Establishment And Application Of The Methods To Detect The Main Milk Allergens In Processed Foods

Milk is one of the most common food allergies which can cause serious anaphylaxis in infants and children. Currently, a cure of food allergies is not yet possible, so the strict avoidance of the allergenic food is the only option for patients. Thus, the availability of accurate and sensitive detection methods for food allergens is crucial to ensure the correct labelling of their products in order to protect allergic consumers. In this research, we took the attempt to build three rapid and sensitive methods for milk allergen(b-lactoglobulin, a-lactalbumin and a-s1casein) detection. And then the three methods were applied to detect the main milk allergens in processed foods.1. To establish a Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergen b-lactoglobulin in food, specific primers and taqman probe were designed based on the gene sequence of b-lactoglobulin published in NCBI. Recombined plasmids were constructed as the standard PCR template for real-time PCR. The detection sensitively reached up to 318 copies. Furthermore, the specificity( goat milk and soybean-milk showed no amplification) and stability(The Intra- and inter- validation were all within 5%) of the method were good. Seven foods were detected with b-Lg DNA residue and the results were consistent well with their allergen labels. The electrochemical DNA sensor based on DNA hybridization was then constructed using the probe established by real-time PCR. The gold electrodes(Au E) were modified by nanogolds(NGs) through direct electro-deposition, and the electrochemical impedance spectroscopy were choosed for quantitative determination of complementary DNA. A detection limit of 2.5×10-13 mol/L(S/N=3)was achieved under the optimum assay conditions. The biosensor exhibits good selectivity, as well as acceptable stability and reproducibility. In addition, the DNA sensor possessed the advantages of simple operation, low cost. we also used this novel LNA biosensor to analyze real samples, yielding satisfactory results.2. PCR primers and taqman probe were designed based on the gene sequence of a-lactalbumin published in NCBI for real-time PCR. Plasmids were prepared as the standard PCR template and and standard curve were constructed with DNA copies and Ct. The a-lactalbumin DNA fragment had been cloned successfully and the standard curve had a good linear relationship ranging from 1.12×103 to 1.12×108 copies. The DNA sensor for a-lactalbumin was fabricated by coating a glass carbon electrode with chitosan-multiwalled carbon nanotubes(MWCNTs) composite and then adding the nanogolds via electro-deposition. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue(MB) as an indicator. Optimization of MB accumulation time and NGs deposition time were performed. Results showed that the detection limit greatly improved compared to the b-Lg DNA sensor, an ultrasensitive detection limit of 1.7×10-16 mol/L(S/N=3) were obtained.3. This study aimed to develop and validate an LC-MRM/MS based method to confirm and quantify milk allergens in food products. Emphasis was placed on two whey proteins(α-lactalbumin and β-lactoglobulin) and a-s1 casein that are known to be the main allergic components of milk. At first, five marker peptides(two for β-lactoglobulin, one for α-lactalbumin and two for a-s1 casein,) from the digestion of standard milk proteins were identified by MALDI-TOF/TOF MS and used for absolute quanti?cation of allergenic milk allergens by UPLC-TQD in multiple reactions monitoring(MRM) mode. The assays were then validated for absolute quanti?cation of three milk proteins with satisfactory results. In addition, the LC-MRM/MS method was applied to the expression of levels in daily foods, providing a good results. All the positive samples showed peak in the MRM condition with no interference, on the contrary the wheat meal(blank matrix) and other negative samples showed no detection peak..Three detection methods based on DNA and protein were established in this research. RT-PCR and DNA sensor were aimed to detect the DNA of milk allergens, which could be supplementary methods for ELISA. Another disadvantage of ELISA is that only one allergen per test and the detected Ig E epitopes were unknown. LC-MRM/MS is a direct detection method and can detect multiple allergens in the same analysis. Thus, the three methods developed in this study may play an important role in milk allergen labeling and protecting the safety of comsumers.