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In silico – Sequence homology studies

Proteins sharing similar primary structure and three
dimensional folds may act as cross reacting allergens. These findings form the
basis of in silico studies which
evaluate the allergenic potential of transgenic proteins. Sequence homology
studies compare the transgenic protein sequences with the known allergens to
identify probable homologues18. BLAST19 and FASTA20
are popular alignment tools used for the comparison studies they predict
functional similarity and clinically important cross reactivity among proteins.
The basis of bioinformatic studies is that if proteins possess reasonable
linear sequence similarity, they tend to share three dimensional structural
motifs21 and are more likely to share allergenic cross reactive
epitopes. The criterion recommended by FAO/WHO (2001) and Codex (2003) states
that protein(s) having greater than 35% identity over any segment of 80 amino
acids between the GM protein and any allergen depicts that the query sequence
may be a potential allergen and should be subjected to further testing. Aalberse and group suggests that proteins showing
greater than 70% identity over an 80 a.a. sliding window are more likely to
share IgE cross reactivity than proteins having less than 50% identity22, 23.
According to safety assessment protocols if high degree of similarity is
observed among sequences, IgE serum screening is mandatory to further validate
the safety of the crop. Several allergen databases are available online for
homology studies including Food Allergy Research and Resource Program (FARRP),
Structural Database of Allergenic Protein (SDAP) 24,
Allermatch25 and AlgPred26. 

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Pepsin resistance, in vitro digestibility
assay and thermal stability

(2001), Codex Alimentarius (2003) and ICMR (2008) suggest that transgenic
proteins need to be assessed for simulated gastric fluid (SGF) digestibility
and thermal stability. These tests are designed to mimic the physiological
conditions of gastric digestion and evaluate the allergenic potential of
foreign proteins. Several studies suggest that a correlation exists between the
potential of a protein to act as an allergen and its resistance to SIF
digestion, pepsin degradation and thermal stability. However, numerous reports have
also demonstrated that this correlation is not absolute and that proteins that
are resistant to pepsin degradation or stable to high temperatures might not be
allergenic in normal conditions of exposure whereas labile proteins could be
allergenic27, 28, 29. Pepsin resistance cannot be considered as a strong evidence of
absence of intrinsic allergenicity of the protein or as a demonstration that
the newly expressed proteins is likely to be digested with loss of its toxic or
allergenic potential. However a relationship between digestibility and
allergenicity exists and many food allergens that sensitize through the oral
route display some stability during gastric and/or intestinal digestion in
physiological conditions30, 31, 32. The outcomes of the
digestibility assays should be interpreted in conjunction with results of other
assays and other properties of the protein under consideration. In addition,
the outcomes of the in vitro digestibility tests should be interpreted
with care as they represent model conditions. In vitro procedures
usually do not reflect the fluctuations in pH values and enzyme to protein
ratios that occur in vivo after consumption of a meal. Interpreting the
outcomes of the in vitro digestibility studies on the isolated newly
expressed protein in the light of other factors, such as the abundance of the
protein within the food, interactions with the food matrix and possible
structural/functional alterations occurring during food processing would be
useful. Although digestibility of a newly expressed protein is an important
issue 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 cutaneous

                                 Some of the
commonly used food processing techniques like fermentation, salting, tenderizing and pasteurization render the proteins linearised
and alter the native structure of the proteins thereby affecting the overall
allergenicity of the foods33. Heat treatments leads to disruption of
the native 3-D structure of proteins leading to loss of conformational
epitopes, activation of new epitopes or improving accessibility of cryptic
epitopes34. A
broad temperature range is taken in to consideration for heat treatment
experiments i.e. from 25°C to 95°C for up to 60 minutes14. Thermal
processing includes moist or dry heating of foods that leads to reduced
allergenicity like pollen-related fresh fruits and vegetable food allergens35.
Among non-thermal processing methods, ?-irradiation has been used to control
food borne pathogens, reduce microbial load and insect infestation, inhibit the
germination of root crops and extend the shelf life of perishable products.
Irradiation reduces the antigenicity of ovalbumin, bovine serum albumin and
milk protein and shrimp tropomyosin36, 37, 38.


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