Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: All contributions to this project were disseminated in scientific meetings such as the Institute of Food Technologists and American Chemical Society on the identification, quantification and physicochemical properties of peptides of the different technological process of soybean under study. Other contributions: Modifications and interactions of the constituents of food during technological processes; Characterization and control of food quality; Development of functional ingredients and foods; Enzymatic biotechnology and food microbiology; Food safety; and Development of new processes and products. Rudy Darmawan (Alice Charlotte Biester merit award 2009; inaugural 2009 FSHN Pre-IFT Poster Competition award; 2010 Julia Holmes merit award)obtained his master's degree in Food Science and Human Nutrition. Plaimein Amnuaycheewa (Alice and Charlott Biester merit award 2009)obtained his master's degree in Food Science and Human Nutrition. PARTICIPANTS: Rudy Darmawan obtained his masters degree in the Department of Food Science and Human Nutrition, University of Illinois using this research project as the basis of her research. Plaimein Amnuaycheewa obtained his masters degree in the Department of Food Science and Human Nutrition, University of Illinois using this research project as the basis of her research. Huanyu Zheng accomplished her Post-Doctoral training with the topic Effect of processing and bioactive compounds on soybean allergenicity. TARGET AUDIENCES: The food industry has benefited from this research. An international presentation was given of the topic: Current issues on food allergens: Effect of processing on soybean allergens at the International Symposium on Safety Assessment of Food Product and Processing. Annual meeting of Taiwan Association for Food Science and Technology. November 27, 2009. Presentation at a national scientific meeting was given on the Effect of processing on the reduction of allergens in soybean products. Impact of Processing on Food Allergens. IFT Annual Meeting & Expo, New Orleans, Monday June 13, 2011. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Food allergy has become a public health problem that continues to challenge both the public community and the food industry. Soybean protein products are widely used by the food and feed industry as highly nutritious plant protein sources. The objective was to analyze the molecular size, changes in protein structure and immunoreactivity of ethanol/water extracted soy protein concentrate in comparison with other soybean protein products extracted at different pH, and allergen stability after pepsin hydrolysis. The solubility of soy protein products changed at different pHs. Ethanol/water extraction denatured proteins and reduced protein solubility. Around 33% to 87% of crude protein was extracted at pH 6.8 and was the most effective extraction. Immunoreactivity differed among processed soy products based on ELISA (human plasma) and TNO sandwich ELISA (rabbit antibody). Results provide a better understanding of the mechanism of immunoreactivity reduction in ethanol/water extracted soy protein concentrates. The ethanol/water extraction process changed protein structure, reduced protein solubility, antigenic activity and molecular size of the soluble fractions without significant alteration of total protein.

Publications

• Song, Y.S., Perez, V.G., Pettigrew, J.E., Martinez-Villaluenga, C. and Gonzalez de Mejia E. 2010. Fermentation of soybean meal and its inclusion in diets for newly weaned pigs reduced diarrhea and measures of immunoreactivity in the plasma. Animal Feed Sciences and Technology, 159:41-49.
• Amnuaycheewa, P. and de Mejia, E.G. 2010. Purification, characterization, and quantification of soy allergen profilin (Gly m 3) in soy products. Food Chemistry, 119:1671-1680.
• Darmawan, R., Bringe,N.A. and Gonzalez de Mejia, E. 2010. Antioxidant capacity of alcalase hydrolysates and protein profiles of nine soybean cultivars grown in three different locations in the Midwestern United States. Plant Foods and Human Nutrition, 65 (3):233-240.
• Zhenga, H., Marquez, S., Andler S., Dersjant-Li, Y., Peisker, M. and Gonzalez de Mejia, E. 2013. Molecular size and immunoreactivity of soybean proteins of products processed under different conditions. Journal of Agricultural and Food Chemistry. (Under Consideration).

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The results were presented at the Institute of Food Technology Annual Meeting and Expo, New Orleans, Monday June 13, 2011,Presentation Number: 179-04: Gonzalez de Mejia, E., Lee, Y.-S. and Yang, W. Effect of processing on the reduction of allergens in soybean products. In: Impact of Processing on Food Allergens. PARTICIPANTS: Training was provided to post-doctoral fellow: Dr. Huanyu Zhen. Collaborator: Dr. Young-soo Lee. TARGET AUDIENCES: Food industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The objective was to study the major soy allergens, methods for soy allergen detection and characterization, and non-thermal methods to mitigate soybean allergens and their status of development. Combination of physical, chemical and biological techniques may be an effective approach to maximize the alleviation of allergen and minimize the change in functionality. Soybean is one of the most healthy and yet allergenic foods. Soybean allergens consist of proteins with molecular weights ranging from 7 to 71 kDa. Among the 34 allergic soy proteins identified, Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K are categorized as major soy allergens, while soy hydrophobic protein (Gly m 1a), soy hull protein (Gly m 2), soy profilin (Gly m 3), glycinin (320-360 kDa), &#946;-conglycinin (140-180 kDa) and tripsin inhibitor (20 kDa) are listed as major soy allergenic proteins. The strongest soy allergen among others is Gly m Bd 30k, or known as P34. To date, the majority of detection methods developed for soybean allergens are immunochemical based. The ELISA technique has become a preferred approach for soy allergen detection due to its high precision, simple handling and good potential for standardization. As a newly developed allergen detection technique, the PCR method offers unique features including less cost, faster turnaround and stability of targeted DNA over processing conditions, as compared to ELISA, but its quantitative correlation to ELISA results has so far been low due probably to the fact that nucleic acids are affected differently from allergenic proteins. In contrast, mass spectroscopy has shown a great potential over ELISA and PCR for detection and quantification of soy allergens due to its wide detection concentration range and its capability of detecting a multitude of allergens within one food species or over a number of different species in a composite food system. Fermented soy ingredients showed negligible human immunoreactivity if proteins were < 2 to < 20 kDa. S. cereviseae decreased the most allergenicity, followed by L. plantarum and B. lactis produced less hydrophobic peptides than non-fermented samples. R. oryzae, A. oryzae and S. bacillus were less effective. New methods of production of hypoallergenic foods to prevent allergic responses to soy foods must preserve food quality and food identity.

Publications

• Yang, W., de Mejia, E.G., Zhen, H. and Lee, Y.-S. 2011. Soybean allergen: Occurrence, detection and mitigation. Chapter 20. In Soybean/Book 4. InTech - an Open Access publisher covering the fields of Science, Technology and Medicine. Viena, European Union, p. 433-464.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Conference presentations included: [1] Food and Wellness: Bioactive Peptides and Human Health, FSHNGSA Pioneer Seminar Series. April 14, 2010. University of Illinois, Department of Food Science and Human Nutrition and Division of Nutritional Sciences. [2] 9th International Symposium on the Role of Soy in Health Promotion and Chronic Disease Prevention and Treatment. Washington, D.C. October 16-19, 2010. PARTICIPANTS: One graduate student completed their M.S. thesis based on their participation in this project. TARGET AUDIENCES: Animals scientists and food companies producing soybean products. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
An experiment was conducted to detect antigenic soy proteins in plasma of pigs fed soybean meal (SBM) and fermented soybean meal (FSBM). The suitability of a commercial FSBM to replace SBM in diets for newly weaned pigs was also evaluated. A total of 160 pigs were randomly assigned to five dietary treatments (n = 8): 400 g/kg soybean meal (FSBM-0), its replacement by protein from fermented soybean meal as 1/3 (FSBM-1), 2/3 (FSBM-2), 3/3 (FSBM-3), and no soy protein in diet (PCON). Diets were fed at weaning, with feed intake (ADFI) and weight gain (ADG) recorded after 3 days. Overall, pigs fed PCON had greater (P<0.001) performance and less diarrhea than pigs fed soy proteins. Pigs fed diets with FSBM had less (P<0.001) diarrhea than those fed the FSBM-0 diet. Western blot analysis showed that pig plasma recognized conglycinin and basic subunits of glycinin present in SBM, while acidic subunits of glycinin and P34 (human immunodominant soybean allergen) showed a weak immunoreactivity. Plasma from pigs fed FSBM exhibited reduced immunoreactivity toward subunits of conglycinin and acidic subunits of glycinin due to a partial hydrolysis of those proteins during fermentation. In agreement with that observation, analysis of soy proteins in FSBM by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis showed partial digestion of large proteins with antigenic activity. A linear regression analysis of values from enzyme linked immunosorbent assay (ELISA) did not support the notion that anti-soy immunoreactivity in pig plasma changed significantly as the dietary inclusion of FSBM increased. The FSBM used in this study may be used in substitution of SBM protein without a detrimental effect on pig performance. Moreover, inclusion of FSBM in diets for newly weaned pigs may reduce diarrhea after weaning. However, optimization of the fermentation conditions in the commercial FSBM is still needed to produce a more hypoallergenic soybean product for young pigs.

Publications

• Amnuaycheewa, P. and de Mejia, E.G. 2010. Purification, characterization, and quantification of soy allergen profilin (Gly m 3) in soy products. Food Chem. 119:1671-1680.
• Song, Y.S., Perez, V.G., Pettigrew, J.E., Martinez-Villaluenga, C. and Gonzalez de Mejia, E. 2010. Fermentation of soybean meal and its inclusion in diets for newly weaned pigs reduced diarrhea and measures of immunoreactivity in the plasma. Anim. Feed Sci. Technol. 159:41-49.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Scientific presentations: Amnuaycheewa, P. and De Mejia, E.G. Development of a method to isolate and characterize the allergen profilin (Gly m 3) in soybean products. Poster presented as part of the 237th American Chemical Society National Meeting, Salt Lake City, Utah. 22-26 March 2009. Abstract published in Division of Agriculture and Food Chemistry: The Cornucopia Spring 2009. Gonzalez de Mejia, E. Current issues on food allergens: Effect of processing on soybean allergens. In: International Symposium on Safety Assessment of Food Product and Processing. Annual meeting of Taiwan Association for Food Science and Technology. November 27, 2009. PARTICIPANTS: Plaimein Amnuaycheewa obtained his masters degree in the Department of Food Science and Human Nutrition, University of Illinois using this research project as the basis of his research. TARGET AUDIENCES: The food industry has benefited from this research. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Profilins are pan-allergen proteins present in various plant foods and pollens. The objective was to develop a method for purification and characterization of profilin from soy protein isolate. Furthermore, profilin was quantified in soy products and the effect of processing evaluated. Profilin was purified using poly-l-proline affinity chromatography, dialysis and ultrafiltration, and its quantification was implemented by indirect ELISA. Profilin in soymilks ranged from 4.37 +/- 0.14 to 7.24 +/- 0.30 mg/g protein, while in fermented products profilin ranged from 1.67 +/- 0.02 to 5.47 +/- 0.02 mg/g protein. Pasteurization of soymilk was an ineffective method to completely eliminate profilin. Food matrix influenced thermal stability, at 100 C, beta-sheet and random coil structures were altered, while the alpha-helices remained intact. Induced fermentation of soybean meal by Bifidobacterium lactic, Lactobacillus plantarum and Saccharomyces cerevisiae resulted in 68.3% to 72.7% reduction of soy profilin. Heat treatment, fermentation and hydrolysis effectively reduced soy profilin.

Publications

• Amnuaycheewa, P. and de Mejia, E.G. 2009. Purification, characterization, and quantification of soy allergen profilin (Gly m 3) in soy products. Food Chem. 119 (2010) 1671.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: A presentation was made by Young-Soo Song, Cristina Martinez-Villaluenga, and Elvira Gonzalez de Mejia titled Development and Validation of a Method for Immunoreactivity of Commercial Soy Products to the Institute of Food Technologists, Toxicology and Safety Evaluation conference in New Orleans. This presentation obtained the first award of the Toxicology and Safety Evaluation Division, 2008. PARTICIPANTS: Young-Soo Song obtained her masters degree in the department of Food Science and Human Nutrition, University of Illinois using this research project as the basis of her research. TARGET AUDIENCES: The food industry has benefited from this research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Food allergy has become a public health problem that continues to challenge both the public community and the food industry. The Food Allergen Labelling and Consumer Protection Act of 2004 includes soy in its definition of the "big 8" which comprises those foods that cause the most allergenic reactions. Due to popularity and innumerable health benefits of soybean, its dietary avoidance becomes increasingly difficult for commercial soybean products. In order to minimize the risk of a serious or fatal outcome by inadvertent consumption of non-labelled or mislabelled soybean allergens, a reliable validated immunochemical method to detect and quantify the major soy allergens is vital to comply with food labelling for manufacturers and protect soy-sensitive consumers. The objective of this study was the detection and quantification of human IgE immunoreactive soybean proteins in commercially available soy ingredients and products. Optimum dilutions of primary antibody and antigens as well as detection sensitivity were determined for the implementation of a sandwich ELISA method using plasma from soy sensitive subjects (IgE ranging from 0.35 to 98.7 IU/mL). Human IgE immunoreactivity of commercial soybean ingredients showed that the plasma of subjects with strong allergic reaction to soybean presented proportionally higher immunoreactive response. Soy protein isolate and soy protein concentrate contained less immunoreactive proteins than soy flour and grits. As expected, a hypoallergenic soybean product presented the lowest IgE immunoreactivity. Hydrolyzed and fermented soy ingredients showed negligible human IgE immunoreactivity when proteins and peptides were < 20 kDa. The IgE immunoreactivity of soymilk samples ranged from 3.4 to 68.9 ng IgE/mg extracted protein. Tofu contained about 20-fold higher IgE immunoreactivity than soymilk products (median 171 ng IgE/mg extracted protein). Furthermore, soy cheese products presented twice the IgE immunoreactivity than tofu products (median 359 ng IgE/mg protein). Meat analogues presented considerably high extracted protein concentration (median 67.9mg/g product). The findings of the current investigation demonstrate sandwich ELISA as a reliable immunochemical method with good repeatability, sensitivity, and low detection limit to quantify IgE immunoreactive proteins in soy ingredients and products. Quantitative measurement of specific IgE is likely to become an increasingly valuable tool for soybean industry to comply with food labeling formanufacturers, thus protecting soy-sensitive consumers.

Publications

• Song, Y.S., Martinez-Villaluenga, C. and De Mejia, E.G. 2008. Quantification of human IgE immunoreactive soybean proteins in commercial soy ingredients and products. Journal of Food Science, 73 (6) T90-T99, 2008.
• Wilson, S., Martinez-Villaluenga, C. and De Mejia, E.G. 2008. Purification, thermal stability, and antigenicity of the immunodominant soybean allergen P34 in soy cultivars, ingredients, and products. Journal of Food Science 73 (6): T106-T114, 2008.