Effects of Glucosinolate Extracts from Various Brassicaceae

Species on Salmonella enterica

Weronika Lewkowicz

Thesis Advisor: Dr. Ross Koning

1
 Introduction

Glucosinolates are found in a variety of cruciferous vegetables of the Brassicaceae. The plants

of this family produce glucosinolates as an anti-feeding mechanism against herbivores, which includes

bitter taste and anti-nutritional value (Mithen 2001). There are three main types of glucosinolates

produced by the Brassica family: aliphatic, indole, and aromatic (Lou et al. 2008). Though there are

only three groups, there are over a hundred specific glucosinolates found in various Brassica species.

Glucosinolates are derived from amino acids, and contain a glucose molecule, a thiocyanate bridge,

and a sulfate (Halkier & Gershenzon 2006).

R S
Glc

N
OSO3-
Figure 1. Chemical structure of glucosinolate.

There are many factors influencing glucosinolate accumulation in each plant. These include

genetic, environmental, and developmental factors, which all have an effect on gene expression (Lou

et al. 2008). When expression of these genes is maximized, more glucosinolates are produced. Plants

must weigh the costs and benefits of putting their resources towards growth, defense, or reproduction,

which also influences the levels of glucosinolates produced.

While important for plant survival, glucosinolates also have an impact on humans as many of

the Brassicaceae vegetables are grown commercially for human consumption. While some vegetables

like broccoli and cauliflower are bred towards low glucosinolate levels to reduce bitter flavor, others,

such as horseradish, are prized for their spicy and bitter taste. Over the last fifty years, members of this

family of plants are increasingly used as a source for dietary canola oil. Rapeseed (Brassica rapa)

2
contains glucosinolates in all parts of the plant, and is selectively bred to reduce glucosinolate levels

and provide a sweeter taste in the oils pressed from the seeds (Halkier & Gershenzon 2006).

Besides providing flavoring, glucosinolates induce production of detoxifying enzymes in cells

and act as long-lasting antioxidants (Lou et al. 2008). Such positive findings have stimulated research

into the benefits of glucosinolate consumption on human health. Glucosinolates are antibacterial and

bacteriostatic agents towards various bacteria (Fahey et al. 2002). These findings are increasingly

important as pathogens are evolving antibiotic resistance and as people seek alternatives to the use of

prescription antibiotics.

As previously mentioned, the Brassicaceae includes cruciferous and mustard vegetables. Many

of the plants have economic importance as they have been altered and domesticated throughout the

years by humans (Prakash et al. 2011). The glucosinolate “spiciness” range starts with cabbage as the

mildest, and gets hotter with each vegetable: cauliflower, radish, mustard, horseradish, and wasabi as

the spiciest. All these plants have been bred for their flavoring, being used as spices in many cultures,

and some form of medicinal aid. Horseradish, for example, was not only bred for its uses as a culinary

herb, but has been used to treat everything from the common cold to aches and pains since the Roman

era (Voigt 2004). Wasabi and mustard seed also have a very long historical usage. Wasabi was

believed to have anthelmintic and sterilizing effects since the seventh century (Shimamura 2009).

Nowadays, mustard seeds are mostly known for their use in the culinary world. However, they were

thought to possess medicinal qualities dating back to Hippocrates, according to historical mentions of

belief (Jones 1983); mustard seeds were used as a laxative, emetic, and to draw blood away from

inflamed areas. It is interesting to consider that all these vegetables with high glucosinolates act as an

anti-feeding agent for small herbivores, yet are a desirable flavoring agent and potential medicinal

3
treatment for large herbivores. Though they do not have negative effects on humans, glucosinolates

can potentially have an effect on the exceedingly small bacteria living in gastric juices.

Historically, it was believed that consuming large amounts of spicy foods such as horseradish,

peppers, or mustard caused stomach ulcers, diarrhea and other abdominal discomforts (Voigt 2004).

However, it was discovered that bacteria from ill prepared foods is the cause of diarrhea, vomiting,

and other food-borne illnesses (CDCP 2016). One common bacterium fitting these criteria is

salmonella.

Salmonella enterica is a food-borne pathogen; it is a gram-negative, rod-shaped bacterium,

which causes 99% of human Salmonella infections (CDCP 2016). It is found all over the world in

undercooked meats, eggs, and unpasteurized milk (CDCP 2016). When infected with S. enterica, one

can experience gastroenteritis, enteric fever, bacteremia, diarrhea, and vomiting (PHAC 2011). The

infection is most commonly treated with antibiotics only if gastroenteritis or enteric fever occur,

otherwise fluid and electrolyte replacement are sufficient (PHAC 2011). However, as antibiotic

resistance is increasing, antibiotic treatments against S. enterica are beginning to fail (Seyfarth 1997).

Perhaps glucosinolate use can become an effective alternative to antibiotic use, although previous

studies on the subject have yielded mixed results.

This experiment examined whether glucosinolates extracted from horseradish (Armoracia

rusticana), wasabi (Eutrema japonicum), spicy green mustard (Brassica juncea), and wild wasabi

arugula (Diplotaxus erucoides) plants have a bactericidal or a bacteriostatic effect on Salmonella

enterica growth in vitro. Though there is minor variation in the amount of glucosinolates present in

individual plants, this experiment is mimicking natural consumption of these vegetables, without

quantifying levels of glucosinolates present. I hypothesized that spicy green mustard would have the

greatest effect on the zone of inhibition on Salmonella enterica because it possesses the highest

4
amount of glucosinolates per serving from the range of vegetables used in this experiment

(McNaughton and Marks 2003). The goal of this experiment was to study these vegetables as a

possible dietary supplement with benefits of lowering bacterial growth.

Materials and Methods

Maintenance of Salmonella enterica

Salmonella enterica was obtained from Fisher Scientific. Preparation and growth of bacterium

was completed from a single-cell colony on lysogeny broth (LB) agar plates. The single-cell colony

sample was inoculated into a vial of lysogeny broth and incubated in a shaker for 24 hours at 36oC.

Maintenance of Brassicaceae plants

Horseradish (Armoracia rusticana), wasabi (Eutrema japonicum), spicy green mustard

(Brassica juncea), and wild wasabi arugula (Diplotaxus erucoides) seeds were obtained from third

party sources (the Territorial Seed Company of Cottage Grove, Oregon and Hirt’s Gardens of Madina,

Ohio), planted in FAFARD #2 soil, and fertilized with Osmocote (12.7%N-7.6%P-10.2%K) plant

fertilizer. Three plants of each vegetable, with the exception of wasabi (n=1), were grown in a growth

chamber. All plants received 16h of light and 8h of dark per day and were kept at a constant

temperature of 23oC for 4 weeks.

Extraction and purification of glucosinolates

Glucosinolates were extracted from horseradish root, wasabi stem, spicy green mustard leaves,

and wild wasabi arugula leaves. The process for extraction was carried out as described by Tolra et al.

(2001) and Velasco et al. (2013). Two replicates of 1g samples from each vegetable were homogenized

in 4 mL of 100% methanol, centrifuged at 13,000 G for 3 minutes, and the supernatants were

decanted. The methanol was removed from the supernatant by evaporation in an 85 oC water bath for

50 minutes in a gentle flow of HEPA-filtered air.

5
 Application of glucosinolates on S. enterica lawns using the Kirby-Bauer method

Previously broth-cultured 200µl of S. enterica was spread onto two LB agar plates per extract, using a

sterile wire spreader. Five sterile, 6mm diameter Whatman 3M filter paper discs were placed onto each

LB plate and moistened with 5µl of extracts of glucosinolates from old horseradish root, new

horseradish root, wasabi stem, wasabi root, spicy mustard greens leaf, or wild wasabi arugula leaf. A

total of 12 plates were incubated for 6 days at 36 oC. The inhibition of S. enterica growth was measured

as the radial width (mm) of any clear zone (without bacteria) in the bacterial lawn around 10 discs for

each vegetable glucosinolate extract. Antibacterial activity was measured as the average width of the

zone of inhibition for each vegetable using a Swift instruments SM80BF microscope, at 15x

magnification. Data were graphed and analyzed with ANOVA in Excel (n = 10 reps of 1 extract per

plant).

Figure 2. Kirby-Bauer disk method of inhibiting Salmonella enterica bacteria on a

lysogeny broth agar plate. Five paper discs were soaked in glucosinolate extracts and
placed onto the plate. In this example plate, no zones of inhibition were observed for old
horseradish root extract.

Measuring effects of glucosinolates on S. enterica using a spectrophotometer

Two ml of LB broth were mixed with 10µl of a one-day broth culture of S. enterica, and 150µl of a

glucosinolate extract (one extract per tube) or 150µl sterile distilled water as the control. Vials were

6
placed on a shaker for 24h at 36oC. A Shimadzu UV-1280 spectrophotometer was used to measure

absorbance of the samples at a wavelength of 650nm.

Results

Kirby-Bauer Disk Method

Although the size of zones of inhibition varied slightly between extracts of various

glucosinolate plants (Fig. 3), ANOVA determined there were no significant differences between the

mean size of zones of inhibition from different treatments (n = 10 per plant sample, P = 0.049). Zones

of inhibition varied from 0.061mm to 0.61mm in radial dimension of the cleared zone. However,

extracts of both old and young horseradish root averaged the widest zones of inhibition at 0.305mm

(Fig. 3). The second and third quartiles overlapped among the treatments as further evidence of

minimal differences.

Figure 3. Zones of inhibition on Salmonella enterica plates using the Kirby-Bauer method.
Salmonella enterica was spread on LB plates and 10 paper discs soaked in glucosinolate extracts
from Brassicaceae species were placed onto each plate. Radial width of zones of inhibition were
measured after the plates were incubated for six days at 32 oC. The whiskers represent the range
of the data, x represents the mean, and the box represents the second and third quartiles with the
line as the median. (n = 10 per plant sample, P = 0.049).

7
Effect of glucosinolates on bacterial growth in a broth culture

Spectrophotometer measurements showed higher absorption values for Salmonella broth

cultures treated with glucosinolate extracts (Table 1). The methanol water control had the lowest

optical density at 650nm with a 1.900 reading, while the lowest absorption with a glucosinolate extract

was observed with Horseradish root (Table 1). None of the extracts inhibited the growth of Salmonella

relative to the controls; in every case, the glucosinolate extracts stimulated Salmonella growth,

resulting in higher optical density than the two controls.

Table 1. Effects of glucosinolate extracts on growth of Salmonella enterica in broth culture. 0.15
mL of various glucosinolate extracts or controls were mixed into 2ml of Lysogeny broth and
inoculated with 10 μL of Salmonella enterica broth culture. The bacteria were incubated at 34 oC
for 24h. Optical density was measured at 650nm using a spectrophotometer.

Absorption at
Glucosinolate Extract with Salmonella enterica
650nm
Horseradish root 2.110
Spicy Green Mustard leaf blades 2.210
Wild Wasabi Arugula leaf blades 2.243
Wasabi stem 2.123
(control) dH2O 1.950
(control) Methanol Water 1.900

Discussion

As antibiotic resistance has become a problem in modern medicine, this experiment was

designed to examine whether glucosinolates extracted from horseradish, wasabi, spicy green mustard,

and wild wasabi arugula plants have a bactericidal or a bacteriostatic effect on Salmonella enterica

growth in vitro. The standard Kirby-Bauer disk method was used to test the effects of glucosinolates

on Salmonella enterica (Fig. 2). A broth culture method was also implemented to test any

bacteriostatic effects by measuring cell density with a spectrophotometer (Table 1). The glucosinolate

extracts had no significant effect on the zones of inhibition on Salmonella enterica; all zones were

8
under 1mm in radial dimension (Fig. 3). Absorption measurements actually increased after addition of

glucosinolate extracts when compared to the control (Table 1).

It was originally hypothesized that spicy green mustard extract would have the greatest effect on

the zone of inhibition on Salmonella enterica and the greatest inhibition of cell proliferation in broth

culture because it possessed the highest amount of glucosinolates per serving from the range of

vegetables in this experiment (McNaughton and Marks 2003). However, this hypothesis was not

supported as there were no statistical differences in the zones of inhibition among extracts (see Fig. 3).

Moreover, all of the extracts actually stimulated cell proliferation in the broth cultures relative to

controls.

The finding that crude glucosinolate extracts increased the optical cell density (Table 1)

compared to controls was an unanticipated result of the experiment. The bacteria unexpectedly

benefited from the addition of Brassicaceae plant extracts. This increase seems to indicate that there

are some beneficial properties found in the crude methanol plant extracts. Perhaps the sugars, amino

acids, and other molecules in the extract may act as a food source supplement to the bacteria, which

helped increase the cell density over the 24-hour incubation period. This could potentially explain the

unexpected increase in absorption measurements. The sugar and carbohydrate filled plant extract could

also be a possible explanation for small zones of inhibition (see Fig. 3). The plates were incubated

over 6 days, so it is possible that some of the sugars and carbohydrates helped fuel Salmonella

enterica, so the glucosinolates were not as effective. It would be beneficial to further pursue this

question through repeating the experiment and separating the components extracted from the plants,

and only using glucosinolates in the Kirby-Bauer disk method.

Furthermore, there have been other studies which looked at various bacteria, both gram-

positive and negative, and any bacteriostatic effects caused by glucosinolate extracts. Previous

9
research shows mixed results. Shin et al. (2009) found that glucosinolate hydrolysis extracts from

wasabi did have a significant effect on gram-negative bacteria, while Vig et al. (2009) summarized that

gram-negative bacteria are less susceptible to glucosinolate extracts than gram-positive bacteria. My

results seem to agree with Vig et al. (2009) as the crude glucosinolate extracts, which included other

plant components and molecules, had no measurable effect on the gram-negative Salmonella enterica.

Therefore, the results of the present study were not completely unexpected as previous studies

have shown a lack of antimicrobial activity of intact glucosinolates compared to glucosinolate

hydrolysis products (Aires et al. 2009). Interestingly, a different preparation of wasabi by Shin et al.

(2009) used concentrated allyl isothiocyanate (AIT) extracts, and resulted in bactericidal activity

against Salmonella typhimurium, which is a close relative of the Salmonella enterica I have used in

this experiment. However, I used a simple extraction method that did not purify or concentrate the

glucosinolates, unlike Shin et al. (2009) where they concentrated their extracts to magnify the effects

of specific glucosinolates. The difference in our experiments was that I was looking at consuming

these vegetables as a dietary supplement in the hopes of killing bacteria, and not in a pharmaceutical

matter where one would amplify the pure glucosinolate extracts.

Unfortunately, the effects of crude glucosinolate extracts in this experiment were limited to

Salmonella enterica, unlike some of the other studies with multiple bacteria ( n = 10 per plant sample).

The original intent was to study the effects on growth of Helicobacter pylori cultures. Unfortunately, a

viable culture was not available despite an extensive search. A back-up plan attempted to use its

relative, Campylobacter jejeuni. The organism was purchased but could not be grown in any

reasonable medium; perhaps because it is fastidious in its nutrient requirements (Davis and DiRita

2008). After attempting local cultures that were contaminated, I purchased Salmonella enterica and

10
found it was easily cultured in our limited facilities. Further experimentation with H. pylori and other

bacterial species is needed, especially considering the mixed results of current research studies.

Over all, the results of this study show that glucosinolate extracts are not an alternative to

antibiotic treatment against Salmonella enterica. Though previous studies have identified

glucosinolates extracted from wasabi and horseradish as having antimicrobial activity, the bacteria

responding in the research was Helicobacter pylori and other food-borne bacteria, but not Salmonella

enterica (Shin et al. 2004, and Aires et al. 2009). In the case of treating Salmonella enterica infections,

antibiotics have to remain as the proper treatment method as compared to glucosinolate extracts.

It was hypothesized glucosinolates would be a promising alternative to antibiotics in the hopes

of being able to simply choose to eat these spicy vegetables, without purifying the glucosinolates. This

explains why this project was limited to testing crude methanol-glucosinolate extracts, mimicking

natural consumption of Brassicaceae vegetables. It was unfortunate that the study was limited to one

bacterium, which was not affected by the extracts, however, it does not completely eliminate

glucosinolates as an alternative to antibiotics against other species of bacteria in the future. Further

research should be completed using not only a wider variety of bacteria, but a cleaner extract of

glucosinolates in an amplified form to address the question if these extracts could be used in a

pharmaceutical manner.

References
Aires, A., Mota, V., Saavedra, M., Rosa, E., & Bennett, R. N. (2009). The antimicrobial effects of
glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the
human intestinal tract. J App Microbio, 106(6), 2086–2095.
Bauer, A., Perry D., & Kirby W. (1959). Single disc antibiotic sensitivity testing of staphylococci.
A.M.A. Arch Intern Med, 104, 208–216.

11
Bauer, A. W., Kirby W., Sherris J., & Turck M. (1966). Antibiotic susceptibility testing by a
standardized single disk method. Am J Clin Pathol, 36, 493-496.
Blanchard, T. G., & Nedrud, J. G. (2006). Laboratory maintenance of Helicobacter species. Curr
Protoc in Microbiol, CHAPTER, Unit 8 B.1.
Centers for Disease Control and Prevention. (2016). Salmonella. Retrieved February 6th, 2017, from
https://www.cdc.gov/salmonella/
Davis, L., & DiRita, V. (2008). Growth and laboratory maintenance of Campylobacter jejuni. Curr
Protoc Microbiol, CHAPTER 8, 1–8.
Fahey, J. W., Haristoy X., Dolan P. M., Kensler, T. W., Scholtus I., Stephenson K. K., … Lozniewski,
A. (2002). Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of
Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. PNAS (US) 99:
7610–7615.
Giamoustaris, A., & Mithen, R. (1995). The effect of modifying the glucosinolate content of leaves of
oilseed rape (Brassica napus ssp. oleifera) on its interaction with specialist and generalist
pests. Ann Appl Bio, 126(2), 347-363.
Gandhi, S. D., Kishore, V. K., & Crane, J. M. (2009). Selection for low erucic acid and genetic
mapping of loci affecting the accumulation of very long-chain fatty acids in meadowfoam seed
storage lipids. Genome, 52(6), 547-556.
Halkier, B. A., & Gershenzon, J. (2006). Biology and biochemistry of glucosinolates. Annu Rev Plant
Biol, 57(1), 303-333.
Haristoy, X., Angioi-Duprez, K., Duprez, A., & Lozniewski, A. (2003). Efficacy of sulforaphane in
eradicating Helicobacter pylori in human gastric xenografts implanted in nude mice.
Antimicrob Agents Ch, 47(12), 3982–3984.
Jones, O. R.. (1983). London mustard bottles. Historical Archaeology, 17(1), 69–84.
Koprna, R., Nerušil, P., Kolovrat, O., Kučera, V., & Kochoutek, A. (2006). Estimation of fatty acid
content in intact seeds of oilseed rape (Brassica napus L.) Lines using near-infrared
spectroscopy. Czech J. Genet. Plant Breeding, 42 (4), 132-136.
Kumar, S., Chauhan, J., & Kumar, A. (2009). Screening for erucic acid and glucosinolate content in
rapeseed-mustard seeds using near infrared reflectance spectroscopy. J Food Sci and Tech,
47(6), 690-692.

12
Lou, P., Zhao, J., He, H., Hanhart, C., Carpio, D. P. D., Verkerk, R., … Bonnema, G.. (2008).
Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves. The New Phytol,
179, 1017–1032.
Magrath, R., Bano, F., Morgner, M., Parkin, I., Sharpe, A., Lister, C., . . . Mithen, R. (1993). Genetics
of aliphatic glucosinolates. I. Side chain elongation in Brasica napus and Arabidopsis thaliana.
Heredity, 72(3), 290-299.
Magrath, R., & Mithen, R. (1993). Maternal effects on the expression of individual aliphatic
glucosinolates in seeds and seedlings of Brassica napus. Plant Breeding, 111(3), 249-252.
McNaughton S.A., Marks G.C. (2003). Development of a food composition database for the
estimation of dietary intakes of glucosinolates, the biologically active constituents of
cruciferous vegetables. Br J Nutr 90(3), 687-697.
Mithen R. (2001). Glucosinolates - biochemistry, genetics and biological activity. Plant Growth Regul,
34, 91-103.
Prakash, S., Wu, X., & Bhat, S. R. (2011). History, evolution, and domestication of brassica crops.
Plant Breeding Reviews Janick/Plant Breeding Reviews, 35, 19-84.
Public Health Agency of Canada (2011). Salmonella enterica spp. Retrieved February 6, 2017 from
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/salmonella-ent-eng.php
Seyfarth, A. (1997). Antimicrobial resistance in Salmonella enterica subsp. enterica serovar
typhimurium from humans and production animals. J of Antimicrob Ch, 40(1), 67-75.
Shimamura, N. (2009). Wasabi. Retrieved February 27, 2016, from http://www.tokyofoundation.org/
en/topics/japanese-traditional-foods/vol.-18-wasabi
Shin, I., Masuda, H., & Naohide, K. (2004). Bactericidal activity of wasabi (Wasabia japonica) against
Helicobacter pylori. Inter J of Food Microbio, 94(3), 255–261.
Tolra, R., Poschenrieder, C., Alonso, R., Barcelo, D., & Barcelo, J. (2001). Influence of zinc
hyperaccumulation on glucosinolates in Thlaspi caerulescens. New Phytol, 151(3), 621-626.
Velasco, P., Lema, M., Francisco, M., Soengas, P., & Cartea, M. (2013). In vivo and in vitro effects of
secondary metabolites against Xanthomonas campestris pv. campestris. Molecules, 18(9),
11131-11143.
Vig, A., Rampal, G., Thind, T., & Arora, S. (2009). Bio-protective effects of glucosinolates – A
review. LWT - Food Sci and Tech, 42(10), 1561–1572.
Voigt, C. E. (2004). Horseradish. The Herbarist, 70, 66-72.

13