Food Protection Trends - November/December 2023 - 481

Agar-based disk diffusion assay. Due to the ease of use
and low-cost method, we also performed a disk diffusionbased
assay to evaluate the antifungal activity of SBS, a
method similar to Zabka et al. (34). For this, the PDA
plates were supplemented with various concentrations of
SBS (0, 0.25, 0.5, 0.75, and 1.0%) by adding filtered sterile
SBS solution in agar just before pouring onto the plates.
The mold disks were prepared by dipping sterile paper disks
(diameter: 0.25 in.; Carolina Biological, NC) in 10 ml of
mold broth for 72 h at 25°C. The average concentrations of
A. flavus and F. graminearum used for disk diffusion assay
were 4.91 and 4.02 log, respectively. The disks were lifted
with sterile forceps and placed on the center of the agar
plates, and the plates were incubated for 72 h at 25°C before
measuring the diameter. The assay was done in triplicate for
each strain.
Wheat inoculation study
A 150-g aliquot of autoclave-sterilized hard red winter
wheat for each treatment was transferred to an autoclaved
plastic container. Note that the autoclaving of the wheat
might change the nutrient composition of the food and
possibly behave differently than raw wheat. The moisture
of the autoclaved wheat was measured and found to be
10.7%. For each mold type, a total of five containers were
maintained: three for treatments (0.5, 1.0, and 1.5% final
concentration of SBS in wheat); one for the positive
control (mold only and no SBS); and one for the negative
control (no mold and no SBS). The various treatments were
selected on the basis of the MIC and MFC of SBS. The
working culture of molds (~4 to 5 log) was spot inoculated
in the wheat and mixed thoroughly by closing the lid
of the jars. Due to the nonuniform nature of harvesting
mold spores from the lawn, the initial inoculum level of
the molds tended to vary for each replication, with 4 to
5 log typical for both A. flavus and F. graminearum. After
the mold inoculation, 30 min at 25°C was allowed for the
attachment of molds. After 30 min, aqueous SBS treatments
were applied to the mold and mixed uniformly. The volume
differential for positive and negative controls was adjusted
by adding sterile distilled water. Microbiological analyses
were conducted for each of the containers at various
predetermined times: 0, 6, 12, 18, 24, and 48 h. From each
treatment and control, a 25-g subsample was collected in
sterile-filtered Whirl-Pak bags (Nasco, Fort Atkinson, WI),
mixed in 225 ml of buffered peptone water, and stomached
for 2 min. The mixtures were serially diluted in 0.1%
peptone water and plated on PDA. A total of 1-ml volume
of the diluent was plated on four PDA plates. The plates
were incubated at 25°C for 60 to 72 h, and colonies were
counted. The detection limit for the mold was 1 log CFU/g
of the sample. The mold colonies were counted, and the
results were expressed as log (CFU per gram of the wheat
sample). Each experiment was conducted in triplicate.
Statistical analysis
The first experiment was a 5 × 6 factorial arrangement of
treatments using three SBS concentrations (0.5, 1.0, and
1.5%), the positive and negative controls, and six sampling
intervals. Each treatment consisted of three replicates. Mean
values were analyzed by using the general linear model
procedure using Tukey's test (P ≤ 0.05) comparison. All
statistical analyses were conducted by using SAS statistical
software, version 9.3 (SAS Institute Inc., Cary, NC).
RESULTS AND DISCUSSION
The powdered SBS provided by Jones-Hamilton Co. had
solubility of 28.5 g/liter of water at room temperature. Due to
this limitation, a concentration higher than 1.5% of SBS was
not able to be achieved in the tempered wheat model. To our
knowledge, this is the first study of its type using SBS as an
antimycotic against molds (A. flavus and F. graminearum). In
this study, we reported the MICs of SBS against A. flavus and
F. graminearum to be 0.31 and 0.16%, respectively, and the
MFC of SBS to be 2.5 and 1.25%, respectively. As expected,
the MFCs were higher than the corresponding MICs for both
organisms. The results indicated that A. flavus is much more
resistant compared with F. graminearum. This observation
is consistent with the report by Savi and Scussel (31) who
also noted a higher resistance of A. flavus against ozone (O3)
when compared with F. graminearum. In another study with
organic acids in animal feed, Higgins and Brinkhaus (19)
also discovered that Aspergillus spp. were more resistant than
Fusarium spp.
Table 1 shows the results of agar-based disk diffusion assay.
The diameters of the disk (mold growth) on the PDA plates
were measured after 72 h with complete inhibition for the
0.75% concentration against both mold species.
In this experiment, we simulated the common tempering
condition for wheat grain during the flour milling process.
Though normal tempering is done for an 18- to 24-h period,
we extended the evaluation time point further (up to 48 h)
to evaluate the potential for reduction during extended time
periods. Further, due to the method used for harvesting
molds, the concentrations of molds were not uniform for
each replication, and an average was taken from the three
replications. Similar methods were used in previous studies
(32) involving mold harvesting. Chlorine and chlorine
gas have been traditionally used during wheat tempering
to reduce microbial loads (33). However, this poses safety
concerns among the consumers, as well as for milling
workers. Hence, studies to find safer replacements, such as
O3 and other chemicals have been sought. SBS possessed
antimicrobial effects against E. coli (Shiga toxigenic) in wheat
tempering conditions (7) and against Salmonella in poultry
meals (6) and rendered chicken fat systems (10, 11).
When A. flavus-inoculated wheat was treated with different
concentrations of SBS, the initial (0 h) recovery from the
control wheat (no SBS) was 4.9 log/g. In the wheat samples
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