Food Protection Trends - November/December 2023 - 480

in 125 deaths and 200 illnesses in Kenya in 2004 (27).
Major mycotoxins found in cereals are aflatoxin (produced
by Aspergillus flavus), deoxynivalenol, zearalenone, and
fumonisins (produced by Fusarium graminearum) and
ochratoxin (produced by Aspergillus ochraceus) (4). It is
estimated that about 25% of the global food and feed output
is contaminated by mycotoxins (16).
Minimizing the introduction of the fungal spores in the
grains during preharvest and postharvest periods is the
primary approach implicated to minimize the mycotoxin
introduction in the food chain (26). In addition to various
management practices, physical and physicochemical
methods are also used to decontaminate and degrade
the already formed mycotoxins in grains (17). However,
chemical control of molds in stored grains for human
and animal consumption is not very common. The use of
plant essential oils such as the terpenoids, eugenol and
cinnamaldehyde, were reported to control fungal growth in
cereals (17). Valeric acid, propionic acid, and other organic
acids have also been known to inhibit mold growth (19).
However, the application of organic acids may affect the taste
and smell, especially in foods for human consumption. In
addition, the cost of organic acids required to treat grains in
bulk storage could be a concern. Chlorine compounds are the
most commonly used antimicrobials during wheat tempering
to control bacterial and fungal pathogens (13). However,
due to the occurrence of potentially hazardous residues
of chlorine (24) and the export restriction of chlorinetreated
foods to some countries, safer and widely accepted
intervention strategies must be investigated.
Given the breadth and magnitude of this issue and the
limitation of the existing intervention strategies, identifying
a method that can be deployed in the grain processing
industry to mitigate mold infestation and growth could have
a profound benefit to human and animal health, reduce
grain losses, and have a positive effect on the sustainability
of grain-based food for future generations. Though the
mycotoxin production in the stored wheat typically happens
prior to milling, the presence of the molds during milling
and the subsequent transfer of molds and mycotoxins in flour
is of concern. In wheat milling, tempering is a premilling
stage, when water is added to the raw grain (final moisture
of ~16%) for ~18 h to toughen the bran and mellow the
wheat endosperm, thereby improving the efficiency of the
flour extraction. The occurrence of Aspergillus and Fusarium
in wheat flour (1) and hence the presence of mycotoxins in
wheat flours (23) lead us to design a study for the application
of an antimycotic during the premilling stage and attempt to
control molds and mycotoxins in postmilling fractions.
Sodium bisulfate (SBS; NaHSO4
) is a natural acidulant
and a hygroscopic chemical that has known antimicrobial
activities against Salmonella (9-12) and Escherichia coli
(7, 30) in foods without negative effects on quality. This is
generally recognized as safe for use in human foods and is
480 Food Protection Trends November/December
also approved for use in animal feed as a general purpose
additive (22). The effect of SBS as an antimycotic in stored
wheat products is not known. Therefore, the objective of
this study was to evaluate the postharvest application of SBS
in wheat grains against A. flavus and Fusarium graminearum
Schwabe.
MATERIALS AND METHODS
Sources of mold strains, antimycotic, and wheat grains
Stock cultures of A. flavus (ATCC 15548) and F.
graminearum Schwabe (ATCC 46779) were obtained from
the American Type Culture Collection. A. flavus (ATCC
15548) is known to produce aflatoxin B1 and G1, and F.
graminearum Schwabe (ATCC 46779) is known to produce
deoxynivalenol and zearalenone. The frozen cultures were
maintained in potato dextrose broth (PDB): glycerol (7:3)
at −80°C. SBS was provided by the Jones-Hamilton Co.
(Walbridge, OH). Wheat samples (hard red winter wheat)
were procured from Indigo Agriculture (Boston, MA).
Preparation of the working culture of the molds
Before use, the frozen cultures of the molds were
grown in PDB overnight. The broth was then streaked on
potato dextrose agar (PDA, supplemented with 0.1 g/
liter chloramphenicol) plates in duplicate and incubated at
25°C for 72 h. After 72 h, the mold-grown agar lawns were
scrapped by dislodging the spores by using sterile L-rod
spreaders in 5 ml of 0.1% peptone water (3, 8). The spore
suspension was then transferred to a sterile tube and used
for the wheat inoculation study within an hour. The mold
suspension was used for the MIC, minimum fungicidal
concentration (MFC), and wheat inoculation study.
MIC and MFC assays
The MIC of SBS was determined by broth microdilution
assay according to the method of the Clinical and Laboratory
Standards Institute (5) in PDB. A 100-μl aliquot of PDB
mold culture containing ~5 log CFU/ml was added
to each well of a plate containing 100 μl of decreasing
concentrations of SBS for a final volume of 200 μl per well.
The positive control consisted of A. flavus or F. graminearum
inoculum only (no SBS), and the negative control consisted
of PDB alone. A separately prepared SBS solution with
concentrations from 0.25 to 3%, with a 0.25% increase, were
tested in the MIC assay. The MIC was defined as the lowest
concentration of the antimicrobial agent that inhibited the
visible growth of molds after 72 h of incubation at 25°C (2).
For the determination of the MFC assay, 10-μL content from
the wells showing no visible growth after 72 h of incubations
were plated on PDA plates in duplicate. The plates were
incubated for 72 h at 25°C. The lowest SBS concentration
that caused no visible colony on the agar plate was considered
to be the MFC (28). Each experiment for each strain was
done in triplicate.
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