Novel Detection And Quantification Tools For Peanut Allergens,and Processing Mechanism For Peanut Allergen Reduction

Peanut（Arachis Hypogea）,in addition to being very nutritious food crops,are one of the most common allergic food,which comes in the list of the"big eight"allergen family.Peanut allergy sensitization is increasing rapidly,with estimates that it has doubled in the last decade.Peanut is regarded the most severe form of food allergy to induce lethal anaphylactic shock in highly sensitive individuals,and its severity is also increasing.There are yet insufficiently reliable therapy and effective mitigation measures to combat peanut allergy.Therefore,there is surge of reliable,fast,and high-throughput detection/quantification technique for allergens and hypoallergenic variant of peanut for breeding and genetic engineering program,and processing technique to develop product with reduced allergens.In this work,we characterized 89 key Chinese peanut cultivars for their major allergens quantity features and clustered the peanut varieties using the best clustering model and grouped those varieties based on Hierarchical cluster analysis（HCA）.LC-MS/MS approach and their specific peptides markers based on LC-MS/MS study were investigated for high-throughput detection of peanut allergy.Based on NIRS（Near-Infrared Spectroscopy）and chemometrics patterns,a robust,non-invasive technique for measurement of peanut allergens from its protein extract powder was developed using machine learning algorithm.The alteration in peanut allergens in alternative meat analogues processed from peanut protein was explored using High-Moisture Extrusion（HME）processing with several crosslinking formulae（TGase and various Polysaccharides）.For allergen analysis,a three-stage sequential protein extraction approach was used to examine post-HME processed proteins.The key findings are as follows:1.A novel method for quantifying peanut allergens based on NIRS and chemometrics（Data mining）and machine learning algorithm for NIRS data have been successfully established in terms of protein and proteins subunit quantification.PLSR and PCR models were used to predict the composition（protein and allergens）of extracted peanut protein powder,which were then combined with various spectral pre-treatments and validated by external data sets.The study revealed that NIRS has showed the remarkable tendency to predict the allergens quantity.Based on statistical indications of training and testing samples,the PLSR prediction model performed remarkably well in predicting protein content and distinct allergenic components.For testing samples,the statistical parameters of the optimized model for different variables was;Ara h 6（R²=0.99,RMSE=0.007）>Ara h 3 basic unit（R²=0.97,RMSE=0.13）>Protein content（R²=0.90,RMSE=1.29）>Ara h 2（R²=0.89,RMSE=0.17）>Ara h 3 acidic units（R²=0.79,RMSE=0.78）>Ara h 1（R²=0.64,RMSE=0.47）.In the case of training and testing data with a composition based on allergen percentages of proteins,the PLSR model again showed good performance.Furthermore,the PCR prediction model worked well for estimating protein content,while it underperformed to predict the allergenic constituents for highly varied data with studied sample size.2.The main allergens（Arah 1,Ara h 2,Arah 3 and Arah 6）in different peanut varieties were located and detected by LC-MS/MS method,and their specific short peptide markers were identified which can be further used for studying the quantification of these allergens with the calibration curve development,and stability study of these peptides will be necessary for future studies.The allergenic attributes of 89 distinct peanut cultivars were studied based on SDS-PAGE and densitometry analysis for relative quantification among different varieties.The protein content among the samples were varied from18-35%analyzed using Dumas method.Ara h 3 constitutes the highest amount of peanut proteins which ranges from 50-70%,then Ara h 1 composition ranged from 16-25%.Ara h 2 composition ranged from6.36-11.82%,and Ara h 6 comprised 3.42 to 11.01%of total proteins.In term of absolute quantification,Ara h 3 had the highest average content of 8.95mg/100g,followed by Ara h 1,accounting for 5.26mg/100g,Ara h 2 accounting for 2.47mg/100g,and Ara h 6 accounted for 2.30mg/100g.The allergenic quantity,especially individual allergens varied significantly among studied varieties.The highest variation was reported in allergens Ara h 6 and Ara h 2.Among these samples,none of the cultivars were found to be lacking in any single allergens,while some of the varieties were lacking in 36.5 k Da allergenic subunit of Ara h 3 on SDS-PAGE.Correlation analysis between proteins and allergens and among allergens suggested that there is high correlation between proteins and allergens content except Ara h 6.Additionally,Ara h 6 was least correlated with other allergens,while other allergens was moderate or highly correlated with each other’s.On the basis of allergenic attributes,these varieties were clustered using PCA,t-SNE,and original attributes.Model based on t-SNE performed extremely well for both（SI index 0.413 for percentage based composition and 0.494 for final absolute quantification based composition）to group the varieties based on their allergenicity traits.Finally,the varieties based on their allergenic proteins attributes were found to have 3 optimal clusters using HCA（Hierarchical cluster analysis）dendogram.Thus,this result based on HCA suggests among studied varieties it could be grouped into 3 categories based on their allergenic attributes to low and high allergen categories.3.The impact of High-Moisture Extrusion（HME）processing on peanut proteins was significant during the development of meat substitute products,resulting in significant changes in peanut allergens（Ara h 1,Ara h 2,Ara h 3,and Ara h 6）.HME processing lowered the protein solubility substantially,and the addition of other crosslinking agents during HME processing reduced the solubility even more.The3-stage sequential protein extraction procedure designed in this work significantly increased the protein extraction efficiency and allowed a comprehensive analysis of the proteins for allergenicity reduction.HME processing alone or in presence of different crosslinking agents such as Transglutaminase（TGase）,carrageenan,sodium alginate,and wheat starch revealed that Ara h 1 was the most altered allergen,with reductions of up to 91%（Ara h 1）,61%（Ara h 2）,60%（Ara h 6）,and 55%（Ara h 3）for all three stages.During the first stage extracts analysis（soluble protein extracts）most of the allergens were mitigated on SDS-PAGE,while 2^nd and 3^rd stage extracts（non-soluble protein extracts）resulted in recovery of these allergenic proteins to a certain extent in non-soluble extracts.Monoclonal antibodies based western blot analysis of soluble protein extracts of meat analogues reflected the presence of Ara h 1,Ara h 2,and Ara h 3 in significantly lower amount indicated significant reduction in their Ig E binding strength.HME is a complex processing mechanism which passes the material through different processing zones.During different processing zones,the most significant reduction in allergenic proteins was in the melting zone.The significant alteration in secondary and tertiary structures as a result of crosslinking shearing and degradation of proteins is likely to lead to allergens reduction.