatography. They are then converted into an uncharged molecule with an enzyme, and are separated and quantitatively analyzed by high performance liquid chromatography. With ISO 9167-3 method, instead of an HPLC analysis, the uncharged glucosinolates are furhter hydrolyzed by an enzyme with one molecule of glucosinolate giving one molecule of glucose. Total glucose is then measured spectrometrically, leading to the determination of total glucosinolates. Total glucosinolate content may also be predicted using NIR spectrometer calibrated using the ISO 9167–3: 2007 method.
Glucosinolates are natural components of canola, rapeseed, and mustard seed. These compounds are found in all Brassica vegetables (cabbage, brussel sprouts, radishes, broccoli, cauliflower) and are responsible for the desirable pungent odor and sharp flavor associated with these foodstuffs. Glucosinolates are also natural toxicants, being associated with goiter and liver damage when consumed in large quantities. Brassica seeds such as rapeseed and mustard are particularly rich in glucosinolates while canola seeds by definition have much lower total glucosinolate contents. While high levels of glucosinolates may be desirable in the case of mustard seed destined for condiment use, the high levels of glucosinolates found in rapeseed meal have restricted the use of this seed as a source of protein in compound feeds. Plant breeding to reduce the level of glucosinolates in rapeseed resulted in canola seed. Nutritional studies have demonstrated that substantially more canola meal can be used in compound feeds than rapeseed meal. The current definition of canola requires that only part of the total glucosinolates in a sample (the aliphatic glucosinolates) be measured. Canola seed contains less than 30 micromoles per gram of these aliphatic compounds expressed on an oil-free, 8.5% moisture basis. Nowadays, Brassica napus canola developed in Canada contain less than 18 micromoles/gram of total glucosinolates on a whole seed, 8.5% moisture basis.
Oil content
Oil content is determined by nuclear magnetic resonance (NMR) according to the International Organization for Standardization, reference number ISO 10565:1992(E) Oilseeds—Simultaneous determination of oil and moisture contents—Method using pulsed nuclear magnetic resonance spectroscopy. A Bruker Mq10 Minispec NMR Analyzer calibrated with appropriated oilseed samples extracted with petroleum ether according to the ISO 659:2009 (Oilseeds—Determination of oil content (Reference method)). This method is the reference method for oil content and is recommended by the Federation of Oils, Seeds and Fats Associations Ltd (FOSFA) International in its list of official methods of analysis.
The oil content, according to ISO 659:2009, is measured directly by grinding the seed and extracting the oil using a a 2055 Soxtec Manual Extraction Unit (Soxtec Avanti, Foss). The official method call for repeated solvent extraction steps followed by further grinding until no further oil is removed. It is a very long method; the total analysis time can be in the order of 2.5 days per sample with a maximum throughput of 12 samples per day in our facility. Indirect oil content analysis can be carried out using either NMR or NIR Spectroscopy as described by ISO 10565:1992. The NMR technique measures the resonance energy absorbed by hydrogen atoms in the liquid state of the sample, NMR methods give very accurate and precise results. Oil content can also be predicted using NIR spectrometer, utilizing the absorption of near infrared energy (1100-2500 nm) by the sample. Sample throughput is increased by both NMR and NIR methods, however, the precision of NIR methods is not as good as extraction methods.
Oil content can be defined as the maximum amount of material (lipid) that can be removed from the seed by extraction with organic solvents, hexane or petroleum ether. This test estimates the amount of oil which could optimally be obtained in industrial crushing. An estimated 97% to 99% of the oil content determined by the analytical method may be removed by commercial solvent extraction or prepress solvent extraction systems. Direct pressing (or cold pressing) usually removes about 90% to 92% of the oil. The quality of the oil removed analytically also differs greatly from the quality of the oil removed in industrial processing. Oil removed analytically usually consists of about 99% triacylglycerol molecules (the desired end product of oil processing) and is relatively colorless. Pretreatment of the seed to increase the efficiency of industrial extraction results in a highly colored crude oil which consists of about 96% of the desired triacylglycerol molecules. The undesired material and color is removed by refining and bleaching.
For canola, oil contents are expressed on an 8.5% moisture basis. This convention was adopted in 1970 at the request of the oilseed trade who wished to be able to compare results from our surveys with international trading specifications which were expressed on a telle quelle moisture basis. Canadian trading contracts usually specify a minimum of 41% oil content for canola. At the time, the moisture content of 8.5% was chosen as a reasonable average moisture content for Canadian rapeseed although moisture contents for recent years' exports of canola have ranged from 7% to 8%. For flaxseed and soybeans, oil contents are expressed on a moisture-free basis although trading specifications usually call for a telle quelle moisture basis.
Protein content
Protein content is determined by the AOCS Official Method Ba 4e-93, revised 1995 and re-approved 1997, Combustion method for determination of crude protein. Results are reported as a percentage, N x 6.25, calculated to specified moisture basis. Canola is calculated to an 8.5% moisture basis, whereas flaxseed, soybean and all mustard seeds are calculated on a dry matter basis.
With this method, grain samples are ground and heated at a very high temperature (about 900°C) in presence of oxygen. This combustion leads to the release of carbon dioxide, water and nitrogen. The nitrogen gas is measured and the response of the instrument is converted to nitrogen content (after instrument calibration with a pure compound of known nitrogen concentration). The measured nitrogen result is converted into crude protein content a specific factor which depends on the amino acid sequence of the analyzed grain. For oilseeds, by convention, the factor is 6.25.

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