In silico – Sequence homology studiesProteins sharing similar primary structure and threedimensional folds may act as cross reacting allergens.
These findings form thebasis of in silico studies whichevaluate the allergenic potential of transgenic proteins. Sequence homologystudies compare the transgenic protein sequences with the known allergens toidentify probable homologues18. BLAST19 and FASTA20are popular alignment tools used for the comparison studies they predictfunctional similarity and clinically important cross reactivity among proteins.The basis of bioinformatic studies is that if proteins possess reasonablelinear sequence similarity, they tend to share three dimensional structuralmotifs21 and are more likely to share allergenic cross reactiveepitopes. The criterion recommended by FAO/WHO (2001) and Codex (2003) statesthat protein(s) having greater than 35% identity over any segment of 80 aminoacids between the GM protein and any allergen depicts that the query sequencemay be a potential allergen and should be subjected to further testing. Aalberse and group suggests that proteins showinggreater than 70% identity over an 80 a.a.
sliding window are more likely toshare IgE cross reactivity than proteins having less than 50% identity22, 23.According to safety assessment protocols if high degree of similarity isobserved among sequences, IgE serum screening is mandatory to further validatethe safety of the crop. Several allergen databases are available online forhomology studies including Food Allergy Research and Resource Program (FARRP),Structural Database of Allergenic Protein (SDAP) 24,Allermatch25 and AlgPred26. Pepsin resistance, in vitro digestibilityassay and thermal stabilityFAO/WHO(2001), Codex Alimentarius (2003) and ICMR (2008) suggest that transgenicproteins need to be assessed for simulated gastric fluid (SGF) digestibilityand thermal stability. These tests are designed to mimic the physiologicalconditions of gastric digestion and evaluate the allergenic potential offoreign proteins.
Several studies suggest that a correlation exists between thepotential of a protein to act as an allergen and its resistance to SIFdigestion, pepsin degradation and thermal stability. However, numerous reports havealso demonstrated that this correlation is not absolute and that proteins thatare resistant to pepsin degradation or stable to high temperatures might not beallergenic in normal conditions of exposure whereas labile proteins could beallergenic27, 28, 29. Pepsin resistance cannot be considered as a strong evidence ofabsence of intrinsic allergenicity of the protein or as a demonstration thatthe newly expressed proteins is likely to be digested with loss of its toxic orallergenic potential. However a relationship between digestibility andallergenicity exists and many food allergens that sensitize through the oralroute display some stability during gastric and/or intestinal digestion inphysiological conditions30, 31, 32. The outcomes of thedigestibility assays should be interpreted in conjunction with results of otherassays and other properties of the protein under consideration. In addition,the outcomes of the in vitro digestibility tests should be interpretedwith care as they represent model conditions. In vitro proceduresusually do not reflect the fluctuations in pH values and enzyme to proteinratios that occur in vivo after consumption of a meal. Interpreting theoutcomes of the in vitro digestibility studies on the isolated newlyexpressed protein in the light of other factors, such as the abundance of theprotein within the food, interactions with the food matrix and possiblestructural/functional alterations occurring during food processing would beuseful.
Although digestibility of a newly expressed protein is an importantissue to address when assessing the risk of sensitization via the oral route,it is to be noted that sensitization may also result from respiratory or cutaneousexposures. Some of thecommonly used food processing techniques like fermentation, salting, tenderizing and pasteurization render the proteins linearisedand alter the native structure of the proteins thereby affecting the overallallergenicity of the foods33. Heat treatments leads to disruption ofthe native 3-D structure of proteins leading to loss of conformationalepitopes, activation of new epitopes or improving accessibility of crypticepitopes34. Abroad temperature range is taken in to consideration for heat treatmentexperiments i.e. from 25°C to 95°C for up to 60 minutes14. Thermalprocessing includes moist or dry heating of foods that leads to reducedallergenicity like pollen-related fresh fruits and vegetable food allergens35.
Among non-thermal processing methods, ?-irradiation has been used to controlfood borne pathogens, reduce microbial load and insect infestation, inhibit thegermination of root crops and extend the shelf life of perishable products.Irradiation reduces the antigenicity of ovalbumin, bovine serum albumin andmilk protein and shrimp tropomyosin36, 37, 38.