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Title:Improving the health benefits of broccoli through myrosinase maintenance
Author(s):Dosz, Edward
Director of Research:Jeffery, Elizabeth H.
Doctoral Committee Chair(s):Engeseth, Nicki J.
Doctoral Committee Member(s):Jeffery, Elizabeth H.; Schmidt, Shelly J.; Lee, Youngsoo
Department / Program:Food Science & Human Nutrition
Discipline:Food Science & Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):broccoli: myrosinase
sulforaphane
thermal stability of brassica phytochemicals
Abstract:Broccoli (Brassica oleracea L.) is the most significant source of sulforaphane (SF), a potent cancer-preventative agent in the Western diet. Because SF is unstable; the plant produces a stable thioglucoside precursor, glucoraphanin, and a thioglucoside glucohydrolase (myrosinase EC 3.2.1.147). Glucoraphanin and myrosinase are stored separately in the plant cell. When tissue is crushed, such as in insect damage, glucoraphanin and myrosinase come in contact and form an intermediate that breaks down to form the isothiocyanate SF. Importantly, when broccoli is crushed/chewed before swallowing by the consumer, the same process takes place as long as the isothiocyanate precursors are present. When broccoli is processed commercially for freezing, it undergoes blanching. Blanching is a thermal treatment using steam or hot water to inactivate enzymes which could adversely affect the broccoli during frozen storage. This thermal treatment also inactivates the thermally unstable myrosinase, yielding a broccoli product with no ability to form SF. Human tissues contain no thiohydrolase and although the gut microbiota can form SF, they do so very poorly compared to myrosinase. Much of the current literature focuses on glucoraphanin content of new or improved broccoli varieties, without looking at the larger picture of SF formation and therefore myrosinase activity. The assays that exist to measure myrosinase involve the disappearance of the glucosinolate (GSL) sinigrin (not endogenous to broccoli) or the appearance of glucose, released during sinigrin hydrolysis. For glucose measurement, protein extraction is carried out to prepare tissue for assay. Extraction is incomplete causing an underestimate of activity as compared to the whole food. The objective of this research was multifaceted. First, to determine if frozen broccoli processing methods significantly inhibited the formation of SF as seen in commercially available broccoli products. Results showed that prior to cooking; commercial samples already had a reduced capacity to form SF, due to a lack of activity by the enzyme myrosinase. This loss of activity was found to be due to the thermal treatment during blanching that they all received prior to freezing. When samples were then micorowaved in a bowl or a steamer bag, the resulting food products were all void of SF formation, due to inactivation of myrosinase. The second objective was to determine a way to overcome loss of hydrolyzing activity. Industrial blanching usually aims to inactivate peroxidase, although lipoxygenase plays a greater role in product degradation during frozen storage of broccoli. Blanching at 86 °C or higher inactivated peroxidase, lipoxygenase, and myrosinase. Blanching at 76 °C inactivated 92% of lipoxygenase activity, whereas there was only an 18% loss in myrosinase-dependent sulforaphane formation. We considered that thawing frozen broccoli might disrupt membrane integrity, allowing myrosinase and glucoraphanin to come into contact. Thawing frozen broccoli for 9 h did not support sulforaphane formation unless an exogenous source of myrosinase was added to the surface. Daikon radish root supported sulforaphane formation even when heated at 125 °C for 10 min, a time and temperature comparable to or greater than microwave cooking. Daikon radish (0.25%) added to frozen broccoli that was then allowed to thaw supported sulforaphane formation without any visual alteration to that of untreated broccoli. Finally myrosinase activity was measured in whole food (5 broccoli and 3 kale cultivars) based on formation of allyl isothiocyanate (AITC) and/or glucose formation from exogenous sinigrin. A significant correlation was seen between AITC and glucose formation (r=0.488, n=21, p=0.025). Using exogenous (2 mM sinigrin) or endogenous (glucoraphanin) GSL, appearance at 5 min correlated between AITC and SF, respectively (r = 0.859, n=8, p=0.0062). Unlike glucose appearance, which required a prolonged incubation period for a reproducible measurement, AITC formation could be accurately measured after as little as one minute, providing a quick and reliable estimation of myrosinase activity in whole foods. These results indicate that commercially frozen broccoli lacks the ability to form SF due to the blanching process. Thawing frozen broccoli in the presence of a more thermally stabile myrosinase, daikon radish, allowed for enhanced formation of SF. Separately, the results suggest that use of AITC or SF formation can be used to measure myrosinase activity during short term hydrolysis as it would pertain to human consumption.
Issue Date:2014-05-30
URI:http://hdl.handle.net/2142/49815
Rights Information:Copyright 2014 Edward Dosz
Date Available in IDEALS:2014-05-30
2016-09-22
Date Deposited:2014-05


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