Current Intervention Strategies For The Microbial Safety of Sprouts

2099
Journal of Food Protection, Vol. 76, No. 12, 2013, Pages 20992123
doi:10.4315/0362-028X.JFP-12-437
Copyright G, International Association for Food Protection
Review
Current Intervention Strategies for the Microbial Safety of Sprouts

ADI MD SIKIN,1,2* CLAIRE ZOELLNER,1
1Institute
AND
SYED S. H. RIZVI1
of Food Science, Cornell University, Stocking Hall, Ithaca, New York 14853-7201, USA; and 2Universiti Teknologi MARA, Shah Alam,
Selangor, Malaysia
MS 12-437: Received 28 September 2012/Accepted 12 July 2013
ABSTRACT
Sprouts have gained popularity worldwide due to their nutritional values and health benefits. The fact that their consumption
has been associated with numerous outbreaks of foodborne illness threatens the $250 million market that this industry has
established in the United States. Therefore, sprout manufacturers have utilized the U.S. Food and Drug Administration
recommended application of 20,000 ppm of calcium hypochlorite solution to seeds before germination as a preventative method.
Concentrations of up to 200 ppm of chlorine wash are also commonly used on sprouts. However, chlorine-based treatment
achieves on average only 1- to 3-log reductions in bacteria and is associated with negative health and environmental issues. The
search for alternative strategies has been widespread, involving chemical, biological, physical, and hurdle processes that can
achieve up to 7-log reductions in bacteria in some cases. The compilation here of the current scientific data related to these
techniques is used to compare their efficacy for ensuring the microbial safety of sprouts and their practicality for commercial
producers. Of specific importance for alternative seed and sprout treatments is maintaining the industry-accepted germination rate
of 95% and the sensorial attributes of the final product. This review provides an evaluation of suggested decontamination
technologies for seeds and sprouts before, during, and after germination and concludes that thermal inactivation of seeds and
irradiation of sprouts are the most practical stand-alone microbial safety interventions for sprout production.
Sprouts have been viewed as a health food in the Far

East for more than 5,000 years. As part of the changing
lifestyle in the West toward convenience and health, the
consumption of sprouts in western populations has grown.
In the United States, about 10% of the population eats
sprouts regularly, resulting in a $250 million market.
According to the International Sprout Growers Association,
about 475 U.S. growers produce almost 300,000 tons of
sprouts annually (91). The increase in sprout consumption is
partly attributed to the high nutritional value of this food
(19, 51, 61, 174), which has been documented by many
researchers to play a role in protecting against certain
carcinogens and reducing the risk of developing chronic
diseases (183). More information about the nutritional
values of sprouts was provided in a recent review by Marton
et al. (99).
Despite the healthy image associated with sprouts, they
are one of the most common vehicles for produce-associated
bacterial foodborne illnesses. Sprouts were involved in
about 10% of the produce-associated outbreaks from 1990
through 2002 in the United States (76). The earliest reported
sprout-associated outbreak in the United States occurred in
1973 and involved soybean, cress, and mustard sprouts
contaminated with Bacillus cereus (131). From 1995 to
2003, multiple outbreaks of Salmonella or Escherichia coli
O157:H7 and O157:NM infections have occurred each year
* Author for correspondence. Tel: 607-255-7904; Fax: 607-254-4868;
E-mail: am882@cornell.edu.
throughout the United States. Because of the multiple

sprout-related outbreaks, in 2001 the U.S. Food and Drug
Administration (FDA) considered raw sprouts a potentially
hazardous food in their Food Code (164). The challenges
concerning the reduction of sprout-associated foodborne
illnesses persist, and repeated nationwide outbreaks have
been caused by the consumption of raw alfalfa sprouts
contaminated with Salmonella or E. coli O157:H7 (26).
Sprout-related outbreaks of foodborne illnesses also have
been reported in other parts of the world. An outbreak of E.
coli O157:H7 infection from radish sprouts in Japan in 1996
led to 9,451 illnesses and 12 deaths (all children) and drew
worldwide attention (106). In May 2011, an outbreak of
E. coli infection mainly in Germany and central Europe
claimed 54 lives and was linked to bean and fenugreek
sprouts contaminated with an unusual strain of E. coli
O14:H4 with increased virulence and antibiotic resistance
(78). A compilation of sprout-related worldwide outbreaks
is available (38).
Contaminated sprout seeds have been recognized as the
primary source of pathogens for these sprout-related
outbreaks. Seeds usually harbor native microbiota (102 to
106 CFU/g) and fecal coliforms (102 to 103 CFU/g) from the
environment (45). These bacterial levels can increase during
sprouting, reaching populations as high as 108 to 1011 CFU/
g (59, 127). Thus, treatment of seeds before exposure to
sprouting conditions, which are conducive for microbial
growth, has been generally accepted as the most effective
intervention approach in the sprout cycle (112).
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SIKIN ET AL.
As the last line of defense for elimination of pathogens

that have survived seed treatment and have proliferated
during germination, surface treatment has been commonly
practiced but has limited efficacy. Although plant pathology
research is difficult due to the short growing times for
sprouts (3 to 7 days) (112) and brief life span (3 to 10 days
at refrigeration temperatures) (39), studies have revealed
internalization of pathogens and the presence of biofilms in
sprouts even after postharvest treatment (54, 73). E. coli
O157:H7 was found in the inner tissue of inoculated radish
sprouts during sprouting, and spraying of the outer surface
of sprouts with mercury chloride did not kill the E. coli
O157:H7 located at subsurface locations (73). Similarly, E.
coli cells were recovered from the apoplastic fluid and
extracts of mung bean sprouts that had been surface
sterilized with 20,000 ppm of sodium hypochlorite and
could be visualized using b-glucuronidase staining (179).
Fransisca et al. (54) used scanning electron microscopy to
examine radish sprouts sprayed with 0.04% calcinated
calcium. Some fine weblike structures, a typical feature of
biofilm formation, were observed on all parts of the sprouts,
especially on the cotyledons and roots. Therefore, more
effective safety interventions for the final sprout product are
needed.
This article is a review of the chemical, physical,
biological, and combination intervention strategies for seed
treatment and raw sprout decontamination. The objective
was to identify the effective areas and methods for
decontamination during sprout production. A brief overview
of irrigation water treatment and sanitation as interventions
during germination also is included.
EFFECTIVENESS OF CURRENT PRACTICES
In the United States, the current industry seed
disinfection step follows the recommendation of the
National Advisory Committee on Microbiological Criteria
for Foods (NACMCF) (112, 162). The use of 20,000 mg/
liter (ppm) calcium hypochlorite or an equivalent antimicrobial treatment capable of achieving a 5-log reduction in
the microbial population is suggested for treatment of seeds
intended for sprout production. However, the NACMCF
noted that 20,000 ppm of chlorine treatment has only a
median reduction of 2.5 log CFU/g (108, 155), and efficacy
varies depending on the type of seed due to differences in
surface topography (52). For example, alfalfa seeds have a
thick waxy coat with a uniform surface containing shallow
valleys, whereas broccoli and radish seeds have more
permeable coats and surfaces with deep pits, making
inactivation of pathogens more difficult (52). However,
20,000 ppm of chlorine treatment is generally beneficial for
achieving above 90% germination when applied for 15 to
20 min (23, 53, 92, 96), which is an important consideration
for the sprout industry.
The recommended concentration of chlorine for
washing fresh produce is 50 to 200 ppm (143). However,
200 ppm of chlorine is considered too weak to overcome the
rapid depletion of free chlorine that results from a sudden
surge in organic matter and therefore probably achieves
J. Food Prot., Vol. 76, No. 12
only 1- to 3-log reductions (5). The highest level of

inactivation by postharvest aqueous chemical interventions
without changes in visual quality parameters during storage
at 4uC for 9 days was achieved with chlorous acid treatment
(268 ppm, 10 min): a 5-log reduction of Salmonella and
Listeria monocytogenes in inoculated mung bean sprouts
(93). Similarly, treatment of alfalfa sprouts with 2,000 ppm
of chlorine for 2 or 5 min caused a 5-log reduction in
populations of Salmonella Stanley (58). However, this
concentration of chlorine exceeded the regulatory limit of
200 ppm in wash water for fresh produce. The traditional
safety assurance step is cooking of the product; however,
sprouts usually are consumed after only minimal cooking
and more often are eaten raw. Thus, chlorine-based
interventions during sprout production have major limitations, and alternative treatments are needed.
DEVELOPMENT OF ALTERNATIVE TREATMENTS
An abundance of alternative methods for reducing
bacterial contamination on sprouts have been reported in the
literature of food safety, food microbiology, and food
processing. The number of articles published in this area
continues to grow each year, particularly for seed treatment
(Fig. 1). Many methods have been more effective than the
FDA recommended method of 20,000 ppm of calcium
hypochlorite, but an analysis of published sprout seed
decontamination data by Montville and Schaffner (108)
indicated that no method published to date has been able to
completely remove pathogens from sprout seeds on a
commercial production scale. In the most recent review by
Ding et al. (37), emerging technologies in chemical,
physical, biological, and combination treatments for seeds
were compared with the FDA recommended method, and
some methods achieved greater than 7-log reductions in
microbial populations. Identification of sources of contamination and an better understanding of the survival of
pathogens on seeds and sprouts as reviewed by Yang et al.
(185) is a complimentary endeavor. However, important
considerations in the development of novel decontamination
methods are the implications and practicality in an industrial
setting, i.e., the effects on quality, treatment time, required
equipment, and regulatory compliance. To address the needs
of the sprout industry for promoting consumer confidence
and reducing the incidence of foodborne illness, the utility
of these intervention strategies should be examined across
methods and throughout the production process.
Chemical interventions. The main limitations of
current chlorine-based washes are the possible hazards
associated with producing, transporting, and handling large
amounts of chlorine. On-site generation of antimicrobials
that can easily be incorporated into sprout farming systems
would be preferable. Organic acids, chlorine dioxide (ClO2),
electrolyzed oxidizing water, and ozone have much
potential as preferred chemical interventions for ensuring
sprout quality and safety. Table 1 provides a summary of
the relative effects of these treatments on seed and sprout
microbial safety.
INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS
2101
FIGURE 1. Published journal articles (bars) per year dealing with decontamination of seed sprouts and/or sprouts. The articles were retrieved
from Google Scholar with the results of 607 articles. For 2011, only articles published up to 27 October, 2011 were taken into account.
Chemical interventions: organic acids. Organic acids

such as lactic acid and acetic acid are effective antibacterial
agents (50) and are classified by the FDA as generally
regarded as safe (GRAS) (166, 168). Organic acids are
preferred to synthetic antimicrobials because they are more
environmentally friendly and cost-effective and can be used
by organic producers. Lang et al. (92) compared the efficacy
of lactic acid and acetic acid alone and in combination with the
FDA recommended treatment for E. coli O157:H7 on alfalfa
seeds. The most lethal treatment was 20,000 ppm of active
chlorine for 15 min (6.9-log reduction) followed by 5% lactic
acid (6.6-log reduction) and 5% acetic acid (6.3-log reduction)
for 10 min at 42uC. However, recovery of pathogens during
germination of seeds treated with all of these methods reached
levels of 107 to 108 CFU/g, indicating the importance of
accounting for injured pathogen cells by using selective and
nonselective media and the inadequacy of seed surface
treatments for achieving safety of ready-to-eat sprouts.
Various combinations and concentrations of organic
sanitizers, including organic acids, fatty acids, and surfactants, have been more effective against pathogens on seeds
(130). Because the surface of alfalfa seeds is waxy, nonpolar
fatty acids were selected for their affinity to this coating and
for their action on cell membrane permeability, as indicated
by the slight antimicrobial effect of these acids when used
alone (27). Lactic acid acts as a chelating agent of metal
cations present in the cell membrane of gram-negative
bacteria, and glycerol monolaurate is a natural surfactant
selected for improving the coverage and penetration of the
sanitizer. Consequently, the most lethal combination was a

15| fatty-acid sanitizer composed of 15,000 ppm of 60:40
caprylic:capric acid, 15,000 ppm of lactic acid, and 7,500 ppm
of glycerol monolaurate, all of which are GRAS compounds.
Alfalfa seeds soaked for 3 min in this sanitizer achieved
reductions of 4.77, 6.23, and 3.86 log CFU/g for E. coli
O157:H7, Salmonella, and L. monocytogenes, respectively,
while maintaining 91.5% seed viability. No injured pathogen
cells were recovered after 48 h of incubation.
Since these experiments were conducted, research on
organic acidbased sanitizers for seed treatment has
continued, and the antimicrobial activity of these compounds has been supported. Bari et al. (14) attempted to
enhance the effects of organic acids by using several cycles
of hot and cold sanitizer treatments to wash seeds. Both,
0.05% phytic acid and 3% oxalic acid used independently
for two cycles of 20 s at 75uC and 20 s at 0uC reduced levels
of E. coli O157:H7 on mung beans from 4.38 log CFU/g to
below the detection limit without impacting germination.
The 3% oxalic acid treatment on radish seeds was equally
effective for reducing E. coli O157:H7 by .5 log CFU/g
but had detrimental effects on germination (61%) (14).
Peroxyacetic acid, an organic peroxide formed by acetic
acid and hydrogen peroxide, has minimal effects (,1-log
reduction) on E. coli O157:H7 and Salmonella on alfalfa
and mung bean seeds (23, 96, 120, 133). However, the use
of acetic acid as a gas has greater antimicrobial effects.
Mung bean seeds treated with gaseous acetic acid at 242 ml/
liter of air at 45uC for 12 h reduced Salmonella and E. coli
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Lactic acid (LA)
Malic acid (MA)

Peroxyacetic acid (PAA)
Fatty acids
5.9; 84.3
3.18
4.06
3.57
3.69
2.39
2.74
2.65
3
1.5
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Total aerobic bacteria
E. coli O157:H7
Salmonella Typhimurium
L. monocytogenes
E. coli O157:H7
L. monocytogenes
L. monocytogenes
15| fatty acid sanitizer (15,000 ppm of 60:40 capryliccapric acid

z 15,000 ppm of LA z 7,500 ppm of glycerol monolaurate),
3 min
Dip in 0.05% PA sanitizer for 2 cycles of 20 s at 75uC z20 s at 0uC
Dip in 3% OA sanitizer for 2 cycles of 20 s at 75uC z20 s at 0uC
Dip in 3% OA sanitizer for 5 cycles of 20 s at 75uC z20 s at 0uC
Dry heat (50uC, 17 h) z soaking in 0.03% PA
Dry heat (50uC, 17 h) z soaking in 0.03% PA
Dry heat (50uC, 17 h) z soaking in 1% OA
500 ppm of ClO2 (5 min) z 45uC, 24 h air drying z dry heat

(70uC, 48 h)
0.5% fumaric acid z 50 ppm of ClO2 (10 min)
0.5% fumaric acid z 50 ppm of ClO2 (5 min)
100 ppm of ClO2, 5 min
Broccoli sprouts
Mung bean sprouts
5% LA, 10 min, 42uC

5% LA, 10 min, 42uC z 2,000 ppm of chlorine
10% MA z 1% thiamine dilauryl sulfate (TDS), 20 min
1% PAA, 15 min with agitation
3% PAA, 15 min with agitation
75 mM monocaprylin, 90 min
3; NA
5; NA
6.6; 93
6.1; NA
4.41; 89
1.77; 91
1.34; 88
1.56; 96
2.56; 96
6.23; 91.5
3.86; 91.5
4.77; 91.5
4.38; 98.2
4.38; 99
5.8; 61
5; NA
7.5
5
Salmonella (inoculated on sprouts)

Salmonella (grown from
contaminated seeds)
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella Stanley
Salmonella Stanley
E. coli O157:H7
Salmonella
L. monocytogenes
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
5% AA, 10 min, 42uC

8.7% gaseous AA, 2 h, 55uC
2% AA, 24 h, 24uC, agitation
5% AA, 1 h, 30uC, agitation (50 rpm)
5% AA, 1 h, 30uC, agitation (50 rpm)
6.3; NAb
5; 97.1
5; 98
5; 96.1
5; 96.1
78
Target microorganism
E. coli O157:H7
E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
Salmonella
Application strategya
Microbial reduction
(log CFU/g); %
germination
75
85
84
13
14
130
27
53
23
92
149
122
92
119
Reference
SIKIN ET AL.
Alfalfa sprouts
Phytic acid (PA) and oxalic Mung bean seeds

acid (OA)
Mung bean seeds
Radish seeds
Mung bean, broccoli,
alfalfa seeds
Radish seeds
Mung bean, radish,
broccoli, alfalfa
seeds
Chlorine dioxide (ClO2)
Radish seeds
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Radish seeds
Radish seeds
Alfalfa sprouts
Alfalfa sprouts
Mung bean sprouts
Mung bean sprouts
Cowpea sprouts
Seed or sprout type
Acetic acid (AA)
Chemical sanitizer
TABLE 1. Relative efficacy of alternative chemical intervention strategies for decontamination of seeds and sprouts
2102
Seed or sprout type
Application strategya
800 ppm of ASC, 45 min

1,200 ppm of ASC, pH 2.5, 2 h
1,200 ppm of ASC, pH 2.5, 2 h z 0.75-kGy irradiation
200 ppm of SOC, 28uC, 24 h
ACC, available chlorine concentration; ORP, oxidation reduction potential; RT, room temperature.
NA, not available.
Alfalfa seeds
Mung bean seeds
0.85
2.2
1.92
0.91
0.12
0.94
1.68
4.4
4.8
2; NA
5; NA
2; NA
3; NA
4.24; 74
4.19; 74
4.51
4.11
2.45
2.85
2.91
1.5; NA
3; NA
3.9; 91
2.7; 98
4.7; 97.1
34; 97
34; 97
1.1; 96.3
1.11; 96.3
1.38
0.81
3.7
2.4
1.56; 56
2.72
2.04; 95.4
4.5; 98.4
6; 59.7
5; NA
Microbial reduction
(log CFU/g); %
germination
152
175
148
133
72
189
13
13
153
14
147
80
89
96
120
Reference
Acidified sodium chlorite

(ASC)
Salmonella
E. coli O157:H7
E. coli O157:H7
Stabilized oxycloro complex Mung bean seeds
E. coli O157:H7
(SOC) sanitizer
Salmonella
Acidic electrolyzed water
Alfalfa seeds
84 ppm of ACC, pH 2.4, 24uC; ORP: z1.081 mV, 3 h
Salmonella
(AEW)
Non-Salmonella
Alfalfa sprouts
84 ppm of ACC, pH 2.4, 24uC; ORP: z1,081 mV, 10 min
Salmonella
Non-Salmonella
Alfalfa sprouts
84 ppm of ACC, pH 2.4, 24uC; ORP: z1,081 mV, 10 min z
Salmonella
sonication z seed coats removed
Non-Salmonella
Alfalfa seeds
50 ppm of ACC, pH 2.6, RT; ORP: NA, 64 min
E. coli O157:H7
Alfalfa sprouts
50 ppm of ACC, pH 2.6, RT; ORP: NA, 64 min
E. coli O157:H7
Alfalfa seeds
70 ppm of ACC, pH 2.5, 22uC; ORP: z1,079 mV, 15 min
Salmonella
Mung bean seeds
Unknown ACC sanitizer for 5 cycles of 20 s at 75uC z 20 s at 0uC E. coli O157:H7
Radish seeds
Unknown ACC sanitizer for 5 cycles of 20 s at 75uC z 20 s at 0uC E. coli O157:H7
Radish, broccoli, alfalfa Dry heat (50uC, 17 h) z AEW (unknown ACC)
E. coli O157:H7
seeds
Mung bean seeds
Dry heat (50uC, 17 h) z AEW (unknown ACC)
E. coli O157:H7
Alkaline electrolyzed water Broccoli, alfalfa seeds Dry heat (50uC, 17 h) z alkaline electrolyzed water (unknown ACC) E. coli O157:H7
Mung bean seeds
Dry heat (50uC, 17 h) z alkaline electrolyzed water (unknown ACC) E. coli O157:H7
Radish seeds
Dry heat (50uC, 17 h) z alkaline electrolyzed water (unknown ACC) E. coli O157:H7
Slightly acidic electrolyzed Mung bean seeds
80 ppm of ACC, pH 5.986.38; ORP: z817889 mV, 15 min
E. coli O157:H7
water (SAEW)
Salmonella Enteritidis
Mung bean sprouts
120 ppm of ACC, pH 5.986.38; ORP: z817889 mV, 5 min
E. coli O157:H7
Salmonella Enteritidis
Radish sprouts
20 ppm of ACC, pH 5.8, RT; ORP: z900 mV, 5 min
Mesophilic bacteria
E. coli
Salmonella
Ozone
Alfalfa seeds
Ozone gas (100% ozone, 100% relative humidity, 20 psi), 25uC, 24 h Salmonella
Ozone gas (100% ozone, 100% relative humidity, 20 psi), 25uC, 24 h Salmonella
z 1% PAA, 25uC, 20 min
Alfalfa sprouts
20 ppm of ozone, 4uC, 64 min
E. coli O157:H7
20 ppm of ozone, 4uC, 64 min z sparging
E. coli O157:H7
20 ppm of ozone, 4uC, 64 min z sparging z pressure (12 psi, 5 min) E. coli O157:H7
Alfalfa sprouts
23 ppm of ozone, 4uC, 2 min
L. monocytogenes
Aerobic microorganisms
20 ppm ozone z sparging, 4uC, 20 min
L. monocytogenes
Aerobic microorganisms
Radish sprouts
2 ppm of ozone z 2% MA soak, 5 min, RT
Shigella spp.
Mung bean sprouts
2 ppm of ozone z 2% MA soak, 5 min, RT
Shigella spp.
Chemical sanitizer
TABLE 1. Continued
2103
2104
SIKIN ET AL.
to below the detection limit, even after enrichment (35).

Treatment of alfalfa and radish seeds with 8.7% acetic acid
in the gaseous phase at 55uC for 3 h resulted in greater than
5-log reductions of E. coli O157:H7 and Salmonella, with
96 to 98% germination (119). Malic acid is a low-cost,
effective organic acid antimicrobial. Fransisca et al. (53)
used 10% malic acid with 1% thiamine dilauryl sulfate (a
surfactant) for treatment of E. coli O157:H7 on alfalfa seeds.
Although a 4.41-log reduction and 89% germination was
achieved in a 10-g sample, upon scale-up to a 50-g sample
size, no significant reduction was achieved and pathogen
proliferation during sprouting occurred (53). Research
indicates that organic acid antimicrobials can inactivate
viable pathogen cells on sprout seeds by greater than 5 log
CFU/g, but the lack of replication and high levels of
pathogens recovered during germination in some studies
indicate that the use of these organic acids alone is less
effective for seed decontamination.
Application of organic acids to sprouts could mitigate
the use of chlorinated compounds and could be easily
adapted for use by sprout growers and consumers. Singh et
al. (149) compared the efficacy of organic acids to that of
chlorine at the maximum concentration of 20,000 ppm.
Soaking cowpea sprouts in 20,000 ppm of chlorine and 5%
acetic acid for 1 h reduced the populations of Salmonella on
surface-inoculated cowpea sprouts by 5 and 7 log CFU/g,
respectively. However, for Salmonella grown from contaminated seeds a lower reduction of only 3 and 5 log CFU/g
was achieved with up to 3 h of chlorination and acetic acid
treatments, respectively. These data indicate that Salmonella
present on seeds may have become internalized during
sprouting; thus, subsequent washing becomes less effective.
The highest concentration of chlorine permitted for seed
treatment is still less effective than acetic acid when applied
to sprouts. Pao et al. (122) explored the potential of acetic
acid for eliminating Salmonella in sprouts based on the
results obtained by Singh et al. (149). Pao et al. found that
immersing contaminated alfalfa and mung bean sprouts in
2% acetic acid for 24 and 48 h, respectively, resulted in a 7to 8-log reduction of Salmonella. However, 5% acetic acid
resulted in the same reduction of Salmonella but required
shorter exposure times of 4 and 16 h for alfalfa and mung
bean sprouts, respectively (122). These results indicate that
soaking raw sprouts in acetic acid solutions may help reduce
Salmonella contamination. However, the very long exposure times needed for a significant reduction in the microbial
load is not a viable commercial option. The characteristic
flavors of the organic acids also may have a negative effect
on the sensory quality of the sprouts. Noticeable discoloration and a strong vinegar odor have been reported after
acetic treatment of parsley (182) and sprouts (152).
Chemical interventions: ClO2 and ASC. ClO2 and
acidified sodium chlorite (ASC) have also attracted interest
as alternatives to chlorine. Treatment with aqueous ClO2
was introduced in place of chlorine-based sanitizers mainly
because of the higher oxidation capacity of ClO2. ClO2 also
is less influenced by pH and the presence of organic matter,
which contributes to its higher efficacy, and it produces
fewer potentially carcinogenic by-products such as trihalomethanes in the presence of organic matter (142). ClO2 is
permitted at a concentration not exceeding 3 ppm for
contact with whole produce in water (167). For ASC, a
much higher concentration of sodium chlorite is allowed
(500 to 1,200 ppm) in conjunction with a GRAS acid to
adjust the pH to 2.3 to 2.9 (173). Although the pH of ASC is
low, the antimicrobial activity is mainly due to the
generation of active ClO2.
A single ClO2 treatment of seeds is ineffective,
resulting in only a 1- to 2-log reduction of bacterial
populations (81, 97, 150). Therefore, combinations of
drying and dry heat have been used to achieve acceptable
microbial reductions. Bang et al. (6) completely eliminated
E. coli O157:H7 from radish seeds by treatment with
500 ppm of ClO2 for 5 min followed by air drying (45uC,
24 h) and dry heat (70uC, 48 h). These authors therefore
recommended these sequential treatments for future studies
on ClO2 for seed treatment before sprout production. ASC
(commercially known as Germin-8-or) alone has not
achieved a .5-log reduction of E. coli O157:H7 or
Salmonella on alfalfa and mung bean seeds (96, 120).
Treatment with 800 ppm of ASC for 45 min reduced
Salmonella on alfalfa seeds by 3.9 log units (96), and
1,200 ppm of ASC for 2 h reduced E. coli O157:H7 on
mung bean seeds by 2.7 log units (120).
All studies on the effect of ClO2 for decontaminating
sprouts suggest that the combination of ClO2 treatment with
other antimicrobial agents is effective; the use of ClO2 is
primarily justified by its higher efficacy compared with
chlorine. Treatment with aqueous ClO2 (100 ppm, 5 min)
reduced populations of Salmonella Typhimurium and L.
monocytogenes by 3 and 1.5 log CFU/g, respectively, on
mung bean sprouts. Subsequent modified atmosphere
packaging helped maintain a high visual quality of sprouts
after 7 days of storage (75). Similarly, the combined
treatment of alfalfa sprouts with ClO2 and fumaric acid
reduced the total aerobic bacteria by 3.18 log CFU/g and E.
coli O157:H7, Salmonella Typhimurium, and L. monocytogenes by 3 to 4 log CFU/g (84). Kim et al. (85) examined
the effects of aqueous ClO2 and fumaric acid on the same
pathogens inoculated onto broccoli sprouts. A 5-min
combined treatment of 50 ppm of ClO2 and 0.5% fumaric
acid reduced the initial populations of E. coli O157:H7,
Salmonella Typhimurium, and L. monocytogenes by 2.39,
2.74, and 2.65 log CFU/g, respectively.
The efficacy of ClO2 is not significantly superior to that
of chlorine when applied to sprouts at concentrations
exceeding the permitted level (167). Furthermore, the U.S.
Code of Federal Regulations for ClO2 (172) requires that all
produce must be washed with potable water following ClO2
treatment. The efficacy of ClO2 gas as an antimicrobial was
tested because this form has greater penetration and has
fewer residual effects (87). However, the efficacy of
treatment of alfalfa sprouts with ClO2 gas (5.0 mg/liter,
20 min, 90 to 95% relative humidity [RH]) was comparable
to that of aqueous ClO2, with only a 2.7-log reduction of
Salmonella (159). The application of ClO2 gas to alfalfa
sprouts was reported to leave high concentrations of by-
products such as chloride and chlorite, above the regulatory

limit of 1.0 mg/liter (159). The sprouts also had significant
discoloration (browning and bleaching), probably due to
chlorophyll oxidation as reported by Singh et al. (150, 151).
Chemical interventions: stabilized oxychloro-based
sanitizer. Stabilized oxychloro complex (SOC) sanitizers
consist of sodium chlorite and trace amounts of chlorate and
ClO2. Unlike ASC, the stabilized sodium chlorite is an
alkaline solution and has low oxidizing power. The
bactericidal action of chlorite is slower and more selective,
particularly through depletion of glutathione from the
bacterial cell (90). Therefore, a much longer treatment time
for inactivating bacteria is required with SOC than with
ClO2 because ClO2 is a more general oxidant. However,
SOC is an attractive alternative to chlorine because sodium
chlorite has no detrimental effect on seed viability over an
extended period of treatment time. Prolonged exposure to
SOC also should allow enough time for the bacteria to
emerge from the protective niches on seeds, which is
beneficial because low levels of surviving pathogens after
treatments may grow to high levels during germination. An
extended soaking time (,16 h) is common as a general
practice to stimulate germination (162).
A 24-h treatment with SOC sanitizer (200 and 400 ppm)
was carried out on a wide variety of commercial seed types
and effectively eliminated E. coli O157:H7 and Salmonella
(not detectable after enrichment) on soybean, alfalfa, cress,
and flax seeds (89). The same 200 ppm of SOC treatment
also resulted in a 3- to 4-log reduction and elimination after
enrichment of these pathogens on mung bean seeds. In a
subsequent study (65), the efficacy of the same SOC
treatment was verified on naturally contaminated mung bean
seeds to confirm this approach as a practical commercial
solution; however, it was ineffective on damaged or
scarified seeds (65). Nevertheless, the effectiveness of
SOC sanitizers on these major types of sprout seeds and
the lack of residual chlorite make SOC a promising
alternative to calcium hypochlorite (89).
Chemical interventions: electrolyzed water. Electrolyzed oxidizing water, a novel antimicrobial agent and
permitted food additive in Japan, is produced by electrolysis
of a diluted salt (NaCl) solution in a cell containing the
anode and cathode electrodes that are separated by a
membrane. Two types of water possessing different
properties are generated from passing electrical current to
the charged electrodes: (i) from the cathode side an alkaline
electrolyzed water, with high pH (10 to 11.5) and low
oxidation reduction potential (ORP) (2800 to 2900 mV)
and containing dilute NaOH, and (ii) from the anode side an
acidic electrolyzed water (AEW), with low pH (2 to 3), high
ORP (.1,000 mV), and a chlorine concentration of up to
50 ppm contributed by chlorine gas, hypochlorite ion,
hypochlorous acid, and hydrochloric acid (67). The
antimicrobial activity of AEW results from the combined
effect of high ORP, low pH, and the relatively high
concentrations of nondissociated hypochlorous acid, which
at acidic pH produce hydroxyl radicals on the cell
2105
membrane and disrupt metabolic process through oxidation

of key metabolic enzymes (2, 16, 71). Hence, the greater
oxidizing strength of AEW is indicated by large positive
ORP values; .1,000 mV is indirectly considered a factor in
bactericidal action (105, 141, 179).
In several studies AEW has been tested at different
combinations of time and concentration for decontamination
of alfalfa seeds, but minimal reduction (,2 log CFU/g) of
pathogens has been achieved (80). The inconsistency was
attributed to the inability of AEW to penetrate the seed coat.
Therefore, treatments for improved absorption have been
investigated, such as sonication, removing the seed coats,
and exchanging the internal gas composition of the seed
with oxygen to create a vacuum, but none have been
effective (80, 153). The effect of AEW with 50 ppm of free
chlorine (pH 2.6) on E. coli O157:H7 on alfalfa sprouts has
reduced the initial population by only 1.05 log CFU/g in
2 min and 2.72 log CFU/g after 64 min (147).
More recent work in this area involves the use of water
collected at both the anode (acidic) and cathode (alkaline)
and in combination with heat to improve pathogen
inactivation on mung bean, radish, broccoli, and alfalfa
seeds (13, 14, 189). Dry heat (50uC) for 17 h followed by
AEW was the most effective treatment for inactivating E.
coli O157:H7 by 5 log CFU/g on radish, broccoli, and
alfalfa seeds, and alkaline electrolyzed water after dry heat
had the same effect on broccoli and alfalfa seeds (13). Five
repeated cycles of hot AEW (75uC) for 20 s and chilled
AEW (0uC) for 20 s reduced E. coli O157:H7 to below the
level of detection in alfalfa seeds but was not effective for
reducing the pathogen to below the level of detection after
24 h of enrichment (14).
The use of AEW has long-term drawbacks because of
its strong acidity, which causes a rapid volatilization of
dissolved chlorine, thus decreasing the bactericidal activity
of the solution over time (94). The adverse effects on human
health and the environment and corrosion of equipment due
to the release of chlorine also may limit AEW applications
(69). The use of slightly acidic electrolyzed water (SAEW),
which is characterized as an effective bactericide even at
low available chlorine concentration (ACC) of 10 to 30 mg/
liter and pH 5 to 6.5, also has been suggested (189). SAEW
(pH 5 to 6.5, 80 mg/liter ACC) for 15 min on mung bean
seeds reduced E. coli O157:H7 and Salmonella by 4.24 and
4.19 log CFU/g, respectively, but also reduced germination
to 74%. For this reason, SAEW has been more commonly
used to treat sprouts and irrigation water (189).
Issa-Zacharia et al. (72) compared the effect of SAEW
with that of a traditional chlorine wash of sodium
hypochlorite on pathogens of radish sprouts. SAEW
treatment (20 mg/liter ACC, pH 5.8, ORP: z900 mV,
5 min) reduced the population of E. coli and Salmonella by
2.85 and 2.91 log CFU/g, respectively. Although these
reductions were not significantly different from those
obtained after treatment with sodium hypochlorite
(100 mg/liter ACC, pH 9.7) alone, direct comparison is
not appropriate because the hypochlorite in SAEW was
applied at or near neutral as opposed to alkaline pH. Zhang
et al. (189) evaluated the efficacy of SAEW (pH 5.98 to
2106
SIKIN ET AL.
6.38; ORP 817 to 889 mV) for reducing the population of E.

coli O157:H7 and Salmonella Enteritidis inoculated on
mung bean sprouts. These authors found that a reduction of
4.29 and 4.37 log CFU/g for E. coli O157:H7 was achieved
by treatment for 3 and 5 min, respectively, with SAEW with
the highest ACC of 120 mg/liter. Similarly, the Salmonella
Enteritidis counts on sprouts were reduced by 4.03 and 4.11
log CFU/g in 3 and 5 min, respectively. No viable cells of
either pathogen were detected in the used SAEW, whereas
approximately 5.1 to 5.9 log CFU/ml was recovered in the
control samples. In addition, the authors highlighted the lack
of product tainting and no sodium residues on the sprouts as
a result of using a chlorine wash alternative such as SAEW.
All of the studies to date (72, 80, 147, 189) generally
supported the use of this technology on seeds and sprouts.
Although AEW alone has not been very effective for seed
decontamination (80), treatment of seeds with electrolyzed
water in combination with heat (13, 14) has improved
inactivation; effects on germination and subsequent sprout
quality require further investigation. The approximately 3log reduction obtained for sprouts in most AEW studies is
considered promising given the infancy of this technique.
Studies on SAEW with seeds and sprouts (72, 189) have
indicated the influential role of high ORP and ruled out the
necessity of low pH for bactericidal action. Thus, a 5-min
SAEW treatment (72) seems to be the most promising
among the electrolyzed water treatments for sprouts because
of its low ACC and short treatment time.
Chemical interventions: ozone. Ozone treatment is
also a potential alternative to chlorine treatment. The FDA
approved ozone as an antimicrobial additive in food in 2001
(167). During applications, the triatomic oxygen (O3) of
ozone is sparged into water and decomposes. This results in
the release of the third unstable atom or reactive oxygen
species (hydroxyl radicals), which may be responsible for
microbial inactivation. Ozone gas can be generated using
different methods such as UV light, corona discharge, and
electrolysis of water. Aqueous ozone produced on site by an
electrochemical process has been used for the rinsing and
washing of sprouts (148, 152, 175), but only one report of
gaseous ozone for seed treatment has been published (133).
In that study, alfalfa seeds were treated in a commercial
chamber with ozone gas (100% ozone, 100% RH, 138 kPa
ozone pressure) at 25uC for 24 h, and Salmonella was
reduced by 1.5 log CFU/g. Because this small reduction was
presumed to be due to injured cells, in subsequent
treatments ozone was combined with 1% peroxyacetic acid
to achieve a 3-log reduction of Salmonella. Increasing the
concentration of peroxyacetic acid did not improve
inactivation, so the lowest concentration was recommended
for practicality.
The effectiveness of ozonized water for decontamination of produce has been poor, with reductions of less than 3
log CFU/g (28, 79, 83). This suboptimal result illustrates the
difficulties of first maintaining ozone concentration in the
aqueous phase and then contacting bacteria embedded in the
produce. In an experiment on the effect of soaking alfalfa
sprouts in ozonized water (20 ppm) for up to 64 min to
reduce the population of E. coli O157:H7 (148), the ozone

treatment was as ineffective as treatment with sterile
deionized water, with only 0.85-log reductions of E. coli
O157:H7 after 64 min. Thus, further studies were carried
out to combine ozonized water treatment with secondary
stress conditions such as continuous ozone bubbling and
pressurization. In general, reductions of E. coli O157:H7
increased with the duration of continuous ozone bubbling in
water but did not exceed 2.2 log CFU/g, and reductions
when pressurization was added were not significantly
different from those obtained with a single unpressurized
ozone treatment.
In another study, the effect of ozone concentration on
populations of aerobic microorganisms and L. monocytogenes was investigated (175). Treatment of inoculated
alfalfa sprouts with water containing 5, 9, or 23 mg/ml ozone
for 2 min resulted in L. monocytogenes reductions of 0.78,
0.81, and 0.91 log CFU/g, respectively. In contrast, the
effect of ozone treatment on the populations of aerobic
microorganisms was almost negligible under the same
conditions. Soaking of sprouts in ozonized water (20 mg/ml)
by continuously sparging ozone gas for up to 20 min
resulted in a 1.68-log reduction of aerobic microorganisms
but only a 0.94-log reduction of L. monocytogenes.
Evaluation of the sensory quality of ozone-treated sprouts
(20 mg/ml, 5 and 10 min) during storage (4uC, 11 days)
revealed negative effects. The panelists stated that sprouts
treated with ozone appeared darker in color and took on a
limp and oily appearance during storage. Scanning electron
microscopic analysis of the untreated alfalfa sprouts
revealed the presence of bacterial cells and biofilm several
millimeters from the root tip. However, the treated sprouts
were not examined microscopically and compared with
untreated sprouts because the efficacy of the ozone
treatment was very low in this study.
Singla et al. (152) reported the antimicrobial effects of
coupling organic acids (e.g., 2% malic acid) with ozone
treatment (2 ppm) on the reduction of Shigella spp. on
radish and mung bean sprouts. The combined treatments
had a significant synergistic effect, reducing pathogen
populations by 4.4 and 4.8 log CFU/g on radish and mung
bean sprouts, respectively. However, the pathogen inactivation in this study may not be primarily the result of ozone
treatment; a single treatment with 2% malic acid alone
significantly reduced Shigella counts by 3 log CFU/g
(complete immersion) and 2 log CFU/g (spraying) on radish
sprouts and by 3 log CFU/g (complete immersion) on mung
bean sprouts. However, complete immersion and spraying
of 2 ppm of ozonated water inhibited pathogen growth by
1.5 and 1 log CFU/g on radish sprouts. Similarly, complete
immersion of mung bean sprouts reduced Shigella counts by
1.8 log CFU/g, whereas spraying reduced the counts by only
0.9 log CFU/g. The combined treatment in this study did not
change the antioxidant levels or sensory quality of the
sprouts.
Ozone in water is extremely unstable because it quickly
dissociates back to oxygen, thereby reducing the antimicrobial effect. Thus, ozone treatment of sprouts may not be
effective unless improved methods for facilitating ozone
delivery to sprouts are found. The combined treatment of

ozone with organic acids may not be a viable option for
seeds or sprouts because ozone was not the primary or even
equal partner in the hurdle effect for controlling pathogens.
However, ozone treatment is still completely chlorine free
because free chlorine is not produced in situ as it is in the
electrolyzed water treatment system.
Biological interventions. Biological interventions
provide another promising strategy for eliminating the use
of synthetic chemicals or reducing their concentrations
when used as preservatives (57). These alternatives also are
appropriate for many sprout growers producing sprouts
intended for the organic market. Biological intervention is
generally achieved by the use of organisms such as
bacteriophages, protective bacterial cultures, yeasts, and
molds. These organisms may produce antimicrobial compounds such as bacteriocins, organic acids, enzymes, and
quorum-sensing molecules that negatively affect the viability of pathogens (70). Table 2 provides a summary of the
relative antimicrobial effects of these treatments on seeds
and sprouts.
Biological interventions: bacteriophages. Studies
concerning phage biocontrol of bacteria on seeds are scarce,
limiting the success of this approach. Phage attachment to
receptors on bacterial cell walls requires the chance collision
of the phage with a bacterial cell; therefore, this initial
interaction can be delayed by physical barriers in seeds.
Kocharunchitt et al. (88) coinoculated bacteriophages with
Salmonella onto alfalfa seed and reported only a 1-log
reduction of the pathogen on the sprouts. Pao et al. (123)
also reported 1.37- and 0.55-log reductions of Salmonella
after 24 h at 25uC when Phage A was inoculated onto
mustard and broccoli seeds, respectively. The use of
multiple phages did not improve the inactivation to .5
log CFU/g. To date, no studies using bacteriophages on
postharvest sprouts have been published. As with seeds, the
binding of phages to plant material was highlighted as a
limitation to the efficacy of bacteriophages for suppressing
the growth of pathogens on sprouts (123). This limitation is
yet another example of the major obstacle associated with
surface treatments, especially because sprouts have a brief
lifespan and pathogens may become internalized.
Biological interventions: protective cultures. The
use of protective strains and cultures has shown more
promise for control of pathogens on sprout seeds (49, 95,
100, 181). The key to effective biological control using this
method is high levels of competition on the seed during
germination, when internalized or injured pathogens are
likely to proliferate (48). Pseudomonas fluorescens 2-79 is
recognized as an effective biological control agent and has
achieved 4.24-log reductions (100) and 4.98-log reductions
(49) of Salmonella on alfalfa seeds and their sprouts after
soaking for 2 h. A diverse competitive culture isolated from
retail market alfalfa sprouts and inoculated onto alfalfa
seeds achieved a 5.48-log reduction of Salmonella on
sprouts after 7 days of germination (100), and other
2107
pseudomonads, such as Pseudomonas jessenii, also have

potential as biological controls (181). To understanding the
antimicrobial effect of P. fluorescens 2-79, Liao (95) looked
at the effects of inoculum size and native microbiota and
concluded that P. fluorescens merely suppressed Salmonella
growth by 2 log CFU/g compared with untreated seeds and
that combinations of native flora or sanitizer treatment
before inoculation with P. fluorescens could improve
pathogen control. In a subsequent study, a combination of
biological control methods with a competitive strain plus
bacteriophages provided acceptable control of pathogens on
mung bean and alfalfa seeds (187). On alfalfa seeds soaked
in an Enterobacter asburiae JX1 suspension and bacteriophage cocktail for 20 min and dried overnight, Salmonella
was completely eliminated and undetectable after enrichment after 4 days. This treatment was not affected by
temperature, did not affect sprout yield, and achieved a .6log reduction on mung beans but did not achieve complete
elimination (187).
Many studies on protective cultures against human
pathogens have examined the use of lactic acid bacteria
(LAB). LAB are commonly used in the food industry
because of their lack of pathogenicity and because they can
produce various antimicrobial agents, including bacteriocins, hydrogen peroxide, and organic acids in vitro (21).
LAB are more widespread on sprout surfaces (24, 126) than
on seeds and therefore have not been considered effective
for outcompeting pathogens on seed surfaces (48). Bennik
et al. (18) compared two LAB strains (Enterococcus mundtii
and Pediococcus parvulus) for their ability to produce a
bacteriocin (pediocin-like mundticin) and then evaluated
their potential to inhibit L. monocytogenes on refrigerated
mung bean sprouts under modified atmosphere packaging.
E. mundtii was selected because it had a significantly higher
maximum specific growth rate than did P. parvulus strains
at CO2 concentrations (,20%) suitable for refrigerated
storage (4 to 8uC) of vegetables under modified atmosphere
packaging. The E. mundtii strain was then evaluated for its
potential for in situ control of L. monocytogenes on mung
bean sprouts and vegetable agar stored under an atmosphere
of 1.5% O2, 20% CO2, and 78.5% N2 at 8uC. The treatment
did not produce the same effect on the pathogen inoculated
on mung bean sprouts as observed on the vegetable agar.
However, the application of pure mundticin (200 AU/ml) to
the sprouts either during a washing step or as part of a
coating procedure with an alginate film was successful
against L. monocytogenes.
Cobo Molinos et al. (32) inoculated Enterococcus
faecalis A-48-32 on soybean sprouts as a protective culture
against B. cereus. This strain produces the cyclic peptide
bacteriocin (enterocin) AS-48. The inoculated sprouts were
stored at 15 and 22uC for up to 7 days to simulate
temperature abuse. The enterococci multiplied rapidly on
sprouts at the two temperatures tested. In both cases,
bacteriocin activity was observed in the sprouts for up to
3 days of storage but not after longer periods. The growth of
coinoculated B. cereus was completely inhibited for the
entire storage period at 15uC. However, this growth
inhibition was observed during only the first 3 days of
25
25
25
25
ppm
ppm
ppm
ppm
of
of
of
of
AS-48
AS-48
AS-48
AS-48
z
z
z
z
0.1% polyphosphoric acid

2% polyphosphoric acid
25 ppm of AS-48 z 40 ppm of PAA, RT, 5 min

25 ppm of AS-48 (soak), RT, 5 min
50 ppm of nisin, 48 ppm of pediocin z 0.02% PA, pH 2.3, 1 min
25 ppm of AS-48 z 1% polyphosphoric acid
Phage A, 108 PFU/ml, 25uC, 24 h

Phage A, 108 PFU/ml, 25uC, 24 h
Phage A z Phage B, each inoculated at 108 PFU/ml onto
contaminated seeds
Phage (SSP6) inoculated at 70 PFU/CFU onto contaminated seeds,
12 h
Bacteriophage cocktail inoculated at 106 PFU/ml onto
contaminated seeds, 4 days
Pseudomonas fluorescens 2-79 inoculated at 108 CFU/ml, 7 days
Market sprout culture (details not given), 7 days
P. fluorescens 2-79 inoculated at 108 CFU/ml, 6 days
P. jessenii LTH 5930 inoculated at 108 CFU/ml onto contaminated
seeds, 48 h
P. jessenii LTH 5930 inoculated at 108 CFU/ml prior to
contamination, 48 h
P. fluorescens 2-79 inoculated at 107 CFU/ml, 6 days
Enterobacter asburiae JX1 (106 CFU/ml), 4 days
E. asburiae JX1 (106 CFU/ml) z bacteriophage cocktail (106
PFU/ml), 4 days
E. asburiae JX1 (106 CFU/ml) z bacteriophage cocktail (106
PFU/ml), 4 days
10,000 AU/g colicin Hu194 (mix & dry), RTb, 1 day
204,000 AU/g colicin Hu194 (mix & dry), RT, 1 day
10,000 AU/g colicin Hu194 (soak), RT, 2 h
25 ppm of AS-48 z chemical preservatives (soak), RT, 5 min
Application strategy
1.37, NAa
0.55; NA
0.57; NA
1; NA
3.41; NA
4.24; NA
5.48; NA
4.98; NA
2
4
2; NA
5.56
6.72
7.62; NA
6; NA
3; NA
5; NA
2.4
2.7
1.3
1.52.4
2.5
2c
2.31
5.5
5.31
4.2
3.7
3.2e
2.3
1.9
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
E. coli O157:H7 ATCC 43890
L. monocytogenes
L. monocytogenes
Bacillus cereus
B. weihenstephanensis
B. cereus
Enterococci
L. monocytogenes
Enterobacter aerogenes
P. fluorescens
Aeromonas hydrophila
Salmonella enterica
E. coli O157:H7
Yersinia enterocolitica
Shigella sonnei
Microbial reduction (log

CFU/g); % germination
Salmonella
Salmonella
Salmonella
NA, not available.

RT, room temperature.
c
Log reduction for enterococci was calculated after 2 days of storage at 10uC.
d
Combined treatment of enterocin AS-48 and polyphosphoric acid was carried out in solutions at pH 9 for 5 min under the temperature of 65uC.
e
Log reduction for E. coli O157:H7 was calculated after 24 h of storage at 6 and 15uC.
Soybean sprouts
Mung bean sprouts
Soybean sproutsd
Alfalfa sprouts
Alfalfa, soybean sprouts
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Mung bean seeds
Mung bean seeds
Mung bean seeds
Alfalfa seeds
Alfalfa seeds
Alfalfa seeds
Mung bean seeds
Mung bean seeds
Alfalfa seeds
Mustard seeds
Broccoli seeds
Broccoli seeds
Seed or sprout type
29
15
32
31
30
111
95
187
49
181
100
187
88
123
Reference
SIKIN ET AL.
Bacteriocin
Protective culture
Bacteriophage
Biocontrol agent
TABLE 2. Relative efficacy of biological intervention strategies for decontamination of seeds and sprouts
2108
storage at 22uC. These studies (18, 32) prove that in situ

bacteriocin production by protective cultures is not always
successful for inhibiting pathogen growth possible because
of the severe effects of the unfavorable food environment,
which impedes the growth and bacteriocin production of the
strain. The use of purified or semipurified preparations may
be more effective because bacteriocin can be added to foods
in a more flexible way than via protective cultures and the
bacteriocin dose can be adjusted to maximize its bactericidal
effects.
Biological interventions: bacteriocins. Bacteriocins
are ribosomally synthesized, extracellularly released lowmolecular-mass peptides or proteins (usually 30 to 60 amino
acids) that are classified by their mechanism of bactericidal
or bacteriostatic effects (1, 74, 86, 156). Similar to
bacteriophage and competitive exclusion, the limited use
of bacteriocins for seed treatment has revealed minimal
biological control but is nevertheless attractive given the
infancy of this intervention strategy. The E. coli Hu194 E2type colicin, previously shown to be effective against 22
strains of E. coli O157:H7, was used in two methods to
control several test strains on alfalfa seeds (111). The mixand-dry method in colicin at 10,000 AU/g of seed reduced
E. coli 43890 from 106 CFU/ml to undetectable levels
within 1 day, while the same method for E. coli 43895 and
E. coli 3081 required 20 times higher concentration of
colicin to achieve a maximum 3-log reduction. The method
of soaking seed in colicin at 10,000 AU/g for 2 h at room
temperature also was effective for eliminating (5 log CFU/g)
E. coli 43895. These results indicate the sensitivity of target
microorganisms to bacteriocins and suggest that this
treatment alone may not be effective for reducing the
incidence of pathogens on sprout seeds. The use of additives
did not improve the treatment, there was no mention of
subsequent germination success, and these test scales are not
yet optimized for application to seeds or produce (111).
Enterocin AS-48 appears to be the most promising
candidate for sprout treatment among those bacteriocins
reported in the literature, although nisin is still the only
bacteriocin permitted by the FDA (161) as a biopreservative
(E234) in many foods. Cobo Molinos et al. (30) examined
the effectiveness of washing with enterocin AS-48 solution
(5 min at room temperature) for treatment of both pathogens
and native microbiota of alfalfa and soybean sprouts. In
general, the effect of enterocin AS-48 treatment was directly
proportional to the bacteriocin concentration (5, 12.5, and
25 mg/ml) and inversely proportional to posttreatment
storage temperature (6, 15, and 22uC). L. monocytogenes
counts in both types of sprouts were reduced by 2 to 2.4 log
CFU/g by the 25 mg/ml enterocin AS-48 washing treatment.
The same washing treatment reduced the viable population
of B. cereus to below the detection level after 1 and 3 days
of storage at 6uC for soybean and alfalfa sprouts,
respectively. Similarly, the population of Bacillus weihenstephanensis was reduced by 1.5 to 2.4 log CFU/g in
soybean sprouts after treatment, and no viable cells were
detected during storage at 6uC for up to 7 days (31).
However, counts of total mesophilic bacteria were reduced
2109
by only 0.5 to 1 log CFU/g, LAB were inconsistently

inhibited, and enterobacteria were almost unaffected,
suggesting that this treatment may not extend the shelf life
of sprouts (29).
Microbial inactivation on sprouts was enhanced when
enterocin AS-48 was combined with various antimicrobials
or sanitizers. An increase in the reduction of L. monocytogenes from 2.4 to 2.7 log CFU/g in alfalfa and soybean
sprouts was obtained by a combination of enterocin AS-48
with chemical preservatives such as lactic acid, sodium
lactate, sodium nitrite, sodium nitrate, trisodium phosphate,
trisodium trimetaphosphate, sodium thiosulfate, n-propyl
p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester,
hexadecylpyridinium chloride, peracetic acid, and sodium
hypochlorite (30). Subsequently, Cobo Molinos et al. (31)
tested the combined treatment of enterocin AS-48 with a
variety of chemicals against B. cereus and B. weihenstephanensis in alfalfa sprouts. Washing with solutions
containing enterocin AS-48 alone reduced the viable counts
of B. cereus and B. weihenstephanensis by 1 to 1.3 and 1.5
to 2.4 log CFU/g, respectively. Combined treatment with
enterocin AS-48 and other antimicrobial agents such as
sodium hypochlorite (100 ppm), peracetic acid (40 ppm),
and hexadecylpyridinium chloride (0.5%) further reduced
the populations of B. cereus by 0.7, 2.5, and 0.7 log CFU/g,
respectively, in alfalfa sprouts, and levels remained below
the detection limit during 1 week of storage at 15uC.
Similarly, a combination of enterocin AS-48 (25 mg/ml) and
hydrocinnamic acid, peracetic acid, hexadecylpyridinium
chloride, or polyphosphoric acid (0.5%) also reduced the
population of B. weihenstephanensis to below the detection
limit after 1 week. Populations below the detection levels in
both sprouts over 7 days of storage at 6 and 15uC indicated
that residual enterocin AS-48 adsorbed by the treated
sprouts was able to provide a protective effect in stored
samples after the initial treatment (30).
Nisin and pediocin individually or in combination with
antimicrobial agents were tested on broccoli and mung bean
sprouts against a bacterial cocktail of five L. monocytogenes
strains (15). Two hours after inoculation with Listeria, the
samples were washed with agitation for 1 min with different
antimicrobial solutions: 0.02 M EDTA, 2% sodium lactate,
0.02% potassium sorbate, 0.02% phytic acid, and 10 mM
citric acid; 50 mg/liter nisin; 48 mg/liter pediocin; or
combinations of bacteriocins and chemical preservatives.
The best result on mung bean sprouts was obtained with the
combination of nisin, pediocin, and phytic acid, which
caused a 2.31-log reduction compared with a washing
treatment with distilled water (0.90 log CFU/g). This
combination treatment also reduced the native microbiota
by approximately 1.4 log CFU/g. However, the proliferation
of surviving bacterial populations was not measured during
further storage of the samples.
In several studies (15, 3032), bacteriocins were active
against many gram-positive bacteria but usually are not
active against gram-negative bacteria. The gram-negative
outer membrane defends against many pathogens, such as
Salmonella and enterohemorrhagic E. coli. However, gramnegative cells can be inactivated by bacteriocins after the
2110
SIKIN ET AL.
cells have been exposed to treatments that increase the

permeability of the outer membrane. Washing soybean
sprouts with enterocin AS-48 alone at neutral pH had no
effect on Salmonella enterica (32). However, increased
bactericidal activity resulting in a 4.7-log reduction was
detected after treatment with alkaline bacteriocin solutions
(25 mg/ml, pH 9.0) heated for 5 min at 65uC and with
washing solutions containing enterocin AS-48 in combination with various chemical compounds (1.5% lactic acid,
1.5% trisodium phosphate, 0.5% trisodium trimetaphosphate, 0.1% polyphosphoric acid, 80 ppm of peracetic acid,
0.5% hexadecylpyridinium chloride, 100 ppm of sodium
hypochlorite, 0.5% n-propyl p-hydroxybenzoate, 0.5% phydroxybenzoic acid methyl ester, and 2% hydrocinnamic
acid). The best results were obtained for combinations of
enterocin AS-48 and lactic acid, peracetic acid, or polyphosphoric acid.
The S. enterica study (32) was then extended to
examine the combined effect of enterocin AS-48 (25 mg/ml)
and polyphosphoric acid in a concentration range of 0.1 to
2% against other gram-negative bacteria on soybean
sprouts. Inoculated sprout samples were stored at 6 and
15uC, and the sensitivity of the inoculated bacteria to the
treatment is listed here in ascending order (with the
corresponding concentration of polyphosphoric acid):
Shigella flexneri (2% polyphosphoric acid), Shigella sonnei
(2%), P. fluorescens (1%), Enterobacter aerogenes (1%),
Aeromonas hydrophila (0.5%), E. coli O157:H7 (0.4%),
and Yersinia enterocolitica (0.1%). The maximum concentration of polyphosphoric acid (2%) in combination with
enterocin AS-48 in the washing treatment that was required
for inactivation of gram-negative bacteria should be
compatible with the allowable daily intake of phosphate
(70 mg/kg of body weight) because only a small percentage
of the phosphate applied in the washing treatment would
remain on the treated food product.
Biological interventions: QS inhibitors. Quorum
sensing (QS) bacteria produce and release chemical signal
molecules (autoinducers) such as N-acyl homoserine
lactones (AHLs or N-AHLs). The AHL helps the bacteria
regulate the production of extracellular hydrolytic enzymes
at high cell densities. Rasch et al. (138) found that bacterial
spoilage of bean sprouts is influenced by QS; an AHLnegative mutant of a spoilage bacterium did not spoil
sprouts to the same extent as did the AHL-positive wildtype. These authors reported that the AHL compound, N-3oxohexanoyl-L-homoserine lactone, was extracted from
samples of bean sprouts undergoing soft-rot spoilage. The
spoilage was identified as a result of proteolytic and
pectinolytic activities of AHL-producing bacteria (Enterobacteriaceae and pseudomonads) isolated from different
batches of commercial bean sprouts. QS inhibitors (e.g.,
signal degrading enzymes or mimics) have been identified
in bean sprouts, carrot, garlic, habanera peppers, and
chamomile (140), indicating that this mechanism forms
part of the plants defense against bacterial colonization.
Rasch et al. (139) also studied the effect of QS inhibitors
(e.g., ProS-AHL, PenS-AHL, and HepS-AHL) as food
preservatives protecting against bacterial spoilage of bean

sprouts. PenS-AHL and HepS-AHL inhibited QS-regulated
protease activity of spoilage bacterial isolate Pectobacterium A2JM in broth cultures but had no effect on bean sprout
spoilage. Although, these QS inhibitors were not effective
in the actual sprout system, this research has laid the
groundwork for potential intervention strategies for preventing food spoilage and enhancing food safety.
Physical interventions. Synthetic chemical disinfectants are hardly associated with the healthy image associated
with fresh produce. This attitude is reflected in the everincreasing consumer preference for the reduction or
elimination of synthesized additives and the enhanced use
of natural preservatives in foods (121). Thus, the development of a physical intervention method that is chemical free
or leaves no residues is a high priority. Because the
chemical and biological treatments discussed above are
limited to surface inactivation, physical methods such as
heat, pressure, and irradiation offer better penetration for
internalized or sheltered microorganisms and may be more
readily adapted for commercialization. Table 3 provides a
summary of the relative efficacies of these physical
treatments on seed and sprout microbial safety.
Physical interventions: thermal inactivation. Dry
and wet heat treatments for decontamination of seeds are
considered easy and cheap methods for sprout processors to
implement before germination (180). Elimination of both E.
coli O157:H7 and Salmonella have been achieved in alfalfa
and mung bean seeds by exposure to dry heat (55uC) for 4
to 6 days, but results vary with inoculation level, and
germination rates differ across seed types (44, 68). Dry heat
alone at 65uC for 10 days eliminated both pathogens
without reducing germination (114). Hot water treatment of
radish and alfalfa seeds at 58uC for 6 min and of mung bean
seeds at 60uC for 10 min achieved .5-log reductions and
did not alter germination significantly (.95%) (180). In
considering commercialization of these treatments, Bari et
al. (9, 10) found an effective hot water treatment (90uC for
90 s) on a lab scale, but it did not hold up at the industrial
scale, even though the current decontamination step in
Japan is hot water pasteurization (85uC for 10 s) (10). The
conflicting results across dry and wet heat research indicate
that effective treatments exist but must be specifically
designed for each seed type to maintain viability. Hot water
treatments also can be used to soften the seed coat and
enhance germination in combination with sanitizers.
Recommended and novel single chemical treatments
have been effective for inactivating surface microorganisms
on seeds, but the persistence of foodborne illnesses associated
with sprouts suggests that these seed decontamination
methods are not sufficient for elimination of pathogens.
Most of the recent work in this area of intervention has
involved combination treatments to enhance the ability of
antimicrobial agents to penetrate the seed coat and eliminate
internal pathogens. The most successful treatments for
adequate inactivation while maintaining germination have
coupled heat with organic and chlorine-based sanitizers.
Because the current commercial method for treating

mung bean seeds in Japan involves hot water soaking, Bari
et al. (10) tested a combination of hot water with the FDA
chlorine recommendation. The combination treatment on
mung bean seeds of 85uC water for 40 s, 0uC water for 30 s,
and 2,000 ppm of chlorine water soak for 2 h reduced E. coli
O157:H7 and Salmonella by 5.69 and 5.84 log CFU/g,
respectively. This treatment was effective for reducing
pathogens to below detectable levels after 24 h of
enrichment and maintained 95% sprout yield after 72 h.
However, treatment at higher water temperature for a shorter
times was not as effective (10). Because recovery of injured
cells often increases and germination is altered when the
treatment sample size increases, this method was further
validated on a commercial scale with a 300-kg sample (9).
However, when tested on mung bean seeds from a different
source, the combination treatment achieved .5 log CFU/g
reduction but was not able to completely eliminate
Salmonella from detection (118).
Bari et al. (13) later tested the impact on decontamination
of a prolonged dry heating step before soaking seeds in
sanitizer solutions. After 17 h at 50uC, low concentrations of
phytic acid (0.03%) and oxalic acid (1%) significantly
reduced viable E. coli O157:H7 (5 log CFU/g) but were not
effective for eliminating the pathogen from seeds; injured
cells recovered during germination (13). Bang et al. (6)
reported that dry heat treatment at 43% RH resulted in 90%
germination of radish seeds, whereas no germination
occurred after wet heat treatment (100% RH) used with
ClO2 for inactivating E. coli. In a subsequent study, Bang
et al. (7) applied sequential treatments of ClO2 (500 ppm,
5 min), air drying (45uC, 23% RH, 24 h), and dry heat (70uC,
23% RH, 48 h) to radish seeds to achieve a 5.9-log reduction
of E. coli O157:H7 and a germination rate of 84.3%.
Thermal treatment of sprouts at elevated temperatures
has been studied, although these studies are less common
than those for seeds. The short duration of heat shock
treatments is generally considered to minimize deterioration,
helping to maintain the nutritional, textural, and physiological quality of sprouts. Dipping alfalfa and mung bean
sprouts in boiling water for 3 and 5 s, respectively, can
eliminate Salmonella by about 7 log CFU/g (122).
However, blanching treatment at the boiling point for 30 s
did not eliminate E. coli O157:H7 from alfalfa sprouts.
Although the color was preserved, the blanched sprouts
were less firm (55). Blanching in hot water (90uC, 60 s)
reduced the total microbial count to 2 log CFU/g in mung
bean sprouts (17) while preserving the vitamin C content. In
contrast, blanching at a temperature above 95uC for 60 s
resulted in 50% loss of vitamin C in mung bean sprouts
(42). The mildest treatment conditions (60uC, 30 s) resulted
in only a 2-log reduction of the total microbial count in
soybean sprouts (125). However, respiration activity was
negligible after heat treatment, which should be considered
when packaging produce for retail purposes.
The sensitivity of plant tissue to heat injury limits the
use of heat treatments necessary to obtain drastic reductions
in the populations of foodborne pathogens. Increasing the
treatment time and temperature has adverse effects on the
2111
textural and nutritional qualities, respectively, of the

products (42, 55). Microorganisms are still capable of
growth in the product after hot water treatment, so avoiding
recontamination and temperature abuse during storage are
critical considerations when using this method. Therefore,
nonthermal alternatives such as irradiation (8, 11, 64, 134,
136, 137, 145, 146, 178) and UV light treatment (62, 84,
137) have been examined.
Physical interventions: irradiation. Radiation dosage, expressed in kilograys (kGy), is a function of the
energy of the radiation source and the time of exposure. One
Gray is equal to one joule of energy absorption per kilogram
of a material. The potential use of an irradiation process
(gamma and beta) is evaluated by determining the radiation
D10-values (dose required to achieve a 1-log reduction in
microorganisms). Typical ionizing radiation facilities use
either gamma rays from the radioactive isotopes 60Co or
137
Cs, electron beams (or beta irradiation), and X rays
generated in electron accelerators. In general, electron
beams are less efficient than gamma irradiation for
inactivation of pathogens because of their limited penetration depth. The FDA has approved irradiation of sprout
seeds at doses up to 8 kGy (163).
Irradiation treatment has been studied on a wide variety
of seeds and D10-values for various pathogens have been
calculated for use in designing treatments to meet the FDA
requirement for seed decontamination methods to achieve
.5-log reduction (134, 176, 177). A radiation dose of
5.9 kGy has been recommended to achieve a 5-log reduction
on broccoli and radish sprouts (176). Kim et al. (82) found
that irradiation was more effective than ultrasound and
AEW water, but an 8-kGy dose was necessary to achieve
.5-log reduction in alfalfa seeds and 4.58-log reduction in
broccoli seeds, with adverse effects on seed viability above
doses of 4 kGy. To compensate for the loss of viability,
Waje et al. (176) noted that electron beam irradiation may
have a benefit over gamma irradiation because of its shorter
penetration depth. Bari et al. (12) combined irradiation with
dry heat treatment (50uC for 1 h) to reduce the effective
dose to 2 kGy for complete elimination of E. coli O157:H7
on alfalfa and radish seeds. Even when inactivation is
effective and safety is improved, decreases in viability and
more importantly length and quality of the resulting sprout
have been reported, which make this approach an
unacceptable commercial option (12, 82, 144, 188).
Research efforts on the application of gamma radiation
to inactivate pathogens in sprouts have been published and
reviewed by Rajkowski and Fan (135). The FDA-approved
dosage of 8 kGy for seeds causes more adverse chemical
changes in raw sprouts because of their higher water content
(36). Any radiolysis by-products formed by irradiation of
the seeds would be diluted during germination and impart
no effect in the grown sprouts (165). Irradiated fresh
produce, including sprouts, should be treated at no more
than 1 kGy to be considered fresh (163), a dosage that has
only been generally identified to extend produce quality.
Low doses of gamma radiation have been widely
reported to be effective against human pathogenic bacteria
Irradiation
Dry heat
Hot water
Physical method
6 days
8 days
17 h
24 h
24 h
4 days
2 kGy gamma
1 kGy gamma
Radish sprouts
Broccoli sprouts
3 kGy gamma
8 kGy gamma
8 kGy gamma
0.75 kGy gamma
1.5 kGy gamma
1.5 kGy gamma
1 kGy gamma
55uC,
55uC,
50uC,
50uC,
50uC,
55uC,
85uC water, 40 sz 0uC water, 30 s z 2,000 ppm of

chlorine, 2 h
70uC, 10 s
80uC, 5 s
90uC, 3 s
100uC, 3 s
70 or 80uC, 20 s
90uC, 10 s
100uC, 5 s
50uC, 1 h
50uC, 1 h
50uC, 1 h
50uC, 1 h z 2.5-kGy irradiation
50uC, 1 h z 2.5-kGy irradiation
55uC, 4 days
5; 97
5; NAa
6.08; NA
5.34; NA
3.69; NA
3.84; NA
5.69 (5% loss in yield)
5.84; NA
5.5; 97.4
5.3; 97.4
7.6
6.9
1; NA
0.9; NA
1.73; NA
5.71; 94
4.56; 98
5; 99
4; 99
5; 76.8
5; 74.6
5; NA
3; NA
5; NA
5.4; 99
1.8; 99
2.7; 91
5.03; 86.5
4.85; 92.5
1.4; 98.2
2.8; 97.2
5.97
5.57
5.48
5.47
5.24
4.88
Salmonella
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella
E. coli O157:H7, Salmonella
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
L. monocytogenes
Microbial reduction (log CFU/g);

% germination

E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
E. coli O157:H7
Salmonella
Salmonella
11
120
134
82
114
13
44
68
12
122
118
10
14
180
Reference
SIKIN ET AL.
Mung bean sprouts
Broccoli seeds
Alfalfa seeds
Broccoli seeds
Mung bean seeds
Alfalfa, broccoli seeds

Mung bean seeds
Radish seeds
Alfalfa seeds
Alfalfa seeds
Mung bean seeds

Radish seeds
Alfalfa seeds
Radish, alfalfa seeds
Mung bean seeds
Mung bean seeds
Mung bean sprouts
Alfalfa sprouts
Mung bean seeds
85uC, 40 s
Mung bean seeds

85uC, 40 s z 2,000 ppm of chlorine, 2 h
58uC, 6 min
60uC, 10 min
90uC water for 90 s, then chilled water for 30 s
Radish, alfalfa seeds

Mung bean seeds
Mung bean seeds
Seed or sprout type
TABLE 3. Relative efficacy of physical intervention strategies for decontamination of seeds and sprouts
2112
NA, not available.
3.3 kGy electron beam

2.3 kGy gamma or 3.65 kGy electron beam
2.45 kGy gamma or 4.80 kGy electron beam
Alfalfa sprouts
Broccoli sprouts
Radish sprouts
600 MPa, 20uC, 2 min

Presoak (60 min) z 600 MPa, 20uC, 2 min
Presoak (15 min) z 600 MPa, 20uC, 15 min
Alfalfa seeds
Barley seeds
28
15
20
15
10
MPa,
MPa,
MPa,
MPa,
MPa,
50uC,
40uC,
45uC,
45uC,
40uC,
60 min
15 min
5 min
10 min
10 min z 150 ml water
300 MPa, 20uC, 15 min
Garden cress seeds
Alfalfa seeds
Alfalfa seeds
600 MPa, 20uC, 25 min

60uC, 1 day z 600 MPa, 35uC, 2 min
Alfalfa seeds
Alfalfa seeds
1 kJ/m2 UV-C (254 nm) z 5 min pretreatment with

fumaric acid (0.5 g/100 ml)
Alfalfa seeds
Clover sprouts
0.2 kJ/m2 UV-C (254 nm) z 5 min pretreatment with

0.1% H2O2
1 kJ/m2 UV-C (254 nm), 3.3 min
2 kGy gamma
Soybean, alfalfa sprouts
Radish sprouts
2 kGy gamma
3 kGy gamma
12 kGy gamma
Mung bean, matki,

chickpea, garden pea
sprouts
Seed or sprout type
1
1.02
1.06
0.87
3.03
2.88
2.35
2; 9.0
1.1; 9.0
2.3; 95
5; 91
5; 89
5.2; 98
5.8; 98
4.5; NA
5.2; 99
5.8; 97
6; 69.81
6; 69.81
6; 69.81
1; 90
7; 43
7; 25
7; 32.67
6; 71.4
Total aerobic bacteria

E. coli O157:H7
L. monocytogenes
E. coli O157:H7
L. monocytogenes
E. coli O157:H7
L. monocytogenes
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella
Salmonella
E. coli O157:H7
Salmonella
S. Typhimurium
E. coli
Listeria innocua
E. coli K12
E. coli O157:H7
Salmonella
L. monocytogenes
Penicillium oxalicum spores
,5
6
,5
23
,5
,5
2
4
1
2
,5
Microbial reduction (log CFU/g);

% germination
L. monocytogenes
Aerobic plate counts
Coliforms
Yeast and molds
Staphylococci
L. monocytogenes, Salmonella
Enteritidis, Staphylococcus
aureus
L. monocytogenes
Typhimurium, L.
monocytogenes, B. cereus
Typhimurium, L.
monocytogenes, B. cereus
Shigella sonnei
124
102
77
184
114
113
117
84
132
146
176
66
145
Reference
Supercritical carbon
dioxide (SC-CO2)
High pressure
Ultraviolet light
Physical method
TABLE 3. Continued
2113
2114
SIKIN ET AL.
across sprout types. Elimination (.5-log reduction) of

Salmonella and strains of E. coli O157:H7 from radish and
mung bean sprouts to below the limit of detection by
enrichment is commonly achieved at doses of 1.5 to 2 kGy
(8, 136); 4- to 5-log reductions of both Salmonella and
E. coli O157:H7 were obtained when alfalfa sprouts were
irradiated at 2 kGy (136). Irradiation of broccoli and mung
bean sprouts at 1 kGy resulted in approximately 4.88- and
4.57-log reductions of L. monocytogenes, respectively, with
average D10-values of 0.20 and 0.22 kGy, respectively (11).
Saroj et al. (145) conducted an extensive study to evaluate
the effectiveness of irradiation for eliminating Salmonella
Typhimurium and L. monocytogenes from a much wider
variety of sprouts (mung bean, dew gram, chickpea, and
garden pea). They found that the radiation D10-values
ranged from 0.192 to 0.208 kGy for Salmonella Typhimurium and from 0.526 to 0.588 kGy for L. monocytogenes in
all the tested sprouts. These ranges were very narrow and
not significantly different for each of the pathogens in all
types of sprouts. Thus, Saroj et al. further studied the effect
of irradiation on the growth of pathogens inoculated on only
mung bean sprouts. Treatment with 2 and 3 kGy was
sufficient to achieve a 5-log reduction of Salmonella
Typhimurium and L. monocytogenes, respectively. However, recovery of these pathogens occurred when these treated
sprouts were stored at 4 and 8uC. Thus, a higher dose of
radiation, .2 kGy for Salmonella Typhimurium and
.3 kGy for L. monocytogenes, is required to achieve a 5log reduction and inhibit the recovery of surviving pathogen
cells following the irradiation treatments. Horak et al. (66)
studied the effects of irradiation on soy sprouts and alfalfa
sprouts inoculated with L. monocytogenes, Salmonella
Enteritidis, and Staphylococcus aureus and stored at 4uC.
The most radiation-resistant microorganism on the sprouts
was L. monocytogenes. Based on the application of five
times the D10-value, the minimum disinfection dose
proposed was around 2 kGy for soy and alfalfa sprouts.
Schoeller et al. (146) reported that a beta irradiation
(electron beam) dose of 3.3 kGy was effective for a 6-log
reduction of L. monocytogenes on alfalfa sprouts without
any adverse effect on quality. The only study comparing the
use of electron beam and gamma irradiation to reduce
pathogen populations was carried out by Waje et al. (176).
They exposed broccoli and radish sprouts inoculated with
E. coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and B. cereus at radiation dose of 0, 0.25, 0.5, 1.0,
1.5, 2.0, 2.5, and 3.0 kGy. Salmonella Typhimurium had the
highest radiation sensitivity with the lowest D10-values of
0.13 to 0.30 kGy. In contrast, E. coli O157:H7 (0.46 to
0.73 kGy) and B. cereus (0.49 to 0.96 kGy) in broccoli and
red radish sprouts, respectively, were more resistant to
irradiation. The D10-value of these microorganisms in
sprouts was lower for gamma irradiation than for electron
beams. In broccoli sprouts, irradiation at 2.30 and 3.65 kGy
with gamma rays and electron beam are required to achieve
a 5-log reduction in pathogens, whereas the calculated five
times the D10-value for gamma irradiated and electron
beamtreated radish sprouts were 2.45 and 4.80 kGy,
respectively.
Effective reductions of aerobic bacteria and total

coliforms, in addition to pathogens, are of interest for
increasing the shelf life of sprouts. For example, a 2-log
reduction of aerobic bacteria and total coliforms achieved on
alfalfa and broccoli sprouts after a 2-kGy treatment
increased their shelf life by 10 days without affecting
nutritional qualities (134, 136, 137). The effect of lower
doses of radiation on the native microbial flora was also
investigated for increasing the keeping quality of different
varieties of sprouts. Irradiation of mung bean sprouts with 1
and 2 kGy resulted in a reduction in aerobic plate counts and
coliform counts by 2 and 4 log CFU/g, respectively; the
yeast and mold counts and staphylococci counts decreased
by 1 and 2 log CFU/g, respectively (145). The recovery of
the microorganisms was not observed during storage for up
to 12 days at 4 and 8uC. In addition, the 2-kGy irradiation
treatment did not affect textural, nutritional, or organoleptic
qualities of the sprouts, but the data were not shown (145).
Similarly, a 4-log reduction of both aerobic plate counts and
coliform counts in dew gram and chickpea sprouts was
observed at 2 kGy. The yeast and mold counts and
staphylococci counts were also reduced by 1.5 log CFU/g
(110). These studies (110, 145) revealed that gamma
irradiation can successfully extend the shelf life of sprouts
at temperatures commonly used for storage and sale in
supermarkets. However, because these sprouts were geographically confined to India the response of other varieties
of sprouts to irradiation needs further study.
Qualitative analyses have been conducted to assess the
appropriateness of effective irradiation treatments on sprouts.
Typically firmness, color, vitamin C, and total carotenoids in
the sprouts are evaluated after irradiation (0.1, 1, 1.5, 2, 2.5,
and 3 kGy) and subsequent storage at 4 to 8uC (8, 11, 40, 41,
64, 66). In one study, the firmness of sprouts was affected by
increased storage time after irradiation; mung bean sprouts
became firmer, whereas radish sprouts became softer (8). An
increase in yellow color with storage time has been observed
for mung bean, radish, and alfalfa sprouts at all treatment
doses (8, 40), although this change was deemed acceptable at
doses of 2 and 1.5 kGy for radish and mung bean sprouts,
respectively (40). Conflicting evidence of the effects on
vitamin C content due to irradiation and subsequent storage
(8, 40, 41, 64) has been reported, indicating that the
nutritional implications of this technology are still unclear.
Total carotenoid content remained almost unchanged in both
control and irradiated mung bean and alfalfa sprouts during
storage (41, 64).
Sensory tests have been conducted to verify quality
implications and determine the acceptability of irradiated
sprouts. Evaluations of appearance, color, texture, taste, and
overall acceptability were made for each type of sprout
several days after irradiation at various doses (11, 41, 66).
These testing panels have concluded that radiation doses of
1 to 2.5 kGy on broccoli, mung bean, alfalfa, and soy
sprouts will not altered consumer acceptance of the product.
In some cases, reevaluation after longer storage is needed,
but the organoleptic quality was maintained for 7, 5, and
14 days for broccoli, mung bean, and alfalfa sprouts,
respectively (11, 41).
The irradiation of sprouts even at low doses has been

effective for inactivation of pathogens and spoilage bacteria
while maintaining consumer acceptability. In spite of the
large body of research and the proven effectiveness for
seeds and sprouts, irradiation still has not been commercialized. The capital cost to build a commercial cobalt-60
food irradiation plant is $3 million to $5 million (34).
Although this amount is within the range of plant costs for
other technologies, the sprout industry consists of mostly
small firms with insufficient throughput to justify investing
in irradiation technologies. The FDA considers sprout
producers food processors rather than farmers (157). The
commercial sprouting operations in the United States are
usually small, with less than 10 employees (158), and the
use of a pricey irradiation facility is not economically
attractive to them.
Physical interventions: UV light. Another physical
intervention consists of treating sprouts with UV light.
Unlike gamma and beta radiation, UV light covers a wide
range of wavelengths in the nonionizing region of the
electromagnetic spectrum. UV light can be divided into
three parts: UV-A (315 to 400 mm), UV-B (280 to 315 mm),
and UV-C (,280 mm). UV-C is germicidal and has the
shortest wavelength but highest energy (107). UV-C
radiation exerts its bactericidal effect by inducing formation
of pyrimidine-base dimers and DNA-protein crosslinks,
which results in decreased viability and cell death (20). FDA
has approved UV radiation (254 nm UV-C) as a technology
that can be used for decontamination of food and food
contact surfaces (171). Inactivation studies using the 254nm wavelength have been effective for reducing pathogenic
bacteria and spores in vegetables and fruits (3, 60).
A combination of UV light and hydrogen peroxide
(H2O2) works as a sanitizing method based on the principle
of advanced oxidation processes. These processes rely on
the synergy of high-energy oxidants such as ozone and
H2O2 with UV photolysis to generate highly reactive
intermediates, particularly the hydroxyl radicals (109). The
result is the on-site oxidation of organic compounds or
microorganisms without the generation of residues (63).
Rajkowski (132) compared a single treatment of UV-C (185
and 254 nm) with a treatment of UV-C combined with a 5min prewashing step (e.g., 0.1% H2O2, distilled water, and
ozone water) in radish sprouts to reduce the populations of
inoculated S. sonnei. The reduction by UV-C radiation alone
was ,1 log CFU/g at a dose of 0.2 kJ/m2. Similarly,
pretreatments with distilled water and ozone water before
UV-C radiation were not effective, resulting in 0.68- and
0.14-log reductions, respectively. A 2-log reduction of S.
sonnei was obtained in sprouts pretreated with 0.1% H2O2.
Hence, enhancing H2O2 efficacy through generation of
advanced oxidation processes seems theoretically feasible
but requires more data for practical applications in foods.
In another study, UV-C treatment (254 nm) was applied
to clover sprouts to inactivate inoculated pathogenic bacteria
such as E. coli O157:H7, Salmonella Typhimurium, and L.
monocytogenes (84). These three pathogens were reduced
by 1.02, 1.06, and 0.87 log CFU/g, respectively, after
2115
irradiation at 1 kJ/m2. UV-C radiation was used in

combination with fumaric acid to realize the maximum
benefit. The combined treatment of fumaric acid (0.5 g/
100 ml) and UV-C radiation (1 kJ/m2) reduced the
populations of E. coli O157:H7, Salmonella Typhimurium,
and L. monocytogenes by 3.03, 2.88, and 2.81 log CFU/g,
respectively. This hurdle treatment was the most effective,
followed by treatment with fumaric acid alone and with UVC radiation alone, in that order. The authors recommended
that further studies be conducted to assess the sensory
properties of the sprouts after treatment.
Goyal et al. (62) examined the effect of UV radiation on
the functional properties and shelf life of mung bean sprouts
during storage. However, this investigation was not carried
out in conjunction with microbial inactivation on sprouts.
UV-C irradiation (10 kJ/m2, 1 h) significantly increased the
ascorbic acid concentration in mung bean sprouts during
storage at 25 and 7uC for up to 72 h. The total phenolic and
ascorbic acid concentrations increased immediately in the
sprouts after UV irradiation and remained high during
storage at 25uC for 48 h. UV-C irradiation affects
phenylpropanoid metabolism; it increases the activity of
phenylalanine ammonia-lyase (a key enzyme in phenol
biosynthesis) and thus the phenolic concentration. Antioxidant activity depends upon the concentrations of phenols
and ascorbic acid. This process was illustrated by a 3%
increase in scavenging of free radical 2,2-diphenyl-1picrylhydrazyl in UV-treated sprouts as compared with
control sprouts after 48 h of storage at 7 and 25uC. A
general decrease in polyphenol oxidase activity occurred in
sprouts after UV-C treatment. However, the polyphenol
oxidase activity gradually increased with longer storage
at 25uC.
Treatment with UV radiation offers an attractive
alternative for food processors because it does not leave
residues, does not have legal restrictions, and does not
require extensive safety equipment (186). However, this
technology is hampered by the poor penetration of UV-C
light, thus limiting its application to surface decontamination of food products. A combination of UV radiation with
other antimicrobial agents could be useful for improving the
decontamination of sprouts.
Physical interventions: high pressure and supercritical carbon dioxide treatments. High-pressure technology has been proven effective for eliminating pathogenic
microorganisms in seeds. In principle, the effectiveness is
attributed to the instantaneous and uniform action of
pressure throughout the mass of a sample unit regardless
of size or shape (43). However, high pressures may be
responsible for changes in biochemical reactions and
denaturation of proteins including key enzymes, leading to
impaired germination in seeds (98). The application of high
pressure for seed disinfection has had limited success, with
low seed viability and delayed germination (4, 184). In one
study (116), treatment at 650 MPa and 20uC for 15 min
succeeded in achieving an approximately 5-log reduction of
E. coli O157:H7 with 93% germination but only after 8 days
of sprouting. Because long duration high-pressure treat-
2116
SIKIN ET AL.
ments are not commercially feasible, milder continuous

high-pressure treatments (475 MPa, 40uC, 8 min; 600 MPa,
20uC, 2 min) have been studied but resulted in only a 2-log
reduction of E. coli O157:H7 in alfalfa seeds (4, 117). Thus,
more research has been dedicated to optimizing the
treatment by incorporation of a pretreatment step to increase
the bactericidal efficacy at milder conditions while maintaining seed viability (113, 114, 117, 128, 129).
Soaking of alfalfa seeds to increase water activity
improved the antimicrobial effect of pressure and completely eliminated E. coli O157:H7 but compromised germination (117). The application of high pressure at a reduced
level in combination with mild heat (500 MPa, 45uC, 2 min)
was able to eliminate 5 log CFU/g for E. coli O157:H7
(115) and Salmonella (113) on alfalfa seeds. Seed
germination was maintained at 98%, but a marked decrease
in sprout length was observed. Thus, sprout yield ratio (the
weight of sprouted seeds divided by weight of total seeds)
was added as an important index of seed viability in addition
to the percentage germination in the subsequent study.
Neeto et al. (114) reported that a dry heating step (60uC,
24 h) in combination with high pressure (600 MPa, 35uC,
2 min) resulted in a 5-log reduction of E. coli O157:H7.
Although the germination rate was not significantly
changed, the treatment reduced sprout yield by 5% (123).
Overall, these extreme process conditions result in high
percentages of seed germination but raise sprout yield
issues. In addition, treatment at pressures of 300 to 700 MPa
face some serious limitations, such as the difficulty of
controlling and managing such high operating pressures,
high equipment cost, and safety.
Supercritical carbon dioxide (Sc-CO2) applications at
pressures less than 20 MPa have drawn attention as an
alternative pasteurization or sterilization technique. Unlike
high pressure, the chemical nature of Sc-CO2 fluid (due to
its molecular behavior in water) has a lethal action on
bacteria, making the treatment not entirely dependent on
pressure. However, the more limited diffusion of carbon
dioxide into solid matrices compared with liquid food
systems restricts the application of Sc-CO2 for solid foods,
including seeds. The lack of information on the bactericidal
action of carbon dioxide in seeds is reflected by the
existence of only three published articles to date. Mazzoni et
al. (102) demonstrated the efficacy this treatment on the
indigenous aerobic microorganisms of alfalfa seeds. However, the Sc-CO2 treatment conditions (27.6 MPa, 50uC) for
60 min resulted in only a 1-log reduction. Subsequently, an
extensive study was conducted by Jung et al. (77), who
contaminated alfalfa seeds with E. coli O157:H7, L.
monocytogenes, and Salmonella Typhimurium. Multiple
Sc-CO2 treatments performed resulted in greater than 7-log
reductions of the three tested pathogens; however, the
germination rates were low. The use of water as a cosolvent
imparted similar inactivation of spores in barley seeds with a
less detrimental effect on germination (124).
Sc-CO2 has a liquid-like density, gas-like diffusivity
and viscosity, and a zero surface tension (104). Therefore,
this molecule can penetrate into complex structures, which
increases the possibility for inactivation of pathogens
embedded within complex produce surfaces and internal

tissues (190). However, the more limited diffusion of CO2
into solid matrices explains the extreme scarcity of
published reports on the feasibility of Sc-CO2 for decontamination of sprouts. Matsufuji et al. (101) was the only
research team who briefly investigated the effect of nearcritical carbon dioxide among other processes on the
inactivation of microorganisms and antioxidants in 10 cut
vegetables, including bean sprouts. Their results indicated
that near-critical carbon dioxide (6 MPa, 35uC, 10 min)
resulted in only a 0.5-log reduction of E. coli K-12 in bean
sprouts, with losses in vitamin C and phenol concentrations.
However, the Sc-CO2 experiment was not carried out
because the vegetable samples became frozen in the
decompression procedure, obviously because of a lack of
precise control of flow rate and temperature.
INTERVENTIONS DURING GERMINATION
The production of sprouts is generally a water intensive
process because dirt, pathogens, and residual sanitizers must
be washed from the seeds before the pregermination soak
and the hydroponic germination step. Growing sprouts are
regularly irrigated to provide hydrocooling to counteract the
heat released from the sprouting seedbeds and prevent
desiccation. Harvested sprouts are also often washed to
remove seed debris and residual chemicals when a
postharvest decontamination step is used. Water quality
will directly affect the likelihood and spread of contamination (170); however, regulations and guidance documents
for safe production of sprouts mention only recommendations for the use of potable water for rinsing and irrigating
seeds and sprouts (33, 169). Therefore, addition of
antimicrobial compounds to production water could be a
viable intervention strategy to counteract the favorable
conditions for microbial growth at sprouting facilities. Very
few reports in this area have been published compared with
the number of studies on potential interventions for seeds
and sprouts, and the use of municipal water is generally
regarded as the best practice (169).
Several treatments during germination have utilized the
potential of chlorine or chlorine compounds applied at
100 mg/liter, which resulted in pathogen reductions of only 2
log CFU/g when sprayed over germinating alfalfa and rice
sprouts because of the depletion of free chlorine (25, 59, 127).
In addition to controlling pathogens, sanitizers in rinse and
irrigation water were tested, but were not successful for
reducing native microbiota that contributes to the short shelf
life of fresh sprouts (46). Fett (47, 48) indicated that addition
of other antimicrobials, such as ozonated water and essential
oils (151), to the irrigation water has not been effective and
concluded that this is not the best approach for intervention.
This ineffectiveness of germination interventions is partly
due to the inability of antimicrobial compounds to reach
viable pathogens internalized within sprout tissue and the
unique challenge of being effective against microorganisms
without imparting phytotoxicity (112).
Most reports on interventions during germination
involve appropriate testing and sampling methods for
detection of viable pathogens after decontamination. During

sprouting, germinated seeds are known to release nutrients
(e.g., reducing sugars and other organic molecules) from
endosperm breakdown into the irrigation water (22). As a
result, this spent irrigation water flowing over and through
the sprouts becomes a rich growth medium for microorganisms and can gather more microorganisms as it passes
through the sprouting seeds. Because the treatments of seeds
and sprouting seeds do not completely eliminate pathogens,
the FDA has recommended that spent irrigation water be
collected 48 h after starting production and tested for E. coli
O157:H7 and Salmonella (162). The testing of spent
irrigation water is preferred over direct screening of sprouts
because of uniformity and ease of sample collection and
analysis (162). Spent irrigation water also provides a more
representative assessment of the microbiological status of
the sprouting bed compared with that obtained from
individual sprout samples.
Attempts have been made to enhance the efficiency of
testing spent irrigation water in commercial settings.
Composite sampling of spent irrigation water from various
sites was proposed to address the issue of nonhomogeneous
distribution of pathogens in sprouting seedbeds. However,
this sampling method from multiple points under sprouting
seedbeds does not increase the probability of detecting
contamination (103). Despite efforts to find ways for better
monitoring of the microbial safety of sprouts, internalization
of bacteria remains a challenge. Treatment during germination by addition of antimicrobial compounds to the
irrigation water may prevent accurate assessment of levels
of existing pathogens because those rinsed from the sprout
surface would be more easily inactivated than those still
tightly attached to the subsurface of the sprouts (158). For
example, populations of Salmonella and E. coli O157:H7
recovered from spent irrigation water were approximately
1-log lower than populations recovered from the sprouts
themselves (56, 154), although a high level of correlation
was observed between populations in spent irrigation water
and those on germinating seeds. The testing of spent
irrigation water at 48 h after sprouting, as recommended by
the FDA, is considered appropriate for detecting the highest
level of pathogens that may remain after seed decontamination and for assessing the overall safety of the final
product (47, 158, 160).
The current literature and regulations provide no
motivation for sprout producers to add an antimicrobial
treatment to the potable and clean irrigation water used
during sprout production. Several additives have been
ineffective for controlling pathogenic or spoilage microorganisms and may have phytotoxic effects on the germinating sprout. Although an irrigation water intervention could
be seen as the perfect hurdle treatment along with seed and
sprout decontamination, chlorine compounds applied to
sprouting seeds might reduce the accuracy of pathogen
detection in spent irrigation water. Therefore, the best
interventions during germination for improved sprout safety
are the use of potable and clean water and the testing of
spent irrigation water, as recommended by the FDA (162).
2117
CONCLUSIONS
Based on the number of published articles on sprout
safety and the persistence of related foodborne illness, no
clear approach to product safety has been found for this
industry, and the next outbreak cannot be predicted. Sprout
producers are presented with a plethora of data and
regulations that promote the implementation of preventive
practices throughout the process of seed harvest, seed
germination, and postharvest sprout storage. Several
conclusions can be reached regarding the efficacy of
intervention methods to improve the safety of sprouts.
(i) Because the main goal of research on sprout safety
is to provide viable options for the industry, an effective
treatment must strike a balance between the need to produce
safe sprouts for consumption and the need to preserve their
delicate nature. Studies on the functional and sensorial
impacts of all treatments (except irradiation) on seeds and
sprouts have not been conclusive. The effect of reported
treatments on quality attributes has not been given much
attention because the focus has mainly been on microbial
inactivation. However, the technology must be transferred to
actual producers to assess the potential of these strategies
under practical conditions. Although evaluation and enhancement of sanitizer alternatives are still very promising,
chlorine continues to be the most widely used sanitizer.
(ii) Most of the 5-log reduction data vary both within
studies depending on seed or sprout type, target microorganism, sample size, and inoculum level and between
studies depending on methods and materials used. Thus,
identification of a single method that can be effective across
the challenges and products in this industry is difficult.
Because seed surface characteristics and sprout size affect
the inactivation of pathogens, a marker system may be
beneficial for testing the efficacy of any decontamination
treatment. Replication of alternative methods is required to
achieve consensus on their efficacy.
(iii) Effective pre- and postharvest decontamination of
seeds and sprouts, respectively, is a challenge because of the
infiltration of pathogens into inner tissues and the ability of
pathogens to form biofilms and/or to coexist with native
microflora in biofilms. The primary obstacle to disinfecting
sprouts may be the ability of the treatments to reach the
pathogens; therefore, current research has been focused on
optimizing contact of antimicrobial compounds with
internalized microorganisms through hurdle treatments.
However, reported reductions of initial surface decontamination should include indication of recovery of injured or
internal pathogen cells. For future work, emphasis should be
placed on reporting treatments that consistently eliminate
pathogens from detection even after enrichment or exposure
to germination conditions.
(iv) Of all the areas for intervention, seed treatment
remains the most crucial in delivery of a safe final sprout
product. Thermal inactivation treatments for seeds best
combine the desirable properties of consistent microbial
inactivation and maintenance of seed viability and are well
suited for the commercial setting. Postharvest sprout
treatment is limited by the inability to impart a harsh
2118
SIKIN ET AL.
enough treatment for microbial inactivation while maintaining sprout quality. To date, irradiation holds the most
promise for pathogen control and/or extension of the shelf
life of sprouts with minimal or no loss of quality, whereas
in practice water disinfection remains an essential step in
the small industry of fresh sprout producers. Treatment of
irrigation water during germination has not reduced
pathogen levels on seeds or subsequent sprouts but may
be considered a good manufacturing practice for mitigating
cross-contamination.
The appropriate next steps for ensuring sprout safety
should focus on treatments easily integrated to the existing
production system. Various technical, economic, and
regulatory challenges must be considered when evaluating
the best interventions for preventing future incidences of
foodborne outbreaks related to sprouts. The overview of
these challenges and the current research presented here
should provide a platform for collaborative work by
academia, industry, and regulatory authorities for speedy
commercialization of the relevant technologies.
12.
13.
14.
15.
16.
17.
18.
ACKNOWLEDGMENTS
A. M. Sikin thanks the Ministry of Higher Education, Malaysia for
their financial aid. The authors also acknowledge Dr. Randy Worobo for his
review of the manuscript.
19.
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2099

Current Intervention Strategies for the Microbial Safety of Sprouts

Sprouts have been viewed as a health food in the Far

throughout the United States. Because of the multiple

As the last line of defense for elimination of pathogens

J. Food Prot., Vol. 76, No. 12

only 1- to 3-log reductions (5). The highest level of

J. Food Prot., Vol. 76, No. 12

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

Chemical interventions: organic acids. Organic acids

sanitizer. Consequently, the most lethal combination was a

Lactic acid (LA)

Malic acid (MA)

15| fatty acid sanitizer (15,000 ppm of 60:40 capryliccapric acid

500 ppm of ClO2 (5 min) z 45uC, 24 h air drying z dry heat

0.5% fumaric acid z 50 ppm of ClO2 (5 min)

100 ppm of ClO2, 5 min

Mung bean sprouts

5% LA, 10 min, 42uC

Salmonella (inoculated on sprouts)

5% AA, 10 min, 42uC

Phytic acid (PA) and oxalic Mung bean seeds

Seed or sprout type

Acetic acid (AA)

Seed or sprout type

800 ppm of ASC, 45 min

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

Acidified sodium chlorite

to below the detection limit, even after enrichment (35).

J. Food Prot., Vol. 76, No. 12

J. Food Prot., Vol. 76, No. 12

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

products such as chloride and chlorite, above the regulatory

membrane and disrupt metabolic process through oxidation

6.38; ORP 817 to 889 mV) for reducing the population of E.

J. Food Prot., Vol. 76, No. 12

reduce the population of E. coli O157:H7 (148), the ozone

J. Food Prot., Vol. 76, No. 12

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

delivery to sprouts are found. The combined treatment of

pseudomonads, such as Pseudomonas jessenii, also have

0.1% polyphosphoric acid

25 ppm of AS-48 z 40 ppm of PAA, RT, 5 min

Phage A, 108 PFU/ml, 25uC, 24 h

Microbial reduction (log

NA, not available.

Alfalfa, soybean sprouts

Mung bean seeds

Mung bean seeds

Seed or sprout type

J. Food Prot., Vol. 76, No. 12

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

storage at 22uC. These studies (18, 32) prove that in situ

by only 0.5 to 1 log CFU/g, LAB were inconsistently

cells have been exposed to treatments that increase the

J. Food Prot., Vol. 76, No. 12

preservatives protecting against bacterial spoilage of bean

J. Food Prot., Vol. 76, No. 12

INTERVENTIONS FOR MICROBIAL SAFETY OF SPROUTS

Because the current commercial method for treating

textural and nutritional qualities, respectively, of the

85uC water, 40 sz 0uC water, 30 s z 2,000 ppm of

Microbial reduction (log CFU/g);