IMPROVING THE HEALTH BENEFITS OF BROCCOLI THROUGH MYROSINASE
MAINTENANCE
BY
EDWARD BARRETT DOSZ
DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Food Science and Human Nutrition
with a concentration in Food Science
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2014
Urbana, Illinois
Doctoral Committee:
Professor Nicki J. Engeseth, Chair
Professor Emerita Elizabeth H. Jeffery, Director of Research
Professor Shelly J. Schmidt
Assistant Professor Youngsoo Lee
ii
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
iii
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
iv
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.
v
ACKNOWLEDGMENTS
I would like to express my most sincere appreciation to my mentor Dr. Elizabeth Jeffery. Her
support and encouragement over the past four years have proven invaluable and what I have
learned from her will continue to be useful throughout my career. I would like to thank Dr. Nicki
Engeseth, Dr. Shelly Schmidt, and Dr. Youngsoo Lee for being members of my doctoral
committee. Whether it was in the classroom or during our committee meetings I felt a genuine
sense of support and guidance.
I would like to thank my past and present lab mates, especially Dr. Sonja Volker, Dr. Jenna
Cramer, Dr. Donato Angelino, Yung-Ju Chen, and Molly Black, who not only taught me the ins
and outs of our lab, but became great friends in the process.
I would like to thank my family; Mom, Gram, Dale, and Dean for their never-ending support and
love. I would especially like to thank my mom for inspiring my love of food and the science
behind it, and for always fitting a week worth of dinners into a two day weekend whenever I
visit.
Finally, I would like to thank Sarah Bluhm, my loving fiancée, for her encouragement,
understanding, and support. To have her standing by my side for the past four years has made
this journey all the more meaningful.
vi
TABLE OF CONTENTS
CHAPTER 1 Introduction............................................................................................................... 1
1.1 Significance ........................................................................................................................... 1
1.2 Specific aims ......................................................................................................................... 4
1.3 References ............................................................................................................................. 6
1.4 Figures ................................................................................................................................... 8
CHAPTER 2 Literature Review ..................................................................................................... 9
2.1 History of Brassicaceae ......................................................................................................... 9
2.2 Nutritional importance of broccoli ...................................................................................... 10
2.3 Broccoli consumption ......................................................................................................... 12
2.4 Synthesis of glucosinolates and their hydrolysis products .................................................. 13
2.5 Glucosinolate and myrosinase compartmentalization ......................................................... 14
2.6 Health benefits of sulforaphane........................................................................................... 16
2.7 Frozen broccoli processing and handling ............................................................................ 18
2.7.1 Blanching ...................................................................................................................... 18
2.7.2 Blanching methods ....................................................................................................... 19
2.7.3 Inactivation of peroxidase ............................................................................................ 20
2.7.4 Lipoxygenase as an indicator enzyme .......................................................................... 20
2.7.5 Freezing methods .......................................................................................................... 21
2.7.6 Quality changes due to freezing ................................................................................... 22
2.8 Cooking and its effects on the glucosinolate/myrosinase system ....................................... 23
2.8.1 Myrosinase.................................................................................................................... 23
2.8.2 Glucosinolates .............................................................................................................. 24
2.8.3 Sulforaphane and sulforaphane nitrile .......................................................................... 26
2.9 Myrosinase activity ............................................................................................................. 27
2.9.1 Myrosinase activity measurements ............................................................................... 29
2.9.2 Direct spectrophotometric assay for sinigrin disappearance ........................................ 30
2.9.3 Spectrophotometric coupled enzyme assay .................................................................. 32
2.9.4 pH-Stat assay ................................................................................................................ 33
2.10 References ......................................................................................................................... 35
vii
2.11 Tables and Figures ............................................................................................................ 49
Chapter 3 ....................................................................................................................................... 54
Commercially Produced Frozen Broccoli Lacks The Ability to Form Sulforaphane .................. 54
Chapter 4 ....................................................................................................................................... 55
Modifying the Processing and Handling of Frozen Broccoli For Increased Sulforaphane
Formation ...................................................................................................................................... 55
Chapter 5 ....................................................................................................................................... 57
Total Myrosinase Activity Estimates in Brassica Vegetable Produce .......................................... 57
5.1 Introduction ......................................................................................................................... 57
5.2 Methods and materials ........................................................................................................ 60
5.2.1 Sample collection and preparation ............................................................................... 60
5.2.2 Myrosinase activity using allyl isothiocyanate and sulforaphane formation ............... 60
5.2.3 Allyl Isothiocyanate and Sulforaphane Measurement .................................................. 61
5.2.4 Determination of Myrosinase Activity Using Glucose Release ................................... 61
5.2.5 Soluble and Insoluble Myrosinase Activity.................................................................. 62
5.2.6 Determination of Interference by Antioxidant Compounds in the Glucose Assay ...... 62
5.2.7 Statistical Analysis ....................................................................................................... 63
5.3 Results and discussion ......................................................................................................... 63
5.4 Conclusion ........................................................................................................................... 68
5.5 References ........................................................................................................................... 70
5.6 Tables and figures ............................................................................................................... 73
Chapter 6 ....................................................................................................................................... 78
Conclusion and Future Directions ................................................................................................ 78
6.1 Conclusion ........................................................................................................................... 78
6.2 Future directions .................................................................................................................. 80
6.2.1 Future work to improve the health benefits of processed broccoli ............................... 80
6.2.2 Future work for myrosinase thermal stability ............................................................... 81
6.2.3 Future work with myrosinase activity assay ................................................................. 81
6.3 References ........................................................................................................................... 83
Appendix A ................................................................................................................................... 84
Appendix B ................................................................................................................................... 88
1
CHAPTER 1
Introduction
1.1 Significance
Fresh broccoli contains myrosinase, an enzyme which, in the presence of water, converts the
glucosinolate glucoraphanin (GRP) into the bioactive compound sulforaphane (SF) (Figure 1.1).
Sulforaphane has been shown to be a potent cancer-preventative compound acting as a strong
inducer of phase II detoxification enzymes (1, 2). Recent work has also shown that broccoli
sprouts, which produce high concentrations of SF upon crushing, can assist in the management
of inflammation caused by type 2 diabetes (3).
Commercially frozen vegetables undergo blanching prior to freezing, a process utilizing hot
water or steam to inactivate enzymes that otherwise cause degradative changes and, limit shelf
life severely (4). Destruction of the thermally stable enzyme peroxidase is most frequently the
endpoint used in determining choice of temperature and time for the blanching process (5, 6).
However, using peroxidase as an indicator enzyme is controversial, due to the fact that it often
has no role in causing or enhancing degradation during storage of frozen product (7). With an
increase in blanching time and temperature, not only does the cost increase, but there is also
greater loss of nutrient content (8). This suggests a potential benefit to the use of lower
temperatures during processing. The less thermally stabile enzyme lipoxygenase may play a far
greater role than peroxidase in degradation and therefore inactivation of lipoxygenase might be a
potential blanching endpoint for some products (9, 10).
Nutrient availability from foods is sometimes dependent upon enzymatic activity, requiring a
balance between destruction of enzymes with negative impact on food quality, while maintaining
2
beneficial enzymes (8). An example of this is seen in broccoli, where the enzyme myrosinase
releases bioactive SF from its parent glycoside GRP (11). This thermally labile enzyme shows
90% degradation when held at 60 °C for 10 min (12). Yet typical blanching protocols for
processing broccoli prior to freezing often exceed the limit of myrosinase stability (13). It has
been determined that commercially frozen broccoli lacks the ability to form SF pre- and post-
cooking (14).
During the freezing of broccoli, water expands forming ice crystals. Water, in a solid state, forms
a lattice network that contains more empty space than liquid water. Depending upon the
temperature to which water is frozen, crystallization can be destructive to the structure of the
plant cell or tissue within which crystals form. Expansion of up to 9% can occur under
conditions where water is frozen slowly, since there will be few points of nucleation and the
crystals formed will be large in size and can damage cell walls (15). The lower the temperature,
the faster freezing takes place, resulting in smaller and less destructive crystal formation. Yet,
freezing under extremely cold conditions is also associated with damage, due to internal
contraction followed by expansion causing cracking (16). Damage to cell membranes caused by
expanding and/or contracting water might cause myrosinase to come into contact with GRP,
releasing SF and thus, improving the nutritional value of frozen broccoli. Addition of exogenous
myrosinase from daikon radish is known to give a far greater yield of SF during GRP hydrolysis
in broccoli (17). Therefore, we chose to compare the temperature sensitivity and resulting
activity of endogenous myrosinase in blanched and frozen broccoli with exogenous myrosinases
from freeze dried broccoli root or daikon radish. In previous work, freeze drying was shown not
to affect the content of GSL or SF formation during a 24 h hydrolysis, excluding the possibility
3
that freeze drying during sample preparation was the cause of any variation during these studies
(18).
To measure the potential available dose of SF in a product many studies measure GRP (19, 20).
Glucoraphanin however is not a direct representation of SF formation. When humans consume
cruciferous vegetables the only sources of myrosinase activity are from plant endogenous
enzymes and to a much lesser extent from intestinal microflora (21, 22). Myrosinase is more
thermally labile and therefore more susceptible to destruction from processing than is GRP.
Therefore it is important to understand the activity of myrosinase to determine the potential for
isothicyanate formation. There are currently three commonly used methods to determine the
activity of myrosinase in broccoli.
Method one is the direct spectrophotometric assay (23, 24). This method involves measuring the
disappearance of sinigrin by monitoring the decrease in absorbance at 227 nm. This protocol is
not very sensitive, as the wavelength is low and the fraction of sinigrin being hydrolyzed is
small. Second is the pH-stat assay where, under very controlled conditions and in a
thermostatically controlled cell, NaOH is added at a specific rate and the acid release rate from
the reaction between myrosinase and sinigrin is measured. This protocol however requires the
use of specialized equipment as well as suffering from interference if the sample being analyzed
contains other GSLs which could then participate in the reaction with myrosinase. Third is the
spectrophotometric coupled enzyme assay (25, 26). This method involves the removal of
endogenous broccoli glucose through dialysis or rinsing with a buffer. Samples are then spiked
with sinigrin and released glucose from the hydrolysis is measured (Figure 2).
This method can be confounded by interfering glucose if it is not all removed, as well as with the
continued formation of glucose as other GSLs are being hydrolyzed and releasing glucose.
4
Although this technique is most commonly used, it also has the problem that some soluble and
insoluble myrosinase may be lost during this rinsing process.
1.2 Specific aims
Long Term Goal: To improve the health benefits of broccoli through the maintenance of the
enzyme myrosinase in broccoli food products.
Central Hypotheses:
1) Modified processing and/or fortification with alternative vegetable materials can
result in broccoli products that maintain the ability to form SF.
2) A new less cumbersome assay is needed to measure myrosinase activity in whole as
well as processed foods.
Aim 1: To evaluate the ability of three commercially available frozen broccolis to form SF
prior to and post further cooking.
Hypothesis: The ability of myrosinase to catalyze the formation of SF within commercially frozen
broccoli samples is destroyed, due to the thermal processing it received.
1.1: To determine the effect that blanching prior to cooking has on the ability of broccoli to form
SF during a 20 min and 24 hr hydrolysis of freeze dried sample.
1.2: To determine the effect of thermal process of previously blanched and frozen broccoli,
specifically microwave heating and microwave steaming, suggested on the three commercial
frozen broccoli samples.
5
Aim 2: Determine if thawing frozen broccoli with and without the addition of a more
thermally stable myrosinase source will allow increased SF formation.
Hypothesis: Thawing frozen broccoli under refrigerated temperatures with and without the
presence of exogenous myrosinase will result in SF formation due to structural cell damage
caused by the freeze/thaw process that brings GRP and myrosinase together.
2.1: To determine the effect of blanching until the inactivation of peroxidase or lipoxygenase has
any effect on SF production.
2.2: To search for a more thermally stable myrosinase by determining the thermal stability of
broccoli root and daikon radish myrosinase as compared to the endogenous myrosinase found in
broccoli floret.
2.3: To determine if thawing a blanched, frozen broccoli tissue with and without the addition of
an unheated freeze dried daikon radish powder will allow for SF formation.
Aim 3: Develop a rapid myrosinase assay applicable to fresh, frozen, and cooked brassica.
Hypothesis: To determine if an assay that uses the formation of an isothiocyanate such as allyl
isothiocyanate (AITC) from sinigrin or SF from GRP can yield an accurate, rapid measure of
myrosinase activity.
3.1: To determine the relative differences in myrosinase activity amongst 5 broccoli and 3 kale
cultivars using AITC formation to calculate myrosinase activity.
3.2: Relate the myrosinase activities of broccoli and kale cultivars to those measured using the
appearance of glucose from glucosinolate hydrolysis.
6
1.3 References
1. Zhang, Y.; Talalay, P.; Cho, C.-.; Posner, G.H. A major inducer of anticarcinogenic protective
enzymes from broccoli: Isolation and elucidation of structure. Proc. Natl. Acad. Sci. U. S. A.
1992, 89, 2399-2403.
2. Jeffery, E.H.; Araya, M. Physiological effects of broccoli consumption. Phytochemistry
Reviews 2009, 8, 283-298.
3. Mirmiran, P.; Bahadoran, Z.; Hosseinpanah, F.; Keyzad, A.; Azizi, F. Effects of broccoli
sprout with high sulforaphane concentration on inflammatory markers in type 2 diabetic patients:
A randomized double-blind placebo-controlled clinical trial. Journal of Functional Foods 2012,
4, 837-841.
4. Andress, E.L., & Harrison, J.A. So easy to preserve. 5th Edition. Cooperative Extension
Service, University of Georgia: Athens, GA, 2006; pp. 375.
5. USDA Technical inspection procedures for the use of USDA inspectors. U.S. Dept. of
Agriculture, Agricultural Marketing Service. 1975, 2013,
6. USDA Technical Procedures Manual—U.S. Dept. of Agriculture, Agricultural Marketing
Service. 2013, 2013,
7. Barrett, D.M.; Theerakulkait, C. Quality indicators in blanched, frozen, stored vegetables:
Lipoxygenase, rather than peroxidase.. J. Am. Diet. Assoc. 1995, 95, 823.
8. Lim, M.; Velasco, P.; Pangborn, R.; Whitaker, J. Enzymes involved in off-aroma formation in
broccoli. ACS Symp. Ser. 1989, 405, 72-83.
9. Williams, D.C.; Lim, M.H.; Chen, A.O.; Pangborn, R.M.; Whitaker, J.R. Blanching of
Vegetables for Freezing - which Indicator Enzyme to Choose. Food Technol. 1986, 40, 130-140.
10. Sheu, S.C.; Chen, A.O. Lipoxygenase as blanching index for frozen vegetable soybeans. J.
Food Sci. 1991, 56, 448-451.
11. Jeffery, E.H.; Keck, A.-. Translating knowledge generated by epidemiological and in vitro
studies into dietary cancer prevention. Molecular Nutrition and Food Research 2008, 52, S7-
S17.
12. Van Eylen, D.; Oey, I.; Hendrickx, M.; Van Loey, A. Kinetics of the stability of broccoli
(Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during
pressure/temperature treatments. J. Agric. Food Chem. 2007, 55, 2163-2170.
13. Lund, D.B. Maximizing Nutrient Retention. Food Technol. 1977, 31, 71-&.
7
14. Dosz, E.B.; Jeffery, E.H. Commercially produced frozen broccoli lacks the ability to form
sulforaphane. Journal of Functional Foods 2013, 5, 987-990.
15. Durance, T. Handbook of Food Preservation: Edited by M. Shafiur Rahman, Marcel Dekker,
Inc., New York, 1999. pp 809, ISBN: 0-8247-0209-3. Food Res. Int. 2002, 35, 409.
16. Hung, Y.-.; Kim, N.-. Fundamental aspects of freeze-cracking. Food Technol. 1996, 50, 59-
61.
17. Matusheski, N.V.; Swarup, R.; Juvik, J.A.; Mithen, R.; Bennett, M.; Jeffery, E.H.
Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the
anticancer agent sulforaphane. J. Agric. Food Chem. 2006, 54, 2069-2076.
18. Keck, A.-.; Qiao, Q.; Jeffery, E.H. Food matrix effects on bioactivity of broccoli-derived
sulforaphane in liver and colon of F344 rats. J. Agric. Food Chem. 2003, 51, 3320-3327.
19. Sasaki, K.; Neyazaki, M.; Shindo, K.; Ogawa, T.; Momose, M. Quantitative profiling of
glucosinolates by LC–MS analysis reveals several cultivars of cabbage and kale as promising
sources of sulforaphane. Journal of Chromatography B 2012, 903, 171-176.
20. Traka, M.H.; Saha, S.; Huseby, S.; Kopriva, S.; Walley, P.G.; Barker, G.C.; Moore, J.; Mero,
G.; van den Bosch, F.; Constant, H.; Kelly, L.; Schepers, H.; Boddupalli, S.; Mithen, R.F.
Genetic regulation of glucoraphanin accumulation in Benefort broccoli. New Phytol. 2013, 198,
1085-1095.
21. Cramer, J.M.; Jeffery, E.H. Sulforaphane absorption and excretion following ingestion of a
semi-purified broccoli powder rich in glucoraphanin and broccoli sprouts in healthy men. Nutr.
Cancer 2011, 63, 196-201.
22. Clarke, J.D.; Hsu, A.; Riedl, K.; Bella, D.; Schwartz, S.J.; Stevens, J.F.; Ho, E.
Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming
broccoli sprouts or broccoli supplement in a cross-over study design. Pharmacological Research
2011, 64, 456-463.
23. Palmieri, S.; Leoni, O.; Iori, R. A steady-state kineties study of myrosinase with direct
ultraviolet spectrophotometric assay. Anal. Biochem. 1982, 123, 320-324.
24. Palmieri, S.; Iori, R.; Leoni, O. Comparison of methods for determining myrosinase activity.
J. Agric. Food Chem. 1987, 35, 617-621.
25. Wilkinson, A.P.; Rhodes, M.J.C.; Fenwick, G.R. Determination of myrosinase (thioglucoside
glucohydrolase) activity by a spectrophotometric coupled enzyme assay. Anal. Biochem. 1984,
139, 284-291.
26. Wilkinson, A.P.; Rhodes, M.J.C.; Fenwick, G.R. Comments on comparison of methods for
determining myrosinase activity [1]. J. Agric. Food Chem. 1988, 36, 871.
8
1.4 Figures
Figure 1.1. The myrosinase catalyzed hydrolysis of glucoraphanin leads to the production of
sulforaphane.
Figure 1.2. The myrosinase catalyzed hydrolysis of sinigrin leads to the production of allyl
isothiocyanate.
9
CHAPTER 2
Literature Review
2.1 History of Brassicaceae
The specific origin and domestication of the Brassicaceae family is a debatable topic.
Historically they have been mentioned in literature as being important staples for human and
animal feed since before 1500 BC (1). The genus is known for providing some of the most
diverse products, ranging from not only use in fresh and processed foods, but as an oil crop,
flavoring, and a source of green manure (2). Originally called the Cruciferae, named by Carolus
Linnaeus based on the flowers found by all the members of the family, in the shape of a cross
(3). The family was then changed to the brassicaceae to fall within the standard naming
procedure ending in -aceae for family names and to incorporate the genus brassica within the
name (4). Brassica means to cut off the head and is derived from Latin (5). Cruciferous
vegetables fall within the brassicacea family and have played a role in health promotion since
before the fifth century BC (6). Cabbage was described as being a cure all for all things;
including organ pain, snake bites, and gout (7). Ironically crucifers consumed in excessive
quantities can act as goitrogens, preventing the uptake of iodine and therefore improper function
of the thyroid gland (8). After this was first described much effort went into breeding crops to
reduce glucosinolate (GSL) concentration to reduce risk of causing cancer and thyroid problems
in animals that were fed brassica crops (9). Recently however studies have shown that even with
high intake of crucifers that no relationship to thyroid cancer exists (10).
Initial research into the glucosinolates-myrosinase system was first evaluated in mustard seeds
(11). The species Brassica oleracea, which includes broccoli, is thought to have originated and
10
been cultivated in the Mediterranean area/and or Asia Minor, stemming from the breeding of
multiple wild brassica species (12). This would have occurred many hundreds of years ago with
the Greeks growing kales as early as 600 B.C. (12). Broccoli in particular originated
phylogenetically from wild cabbage (5). A theory called the triangle of U was developed by a
Korean botanist named Woo Jang-choon. This theory explains how three ancestral species of
Brassica; B. oleracea, B. nigra, and B. rapa which were diploids, interbred with one another to
form three further species that are common crops today, B. carinata, B. juncea, and B. napus
which are amphidiploids, meaning they contains genes from both of their originating species
(13).
2.2 Nutritional importance of broccoli
Broccoli contains many nutrients (Table 1) (14).The processing of foods can have a great effect
on the nutritional content. In broccoli, thermal processing is typically detrimental, having a
significant negative impact on nutrient content. The content of broccoli as described here will be
for 156 g, this is to standardize the serving size by weight since 1 cup of raw broccoli is 91
grams where 1 cup of cooked broccoli it is 156 g. Daily values can also be considered as that to
which meets the recommended amount for an adult male. The USDA has a database containing
information for raw and cooked broccoli and the vitamin and mineral content for each (14). Raw
broccoli has a high content of many necessary micronutrients, while contributing only 53
calories per 156 g to our total caloric intake. This is in part due to the water content of over 91%
of total product weight (15). Broccoli contains very little fat which account for 2.8 of the total
calories (14). The majority of calories in broccoli are from carbohydrates, which account for over
40 of the 53 calories. Fiber is widely accepted as being helpful in the support of bowel regularity,
maintenance of normal cholesterol levels, and useful in regulating body weight (16). Broccoli is
11
a good source of fiber providing 16% of the daily recommended intake. Raw broccoli (156 g)
contains over 100% of the daily recommended intake of vitamin K and vitamin C with 200% and
155% respectively. Broccoli is also considered a good source of vitamin E, supplying 8% of the
RDA for the nutrient per serving. Folate is another vitamin which is a necessary component of
our daily diets, required for synthesis of DNA. Not usually plentiful in our diet, the Food and
Drug Administration began requiring folate fortification of grains and cereals in 1998 (17). Raw
broccoli (156 g) contains 25% of the recommended dietary allowance for folate. Broccoli is also
a source of many minerals; including calcium, magnesium, and manganese. On a caloric basis
broccoli contains more calcium then whole milk. Chromium is an important trace nutrient
needed to control the metabolism and storage of carbohydrates, fat, and protein in the body (18).
Broccoli is one of the highest dietary sources of chromium accounting for 62% of our daily
adequate intake (19). Along with the vitamins and minerals, broccoli also contains many
secondary metabolites and phytonutrients which have been shown to provide health benefits.
Broccoli is a rich source of the flavonoids quercitin and kaempferol which have been shown to
reduce inflammation (20) and incidence of heart disease (21), respectively. Broccoli also
contains a family of unique sulfur-containing defensive compound called GSLs. There are over
120 naturally occurring GSLs across the range of Brassicaceae species (6). The main GSL found
in broccoli is glucoraphanin (GRP) (22, 23). Along with GSLs comes an enzyme thioglucoside
glucohydrolase, named myrosinase (EC 3.2.1.147), which has been found in all GSL-containing
plants studied thus far (9). When brought together, myrosinase will hydrolyze GRP and typically
form the isothiocyanate sulforaphane (SF). This reaction is strongly impacted by the presence of
cofactors, or modifier proteins, such as Epithiospecifier Protein (ESP) and Epithiospecifier
Modifier Protein (ESM1). In the presence of ESP the reaction can be directed to form the non-
12
bioactive compound sulforaphane nitrile (SF nitrile), while in the presence of both ESP and
ESM1, or when no modifier proteins are present, the reaction favors SF formation (24).
Thermal processing of broccoli can play a large role in effecting the nutritional benefits.
Currently, the USDA presents nutrient data for broccoli cooked boiled without other beneficial
information such as cooking time or cultivar of broccoli used. Nutritional content in broccoli is
largely dependent on cultivar and growing environment (25, 26). Method of cooking can also
play a great role on the nutritional content due to destruction or leaching that can occur during
the cooking process, this is outlined in greater detail in section (2.8). Boiling broccoli causes a
loss of up to 33% of ascorbic acid concentration (27). The thermal stability of folate is limited
under boiling conditions and broccoli will lose up to 66% under these conditions. Chromium
decreased by 20% during cooking (14). The thermal stability of the phytonutrients mentioned
above are also affected by cooking. Quercetin has been shown to decrease by upwards of 30%
due to leaching into cooking water (28). The effect of cooking on the GSL GRP and the enzyme
myrosinase are explained in more detail in section (2.8), but overall show decreases due to
leaching and thermal inactivation during the cooking process.
2.3 Broccoli consumption
Broccoli demand over the past three decades has dramatically increased from 1.4 pounds per
person in 1980 to 5.6 pounds per person in 2010 (29). There is currently little information on SF
formation or potential for formation from frozen broccoli (30). This is important as the
consumption of frozen broccoli throughout the past two decades has almost doubled, rising from
1.5 pounds to 2.5 pounds consumption per person (29). Processed frozen broccoli typically costs
much less than fresh and has a longer shelf life, supporting its popular use over fresh (31). As the
13
population becomes more health conscience and informed, the use of dietary choices to provide a
health benefit is increasing and that includes an increase in the use of vegetables both with the
meal and as healthy snacks (32).
2.4 Synthesis of glucosinolates and their hydrolysis products
The synthesis of GSLs occurs in three distinct steps. First, side chain elongation of amino acids
with methylene groups. Second, the elongated amino acids are converted to parent GSLs.
Finally, GSLs can be converted or modified due to the presence of endogenous, species specific
enzymes (33). Glucosinolates can be broken into three distinct classes; the aliphatic, aromatic,
and indolic (33). Aliphatic GSLs are derived from alanine, methionine, valine, leucine, or
isoleucine. Aromatic GSLs are derived from phenylalanine or tyrosine. The indolic GSLs are
derived from tryptophan (33). These amino acid-derived side groups help determine the end
hydrolysis products of the GSLs. Thioglucoside glucohydrolases with the trivial name
myrosinase promote the cleavage of D-glucose from GSLs which results in an unstable aglucone.
This aglucone quickly rearranges to form one of multiple products; isothiocyanates, nitriles,
thiocyanates, epithionitriles, or oxazolidines (Figure 2.1) (33). The most common products are
isothiocyanates formed from a Lossen like rearrangement of the unstable aglucone which also
results in the release of sulphate ions (34). The composition and breakdown products will depend
on the starting GSL as well as pH, presence of metal ions, and modifier proteins such as ESP
(34-36). The research discussed in this literature review and dissertation focuses on SF, as it is
well known for possessing anti-cancer activity as well as originating from GRP, the most
prevalent GSL in broccoli (22, 23, 37). Glucoraphanin is an aliphatic GSL, which in the presence
of myrosinase forms the bioactive isothiocyanate SF (Figure 2.2) (38). In the presence ESP, a
14
modifier protein, the hydrolysis can favor the formation of non-bioactive SF nitrile (Figure 2.2)
(38).
2.5 Glucosinolate and myrosinase compartmentalization
The enzyme myrosinase catalyzes the formation of multiple products from GSLs, such as
isothiocyanates, epithionitriles, thiocyanates, nitriles, and oxazolidine-2-thiones (33). To control
this interaction, myrosinase and GSLs are stored separately within the plant (39). Myrosinase has
been found in all tissues of B. napus plants, primarily in idioblasts, specifically named myrosin
cells (40, 41). Within myrosin cells are several vacuoles which contain myrosinase; these
vacuoles are denoted as myrosin grains (40). The presence of myrosin grains has been studied in
four species of Brassicaceae; B. oleracea, B. napus, S. alba, and R. sativus using immunogold
labeling with electron microscopy (40). Through the use of confocal microscopy in B. napus it
was determined that the myrosin grains form a continuous reticular system within a cell (42).
Studies looking at the content of myrosinase in B. napus show that all tissues contained
myrosinase, with the highest activity being found in seedlings, and with the exception of the
roots the fully grown plant contains relatively low myrosinase activity (43). Environmental
conditions can play a role in the different activities of myrosinase found in different organs of
broccoli. One study found that leaves and stems, when grown at 12 °C produced myrosinase
activities of 147 and 186% respectively, of broccoli grown at 22 °C (44). A second study that
looked at ten cultivars of B. oleracea, found that myrosinase activity was linearly correlated to
temperature as well as to photosynthetic photon flux, but that overall specific cultivars varied
greatly (26). To fully exploit the hydrolyzing capabilities of myrosinase, specific cultivar and
season need to be considered.
15
Glucosinolates are water soluble, making conventional methods of tissue fixation and
dehydration to pinpoint precise cell location challenging (39). Glucosinolates have been known
to accumulate in vacuoles (39). During GSL biosynthesis, the final stage, involving the
modification of side chains, takes place in the cytosol (45). Through the use of x-ray
microanalysis looking for high concentrations of sulfur, GSLs were found in S-cells located
between the phloem and endodermis (46). Climactic effects can cause great changes in the GSL
content of Brassica crops. Studies have shown that higher temperatures, high light intensity, or
low water availability all tend to positively influence the accumulation of GSLs (47-49). This
relationship of GSL with the environment can potentially be explained by what appear to be the
major function of the GSLs in the plants. Glucosinolates are a class of secondary compounds
related to cyanogenic glucosides (35). Cyanogenic glucosides can act as storage compounds but
are primarily involved in plant defense (50, 51).
Numerous studies have demonstrated that intake of GSLs is toxic and deters feeding from
insects, nematodes, and bacteria (52-54). An even greater deterrent from the consumption of
brassica by herbivores and infection from pathogens is the formation of the toxic hydrolysis
products from parent GSLs (55, 56). Isothiocyanates are found to be more toxic then nitriles and
thiocyanates, functioning by cleaving disulphide bonds in proteins (57-59). Just as some insects
are susceptible to the effects of GSLs and hydrolysis products, others are immune or even use the
toxicity of the compounds in their own defense (60-62). The diamond back moth is a prime
example of an insect that has developed a method to prevent the formation of isothiocyanates.
The moth possesses an enzyme called GSL sulfatase which prevents GSL hydrolysis (60). This
is quite intriguing in that sulfate removal is nowadays used to stabilize the GSL backbone during
analysis (63). The cabbage white butterfly manages to also redirect the formation of
16
isothiocyanates by possessing an internal nitrile specifier protein which redirects the GSL
hydrolysis to a nitrile which is much less toxic (61). Finally, the turnip sawfly stores GSLs and
excretes them when attacked to present an unpalatable taste to predators, making them a less
appealing food source (62).
2.6 Health benefits of sulforaphane
Epidemiological studies indicate a strong relationship between consumption of cruciferous
vegetables and reduction in cancer risk (64-69). Broccoli may play a role in the defense against
many cancers, in particular colorectal, liver, prostate, breast, and lung (62-67). Cancer is the
second leading cause of death in the Unites States after cardio vascular disease (70). Reduction
in cancer risk was found to be related to the number of servings a week broccoli was consumed
(65, 66). For example, intake of greater than 3 servings a week was seen to reduce prostate
cancer risk by 41% (65), and in another study, 5 or more servings a week reduced relative risk of
bladder cancer by 51% (66). Sulforaphane is considered to be responsible for these decreases in
cancer risk (37), although there is controversy over whether indolic GSL add or subtract to this
risk (71). Sulforaphane is formed from the parent GSL GRP and the enzyme myrosinase which
catalyzes the hydrolysis to SF through the cleavage of the -thioglucosidic bond (34). The
intestinal flora can perform this hydrolysis but to a far lesser extent, around 1/10th
that of plant
myrosinase (72). Sulforaphane however, is not the only product formed from the hydrolysis of
GRP, the nitrile is another common product whose formation is mediated by the presence of ESP
as well as presence of iron (73). Sulforaphane nitrile does not confer the same bioactivity as SF
(74).
17
The mechanisms by which SF reduces risk of cancer development are multiple. One method is
through the down-regulation of cytokines responsible for triggering chronic inflammation. The
transcription factor nuclear factor kappa B (NFkB) mediates inflammation, promotes cell
proliferation, and cell survival, which are key steps in tumor formation and progression (75).
Sulforaphane appears to down-regulate activation of NFkB both by preventing phosphorylation
of the inhibitory subunit it is normally bound to and by activating the transcription factor nuclear
factor (erythroid-derived 2)-like 2 (Nrf2), thought to directly inhibit NFkB (75).
Another mechanism in which SF is found to reduce cancer risk is through the modulation of
phase 1 and phase 2 detoxification metabolism (76). In phase 1, enzymes such as those found in
the cytochrome P450 (CYP) family, incorporate reactive and polar groups onto xenobiotic
compounds through the processes of oxidation, reduction, and hydrolysis (77). Despite normally
functioning to reduce the activity of xenobiotic compounds, there are cases when it forms more
reactive intermediates, such as oxidative activation of polycyclic hydrocarbon carcinogens (78,
79). Sulforaphane has been shown to be an inhibitor of many of these CYP enzymes which
participate in the carcinogenic activation of many compounds (80, 81). Sulforaphane upregulates
many phase 2 enzymes. Phase 2 metabolism involves the conjugation of compounds, some of
which are products of phase 1 metabolism, with compounds such as glutathione, sulfate, glycine,
and proteins (82). This conjugation normally produces less toxic and more readily excreted
compounds. Some proteins that play a role in the detoxification system include quinine
oxidoreductase and glutathione S-transferase. Therefore an increase in these proteins could help
reduce the occurrence of certain types of cancer by removing chemical carcinogens.
Sulforaphane has been shown to increase many of these phase 2 enzymes, which could be
responsible for some of the reduced risk in different cancer types seen in epidemiological studies
18
(83). The mechanism of upregulation involves the transcription Nrf2 which, once activated,
travels to the nucleus to bind the promoter region of these phase 2 genes (71).
A most recently identified mechanism of SF action on the reduced risk of cancers is as an
epigenetically targeted dietary compound (84). Epigenetics is the study of direct interaction of
compounds with the DNA or DNA regulatory proteins that activate or deactivate parts of the
genome at strategic times (85). It is possible that tumor suppressor genes are silenced in the
presence of SF due to epigenetic changes (86). Sulforaphane has been shown to inhibit histone
deacetylase which could be responsible for gene silencing and is overexpressed in cancer (87-
90). A second epigenetic action of SF is through the decrease of a DNA methyltransferase, an
enzyme associated with the silencing of tumor suppressor genes by altering the methylation of
DNA bases (87, 91). These health promoting and cancer reducing steps highlight the importance
of focusing on and maintaining the bioactive compounds in broccoli throughout the processing
and cooking steps typically encountered prior to ingestion.
2.7 Frozen broccoli processing and handling
2.7.1 Blanching
Blanching is the process of exposing vegetables or fruits to hot water, steam, or microwaves to
inactivate degradative enzymes which could affect the quality of the product during frozen
storage. Initial studies looking at the effect of blanching on product preservation prior to freezing
were performed in the late 1920s and 30s (92-94). Blanching can significantly increase the shelf
life of a product, extending it to years during frozen storage (95). Since 1975 peroxidase has
been used as the recommended indicator enzyme for blanching completeness (96). Peroxidase is
one of the most thermally stabile enzymes that is present in most plant tissue (97, 98).
19
Inactivation of peroxidase ensures that all other degradative enzymes are also inactivated; these
enzymes can be responsible for off flavor, aroma, and color development, as well as nutrient loss
(99-101).
2.7.2 Blanching methods
Traditional common methods for blanching use submersion into hot or boiling water or the
exposure of the product to active steam. A development over the past few decades is the use of
microwaves to produce a product which requires the use of little to no water which should
therefore increase the retention of nutrients otherwise removed due to water solubility or
leaching (102, 103). Water blanching is typically performed in a vat conveyor setting where a
chain or screw will pull the product through water ranging in temperature from 70-100 °C (104).
The time varies depending on the size and age of the product being processed; the typical
endpoint however, is peroxidase inactivation. Broccoli on average is steam blanched for 5-6 min
while blanching in hot water takes 3-4 minutes (105, 106). Steam blanching is performed using
food grade steam of approximately 100 °C. Steam is forced over the product with the use of fans,
making the process more energy efficient than hot or boiling water. This process can suffer from
overheating of the surface of the product in order to get the center heated to a high enough
temperature. Microwave heating can be performed using no additional water or with the
combination of hot water. Without the addition of water to the product, microwave blanching
has been shown to suffer negative textural differences from internal water vaporization and
associated damage (107, 108). The use of water with microwave blanching reduces these
problems but reintroduces leaching of water soluble nutrients.
20
2.7.3 Inactivation of peroxidase
A study looking at the effects of the enzyme peroxidase on quality deterioration points out that
with the exception of lignin formation in asparagus there are no quality effects directly related to
peroxidase (109). When a blanched broccoli sample void of enzymatic activity had the enzyme
peroxidase added to it there was no significant difference in aroma compounds between it and
the blanched reference (109). Peroxidase inactivation requires extensive heating of broccoli
material; this heating can be responsible for more destruction than just that of peroxidase.
Ascorbic acid has been shown to decrease to a greater extent during the inactivation of
peroxidase than that of lipoxygenase, a less thermally stabile degradative enzyme found in
broccoli and many other vegetables. A similar trend was seen when looking at the retention of
chlorophyll (110). One of the goals during inactivation of peroxidase is to preserve color similar
to that found in fresh tissue, but the act of blanching to a peroxidase endpoint reduces
chlorophyll content, causing a yellowing of color (110).
2.7.4 Lipoxygenase as an indicator enzyme
Lipoxygenase has been suggested as a better indicator enzyme than peroxidase (111).
Lipoxygenase is strongly involved in off-flavor development during frozen storage due to lipids
which are oxidized to aldehydes, ketones, and alcohols (112). Because lipoxygenase is less heat
stabile then peroxidase, blanching less time to inactivate lipoxygenase could also help prevent
the formation of dimethyl sulfide which has a strong grassy odor which occurs in broccoli if
blanched for an extended length (113). Off flavor production during frozen storage is the primary
limiting factor; this off flavor is thought to relate to the presence of lipoxygenase in the storage
of corn, green beans, and green peas (113). A reduction in heating time or temperature from that
21
needed to inactivate peroxidase to that needed to inactivate lipoxygenase would not only
preserve nutrients, but also use less energy and therefore reduce cost.
2.7.5 Freezing methods
Freezing is a much older method of food preservation than it is normally credited with. Ice
cellars were used to preserve a variety of foods as early as 1000 BC in China (114).
Commercially available frozen vegetables became popular in the 1930s, in part thanks to the
work of Clarence Birdseye who founded Birdseye frozen foods (115). His work showed that the
rapid freezing of a food created smaller ice crystals which in turn produced better results after
thawing or cooking. Many methods exist for the freezing of broccoli after blanching, ranging
from the use of forced air as a cooling medium, to the use of cryogenic liquids for a more
efficient heat transfer (116). Forced air freezers use chilled air (-30 to -40 °C) moving from 2-6
m/s in co-current or counter-current to the product being chilled. Forced air coolers ensure the
product is frozen in a relatively uniform manner. A common principle used in the processing of
broccoli is the 7/8 cooling time, which describes the time it takes to remove seven eighths of the
temperature difference between the starting product and the temperature of the cooling medium
(air). Broccoli is considered a highly perishable crop and therefore its 7/8 time is recommended
to be less than 90 min to prevent excessive respiration and moisture loss (117). One common
type of air-blast freezer is the fluidized bed freezer. In this processing technique products are
placed on a conveyor belt with a perforated bottom allowing the passage of air from beneath the
product, causing it to tumble. This tumbling ensures even temperature distribution, quick
freezing, and prevents the product from sticking together. The tumbling also introduces the risk
of product damage (118). Cryogenic freezers are a relatively recent development in processing
22
techniques and use began in the sixties with the increase in availability of cryogens such as
nitrogen and carbon dioxide (119). Systems using these freezing components can vary greatly
due to the lack of a refrigeration system. The cryogen is shipped and delivered to the freezing
location. This lowers the initial cost of this set up compared to other equipment uses, but the
long term operating costs are fairly expensive, due to reliance on a second company for
continued delivery of cryogen (115). Liquid nitrogen has a boiling point of -196 °C. The liquid
can be sprayed on the surface of broccoli as it moves through a conveyor. The liquid evaporates
and the remaining gas continues to freeze the product. The consumption of refrigerant is on
average 1.2 kg per kg of product (115) depending on product density and starting temperature.
Liquid carbon dioxide can be used independently as a freezing medium, with a temperature of -
70 °C, or as a pre-chilling method prior to nitrogen freezing (120). Use as a pre-chilling medium
allows for the product to be frozen more gradually, reducing cell contraction and therefore
cracking. It can also reduce liquid nitrogen consumption during processing.
2.7.6 Quality changes due to freezing
During the freezing of broccoli, water expands forming ice crystals. Water, in a solid state, forms
a lattice network that contains more empty space than liquid water. Depending upon the
temperature and speed at which water is frozen; crystallization can be detrimental to the structure
of the plant cell or tissue within which crystals form. Expansion of up to 9% can occur under
conditions where water is frozen slowly, since there will be few points of nucleation and the
crystals formed will be large in size and can damage cell walls (121). The lower the temperature,
the faster freezing takes place, resulting in smaller and less destructive crystal formation. Yet,
freezing under extremely cold conditions is also associated with damage, due to internal
23
contraction followed by expansion causing cracking (119). Even products which are frozen
properly can undergo the same tissue cracking and membrane damage if recrystallization occurs
(122). Recrystallization is the continued growth of ice crystals due to temperature gradients in
the product during freezing and thawing or due to increases in temperature during storage or
transportation (123).
2.8 Cooking and its effects on the glucosinolate/myrosinase system
2.8.1 Myrosinase
As discussed earlier, cooking can have a significant effect on the nutritional content and value of
broccoli. While looking specifically at the effect cooking has on the activity of myrosinase, the
key component responsible for the enzymatic formation of isothiocyanates, we see that the
cooking time and method plays a large role. Even before cooking, frozen broccoli has already
undergone the process of blanching, whether it be with hot water or steam, that greatly reduces
or completely inactivates myrosinase activity (30). Blanching alone, for 105 sec at 91.8 °C
caused over 93% decrease in myrosinase activity as compared to fresh (124). Boiling broccoli
for 3 min resulted in 86% lower myrosinase activity than in fresh tissue (124). Stir frying
broccoli for 2 min in a heated wok followed by 2 subsequent min covered with the addition of
water resulted in an 83% decrease in myrosinase activity (124). Reports of the effect of other
cooking methods on myrosinase activity are scarce; interpretations from other members of the
brassica family exist however and give insight into the effects of these cooking methods.
Microwave cooking of cabbage for 2 min effectively inactivated 100% of myrosinase, whereas
steaming for 2 min had no effect on myrosinase activity. Microwaving for 2 min successfully
managed to heat the cabbage to 91 °C while steaming for 7 min only brought the temperature of
the cabbage to 68 °C (125). This significant difference in temperature is caused by the different
24
method of heat transfer occurring in each. Steaming heats the exterior surface of the cabbage,
with heat moving inward via conduction, whereas microwaving heats through radiation
occurring throughout the tissue concomitantly. In many studies of the thermal stability of
myrosinase, activity is measured as the formation of SF. This may be because the assay is more
straightforward then some others. Sulforaphane measurement can give insight into the
myrosinase activity, but does not measure it directly, as a rate. One study looked at the effect of
boiling, microwaving, and steaming on the SF formation of four different broccoli cultivars
(126). The results showed that some samples preferentially formed SF nitrile due to the presence
of ESP, but also that brief periods of heating by any method has the potential to inactivate ESP, a
more heat-sensitive protein, leaving more SF formation. In that study, steaming for 3 min
proved to be the most beneficial and leave the most myrosinase active (126). Since myrosinase is
found in all members of the brassica genus looking at the possibility of a more thermally stable
form from another plant is reasonable. Both daikon radish and mustard seed myrosinase have
been shown to possess greater thermal stability than myrosinase found in broccoli (127, 128); the
mustard seed up to 70 °C for 10 min (128) and the daikon radish of up to 125 °C for 10 min
(127). This variation again supports the idea that myrosinase vary in their thermal stability and
that more work needs to be done to look at the differences in myrosinase content and stability
from other sources.
2.8.2 Glucosinolates
Glucosinolates are recognized as being more thermally stable then myrosinase during thermal
processing (129). However, the postharvest process that has the greatest effect on GSL content is
still cooking. Glucosinolates degrade under cooking conditions as well as leaching into the
cooking medium, such as water. The aliphatic GSLs, which include GRP, are prone to leaching
25
during conventional methods of cooking such as steaming or boiling (130, 131). Up to 43% of
the aliphatic GSLs leached from broccoli during a 5 min boiling in a 1:1 quantity of water (130).
There may be even greater losses of GSL content if larger quantities of water are used during
cooking, as water and GSL content are inversely correlated (132, 133). Unlike the steaming or
boiling cooking methods mentioned above which use conduction or convection, microwave
heating uses microwave radiation which causes water in food to continuously readjust its dipole
to match the oscillating electric field; this activity produces heat (134). The temperature during
microwave cooking can greatly exceed that seen in any of the more traditional heating methods.
Some studies found that microwaving is much more destructive then steaming or boiling and
causes an 80% loss in the aliphatic GSLs, 29% of which was GRP (130). Another study
comparing the effect of cooking on the major GSLs in broccoli saw no significant loss from
microwaving as long as cooking period was only 2 min; in contrast, 5 min microwaving led to a
significant decrease in GRP content (135). It should be noted that some studies estimating the
GSL content of other members of the brassica family found increased quantities of GSLs after
microwave cooking for over 4 min at 900 W. They found total GSL values increased upwards of
78% (136) and attributed this to the ease of extraction after microwave cooking. The effect on
particular GSLs in this study varied greatly, showing that a difference in thermal stability exists
amongst the GSLs. Stir frying is one of the most commonly used cooking methods in Eastern
countries and is becoming more popular worldwide. Unlike myrosinase which was more
thermally labile, stir frying did not significantly decrease the content of any of the main GSLs
found in broccoli (137, 138).
26
2.8.3 Sulforaphane and sulforaphane nitrile
The isothiocyanate SF is the major bioactive compound formed in broccoli after myrosinase
mediated hydrolysis of GRP. Typically cooking precedes chewing and thus the formation of the
isothiocyanate. However studies looking at the thermal stability of SF still exist. The cooking
methods mentioned above; steaming, boiling, microwaving, and stir frying all have an indirect
effect on the concentration of SF in the broccoli, by decreasing either GRP levels or myrosinase
activity which in turn ensures less formation of SF. Sulforaphane has an inherently short half-life
of only 24 hr in a food matrix held under refrigerated conditions. One study found that SF suffers
a 40% decrease after only 20 min at 60 °C (139). Sulforaphane is a volatile oil compound,
which can undergo oxidation, this process has been shown to accelerate at higher temperatures
and higher pH (140). When looking at the specific stability of SF in an aqueous solution,
degradation is greatly influenced by temperature. The thermal breakdown products after 1 hr of
heating at either 50 or 100 °C shows that dimethyl disulfide, among other volatile byproducts, is
readily formed upon extended heating of SF (Table 2) (141). Dimethyl disulfide is often
described as having a very potent garlic-like and fecal odor (142). Cooking to this extent had a
negative impact on the formation of bioactives in broccoli, but light or short cooking methods
can actually play a role in increasing SF formation. Sulforaphane nitrile, the formation of which
is moderated by ESP, is a non-bioactive compound which is formed from the hydrolysis of GRP
as an alternative to SF. The content of ESP can be dependent on the specific cultivar of broccoli
as well as the environmental conditions in which it was grown; such as weather, soil conditions,
and insect presence (126, 143). Epithiospecifier protein is more thermally labile than the enzyme
myrosinase with inactivation occurring after 5 min of heating at 60 °C in broccoli florets (144).
27
With more brief and lower temperature cooking, broccoli florets can form more SF and less SF
nitrile (Figure 2.3) (144).
2.9 Myrosinase activity
The formation of isothiocyanates requires the presence of the enzyme myrosinase. Therefore
estimating at the GSL alone does not confer the actual amount of health promoting compounds
provided by a particular broccoli sample, nor would it be considered a direct measurement. There
have been studies performed in the past as well as recently that look at increasing the amount of
GSL in a product and linking this to a claim that GSL hydrolysis products have been shown to be
beneficial (83, 145). A prime example of this is found in Beneforte broccoli, where the
manufacturers make a content claim for GRP being 2.7 fold higher than regular broccoli. The
package claim states that GRP is supportive of your body’s antioxidant enzyme levels. This basis
of this statement is that “your body converts GRP to SF which naturally boosts our body’s
antioxidant enzyme levels” (146). Without a focus on whether there is sufficient myrosinase, the
enzyme responsible for this conversion to SF, the consumer may not get a greater SF content out
of a broccoli containing excess GRP. The human body is a poor converter of GRP to SF. Studies
have shown that microflora of the mammalian colon are capable of hydrolyzing GRP and contain
myrosinase-type activity (72, 147). A study looking at the gastrointestinal digestion of GRP
showed that no digestive enzymes caused GRP loss (72). Therefore lack of SF formation would
not be due to degradation during consumption, but rather inability to fully convert GRP to SF.
Multiple studies have evaluated how well gut microbiota can hydrolyze GRP and found fairly
poor GSL hydrolysis. A study involving the direct feeding of broccoli GSLs void in myrosinase
activity found that only 10-20% of hydrolysis occurred (147). This same study found that upon
28
destruction of the intestinal microflora through the use of antibiotics, the conversion of GSLs to
metabolites further decreased 8.7 fold (147). Another study focusing on broccoli sprouts fed
either pre formed isothiocyanate or extracted GSLs and found a similar trend where only
between 15-20% isothiocyanate metabolites were formed in the GSL group, while the direct
isothiocyanate group recovered over 70% of the dose (148). Finally a study looking at the
bioavailability of fresh compared to steamed broccoli found that fresh broccoli had three times
greater isothiocyanate formation as compared to the broccoli that was steam cooked for 15 min,
where the enzyme myrosinase was inactivated (131). All of these studies point to the conclusion
that even with increased GSL content, broccoli is incapable of releasing its main health
promoting compound SF without the presence of the enzyme myrosinase.
Myrosinase has been identified in all GSL containing plants studies thus far. Not only is it found
in GSL containing plants, but enzymes with similar thiohydrolase activity to myrosinase have
been found in different types of fungi and bacteria, as well as certain insects (149-152). Fungi,
particularly those in the aspergillus genus have been shown to convert sinigrin into AITC during
a 48 hour cultivation (149). Bacteria, possibly including some of those commonly found in the
human gut, such as Paracolobactrum, were found to convert the GSL progoitrin to goitrin (152).
Insects have developed innovative ways to avoid the toxicity of isothiocyanates, which include
the formation of the less toxic nitriles via a nitrile specifier protein located in the gut (153).
Genetic information collected from Brassica napus and Sinapis alba has verified the existence of
at least three different types of myrosinase gene families designated as MA (Myr1), MB (Myr2),
MC (35, 154). The existence of other myrosinase subfamilies which do not fit within the MA,
MB, or MC families has been identified in horseradish root Armoracia rusticana (155). Within
each of the families there are multiple genes expressed (156). Western blot analysis of B. napus
29
seeds show the presence of all three myrosinase subunits with molecular sizes of 75, 70, and 60
kDa for myrosinases encoded by MA, MB, and MC respectively (154, 157). Myrosinase
subfamily A has been designated as soluble due to ease of water-based extraction (158). The
other myrosinase families MB and MC have a tendency to form complexes with modifier
proteins and myrosinase binding proteins, increasing molecular weights up to 800 kDa; These
appear to be insoluble unless denaturing agents are used (154, 158). When analyzing a water
extraction of B. napus only MA weighing 75 kDa occurred as free dimers, the others being
bound to protein complexes (159). All plant myrosinases are glycosylated, although to different
extents, where the carbohydrate side chain accounts for 10-20% of the total molecular mass (35).
Myrosinase families specific to different tissues or organs have been studied in B. napus and S.
alba where genes of all myrosinase sub-families are actively transcribed and detectable 20-25
days after pollination (160, 161). Myrosinase activity has been identified in all portions of a
plant, specifically within myrosin cells (43, 162). In B. napus and S. alba, MA genes were found
in the embryo while MB were specifically seen in the cotyledons (160, 161). In B. napus MC
was also found exclusively in the embryo (161). These differences in myrosinase content seen
across the plant family Brassicaceae show that conditions such as environment, insect presence,
and species can all factor in to the myrosinase activity in a plant. They also point to the fact that
information is incomplete.
2.9.1 Myrosinase activity measurements
There are currently three main methods that are used to measures myrosinase activity. Each of
these methods has advantages and disadvantages for giving an accurate and timely measurement
of myrosinase activity. Current methods for analyzing myrosinase activity measure either
30
substrate disappearance or product formation, following the addition of an exogenous substrate,
sinigrin. Sinigrin is a member of the GSL family found as the predominant GSL in Brussels
sprouts, cabbage, cauliflower, kale, as well as horseradish root (163). However, it is not a
common GSL in broccoli, often found at low levels or not at all. Using sinigrin to measure
myrosinase activity in a vegetable that is not known for having quantifiable levels of this
substrate could cause skewed results due to enzymatic substrate specificity. Substrate specificity
of myrosinase has been studied to a small extent with one study in Crambe abyssinica seeds,
where endogenous myrosinase was found to be highly specific for the GSL epi-progoitrin, the
major GSL in C. abyssinica (164). When exogenous myrosinase purified from white mustard
seeds was incorporated, the specificity for epi-progoitrin was no longer present and other GSLs
were hydrolyzed equally (164). Therefore using sinigrin as the GSL to determine the activity of
myrosinase in broccoli could produce an incorrect value, if broccoli myrosinase is specific for
GRP. Myrosinase activity is in fact defined in terms of sinigrin, where a unit of activity is
defined as that which hydrolyzes one micromole of sinigrin per minute.
2.9.2 Direct spectrophotometric assay for sinigrin disappearance
The most commonly used method to measure myrosinase activity is monitoring the
disappearance of sinigrin at 227 nm (165). This was the first assay which claimed to measure the
kinetic activity of myrosinase (165). The method is carried out during hydrolysis while the GSL
sinigrin is being converted into its corresponding isothiocyanate AITC by the actions of
myrosinase (Figure 2.4). The wavelength (227 nm) is in the shortwave ultra violet (UV) range.
Short wave UV is known to experience photon noise, stemming from the light source being used
to produce it (166). At 227 nm all GSLs present can be monitored (167); therefore a strong
31
interference in signal may occur when testing a sample rich in endogenous GSLs. This is
particularly true because GSL vary in their response factors when measured
spectrophotometrically. For instance neoglucobrassicin, an indole GSL can give a response
factor of 0.2 as compared to sinigrin which under the same conditions would give a 1 (168). To
prevent interference from endogenous GSLs, it is common practice to perform protein semi
purification, running samples through a Sepharose column (169, 170). This filtration step often
occurs after a centrifugation step, where insoluble tissue is removed (169-171). When removing
insoluble material it is possible to inadvertently discard insoluble myrosinase, which may be
responsible for up to 36% of myrosinase activity (172). Not only do GSLs cause interference
during measurement, but ascorbic acid, ascorbic acid decomposition products, and other proteins
also cause interference (173, 174). As ascorbic acid is not only endogenous to broccoli (175), but
is also added as a cofactor to myrosinase in many studies (176, 177) its presence could play a
significant role in the determination of myrosinase activity. Incorporating ascorbic acid at a
concentration above that found in broccoli will increase myrosinase activities above that which
would be expected endogenously. Some studies have had to use 30 min incubation times to make
initial readings for myrosinase activity (177, 178). It is important to measure enzymatic activity
when it is linear, and by 30 min it is possible the reaction will have slowed down before initial
measurements were taken. Despite the necessity of using a quartz cuvette or multi-welled plate,
to not cause interference at a UV wavelength, which might prove cost prohibitive, this method
can easily be automated and used to measure multiple samples at once.
32
2.9.3 Spectrophotometric coupled enzyme assay
Another common method used to measure myrosinase activity is by monitoring the release of
glucose during GSL hydrolysis (179, 180). In these measurements sinigrin is still used as the
exogenous GSL, being added in excess to determine myrosinase activity. The reaction is
performed in one of two ways using a coupled enzyme reaction (Figure 2.5). The principle of the
first is that in the presence of adenosine triphosphate (ATP), glucose is phosphorylated by
hexokinase into glucose 6-phosphate (G6P). The G6P is then oxidized by glucose 6-phosphate
dehydrogenase in the presence of nicotinamide adenine dinucleotide phosphate (NADP+) to yield
gluconate 6-phosphate, a hydrogen ion, and the reduced form of NADP+
(NADPH). The
NADPH appearance is monitored at 340 nm and is formed at a stoichiometric ratio with glucose
(179). This method suffers from interference by endogenous glucose which is reported in
broccoli at the high concentrations of 446 mg per 91 g serving, or 4.9 mg/g (14). This method
also suffers from interference from the endogenous flavonoids quercetin and kaempferol and
decompositions products of ascorbate which absorb at 340 nm (173, 174). A second, similar,
method commonly used in the measurement of glucose is the glucose oxidase-peroxidase method
(Figure 2.5). Here glucose reacts with glucose oxidase, yielding gluconic acid and hydrogen
peroxide. In the presence of peroxidase, hydrogen peroxide can be stoichiometrically utilized to
oxidize a chromophore such as o-dianisidine or more commonly 2,2′-azinobis-(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS), which then are stoichiometrically converted to a
colored (oxidized) products measured at 540 or 630 nm, respectively (Figure 2.6) (181-183).
However, this method also suffers interference from endogenous glucose and antioxidant
compounds, including ascorbic acid (173, 174, 181). The presence of ascorbic acid and other
antioxidants causes a dose-dependent color formation with concentrations greater than 333 µM
33
(181). This is due to the assay relying on the oxidation of glucose to form hydrogen peroxide,
since antioxidants reverse this process. Both of these glucose assays use samples which typically
utilize a filtering step to remove endogenous glucose, utilizing multiple rinsing steps or dialysis
(128, 139, 158). During both of these assays, the potential to lose some soluble and potentially
all insoluble myrosinase exists. Although these assays have been utilized to study the
biochemistry of the enzyme, this would make them poor methods to identify total myrosinase
activity within a sample intended to be consumed. All the methods mentioned above are
performed spectrophotometrically and can be used to measure multiple samples concomitantly,
using a uv-vis plate reader. However, spectrophotometry requires a clear solution, difficult to
attain if the substrate is whole food.
2.9.4 pH-Stat assay
One additional method used to determine myrosinase activity measures the acid release rate from
the hydrolysis of sinigrin to AITC; it is commonly referred to as the pH-Stat assay (184, 185). In
the pH-Stat assay, sodium hydroxide (NaOH) is added to the reaction medium of sinigrin and
myrosinase at a controlled rate to keep the pH at 6.5. This titration occurs in a thermostatically
controlled cell. The rate at which NaOH is added is recorded and is then used to calculate GSL
breakdown. Glucosinolate breaks down at a 1:1 ratio yielding a hydrogen sulfate ion (Figure 2.1)
(184, 186). It is common when using this assay to use a defatted crude protein extract or even
purified myrosinase (171, 179). Since this method is not based on spectrophotometric results, the
color or transparency of the samples are not stipulations to their use (187). This method requires
the use of an automated titrator equipped with a pH-stating option, which may not be accessible
to all laboratories. Also, this titrator would limit analysis of myrosinase activity to single samples
34
at a time. Unlike measuring glucose release, this is a real-time measurement and not an
endpoint, acid release is being monitored in real time. This would be a disadvantage during
analysis of multiple samples, as automation of this titrator does not exist.
Development of a direct measure of activity that can be used in whole food would be of
significant advantage during development of potentially bioactive brassica food products.
35
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49
2.11 Tables and Figures
Table 2.1. Nutritional content of raw broccoli based on 156 g serving. Adapted from (14, 23) .
compound 50 °C
(µg/g SF) 100 °C
(µg/g SF)
S-methyl methylthiosulfonate 26.6 174
S-methyl methylthiosulfinate 17.2 184.8
dimethyl disulfide 2.1 309 *
methyl (methylthio)methyl disulfide 4.9 38.7
1,2,4-trithiolane 6.5 23
4-isothiocyanato-1-(methylthio)-1-butene 13.2 134.9
Table 2.2. Products formed during thermal degradation of sulforaphane at 50 and 100 °C for 1
hr. The * indicates dimethyl disulfide the primary volatile decomposition product. Adapted from
(141).
Raw Broccoli
(156 g) Content Unit
Calories 53.0 kcal
Water Content 139.0 g
Carbohydrate 10.4 g
Lipid 0.5 g
Fiber 4.1 g
Vitamin K 160.0 µg
Vitamin C
(ascorbic acid) 140.0 mg
Vitamin E
(α-tocopherol) 1.2 mg
Folate 99.0 µg
Calcium 74.0 mg
magnesium 33.0 mg
manganese 0.3 mg
chromium 35.0 µg
Total
Glucosinolates 0.25-0.83 mmol
50
Figure 2.1. The glucosinolate-myrosinase system. Examples of variation in R-group seen
between aliphatic, aromatic, and indolic glucosinolates. Enzymatic reaction of myrosinase
yielding an unstable intermediate and D-glucose. The unstable aglucone immediately rearranges
to yield hydrogen sulfate and one or more final decomposition products. Adapted from (33).
51
Figure 2.2. The myrosinase catalyzed hydrolysis of glucoraphanin leads to production of
sulforaphane and sulforaphane nitrile. ESP: Epithiospecifier proteins. Adapted from (38).
52
Figure 2.3. Promotion of sulforaphane formation during heating due to inactivation of
epithiospecifier protein which directs hydrolysis products to form sulforaphane nitrile.
Adapted from (144).
Figure 2.4. The myrosinase catalyzed hydrolysis of sinigrin leads to the production of allyl
isothiocyanate and D-glucose.
53
Figure 2.5. Scheme of hexokinase based glucose assay to determine myrosinase activity.
G-6-PDH: glucose 6-phosphate dehydrogenase
Figure 2.6. Scheme of glucose oxidase based glucose assay to determine myrosinase assay.
ABTS: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
54
Chapter 3
Commercially Produced Frozen Broccoli Lacks The
Ability to Form Sulforaphane1
Abstract
Sulforaphane is produced from the hydrolysis of the glucosinolate glucoraphanin in the presence
of the endogenous enzyme myrosinase. Sulforaphane has been shown to provide cancer
prevention through a number of mechanisms including the upregulation of detoxification
enzymes and epigenetic changes. Optimal temperature and pH for sulforaphane formation from
broccoli was determined. Sulforaphane formation was measured in three commercially frozen
broccoli samples pre- and post-cooking. The results show that in these products, there was very
little potential to form sulforaphane prior to cooking and essentially none after the recommended
cooking method was performed. Research is needed towards improved processing methods.
1This chapter appeared in its entirety in the Journal of Functional Foods. Dosz, E.B.; Jeffery, E.H. Commercially
produced frozen broccoli lacks the ability to form sulforaphane. Journal of Functional Foods 2013, 5, 987-990. This
article is reprinted with permission from the publisher, and can be found in Appendix A.
55
Chapter 4
Modifying the Processing and Handling of Frozen Broccoli For Increased
Sulforaphane Formation1
Abstract
Frozen broccoli can provide a cheaper product, with a longer shelf life and less preparation time
than fresh broccoli. We previously showed that several commercially available frozen broccoli
products do not retain the ability to generate the cancer-preventative agent sulforaphane. We
hypothesized that this was because the necessary hydrolyzing enzyme myrosinase was destroyed
during blanching, as part of the processing that frozen broccoli undergoes. This study was carried
out 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. Thermal stability
studies showed that broccoli root, as a source of myrosinase, was not more heat stable than
1This chapter appeared in its entirety in the Journal of Food Science. Dosz, E.B.; Jeffery, E.H. Modifying the
processing and handling of frozen broccoli for increased sulforaphane formation. J. Food Sci. 2013, 78, H1459-
H1463. This article is reprinted with permission from the publisher, and can be found in Appendix B.
56
broccoli floret. Daikon radish root supported some 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. Practical
Application: Microwave-cooked frozen broccoli currently lacks sulforaphane content. Processing
of frozen broccoli was modified to permit sulforaphane formation, to provide a product with
similar content to that of fresh broccoli.
57
Chapter 5
Total Myrosinase Activity Estimates in Brassica Vegetable Produce
5.1 Introduction
Brassica vegetable crops contain diverse health-promoting compounds including glucosinolates
(GSL), flavonoids, carotenoids and vitamins. Glucosinolates themselves are not bioactive, but
are converted into multiple hydrolysis products including isothiocyanates and nitriles, through
the action of the endogenous enzyme myrosinase (1). The health benefits of broccoli are strongly
associated with isothiocyanates including sulforaphane (SF), phenethyl isothiocyanate (PEITC),
and allyl isothiocyanate (AITC). The chemical structure of hydrolysis products depends on the
structure of the GSL side chain and reaction conditions, including presence of myrosinase co-
factors such as epithiospecifier protein (ESP). Abundance of ESP favors formation of nitriles,
which not only lack anti-cancer activity, but are alternative hydrolysis products to
isothiocyanates, thus lowering isothiocyanate levels (1, 2).
Recent studies report that destruction of myrosinase during processing of commercially available
frozen broccoli products was the probable cause for little to no formation of the anticancer
compound SF, upon crushing the material (3, 4). We and others find that in the absence of
myrosinase, only about one tenth of the glucoraphanin present in a supplement or processed
broccoli product appears as SF and its metabolites in plasma and urine (5, 6). A larger study
found that SF absorption from a broccoli product lacking myrosinase may vary between <1%
and approximately 40% of the starting dose of glucoraphanin (7). Therefore, if consumers are to
gain the full health benefit of brassica foods, research is needed to find ways to enhance GSL
58
hydrolysis in brassica vegetable produce. A first step in such an endeavor is the accurate
measurement of myrosinase activity contained within a brassica vegetable sample.
Much of the work on myrosinase to date has been focused on the biochemistry of the enzyme,
rather than measurement in unextracted plant samples (1). Current methods for analyzing
myrosinase activity measure either substrate disappearance or product formation, following
addition of an exogenous substrate, sinigrin. Myrosinase activity is typically reported in terms of
hydrolysis of sinigrin, where a unit of activity is defined as that which hydrolyzes one micromole
of sinigrin per minute. Activity is typically measured by either sinigrin disappearance or glucose
release. Each has benefits as well as limitations, impacting the calculated enzymatic activity.
Disappearance of exogenous sinigrin, measured at 227 nm, suffers from lack of sensitivity as
well as interference from other components in the plant sample, including ascorbic acid,
endogenous GSL and proteins (8). Due to the variation in response factors of different GSL at
227 nm, myrosinase activity can be misinterpreted when endogenous GSL are present.
Neoglucobrassicin, an indole GSL, has a very different response factor compared to sinigrin: 0.2
and 1, respectively when measured at 227 nm (9). Glucose release during GSL hydrolysis is
typically measured as a coupled enzyme assay, either glucose oxidase-peroxidase with a
chromophore such as 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) and 4-
hydroxybenzoic acid or hexokinase-glucose-6-phosphate dehydrogenase measuring NADPH at
340 nm. The glucose oxidase-peroxidase procedure is sensitive, yet suffers from interference
from endogenous glucose and antioxidant compounds including ascorbic acid and polyphenolics
(8, 10). Hexokinase-glucose-6-phosphate dehydrogenase similarly suffers from interference by
endogenous glucose, flavonoids, and decomposition products of ascorbate (8, 10).
59
A key concern in measurement of myrosinase using either glucose release or sinigrin
disappearance is endogenous glucose and GSL in brassica vegetable samples. Typically,
endogenous glucose and GSL are removed by aqueous extraction followed by semi-purification
of the crude myrosinase protein – which is both time-consuming and alters the protein content –
and thus the reported myrosinase specific activity. Additionally, myrosinase exists in both
soluble and insoluble forms in plant material, so extraction could lead to loss of insoluble
myrosinase due to discarding the pellet following aqueous extraction, and even loss of some
soluble myrosinase, due to multiple rinses (11-13). Therefore it appears that myrosinase activity
estimated through purified protein may not directly reflect myrosinase presence in unextracted
brassica vegetable tissue samples.
A less common measure of myrosinase activity is by the measurement of the acid release rate, by
titrating the sample with 1 mM NaOH, using a dedicated pH meter coupled with a dispenser in a
thermostatically controlled cell (14). This may overcome some of the concerns mentioned
above, but requires specialized equipment, not available in all labs.
With these aspects in mind, glucose release and isothiocyanate formation assays may be more
appropriate for measurement of total myrosinase activity in brassica vegetables, than other
methods. Since it is necessary to purify myrosinase and use exogenous sinigrin during the
sinigrin disappearance assay, that assay is less likely to be able to measure total myrosinase
activity. Glucose release-based assays may be able to be modified for measurement of total
myrosinase activity. This manuscript endeavors to evaluate total myrosinase activity based on
isothiocyanate formation as it occurs in brassica vegetable produce, during a meal, and compare
this to measures of myrosinase activity using a modified glucose release assay.
60
5.2 Methods and materials
5.2.1 Sample collection and preparation
Broccoli florets, leaves, and roots (Brassica oleracea L. cv. ‘Sultan’, ‘Marathon’, ‘Patron’,
‘Pirate’, ‘Maximo’) and kale leaves and roots (Brassica oleracea L. cv. ‘Red Winter’, ‘Red
Russian’, ‘Dwarf Blue Curled Vates’) were grown at the University of Illinois in 2013, harvested
at commercial maturity, frozen in the field using liquid nitrogen, and transported to the lab on
dry ice for further processing. These 21 bulk tissue samples were freeze-dried for later analysis
of myrosinase activity. Three subsamples from each leaf, root and floret sample were used for
replication in each of the following assays. In previous work, freeze drying was shown not to
affect the content of glucosinolates or sulforaphane formation during hydrolysis (15). GSL were
measured as previously described (16).
5.2.2 Myrosinase activity using allyl isothiocyanate and sulforaphane formation
Myrosinase activity is traditionally measured as sinigrin hydrolysis (17, 18). However, broccoli
contains little or no sinigrin (16, 19), so we developed an assay that could compare hydrolysis of
exogenous sinigrin with endogenous glucoraphanin, by monitoring the concomitant formation of
AITC and SF. Myrosinase activity was initiated by the addition of sinigrin (2 mM final
concentration; Sigma, St. Louis, MO) to 100 mg of freeze-dried broccoli or kale tissue in 5.0 mL
water and incubated at 25C. Samples (0.5 mL) were removed at 5 min, spiked with an internal
standard of benzyl isothiocyanate (Sigma, St. Louis, MO), then immediately extracted into
dichloromethane (0.5 mL), centrifuged for 1 min at 16,000 g and analyzed for AITC and SF
content (see below). In later studies, samples were removed from the incubation at different
times, as shown in the figures. For calculation of myrosinase activity, the first three minutes were
used based on linearity of isothiocyanate generation.
61
5.2.3 Allyl Isothiocyanate and Sulforaphane Measurement
Using an Agilent model 7683B series auto sampler, 1 μL dichloromethane extract was injected
onto an Agilent 6890N gas chromatography system equipped with a single flame ionization
detector (Agilent Technologies, Santa Clara, CA). Samples were separated using a 30 m × 0.32
mm J&W HP-5 capillary column (Agilent Technologies). After an initial hold at 40 °C for 2
min, the oven temperature was increased by 10 °C/min to 260 °C and held for 10 min. Injector
temperature was 200 °C; detector temperature was 280 °C. Helium carrier gas flow rate was 25
mL/min. Results were quantitated against standards of SF (LKT Laboratories, St. Paul, MN) and
AITC (Sigma, St. Louis, MO).
5.2.4 Determination of Myrosinase Activity Using Glucose Release
The glucose release assay has traditionally utilized an aqueous extract of plant material. Because
we were interested in the activity in the whole food, we chose to run the assay using freeze-dried
plant material. In other aspects, the assay was similar to published methods (17, 20). Briefly,
freeze-dried, powdered plant material (50 mg) was weighed in duplicate into 2 mL tubes and one
mL sinigrin (10 mM) was added to each tube. After 10 sec of vigorous vortexing, one of the
paired samples was put directly into a heating block (95 ˚C) for 10 min to inactivate the
myrosinase enzyme (zero time blank). The second sample was incubated at 40˚C for 30 min and
then inactivated as outlined above. After inactivation, samples were cooled on ice for 5 min then
centrifuged at 16,000 g for 2 min. The supernatants were diluted 96-fold and aliquots (30 μL) or
glucose standard were added in a 96 well plate and followed by adding 200 µL of an ABTS-
glucose solution [2.7 mM ABTS, 1,000 units peroxidase (Type VI-A, Sigma), and 1,000 units
62
glucose oxidase in 100 mL], incubated for 20 min and absorbance measured at 630 nm in a
µQuant plate reader (Bio-Tek instruments, Winooski, VT). Myrosinase activity was also
measured for different incubation times (5, 10, 20, 30, and 40 min), as shown in the Figure 5.2A.
In an additional replicated set of the 21 plant samples, myrosinase activities was measured by
adding water in place of sinigrin, in order to compare myrosinase activity in the presence of
exogenous GSL (sinigrin) and endogenous GSL (Figure 5A).
5.2.5 Soluble and Insoluble Myrosinase Activity
Because we chose to measure myrosinase activity in plant material, we were interested to
compare myrosinase activity between freeze-dried brassica vegetable tissue and a crude protein
plant extract more typical of published measurements: aqueous extracts were generated by
extracting plant material into solutions containing 10 mM sinigrin, followed by centrifugation at
16,000 g for 1 min. Supernatants containing soluble myrosinase were transferred to new tubes,
brought to a final volume of 1 mL using sinigrin in water (10 mM), and either immediately
heated (zero time blank) or incubated at 40 ˚C for 30 min. Myrosinase activity of the centrifugal
pellet after crude protein extraction containing insoluble or bound myrosinase was also evaluated
by adding 1 mL of 10 mM sinigrin, vortexing and then heating immediately (zero time blank) or
incubating at 40 ˚C for 30 min, then heating.
5.2.6 Determination of Interference by Antioxidant Compounds in the Glucose Assay
In order to measure interference to the ABTS-glucose assay by endogenous antioxidant
compounds from plant samples, eight different concentrations of gallic acid ranging from 1.17 to
150 µM were incubated with glucose (5 µg fixed concentration) and the ABTS-glucose assay
63
was conducted. Interference of glucose measurement by endogenous antioxidant compounds in
the plant extracts (‘Marathon’ root, leaf, or floret), was also measured using the ABTS
antioxidant assay (21). Briefly, plant tissue extracts (30 µL of 50 mg/mL or 96-fold diluted
samples) were added to 200 µL of ABTS in 96 well plates (absorption at 630 nm = 1.0) and
measured absorbance after 6 min.
5.2.7 Statistical Analysis
Statistical analysis was conducted using the JMP 10 software (SAS institute Inc., Cary, NC).
Student’s T-tests were used for comparing treatment groups. Fisher’s Least Significant
Difference (LSD) test was conducted to compare means; P < 0.05 was accepted as significant.
Pearson’s correlation was conducted where pairs of myrosinase activities were compared.
5.3 Results and discussion
As shown in Figure 5.1, myrosinase activity of whole, freeze-dried broccoli floret tissue has 63%
greater activity than that of the corresponding aqueous extract. The freeze-dried broccoli floret
powder best represents total myrosinase activity in a serving of broccoli. Interestingly, when the
value for the crude protein extract (soluble myrosinase) was added to the value for myrosinase
remaining in the pellet (insoluble myrosinase), this equaled the value obtained for the
unextracted plant tissue, suggesting that this was a reasonable measure of soluble plus insoluble
myrosinase activity. In a previous study measuring insoluble myrosinase from Sinapis alba
seeds, the authors found that after three extractions, when the centrifugal pellet was resuspended
and assayed for activity, it was highly active, in agreement with our present results for floret
64
tissue. This suggests that measurement of myrosinase activity in an aqueous extracts greatly
underestimates activity of the unextracted tissues.
Aqueous extraction can be expected to adversely impact myrosinase activity measurement in
several additional ways. For example, isolating crude protein is often preceded by defatting,
which could alter myrosinase activity by changing its structure (22). Protein extraction buffers
often contain EDTA, which is known to chelate iron, as well as many other cations. Because the
iron at the active center of ESP is relatively labile, EDTA treatment has been found sufficient to
completely inhibit ESP-dependent nitrile formation, in favor of the generation of isothiocyanate
(23). Whereas this would not alter measurement of myrosinase activity by sinigrin disappearance
or glucose appearance, inclusion of EDTA could optimize measurement of activity measured as
ITC appearance. Endogenous ascorbic acid and magnesium chloride may also be removed
during extraction, particularly if extraction is followed by fractionation for semi-purification of
the enzyme away from endogenous glucose (17). The use of unextracted plant tissue when
measuring total myrosinase activity reduces the risk of excluding insoluble myrosinase and of
discarding important cofactors.
The results shown in Table 5.1 are comparisons of myrosinase activity in 5 broccoli and 3 kale
varieties measured independently using either glucose release or the proposed method of AITC
formation. The myrosinase activities from the glucose release assay are much lower values than
those from the AITC formation-based assay. Furthermore, although the two assays were in
relatively good agreement in ranking activity across plant parts (leaf, floret, root) within a
cultivar, there were several striking inconsistencies between assays. For example, ‘Red Russian’
kale root tissue had 85% of leaf activity levels by the glucose assay, but 145% of leaf activity by
the AITC assay. Conversely, for ‘Red Winter’ kale, both assays showed root tissue with 50% of
65
the activity of leaf samples. In all, when myrosinase activity from all tissues was compared
across the two assays, there was a significant, positive correlation between glucose appearance
and AITC formation (r = 0.488, n = 21, P = 0.0252). This suggests that the two assays measure
the same activity, although not optimally. When measuring an enzymatic activity it is important
to ensure that during the period measured, the rate is linear. Therefore we chose to evaluate
myrosinase activity at shorter time points, to determine if rates were more similar at earlier
timepoints.
In reviewing change in enzyme rates over time, Figure 5.2 shows that rates were initially linear,
but no longer linear by 5 min, whether using appearance of glucose (Figure 5.2A) or AITC
(Figure 5.2B).
At 5 min, there was insufficient glucose formed to differentiate glucose production from
background glucose present in the whole tissue sample, causing great variability in repeat
measures (Figure 5.2A). Thus it was not until 30 min that leaf samples showed significantly
greater activity than root samples – and not until 40 min that floret samples showed significantly
greater activity than root samples (Figure 5.2A). Both assays identified leaf as having the
greatest myrosinase activity per g DW of tissue and root the least, even though under the
conditions used, linearity was possibly only present for the first 3 min of incubation. In leaf
tissue, neither glucose nor AITC appearance at 5 min were significantly different from their
appearance at 30 min, showing that the reaction had almost stopped by 5 min under these
conditions.
In the literature the glucose release or sinigrin disappearance assays is typically run for 15 min or
more (8, 18, 20, 24), and assumes linearity of glucose formation during the entire assay (17),
which could greatly underestimate myrosinase activity and not accurately reflect the real time
66
activity of myrosinase, if the reaction slows in extracts in the same manner as we saw it slowing
in the unextracted tissue. We considered the possibility that substrate loss, when sinigrin is
converted to hydrolysis products, could be responsible in part for the change seen in linearity of
myrosinase activity. However, based on product formation at 30 min, over 40% substrate
remained in the samples from the AITC protocol. A previous study suggested that AITC can
react with protein, resulting in inhibition of enzymatic activity due to modification of amino acid
profile (25). This could also explain part of the decrease in myrosinase activity that occurred
over the 30 min measurement period. We further considered whether the steep decline in rate
from 5 to 30 min for the AITC assay (Figure 5.2B) could be due to reaching the limit of water
solubility for AITC. However, AITC solubility is 1.3 mg/mL, and the 30 min values for AITC
were an order of magnitude less than this (0.118 mg/mL) (26).
Use of the AITC formation assay (Figure 5.2B) gave a value closer to linear, particularly when
shorter incubation times were used (3 min), an option that is not possible for the glucose
appearance assay, if using unextracted plant tissue samples, because of variation due to the high
glucose background interference (Figure 5.2A). However, use of AITC formation could also
underestimate myrosinase activity, by ignoring formation of nitriles formed due to the presence
of ESP (1). The inclusion of EDTA in the reaction solution could possibly prevent formation of
nitriles, channeling hydrolysis toward isothiocyanate formation (23). However as with EDTA
removal of iron, this would actually overestimate the formation of isothiocyanates during food
consumption, which is important when considering the role for myrosinase-dependent
isothiocyanate release in health-promoting bioactivity.
We considered that antioxidant compounds naturally present in plant tissues might also interfere
with glucose estimation in the glucose appearance assay (8, 10), since the ABTS-glucose assay is
67
dependent on the oxidation of ABTS (Figure 5.3). When increasing concentrations of gallic acid
were added to the assay medium in the presence of 5 μg glucose, the appearance of oxidized
ABTS was inhibited in a dose-dependent manner, with 150 µM gallic acid causing a greater than
93% inhibition (Figure 5.4A). In order to avoid this interference for the ABTS-glucose assay, we
evaluated 96-fold diluted sample extracts to estimate glucose. At this dilution, plant endogenous
antioxidant compounds interfered with glucose generation less than 1, 6, and 3 % for root, leaf,
and floret tissues, respectively (Figure 5.4B). This dilution not only allows us to minimize the
potential interference from antioxidant compounds, but also to decrease the concentration of
endogenous glucose within the capacity range of the ABTS-glucose assay.
Published myrosinase activity assays typically use the addition of excess exogenous GSL; in
most cases sinigrin is used and activity is reported in terms of sinigrin hydrolysis (18, 24). Often
however, myrosinase activity is being measured in samples where other GSL or isothiocyanates
are the compounds of interest. The use of a GSL not endogenous or not found in high
concentrations in a cruciferous vegetable may skew results, particularly if substrate specificity
exists between myrosinase and endogenous GSL, as previously shown for Crambe abyssinica
seeds (27). In this respect we hypothesized that using endogenous GSL to measure myrosinase
activity should also be possible if the sample contains sufficient endogenous substrate to ensure
that myrosinase (and not substrate) is rate limiting. Hydrolysis of endogenous GSL was
measured as appearance of glucose in 21 test samples. The tissue total GSL concentrations
ranged from 1.86 µmole/g to 70.9 µmole/g (concentration during the assay, 0.093 mM – 3.54
mM). There was a highly significant correlation between myrosinase activity using endogenous
GSL and exogenous GSL (10 mM sinigrin) measured as glucose appearance (r = 0.744, n = 21,
P < 0.0001, Figure 5.5A). This shows that myrosinase activity can be measured using
68
endogenous GSL over this naturally-occurring GSL concentration range. The GSL concentration
varied significantly from tissue to tissue; averaged values of total broccoli floret and root GSL
concentration from 5 cultivars were 16.2 and 14.8 µmol/g DW, respectively which were
considerably higher concentrations than those observed in leaf tissue (4.0 µmol/g DW).
Averaged values of kale root and leaf tissue GSL concentrations were 44.3 and 10.0 µmol/g DW,
respectively. However, this method is only feasible to use for short time measurements and for
screening plant tissues which contain sufficient GSL. Since glucoraphanin is usually the
predominant GSL found in broccoli, we hypothesized that its hydrolysis would correlate to the
myrosinase activity determined previously. We found myrosinase activity using the formation of
either AITC or SF to be significantly correlated (r = 0.860, n = 8, P = 0.0062; Figure 5.5B). This
suggests that the ITC formation-based myrosinase assay described here can be utilized to
measure myrosinase activity using endogenous, as well as exogenous glucosinolates. For
example, myrosinase activity in broccoli, watercress, and horseradish might be measured by SF,
PEITC, and AITC formation, respectively. However, it is necessary to remember that whereas
this may provide an accurate measure for ingested foods, presence of ESP, and possibly other
myrosinase protein co-factors, will cause underestimation of absolute myrosinase activity.
5.4 Conclusion
Evaluating myrosinase activity from multiple broccoli varieties, we find that leaf and floret tissue
have relatively greater activity than root tissue, on a per g DW basis. Kale varieties exhibited the
same trend, with leaf tissues possessing greater myrosinase activity than root. Myrosinase
activity based on formation of AITC can be useful in determining short-term enzymatic rates of
myrosinase, with incubation times limited to one to three minutes. Samples can be run and
69
hydrolysis products extracted rapidly, allowing a quick turn-around time for a few activity
estimations. When using brassica vegetable tissues the glucose release assay cannot be run for
such short times, due to high baseline interference by endogenous glucose: on average, 30 min
was required to reliably measure glucose formation. For limited sampling, the glucose assay also
takes longer to perform, with multiple steps and incubations. However, if there are a large
number of samples, performance time per sample is reduced radically, making glucose release a
better high-throughput method, as long as samples are prepared in such a way that background
glucose does not interfere, such as the use of a crude protein extract in place of tissue samples.
However, we found that up to 36% of myrosinase activity was discarded in the pellet during
generation of the crude protein extract. Whether soluble and insoluble myrosinases are similar
with regards to activity or even substrate specificity is not known, although we found that
changing substrates from sinigrin to the endogenous substrate glucoraphanin did not alter
myrosinase measurements using the AITC method.
The proposed AITC formation assay can measure activity during the short-term linear phase of
myrosinase activity (~ three min), when used for brassica vegetable samples. Whereas this assay
appears most relevant for estimating myrosinase activity in terms of food consumption, further
studies are needed to understand if soluble and insoluble myrosinase enzymes are identical in
activity.
70
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5.6 Tables and figures
Cultivar Tissue Glucose assay AITC assay
Mean ± SD (Unit/g DW) Mean ± SD (Unit/g DW)
Sultan Floret 1.75 ± 0.08 15.62 ± 2.43
Sultan Root 0.83 ± 0.05 4.44 ± 0.53
Sultan Leaf 1.85 ± 0.11 20.60 ± 2.27
Marathon Floret 0.69 ± 0.12 13.64 ± 1.25
Marathon Root 0.73 ± 0.01 10.68 ± 0.44
Marathon Leaf 1.53 ± 0.12 19.01 ± 1.94
Patron Floret 2.04 ± 0.16 13.31 ± 1.49
Patron Root 0.86 ± 0.08 9.13 ± 0.25
Patron Leaf 1.27 ± 0.11 15.49 ± 2.68
Pirate Floret 1.63 ± 0.24 14.46 ± 1.10
Pirate Root 0.56 ± 0.03 3.62 ± 0.20
Pirate Leaf 1.66 ± 0.11 14.72 ± 2.39
Maximo Floret 1.77 ± 0.19 13.08 ± 1.41
Maximo Root 0.80 ± 0.03 9.00 ± 1.29
Maximo Leaf 1.38 ± 0.05 17.87 ± 1.74
Red Russian Root 1.68 ± 0.02 9.94 ± 0.32
Red Russian Leaf 1.96 ± 0.05 6.87 ± 0.96
Red Winter Root 1.09 ± 0.05 6.37 ± 0.58
Red Winter Leaf 2.21 ± 0.24 13.04 ± 0.46
Dwarf Blue Curled Vates Root 1.47 ± 0.07 15.30 ± 0.69
Dwarf Blue Curled Vates Leaf 2.39 ± 0.14 14.70 ± 0.94
Table 5.1. Comparison of myrosinase activity assays.
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Figure 5.1. Myrosinase activity in whole freeze-dried Marathon broccoli floret samples, crude
protein extract (soluble myrosinase), centrifugal pellet after crude protein extraction (insoluble
myrosinase activity), and total myrosinase activity (soluble + insoluble activity), measured as
glucose appearance after 30 min incubation. Mean ± SD (n = 3). Different superscripts indicate
significantly different values based on LSD test (P < 0.05). A unit is defined here as one µmol
glucose formed per minute, using 10 mM sinigrin as substrate.
75
Figure 5.2. Glucose and allyl isothiocyanate (AITC) time-dependent formation from ‘Marathon’
broccoli floret (filled diamond), root (filled square) or leaf (filled triangle) tissue samples
supplemented with sinigrin. (A) Glucose release, (B) AITC formation. (Insert: Linear range of
AITC formation used for myrosinase activity calculations). Values are mean ± SD (n = 3).
Different superscripts indicate significantly different values based on LSD test (P < 0.05).
Figure 5.3. Scheme of ABTS-glucose assay showing site of potential antioxidant interference.
76
Figure 5.4. Endogenous antioxidant interference of glucose measurement when using the ABTS-
glucose assay. A) interference of gallic acid (1.17-150 µM) on measurement of glucose (fixed at
5 μg). B) Percent interference of endogenous antioxidant compounds in different ‘Marathon’
broccoli tissues extracts (root, leaf, and floret) undiluted and diluted 96-fold. (A) ** significantly
different from control (no added gallic acid; P < 0.01). (B) Mean ± SD (n = 3). Different
superscripts indicate significantly different values based on LSD test (P < 0.05).
77
Figure 5.5. Correlation between myrosinase measurements using exogenous (singirin) or
endogenous glucosinolate in 21 broccoli and kale tissue samples. A) sinigrin (10 mM) or no
glucosinolate addition; glucose assay. B) sinigrin (2 mM) or no glucosinolate addition; AITC and
SF assay. Pearson’s correlation was calculated based on mean activity for each assay.
78
Chapter 6
Conclusion and Future Directions
6.1 Conclusion
The overall conclusion of this research project is that through fortification of frozen broccoli
with an exogenous source of myrosinase SF formation can be enhanced, even when broccoli is
cooked in a microwave oven. Current blanching practices used in frozen broccoli manufacture
destroy myrosinase activity, yielding a product that without exogenous myrosinase or
modification of the blanching protocol, is void of sulforaphane (SF) formation. Decreasing
blanching temperatures by 20 degrees to 76 °C can successfully prolong shelf life by inactivating
lipoxygenase, while preserving 82% of broccoli’s capability to form SF. The addition of an
exogenous myrosinase source, specifically daikon radish, a member of the Brassicaceae family
(Raphanus sativus), proved to yield exceptional thermal stability, maintaining 11% hydrolyzing
activity even after heating at 125 °C for 10 min. This time and temperature would far exceed
those applied during blanching as well as an average cooking process. The rate at which SF was
formed during thermal stability trials using two exogenous myrosinases, (10% w/w broccoli root,
or 10% w/w daikon radish) showed large variation in SF yield. In both instances where
exogenous myrosinase was present, SF formation increased by a factor of 10 compared to
endogenous myrosinase only. The commercial use of radish would be cost effective and timely
due to its quick growth rate. In addition, daikon is considered beneficial to marginal agricultural
farm land. Daikon radish is recommended as being used as a cover crop to penetrate tightly
packed soil which might normally be unsuitable for crop growth (1). Estimates of myrosinase
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activity from 5 different broccoli and 3 kale cultivars showed that floret and leaf tissue provide
greater activity then roots on a per g DW basis.
In order to perform this work it was necessary to develop an assay suitable for samples of whole
plant material. The newly developed allyl isothiocyanate (AITC) assay proved to be suitable to
determine the enzymatic rate of myrosinase in a whole food system. It was not necessary to
make a crude protein extract, since AITC measurement is not interfered with by other
endogenous compounds and, not being a spectrophotometric method, sample transparency does
not affect results. Furthermore, measurements can be made in as short as one or two minutes.
Often previously existing methods to measure myrosinase activity require extended incubation
times, which then greatly underestimate the true myrosinase activity because maximum
myrosinase activity rates were observed within the first five minutes (2, 3). Previously existing
methods that measure myrosinase often employ the use of crude protein extractions (4-6), which
do not give an accurate measure of activity in the whole food because myrosinase exists in both
soluble and insoluble forms. Finally, all past studies have used exogenous sinigrin as a substrate,
yet broccoli and many other brassica do not contain sinigrin. The use of substrates other than
sinigrin, such as glucoraphanin the glucosinolate in broccoli provided substantial correlation
between myrosinase activities. The use of an exogenous substrate to measure an endogenous
process can be avoided if samples contain enough of a key glucosinolate of interest, which could
provide better insight into the actual glucosinolate myrosinase system occurring in a specific
plant. Both the formation of AITC and the release of glucose proved to be important assays for
the measurement of myrosinase activity. Glucose release is not affected by the presence of
myrosinase modifier proteins such as Epithiospecifier protein (ESP) which have their action after
glucose release, but ignores the fact that some glucosinolate is converted to nitrile, which is not
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biologically beneficial. Furthermore, being a spectrophotometric assay, it is carried out on an
extract rather than the whole food. Multiple samples can be prepared at one time, making it
useful in the analysis of multiple sets of samples or field studies where hundreds of samples will
be compared to one another. The formation of AITC takes into account formation of nitriles
from ESP activity, in that only that activity resulting in isothiocyanates is recorded, and is
capable of measuring myrosinase activity at its peak rate with no interference from endogenous
compounds. For small sample sets it provides the means to prepare a sample in under five
minutes, but samples must be prepared individually. Perhaps the most important aspect of this
assay is that insoluble myrosinase activity is not ignored as it is in other methods.
6.2 Future directions
6.2.1 Future work to improve the health benefits of processed broccoli
This work is the first step in furthering the health promoting benefits found in frozen broccoli
and frozen broccoli products by optimizing myrosinase-dependent hydrolysis, and more work is
needed to ensure translation to commercialization. Since commercially available frozen broccoli
currently lacks any myrosinase activity and therefore lacks SF production, the modification of
current blanching practices should be reconsidered. The use of lipoxygenase as a blanching
indicator enzyme allowed formation of SF after a 24 hr hydrolysis, but not during a 9 hr thaw.
The active myrosinase enzyme required to form SF survived the redesigned blanching protocol
but structural damage was not severe enough to bring myrosinase and glucoraphanin in to
contact with one another. A study to look in more detail at the effect freezing and thawing has on
the structural integrity of broccoli could be performed to determine why glucosinolates but not
myrosinase were released during thawing. A sensory study should be performed to determine the
palatability of broccoli before and after the addition of an exogenous myrosinase source, such as
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daikon radish. The sensory study should also focus on any changes that occur during frozen
storage of broccoli and exogenous myrosinase, with a focus on shelf life changes. These studies
could help commercial frozen broccoli producers implement these changes with less resistance
from process and storage differences.
6.2.2 Future work for myrosinase thermal stability
Further research could be conducted to look at the differences in thermal stability that exist
among a full range of the brassica genus. Differences in myrosinase isoenzymes have been noted
for years across many members of the genus (7-9), although mostly the studies evaluated
physical characteristics, not activity. It is currently not known if certain subfamilies of
myrosinase show substrate specificity, although we do know that both soluble and insoluble
myrosinase are able to perform sinigrin hydrolysis to isothiocyanates. It is possible however;
those differences between myrosinase subfamilies A, B, C, or other less studied subfamilies
could exhibit different substrate selectivity and a different pattern of hydrolysis products. They
also could differ in their interaction with modifier proteins. Taking advantage of differences
could be the basis for hybridization of plants with myrosinase subfamilies which display a higher
thermal stability and favor the formation of isothiocyanates over nitriles.
6.2.3 Future work with myrosinase activity assay
Using AITC to measure myrosinase activity has proven to be a useful method in measuring the
total tissue activity of myrosinase, as it applies to a whole food system. This system should be
validated in other cruciferous vegetables, comparing activity using the addition of sinigrin as a
substrate to that of an abundant endogenous glucosinolate. Measuring isothiocyanate formation
suffers from not estimating any activity associated with ESP, which directs glucosinolate
hydrolysis to a non-bioactive nitrile. While working with a food system, this measure is pertinent
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to consider, since it is of value to understand the myrosinase-dependent isothiocyanate release,
and thus health promoting effects. To estimate the extent that ESP confounds the assay for any
given sample, samples could be chelated via the addition of EDTA which can bind to iron
located at the ESP active site (10). The addition of EDTA would give a maximum myrosinase
activity, as no other product formation should occur. This would allow a comparison between
total isothiocyanate formation with the actual isothiocyanate formation. Finally a study to look at
substrate specificity across the family of glucosinolates needs to be evaluated. Previous work
showed that substrate specificity existed in Crambe abyssinica seeds (11). If such a substrate
specificity was seen in other brassica vegetables it could point to the need to use endogenous
glucosinolates during the myrosinase assay, instead of sinigrin, for a true measure of myrosinase
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6.3 References
1. Chen, G.; Weil, R.R. Penetration of cover crop roots through compacted soils. Plant & Soil
2010, 331, 31-43.
2. Kim, H.-.; Chen, F.; Wang, X.; Choi, J.-. Effect of methyl jasmonate on phenolics,
isothiocyanate, and metabolic enzymes in radish sprout (Raphanus sativus L.). J. Agric. Food
Chem. 2006, 54, 7263-7269.
3. Xian, L.; Kushad, M.M. Correlation of glucosinolate content to myrosinase activity in
horseradish (Armoracia rusticana). J. Agric. Food Chem. 2004, 52, 6950-6955.
4. Dong, A.; Meyer, J.D.; Kendrick, B.S.; Manning, M.C.; Carpenter, J.F. Effect of secondary
structure on the activity of enzymes suspended in organic solvents. Arch. Biochem. Biophys.
1996, 334, 406-414.
5. Van Eylen, D.; Oey, I.; Hendrickx, M.; Van Loey, A. Kinetics of the stability of broccoli
(Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during
pressure/temperature treatments. J. Agric. Food Chem. 2007, 55, 2163-2170.
6. Palmieri, S.; Iori, R.; Leoni, O. Comparison of methods for determining myrosinase activity.
J. Agric. Food Chem. 1987, 35, 617-621.
7. MacGibbon, D.B.; Allison, R.M. A method for the separation and detection of plant
glucosinolases (myrosinases). Phytochemistry 1970, 9, 541-544.
8. Henderson, H.M.; McEwen, T.J. Effect of ascorbic acid on thioglucosidases from different
crucifers. Phytochemistry 1972, 11, 3127-3133.
9. Buchwaldt, L.; Larsen, L.M.; Plöger, A.; Sørensen, H. Fast polymer liquid chromatography
isolation and characterization of plant myrosinase, β-thioglucoside glucohydrolase, isoenzymes.
Journal of Chromatography A 1986, 363, 57-69.
10. Burow, M.; Markert, J.; Gershenzon, J.; Wittstock, U. Comparative biochemical
characterization of nitrile-forming proteins from plants and insects that alter myrosinase-
catalysed hydrolysis of glucosinolates. FEBS Journal 2006, 273, 2432-2446.
11. Bernardi, R.; Finiguerra, M.G.; Rossi, A.A.; Palmieri, S. Isolation and biochemical
characterization of a basic myrosinase from ripe Crambe abyssinica seeds, highly specific for
epi-progoitrin. J. Agric. Food Chem. 2003, 51, 2737-2744.
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Appendix A
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Appendix B
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