Water leached from growing broccoli sprouts is shown to inhibit the growth of E. coli bacteria. Sprouts (green vegetables) have long been known to be a good source of nutrition, but where water supplies are contaminated, growing them can be risky. This finding hints that some sprouts could be safer than others.
The authors have been investigating the medicinal
properties of broccoli sprouts for over 10 years. They report their finding
that spent irrigation water used to grow these sprouts inhibits the growth of
E. coli bacteria. With further investigation, methods might eventually be
developed for growing sprouts as an important nutrition source in places where
vegetables are unavailable or not affordable and water is scarce or
Laboratory testing showed that spent sprout
irrigation water (i.e. leachates), from germinating broccoli sprouts and seeds,
but not from alfalfa sprouts or seeds, inhibited the growth of non-enterohemorrhagic
E. coli DH10B. Growth inhibition was inversely proportional to isothiocyanate
concentration, which reached 192 μM (about 34 μg/ml) in the leachates by the
seventh hour of sprout growth. Sprouts produced from Brassica spp.
(cruciferous) vegetable seeds such as broccoli may offer special advantages in
terms of safety from microbial contamination. If pathogen-free water supplies
can be secured for the production of green sprouts, they may represent a viable
alternative to fresh green vegetables in areas where hostile climate, poor soil
conditions, insect predation, or even a nomadic lifestyle, preclude other
agricultural pursuits such as vegetable gardening, and where trade for fresh
vegetables is not a realistic possibility.
Green sprouts have been part of the human diet for
much of recorded history. Their commercial production has been a small niche
industry in the U.S. for the past 30 or so years. They are much more widely
consumed in a number of Asian countries (e.g. Japan and Korea), where they are
part of mainstream diets. In the past 8 years broccoli sprouts have gained
increased scientific attention due to their high content of phytochemicals that
are involved in protection against cancer and other degenerative diseases (1-6).
Nutritionally, green sprouts are also an excellent source of a variety of
vitamins and minerals. In many ways, they are an ideal fresh vegetable, one
that can be produced in 3 days’ time, in all climates, provided that an
enclosed area with relatively moderate temperature, minimal light (either
natural or artificial), and a source of clean fresh water is available. The
most critical factor is the need for a clean water supply. If food-borne
pathogens are present in the water supply, they will undoubtedly proliferate in
the warm, moist environment that drums or trays of sprouts provide. Thus, we became
interested in investigating the potentiality that some types of sprouts
(certain edible plant species) might serve to antagonize or prevent the growth
of the human pathogens that most frequently become problematic in situations of
potable water contamination. While success in this endeavor should not be
viewed as a go-ahead to produce green sprouts in areas with very poor
sanitation and contaminated water supplies, this approach might ultimately be
combined with other imaginative approaches. With additional creative inputs, it
may be possible to develop local production of a ready source of fresh
vegetables in areas where climate, soil, predation, or even a nomadic lifestyle
might preclude other agricultural pursuits such as vegetable gardening.
Sprouts are grown from seeds placed in
environmentally controlled, hydroponic conditions and incubated in warm, moist
conditions which are ideal environments for microbial growth. If Escherichia
coli or Salmonella spp. are present on the surface of the seed, it is
likely that they will multiply in the sprouting environment. To date, no
practical methods have been developed to check the growth of these contaminants
during germination and sprout growth or processing. They must therefore be
prevented from entering the process. If contamination occurs the affected
final product must be identified and destroyed. It is therefore essential that
seeds to be used for sprouting undergo surface-disinfection by treatment with a
biocide. The efficacy of such agents, most notably calcium hypochlorite, has
been extensively documented in the laboratories of Beuchat and colleagues
(7-11), as well as others (12-14). When these agents are used correctly the
resulting sprouts are safe to eat. A recommendation to use such a surface disinfection
process is now part of a guidance that the U.S. FDA issued in 1999 (15).
In addition to these safeguards, sprouts of
cruciferous or Brassica spp. vegetable seeds have a second-line defense
against contamination in that they produce secondary metabolites, also known as
phytochemicals, which have bactericidal activity. This bactericidal activity of
isothiocyanates has been known for many years. Isothiocyanates and the glucosinolate
/ myrosinase system that leads to their production plays a major role in plant
defense against fungal diseases and pest infestation (16). This activity has
been discussed extensively in the plant and microbial literature (reviewed by
16-18). The antimicrobial effects and mode of action of allyl isothiocyanate
have been examined against an array of bacteria and fungi including E. coli.
(19,20). Over 40 years ago, the antibacterial effects of 15 isothiocyanates
were evaluated on 10 test organisms, including both gram positive and gram
negative organisms and including E. coli (21). Kim et al. (22) utilized allyl
isothiocyanate (from the glucosinolate, sinigrin) as an antimicrobial on cooked
rice. Park et al. (23) recently determined that although allyl isothiocyanate
is a highly effective agent against E. coli 0157:H7 on alfalfa seeds, it
also has negative effects on seed viability. Sulforaphane, the isothiocyanate
of glucoraphanin from broccoli and broccoli sprouts, was originally isolated
from the cruciferous weed hoary cress (Cardaria draba), based upon its
bactericidal activity (24). This antimicrobial activity against a range of
bacteria and fungi was further characterized by Dornberger (25), and it was
recently demonstrated to have potent activity against the human bacterial
pathogen and carcinogen – Helicobacter pylori (2). H. pylori is
the causative agent of much of the world’s gastritis, ulcers, and stomach
Sulforaphane, a compound unique to the cruciferous
species, is a potent bacteriostatic and bactericidal agent against both
reference strains and clinical isolates of H. pylori, including those
that were resistant to multiple synthetic antibiotics. Sulforaphane, in
contrast to allyl isothiocyanate, is much less volatile, and does not have
similar effects upon seed viability. We present herein, evidence of the
bactericidal activity of broccoli seed and sprout leachate (i.e., spent sprout
irrigation water), but not alfalfa seed and sprout leachate, against a
laboratory strain of E. coli. We discuss the potential bactericidal
activity in broccoli and perhaps other cruciferous sprouts (e.g., cabbage, arugula,
kale, radish, mustard, cress and daikon), which may bolster the safety profile
of these sprouts in particular.
Materials and Methods
Levels of glucosinolates and isothiocyanates in seed and sprout leachates
In a laboratory setting, about 600 g of both
broccoli and alfalfa seeds (Caudill Seed Co., Louisville, KY, USA) were placed
in separate, adjacent trays on a commercial-style tray-type sprout cart. Before
being placed on the sprout cart, the broccoli and alfalfa seeds were
surface-disinfected by immersing them in a solution of 25% Clorox® commercial
bleach (ca. 13,000 ppm of sodium hypochlorite) in water with 30 mg/L Alconox®
laboratory detergent for 15 minutes, and then exhaustively rinsing the seeds
with water -- similar to commonly accepted protocols used by the sprouting
industry. The seeds were then placed in an inclined sprouting tray, rinsed
again with spray from an overhead mist nozzle, and then allowed to sit
undisturbed for 1 hour. After one hour, water was delivered via overhead mist
until about 200 ml of liquid per sprouting tray had run off and been collected.
Misting was continued using six 20-second sprays per hour until the next
collection period, at which time the trays were again left undisturbed for 1
hour. After this hour the leachate was rinsed away with fresh water and
The amounts and types of glucosinolates present
were determined by established spectrophotometric and HPLC methods (27-29). Total
glucosinolate + isothiocyanate determinations were made after digestion of leachate
samples with purified myrosinase (0.003 U/ml) and ascorbate (500 µM), by the cyclocondensation
reaction as described by Zhang and colleagues (29).
Effect of seed and sprout leachates on bacterial growth
Leachate collected as described above was
assessed for its effect on the growth in broth culture of E. coli K12
strain DH10B (laboratory collection). Bacterial growth at 37˚C in the dark was
monitored in complete Luria Broth (LB), as well as in a 10-fold dilution of
this medium (LB/10). The culture media were prepared by adding the dry
ingredients (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl for
full-strength medium) to the appropriate volume of water, or to leachate from
collections made following disinfection of the seeds as described above. (Tryptone
and yeast extract were from Difco Laboratories, Detroit, MI, USA; all other
reagents were from Sigma-Aldrich, St. Louis, MO, USA). Once the dry LB or LB/10
ingredients were dissolved, the pH was adjusted to pH 7.5 with NaOH, and the
medium was filter-sterilized and used for the cultivation of E. coli
DH10B. The LB/10 was prepared with leachates collected after the first, fourth,
and seventh hour of broccoli seed germination. Bacterial growth was monitored
according to standard methods by measuring the increase in absorbance at 550 or
600 nm, compared to an appropriate sterile medium control or water, on a UVMax microtiter
plate reader (Molecular Devices Corp., Sunnyvale, CA, USA). In separate
experiments, freeze-dried leachates were added to liquid medium and leachate
was used to prepare agar-solidified medium (1.5% Bacto Difco Agar). Dry weight
determinations were made on leachates by evaporating a subsample and holding it
at 105ºC until a constant weight was attained. Growth on semi-solid medium was
monitored by spot-plating 20 :L drops of an appropriate dilution (calculated to
yield between 3 and 30 CFU/drop) of viable bacterial culture (30) on both
control and leachate-amended media.
Leaching of glucosinolates and their cognate isothiocyanates from
germinating seeds and sprouts
The initial broccoli seed waste/rinse waters,
collectively termed “the leachate,” had a pH of 6.8 and was straw-colored. The
levels of isothiocyanate in the leachate from broccoli seeds and sprouts
increased from about 13 µM after the first hour of
growth following the seed disinfection treatment, to about 102 µM and 192 µM
after the fourth and seventh hour of growth, respectively. Levels of glucoraphanin
(the precursor of sulforaphane) were highest at the fourth hour, and the glucosinolate
concentrations were 148, 242 and 154 µM at the 1, 4 and 7 h collection times,
respectively. Glucosinolates recovered consisted of 99.4% glucoraphanin and
0.6% neoglucobrassicin. In contrast, there were no traces of these compounds
when alfalfa seeds/sprouts were treated in the same manner.
Recovery of total dissolved solids (dry weight)
from the hourly broccoli leachate collections ranged from 3.9 to 5.2 mg/ml. Total
recovery of glucoraphanin from the seeds was as high as 81.5 nmol/g seed/h. When
leaching was continued for 12 h, about 1 µmol of glucoraphanin per gram of
seed, or about 2% of the total glucoraphanin content of the seed used in these
experiments, was thus leached out of them. Leaching of glucosinolates rapidly
falls off as sprouts are grown further, such that the majority of the glucoraphanin
initially present in the seeds can still be found in the sprouts grown from
those seeds (data not presented;3,31).
Effect of broccoli seed and sprout leachates on bacterial growth
The growth inhibitory activity of broccoli seed
and sprout leachate was unaffected by freeze-drying or heating to 50°C, but
such activity was rapidly lost upon mixing with an agar base. There was a
substantial inhibitory effect on bacterial growth of broccoli seed leachate
when amended into Luria Broth (LB; Figs. 1 & 2). In contrast, no inhibitory
effect of the leachate obtained from alfalfa seeds was observed (Fig. 2).
This growth inhibitory effect was even more
dramatic in a low nutrient medium background (LB/10) since bacterial growth was
completely halted (Fig. 3; relative bacterial numbers and viability was
confirmed by drop-plating samples at various time points onto semi-solid Luria
Broth [data not shown]). This low nutrient environment is in fact more
representative of the actual “nutrient conditions” that exist during the
sprouting process, since, as discussed in the previous section, sprout leachates
had total dissolved solids contents of 4-5 mg/L, whereas LB contains 15 mg/L of
extremely rich organic material (10 mg/L tryptone and 5 mg/L yeast extract). In
addition to being more congruent with sprout leachate from a nutrient density
perspective, sulforaphane or other isothiocyanates would likely be more rapidly
complexed or bound in a growth medium as rich as LB. Thus, LB/10 was used for
Effect of leachate collection time on antibacterial effect
LB/10 was prepared with leachates collected after
the first, fourth, and seventh hour of seed germination. There was a potent
inhibitory effect of the broccoli seed/sprout leachate on bacterial growth.
This inhibitory effect increased with time over the germination period (Figure
4). The differential effects were rank-correlated with the isothiocyanate
content and with the glucosinolate-isothiocyanate content of the leachate (see
Fig. 1), and the bactericidal activity had disappeared by the third day of
broccoli sprout growth [data not shown]. There was in fact a stimulatory effect
of alfalfa seed leachates on bacterial growth: a 34% increase in final
bacterial titer was observed after incubation with the 7h alfalfa leachate. None
of the alfalfa sprout leachates showed any growth inhibitory effect on the E.
coli strain tested (Figure 4).
We have evaluated the bactericidal activity of leachates
(i.e., spent irrigation water) from sprouting seeds of broccoli. Leachates from
germinating broccoli sprouts, but not from alfalfa sprouts, have significant
capacity to inhibit the growth of E. coli DH10B in spent sprout
irrigation water. Although we have not tested these leachates against other
isolates or pathovars of E. coli or against Salmonella spp., we
have attempted to highlight the potential bactericidal activity of broccoli and
perhaps other cruciferous sprouts. Antimicrobial activity falls off after 7
hours, perhaps due to reduced leaching or to increased binding of isothiocyanates
to organic matter. Nonetheless, the early activity may provide special
advantages from the perspective of increased protection from microbial
contamination. Further testing against other bacterial strains, and testing of
production-scale drums that have been deliberately inoculated with pathogens,
is of course warranted. When combined with a seed surface-disinfection step,
the bactericidal activity demonstrated herein addresses a separate and
independent avenue for achieving higher levels of food safety in sprouts of
broccoli and perhaps other cruciferous vegetables. With proper attention to
growing conditions and assurance of a pathogen-free water supply, the
advantages of fresh green sprouts might ultimately be safely realized by those
who by virtue of climate, geography, or a host of other social-economic factors
are not able to produce or obtain green vegetables that are part of a healthy
One of the authors (JWF), as well as Johns Hopkins
University, own stock in Brassica Protection Products (BPP), a company whose
mission is to develop chemoprotective food products and which sells broccoli
sprouts in the U.S., Belgium, France, Germany, Japan, Korea, the Netherlands,
and New Zealand. JWF is a co-founder and an unpaid scientific consultant to BPP
and his stock is subject to certain restrictions under University policy. The
terms of this arrangement are being managed by Johns Hopkins University in
accordance with its conflict of interest policies.
This work was supported by unrestricted research
funds generously provided by the Lewis B. and Dorothy Cullman Foundation. We
acknowledge many colleagues, in particular Doug Archer, Arthur Davis, Fred Degnan,
Paul Swidersky, and Paul Talalay, for perceptive, critical reading of early
versions of this manuscript.
A portion of this paper was presented at the FDA, Center
for Food Safety and Applied Nutrition, Public Meeting: 2005 Sprout Safety, May 17, 2005, College Park Maryland, which can be accussed at:
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(Click to enlarge)
Figure 1. Effect of leachate from sprouting broccoli
seeds on the growth of E. coli DH10B in 10% Luria broth (LB/10),
assessed by spectrophotometry of bacterial broth cultures after 5h of
incubation at 37˚C.
(Click to enlarge)
Figure 2. Growth of E. coli DH10B, inoculated
into Luria Broth prepared with distilled water, or with broccoli or alfalfa
seed leachates collected 1 h after surface sterilization of seeds. Bacterial
growth was assessed over 8 h by spectrophotometry of bacterial broth cultures
incubated at 37˚C.
(Click to enlarge)
Figure 3. Comparison of growth of E.coli DH10B
inoculated into either Luria Broth or dilute (10%) Luria Broth prepared with
distilled water (open symbols; LB or LB/10) or with broccoli seed leachate
prepared in these basal media (closed symbols). Bacterial growth was assessed
over 8 h by spectrophotometry of bacterial broth cultures incubated at 37˚C.
(Click to enlarge)
Figure 4. Growth of E. coli DH10B inoculated
into dilute (10%) Luria Broth prepared with water or with broccoli or alfalfa
sprout leachate collected after 1h, 4h, or 7 h of seed/sprout germination. Bacterial
growth was assessed over 5 h by spectrophotometry of bacterial broth cultures
incubated at 37˚C. Open symbols represent alfalfa; filled symbols represent
broccoli; cross () represents controls. 1h (, ); 4h (, ); 7h (, ).