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Title

IMPROVED METHODS FOR DETECTING PARTIALLY HYDROLYZED GLUTEN IN FOOD

Creator

Cao, Wanying

Date

2016, 2016-07

Description

Celiac disease is an autoimmune disorder in susceptible individuals caused by the consumption of gluten, a class of storage proteins present...
Show moreCeliac disease is an autoimmune disorder in susceptible individuals caused by the consumption of gluten, a class of storage proteins present in wheat, barley and rye. There is no cure for celiac disease, and the only effective treatment is strict adherence to a gluten-free diet. Manufacturers who label their products as “gluten-free” must ensure that these products contain < 20 ppm gluten. Analytical methods currently exist for detecting and quantifying gluten in foods. However, quantifying gluten in fermented or hydrolyzed foods presents an analytical challenge. In order to develop reliable and accurate gluten analytical methods, a better understanding is needed with respect to gluten hydrolysis reactions that occur in fermented and hydrolyzed foods. In addition, research is needed to determine ways to control gluten in food production facilities that produce gluten-containing and gluten-free products on the same processing line. The objectives of this project were to: 1) evaluate the effectiveness of different cleaning procedures on removing gluten from a pilot-scale beer brewing line, 2) assess gluten cross-contact from a shared beer brewing line, 3) track the changes in gluten detection in traditionally brewed soy sauce at different stages of production using five commercial gluten ELISA kits and 4) evaluate the effects of an enzyme (a prolyl endopeptidase- Brewers Clarex®) on detecting gluten in beer brewed with barley malt as an ingredient. A pilot-plant scale beer brewing line located at the University of Wisconsin-Madison (UW-Madison) was used to produce sorghum beer (gluten-free beer), barley malt-containing beer and Clarex®-treated barley malt beer. Three cleaning methods (a push-through cleaning treatment, a hot water rinse, and a full cleaning procedure involving the use of an alkaline detergent) were evaluated for effectiveness at removing gluten residue from the pilot-scale brewing line at UW-Madison. Cleaning study results indicated that a hot water rinse and a push-through cleaning treatment with sorghum beer were less effective in removing gluten from equipment in the brewing line than a full cleaning procedure that included use of an alkaline detergent solution followed by a final hot water rinse. Gluten-free sorghum beer samples used to evaluate cross-contact from an inadequately cleaned brewing line were analyzed, and up to 105.1± 9.3 ppm (μg/mL) gluten was detected using the RIDA Competitive ELISA test kit which is designed to detect hydrolyzed gluten. Model soy sauce products were produced in a pilot-plant at Kikkoman R&D Center in Japan, and samples were obtained at different stages of production. Studies that traced gluten in soy sauce products found that high levels of gluten could be detected at the early stages of production prior to fermentation. However, gluten concentrations in soy sauces after fermentation were below the limit of quantitation (LOQ) for all of the gluten ELISA kits evaluated in the study. Use of Clarex® during the production of barley malt beer resulted in substantial reductions in the amount of gluten (intact and partially hydrolyzed) detected in beer compared to the control treatment without added enzyme. Although, gluten was detected at levels >20 ppm in some Clarex®-treated beer samples, filtration treatment further reduced gluten concentrations in these beer samples below 20 ppm gluten. Results of this project indicate that use of adequate cleaning procedures is needed to control gluten in food production facilities that have shared processing lines. Some fermentation and hydrolysis reactions that occur in food result in substantial reductions in gluten content as measured by ELISA. However, more work is needed to determine if celiac-reactive peptides still remain in these products.
M.S. in Food Safety and Technology, July 2016
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Title

IMPROVED METHODS FOR DETECTING PARTIALLY HYDROLYZED SOY IN FOOD

Creator

Kande, Parnavi

Date

2016, 2016-12

Description

Methods are needed to detect soy allergens in hydrolyzed and fermented food. Project objectives included utilizing soy-specific ELISA kits and...
Show moreMethods are needed to detect soy allergens in hydrolyzed and fermented food. Project objectives included utilizing soy-specific ELISA kits and Western blot analysis to a) measure soy protein content of commercial soy sauce products; b) trace soy proteins in model soy sauces during a traditional brewing process, and c) investigate detection of soy proteins in soy protein isolate (SPI) hydrolyzed with trypsin. Twelve different soy sauce products were purchased locally. Traditionally brewed soy sauce formulated with soybeans and wheat was produced by Kikkoman in their pilot plant in Japan. Samples were obtained at the ingredient stage, after the soybeans were cooked, at the koji and moromi mash stages, and during six months of fermentation. Partially hydrolyzed SPI was prepared by treating SPI with trypsin for up to 8 h at 38 ºC. Samples were analyzed in triplicate using six soy-specific ELISA kits: 1) Neogen Veratox for Soy; 2) Ridascreen Fast Soya; 3) Elution Technologies Soy Protein; 4) Morinaga Soya; 5) AgraQuant Soy and 6) ELISA Systems Soy Protein. Western blot analysis was performed using Morinaga Soy ELISA kit antibodies. Of the 12 commercial soy sauce products, soy protein was detected in seven products at concentrations of 7.0 (1.7% CV) - 10.5 (1.8% CV) μg/g using the Elution Technologies kit, and in five samples at concentrations of 0.31 (0.6% CV) - 1.5 (0.3% CV) μg/g with the Morinaga kit. Qualitative Western blot analysis suggested the presence of beta-conglycinin in five commercial brands. Tracking soy proteins in model soy sauce by ELISA resulted >90% decrease in detection after cooking. After addition of koji culture and brine to form the moromi mash, soy protein levels were further reduced. The measured level of soy protein after one month of fermentation was 0.67 μg/g (1.1% CV) and decreased below the limit of quantitation (LOQ, 0.31 μg/g) for samples aged 2-6 months using the Morinaga assay, while other kits resulted in soy protein concentrations below the LOQ. Western blot results showed light bands of proteins for samples after 1-2 months of fermentation suggesting the presence of beta-conglycinin. Trypsin hydrolysis of SPI resulted in a gradual decrease in soy protein bands in SDS-PAGE analysis, while some ELISA kits indicated no changes in detectability of soy proteins after 10 min of hydrolysis. Overall, the results of this study indicate that heating, hydrolysis and fermentation processes can have substantial effects on the detection of soy proteins in food.
M.S. in Food Safety and Technology, December 2016
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