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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.

List of oilseeds 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.
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 destroyed (metabolized) as the seed matures and fully ripe seeds contain very little chlorophyll. Because of the indeterminate nature of the flowering and ripening process in B. napus canola, some canola seed lots contain large amounts of chlorophyll. This causes problems in 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". A level of about 24 mg/kg (ppm) chlorophyll corresponds to about 2% distinctly green seeds. 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. For analysis, chlorophyll is extracted from the seed with suitable solvents and determined by spectroscopy. Approximately 50 samples per day can be analyzed by this method. Near infrared (NI) instruments can be modified to allow rapid chlorophyll determination, increasing the throughput to 200 samples per day.
Fatty acid composition
Fatty acid composition is determined by the International Organization for Standardization method reference number ISO 5508:1990 (E), Animal and vegetable fats and oils-Analysis by gas chromatography of methyl esters of fatty acids.A 15m by 0.32mm column with a 0.25 µm Supelcowax 10 coating is used. 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.
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 (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. In the future the term "canola oil" will be used to describe an oil derived from the seed of the genus Brassica, with less than 1% of all fatty acids as erucic acid. Recent sales of canola oil products in the U.S. have been based upon this oil's low level of saturated fatty acids (mostly palmitic and stearic acid), its high level of mono-unsaturated fatty acids (mostly oleic acid), and its good ratio of polyunsaturated fatty acids (linoleic and linolenic acids). ----- Iodine value, or iodine number, is a measure of the total amount of unsaturated fatty acids in an oil and is a measure of the number of grams of iodine which will combine with 100 grams of the oil. While the method originally involved actually reacting samples of oil with iodine, most laboratories now calculate iodine values from the fatty acid composition. In routine surveys, iodine values for flaxseed may be determined by NIR analysis. In flaxseed, high iodine values (189 or greater) are required for the manufacture of paints and inks. Somewhat lower values (ca. 182) are needed for linoleum manufacture. ----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. Similarly, it is important to know which areas produce canola with levels of iodine value suitable for coating manufacture and which produce canola with levels suitable for linoleum manufacture. ----- Fatty acid composition and iodine values are determined by a process which starts with extraction of a portion of the oil and its chemical conversion into individual fatty acids (taking apart the triacylglycerols). The fatty acids are then converted to methyl esters, compounds which can easily be converted to gases. The different fatty acids are then separated and analyzed on a gas chromatograph. The method is rapid and accurate. Starting from seed, a single sample can be analyzed within 30 minutes. Using automated equipment and overnight operation, complete fatty acid profiles can be obtained on as many as 100 samples per day.
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.
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.
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. ----- Free fatty acids are measured by an acid-base titration of the oil extracted from the seed during an oil content determination. The procedure is time consuming but is really restricted by the analytical extraction capabilities to about 18 samples per day. More rapid methods for free fatty acid estimation are being developed.
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 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 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. ----- Glucosinolates are ionic, that is they are charged molecules. They are determined by first extracting them from the seed into 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. 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. The current definition will be changed in the future so that canola seed will be seed of the genus Brassica which shall contain less than 18 micromoles/gram of total glucosinolates on a whole seed, 8.5% moisture basis. This will allow other more rapid and less expensive methodologies which determine total glucosinolates to be used. Glucosinolates may also be determined by NIR analysis. ----Glucosinolate levels are highly dependent on environment with up to three-fold differences being noted between seed planted and seed harvested. It is important that the glucosinolate level in the crop be monitored closely to determine if the canola specification is being met.
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 NMS 110 Minispec NMR Analyzer calibrated with appropriate oilseed samples extracted with petroleum ether is used. Results are reported as a percentage, calculated to a specified moisture basis. Canola is calculated to an 8.5% moisture basis, and flaxseed, solin, soybean and all mustard seeds are calculated on a dry matter basis. ---- The NMR instrument is calibrated with oilseed samples extracted with petroleum ether according to the F.O.S.F.A. International Official Method “Oilseeds-Determination of Oil Content-Solvent Extraction (Reference Method)” as described in F.O.S.F.A. International, Technical Manual, Part Two, Standard Contractual Methods, pp. 64-71, Federation of Oils, Seeds and Fats Association Limited, London, 2005.
Oil content can be defined as the maximum amount of material (lipid) that can be removed from the seed by extraction with specific solvents (usually 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 estimated analytically 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. ---- Oil content is measured directly by grinding the seed and extracting the oil in a continuous extractor. The official methods call for extracting for periods of 1 to 3 hours followed by further grinding and extraction periods until no further oil is removed. Total analysis time is in the order of 6 to 8 hours per sample with a maximum throughput of 18 samples per day. Indirect oil content analysis can be carried out using either Nuclear Magnetic Resonance (NMR) Spectroscopy or Near Infrared (NI) Spectroscopy. The NMR technique measures the resonance energy absorbed by hydrogen atoms in the liquid state of the sample while NI utilizes the absorption of near infrared energy (1100-2500 nm) by the sample. Sample throughput is increased to about 100 per day for NMR and 200 per day for NIR. While the precision of NIR methods is not as good as extraction methods, NMR methods give very accurate and precise results when calibrated carefully. ------ 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 40% oil content for canola. 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, and flaxseed, solin, soybean and all mustard seeds are calculated on a dry matter basis.

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