Date of Award

12-2015

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Food Science and Human Nutrition

Advisor

Balunkeswar Nayak

Second Committee Member

Denise Skonberg

Third Committee Member

Brian L. Perkins

Abstract

With recent increase in the utilization of soy proteins, there are mounting concerns of the escalation of soy allergies. It is, therefore, important to improve current methods of allergen detection to ensure accurate labeling of foods, produce more reliable and representative results in studies to create hypoallergenic food and establish allergen thresholds. The conditions used to extract proteins from foods are important determinants for appropriate detection and interpretation of the allergenicity of food materials. The extraction of soy proteins for soy allergen detections is conventionally performed with phosphate buffered saline (PBS) buffer for an extraction period of at least 2 h at 23 or 4°C. This has been reported to be inefficient due to time consumption and inadequate protein extraction, resulting in false negative allergen detection and mislabeling of foods containing allergenic proteins.

The objectives of this study were: 1) To improve the extraction of allergenic proteins from soy products using different buffers in combination with selected thermal and non- thermal extraction conditions and 2) To evaluate the efficiency of extraction methods on the extraction of allergenic soy proteins from food matrices. With study one, soy proteins were extracted from raw soy flour, soy protein isolate (SPI) and soy milk using water bath extraction at 60, 70 and 100°C for 5, 15 and 30 minutes, microwave assisted extraction (MAE) at 60, 70 and 100°C for 0, 5 and 10 minutes and ultrasound assisted extraction (UAE) at 4 and 23°C for extraction times of 1, 5 and 10 minutes with three different buffers namely PBS, Laemmli and urea. Extracts were analyzed for total proteins, protein molecular weight profile (SDS-PAGE) and antibody-based detection (ELISA) of soy proteins. Conventional extraction with each of the buffers was used as controls. Overall, protein recoveries using water bath, MAE and UAE methods were significantly higher than recoveries from the controls in all soy matrices. Under all extraction conditions, Laemmli and urea buffer recovered more proteins than PBS. Electrophoretic analysis of proteins showed bands around 75, 50 and 37 kDa indicating the presence of soy allergenic proteins β-conglycinin, glycinin and P34, in all samples. ELISA analyses showed that water bath extraction and MAE as well as the use of urea reduced the ability of the ELISA kit to detect soy proteins. The use of Laemmli buffer with conventional extraction and UAE, however, produced similar or better results with ELISA, compared to conventional extraction with PBS, especially for SPI and soy milk.

These extraction conditions were further studied and their practical use tested on some commercial samples containing soy as well as samples spiked with known concentrations of soy flour in study two. The use of Laemmli buffer with conventional extraction and UAE once again resulted in comparable and in some cases, better outcomes than conventional extraction with PBS suggesting that these extraction conditions may be used as alternative or additional extraction methods that could be employed in the extraction step to improve soy allergen detection.

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