Multiplex-Competitive ELISA for Detection and Characterization of Gluten during Yogurt Fermentation: Effects of Changes in Certain Fermentation Conditions on Gluten Protein Profiles and Method Reproducibility Assessment

Abstract

The protein/peptide profiles of gluten during yogurt fermentation were evaluated using an optimized multiplexcompetitive ELISA by preparing yogurts incurred with gluten at different concentrations and by varying certain fermentation conditions. Analysis indicated that epitope-specific responses with antibody binding to glutenin epitopes decreased less during longer fermentation times or at higher starter culture concentrations relative to gliadins. Incomplete proteolysis was observed after 24 h of fermentation, which became more efficient as fermentation time was increased. Western blot confirmed the results of ELISA. Cluster analysis indicated that out of the investigated parameters, fermentation time is the only parameter that could affect the overall gluten protein/peptide profiles during yogurt fermentation. This parameter needs consideration in evaluating the suitability of calibrant(s) to be used with the multiplex-competitive ELISA or any other methods to ensure accurate quantitation of gluten in yogurts and potentially in other foods with similar fermentation chemistry. A small-scale multilaboratory evaluation indicated that the multiplexcompetitive ELISA has good analytical reproducibility (average interlaboratory % CV of 28−41%).

Evaluation of Gluten Protein Profiles in Hydrolyzed Food Products by a Multiplex-Competitive Enzyme-Linked Immunosorbent Assay

“…Multiplex-competitive ELISA was performed as described previously. , Briefly, 10 μg/mL gluten was coated onto microtiter plates, and the plates were incubated overnight at 4 °C. The plates were then washed (4 times with PBST) and blocked with NFDM (37 °C for 1 h).…”

“…Intact wheat gluten (G5004, Sigma-Aldrich) was used as a calibration standard in the multiplex-competitive ELISA as previously described. , The lot of gluten used in this study had a protein content of 83% (N × 5.7) as per the Certificate of Analysis. This protein content value was used when preparing the gluten standards.…”

Approaches for development of functional and low gluten bread from sorghum: A review

“…Bender and Schönlechner (2020) in their review reported negative impact of protease application on sorghum bread quality in comparison with other gluten-free breads, however further studies needs to be The study highlighted that such techniques may also be exploited for sorghum to control the process parameters to achieve desired flour properties. Method for detection of gluten during each step of the bread development must also be developed, similar to the one performed for meat and pastry (Scharf et al, 2013), beer (Panda et al, 2015) and yogurt (Panda et al, 2021).…”

“…The study highlighted that such techniques may also be exploited for sorghum to control the process parameters to achieve desired flour properties. Method for detection of gluten during each step of the bread development must also be developed, similar to the one performed for meat and pastry (Scharf et al, 2013), beer (Panda et al, 2015) and yogurt (Panda et al, 2021).…”

A label-free aptamer-based colorimetric biosensor for rapid gliadin detection in foods: a focus on pasta, bread and cookies

“…Consequently, the Codex Alimentarius and the European Union have designated 'glutenfree' as a gluten content of 20 ppm or less and 'very low gluten' as a gluten content of less than 100 ppm, respectively. 6,7 Despite the recent development of numerous gluten detection methods, including various enzyme-linked immunosorbent assays (ELISA) [8][9][10][11] and other sophisticated techniques, such as real-time polymerase chain reaction, 12 gel-permeation highperformance liquid chromatography with uorescence, 13 pretreatment method to improve sensing accuracy, bio-sensing pretreatment method, microarray technology, 14 lateral ow immunoassay, 15 and liquid chromatography-mass spectrometry, 16 there remains a need for cost-effective and practical in situ detection methods that do not require complex analytical instruments.…”