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Detection of allergenic proteins in refined oils
It has been assumed that the physical and chemical processes involved in refining edible oils are so rigorous that there is an almost complete removal of proteins which might act as allergens. Nevertheless, there remains the possibility that residual traces could still provoke an allergic reaction in particularly sensitive individuals. With this in mind, Matteo Ramazzotti and colleagues from the University of Florence in Italy have developed methods for the extraction and analysis of any residual proteins in commercial samples of soy, peanut, sunflower and maize oils.
In their study, Ramazzotti et al. optimised a single step protein extraction method using phosphate buffered saline (PBS) at pH 6.5, which they applied to the commercial oil samples. Extracted proteins were quantified by colorimetric assays and amino acid analysis, and preliminary tests were carried out to assess the allergenicity of the proteins which had been extracted.
The authors explain that due to the extremely low protein content of the oils, attention was paid to the use of appropriately cleaned glassware or disposable plastics in order to avoid dust and protein contamination from other sources. Only in the case of soybean oil, for which the PBS/oil separation was poor, was isobutanol added prior to centrifugation. The samples were then dialysed for 48 hours at room temperature to remove the salt, and the salt-free proteins dried in a vacuum-concentrator centrifuge and stored at -20 o C until use. Following acid hydrolysis of the proteins, amino acids were separated and analysed with an amino acid analyser and results used to estimate average total protein content. Electrophoresis of the protein extracts allowed estimates of their molecular weights to be made. Extracted proteins were electro-transferred on to a nitrocellulose membrane by Western blot techniques and then incubated with sera derived from children allergic to soy, maize or peanut allergens. Negative control sera from non-allergic children were also obtained.
The colorimetric assays were able to measure total residual protein content varying from 6.5 - 19 μg/100 ml of oil. By amino acid analysis the figures ranged from 33 - 166 μg/100 ml of oil. Proteins were present in the two commercial samples of soybean oil analysed, with protein band patterns differing, probably due to the different refining methods used. For the two maize oils, similar results were seen, but in this case staining revealed three major proteins bands at 12 kDa, 60 kDa and >200 kDa.
Peanut oils were shown to contain three major protein bands around 30 - 40 kDa region with Coomassie Blue staining. Silver nitrate staining revealed a further two protein bands at 14 and 66 kDa. The band at 66 kDa had a molecular weight similar to the known peanut allergen - Ara h 1. However, results from the tests with the allergic-child sera showed the presence of IgE responsive proteins in refined peanut oil, but with molecular weights of 70 - 80 kDa, which the authors say are not presently known to be associated with peanut allergens.
There was a lack of response to the sera for soybean and maize extracts. IgE levels for soybean and maize were all classifiable in RAST CAP classes 1 and 2, with IgE levels below 17.5 kU/litre.
In conclusion, it is suggested that immuno-based methods should be applied more widely to refined seed oils before it can be confidently asserted they are safe for allergic individuals. (Ramazzotti et al. Food and Chemical Toxicology, article in press, doi:10.1016/j.fct.2008.08.005).
Also in same issue of Food & Chemical Toxicology, Food and Chemical Toxicology, in press doi:10.1016/j.fct.2008.07.021, "Current Codex Guidelines for Assessment of Potential Protein Allergenicity" by G.S. Ladics, DuPont Co, Wilmington, DE .(No abstract available yet).
RSSL's DNA and Protein Laboratory carries out allergen testing using immunological, DNA and distillation techniques, depending on the allergen to be detected. Detection limits are in the range 0.1 - 10 mg allergen/kg of sample for almond, Brazil nut, macadamia nut, peanut, walnut, hazelnut, cashew nut, pistachio nut, pecan nut, pine nut and chestnut. Celery, celeriac, black mustard, lupin and kiwi allergens can also be detected by DNA methods, as can crustacean allergens. The laboratory also uses a range of UKAS accredited immunological procedures for the detection of allergens including gluten, peanut, hazelnut, almonds, soya, egg, milk, lactose, sesame and histamine. Distillation and titration methods are used for the determination of sulphur dioxide and sulphites. For more information please contact Customer Services on Freefone 0800 243482 or e-mail enquiries@rssl.com.
Related RSSL services: http://www.rssl.com/...llergenTesting/
