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Allergenic Protein Content in Apples

Allergenic Protein Content in Apples. Michelle Campeau Chem 4101 December 9, 2011. Background. Oral allergy syndrome causes the body to react negatively to proteins in fresh fruits, vegetables, and nuts. The severity of reactions can range from an itchy mouth to anaphylactic shock .

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Allergenic Protein Content in Apples

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  1. Allergenic Protein Content in Apples Michelle Campeau Chem 4101 December 9, 2011

  2. Background • Oral allergy syndrome causes the body to react negatively to proteins in fresh fruits, vegetables, and nuts. • The severity of reactions can range from an itchy mouth to anaphylactic shock. • Mal d 1 is the primary protein responsible for causing allergic reactions.

  3. Analyte • Mal d 1 is produced in apples when the fruit is under environmental stres. • Breaks down when exposed to heat (60° C) or during digestion due to pepsi. • Protein Sequence: MGVYTFENEFTSEIPPSRLFKAFVLDADNLIPKIAPQAIKQAEILEGNGGPGTIKKITFGEGSQYGYVKHRIDSIDEASYSYSYTLIEGDALTDTIEKISYETKLVACGSGSTIKSISHYHTKGNIEIKEEHVKVGKEKAHGLFKLIESYLKDHPDAYN Figure 1. 3D model of the Mal d 1 protei.

  4. Problem and Hypothesis • Problem: • In apples, the protein Mal d 1 is the protein primarily responsible for triggering allergic reactions in OAS patients. • The amount of Mal d 1 present in an apple varies from species to specie. • Hypothesis: • Mal d 1 content in a crossbred apple, such as the Honeycrisp, is proportional to that of the parent species.

  5. Separation Techniques Considered

  6. Detectors Considered

  7. Sample Preparation Removal of the Apple Core Homogenization of pulp in potassium phosphate buffer (10 mM HP, 10 mM P, pH 7) using a grinde Supernatant drawn off and frozen at -80°C for storage Incubation at room temperature for 4 hours Centrifuged at 4°C for 15 minutes at 5000g

  8. Separation • The four Mal d proteins vary in molecular weight and can be easily separated with size-exclusion chromatograph. • Eluent: phosphate-buffered saline (pH 7.4 Figure 2. Chromatograph of separation of Mal d 1 and

  9. Size-Exclusion Chromatography • Separates analytes based on size, allowing for potential quantification of all four Mal d proteins • Larger molecules elute quickly • Smaller analytes spend more time trapped in the porous column Figure 3. Diagram of a size exclusion chromatography colum

  10. Analysis • Mal d 1 can be derivatized with SYBR Green I in order to be selectively detecte • Excitation λ = 497 nm • Emission λ = 520 nm FLS920-s Spectrometer from Edinburgh Instruments Lt *for the FLS920-s Spectrometer from Edinburgh Instruments Ltd

  11. Conclusion • Best separation and analysis with size-exclusion chromatography and fluorescence spectroscopy • Future possibilities: • Potential to breed apples with low concentrations of Mal d 1, causing it to be more hypoallergenic • Since little is known of Mal d 2, 3, and 4, this analysis could be applied to them as well.

  12. References • Canadian Food Inspection Agency. “Food Allergens – Oral Allergy Syndrome.” Canadian Food Inspection Agency. 07-15-2009. < http://www.inspection.gc.ca/english/fssa/concen/tipcon/orale.shtml >. • " FLS920-s Spectrometer (Spectro-Fluorimeter) from Edinburgh Instruments Ltd." LabCompare. <http://www.labcompare.com/407-UV-VIS-NIR-Spectrometer/42977-FLS920-s-Spectrometer-Spectro-Fluorimeter/>. • Internet Symposium on Food Allergens. Matthias Besler. 2000, Vol 4, No 2. Internet Symposium on Food Allergens. <www.food-allergens.de>. • J. Szamos et all. " Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple." Food Research International (2011): pgs 2674-2678. • Maria Puehringer, Helene et all. "MdAP, a novel protein in apple, is associated with the major allergen Mal d 1." Gene 321(2003): pgs. 173-183. • Matthes, Anne, Schmitz-Eiberger, Michaela . "Apple (Malus domestica) Allergen Mal d 1: Effectof Cultivar, Cultivation System, and Storage Conditions." J. Agric. Food Chem 57 (2009): 10548-10553. • Mogensen, Jesper et all. “The Major Birch Allergen, Bet v 1, Shows Affinity for a Broad Spectrum of Physiological Ligands.” Journal of Biological Chemistry3-1-02 <http://www.jbc.org/content/277/26/23684.full>. • Size-Exclusion Chromatography. University of Adelaide. <http://www.chemistry.adelaide.edu.au/external/soc-rel/content/size-exc.htm>. • Skozo, Eva. “Protein and Peptide analysis by capillary zone electrophoresis and micellar electrokinetic chromatography." Electrophoresis 18 (1997): pgs. 74-81. • University of Texas Medical Branch. "Allergen Mal d 1." SDAP: Structural Database of Allergenic Proteins. 2011. University of Texas Medical Branch. <http://fermi.utmb.edu/cgi-bin/SDAP/sdap_02?dB_Type=0&allid=304>.

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