Charlene E. Wolf-Hall

Evaluation of household sanitizers for reducing levels of Escherichia coli on iceberg lettuce.

Diluted solutions of various household sanitizers (apple cider vinegar, white vinegar, bleach, and a reconstituted lemon juice product) were tested for their effectiveness in reducing counts of inoculated Escherichia coli and naturally present aerobic, mesophilic bacteria on lettuce. Sanitization treatments were carried out at 4 degrees C and at room temperature (ca. 21 degrees C) with and without agitation and at different exposure times (0, 1, 5, and 10 min). Of the sanitizers tested, 35% white vinegar (1.9% acetic acid) was the most effective in reducing E. coli levels (with a 5-log10 reduction after 5 min with agitation and after 10 min without agitation) and in reducing aerobic plate counts (with a >2-log10 reduction after 10 min with agitation). Lettuce samples treated with diluted household sanitizers were analyzed for consumer acceptability by sensory evaluation using a 9-point hedonic scale. The sanitized samples did not differ in acceptability (P > 0.05), except for samples treated with white vinegar. Samples treated with the white vinegar for 10 min were noticeably sour and slightly wilted in appearance. Consumer acceptability was maintained with all sanitization treatments, including those involving 35% white vinegar.

Stability of deoxynivalenol in heat-treated foods.

The effects of high-temperature and -pressure processing of foods spiked with deoxynivalenol (DON) were examined. In extruded corn grits, extruded dry dog food, and autoclaved moist dog food, there were no significant reductions (P < 0.05) in DON after processing. Autoclaved cream-style corn showed a reduction in DON of only 12%. Overall, DON was stabile to the high temperature and pressure processes tested. The use of an alpha-amylase in the extraction method for analysis by an enzyme-linked immunosorbant assay (ELISA) improved the recovery of DON from the spiked extruded and autoclaved products by as much as 26% over the standard ELISA method.

Evaluation of gaseous ozone and hydrogen peroxide treatments for reducing Fusarium survival in malting barley.

The use of Fusarium-infected barley for malting can lead to mycotoxin production and decreased malt quality. Methods for treatment of Fusarium-infected barley might prevent these safety and quality defects and allow use of otherwise good-quality barley. Gaseous ozone and hydrogen peroxide (HP) were evaluated for effectiveness in reducing Fusarium survival while maintaining germinative energy (GE) in barley. Gaseous ozone treatments (GOT) included concentrations of 11 and 26 mg/g for 0, 15, 30, and 60 min. HP treatments included 0, 5, 10, and 15% concentrations with exposure times of 0, 5, 10, 15, 20, and 30 min. For GOT, in naturally Fusarium-infected barley, a statistically significant (P < 0.05) decrease (24 to 36%) of Fusarium survival occurred within 15 min of exposure at either concentration. GE was significantly (P < 0.05) affected by 30 min at both concentrations in naturally Fusarium-infected barley, but not in sound barley. GOT did not cause any significant (P > 0.05) effect on GE in sound barley at either concentration over the full 30-min exposure time. For HP, Fusarium survival was significantly decreased (50 to 98%) within 5 min of exposure. With the exception of two treatments (10 and 15% HP agitated for 20 min), GE was not statistically significantly different from the control in naturally Fusarium-infected barley. In sound barley, HP had no significant (P > 0.05) effect on GE. The results suggest that GOT and HP might have potential for treatment of Fusarium-infected malting barley.

Mycotoxins and fermentation--beer production.

Along with food safety issues due to mycotoxins, the effects of Fusarium infections on malt and beer quality can be disastrous. While some of the Fusarium head blight mycotoxins, such as DON, present in infected barley may be lost during steeping, the Fusarium mold is still capable of growth and mycotoxin production during steeping, germination and kilning. Therefore, detoxification of grain before malting may not be practical unless further growth of the mold is also prevented. Methods for reducing the amount of mold growth during malting are needed. Physical, chemical and biological methods exist for inhibiting mold growth in grain. Irradiation is a promising means for preventing Fusarium growth during malting, but its effects on malt quality and mycotoxin production in surviving mold need to be evaluated in more detail. Chemical treatments such as ozonation, which do not leave chemical residues in beer, also appear to be promising. Although biological control methods may be desirable, the effects of these inhibitors on malt and beer quality require further investigation. In addition, storage studies are needed to determine the effect of biological control on Fusarium viability and malt quality. It may also be possible to incorporate detoxifying genes into fermentation yeasts, which would result in detoxification of mycotoxins present in wort. Development of these types of technological interventions should help improve the safety of products, such as beer, made from Fusarium infected grain.

Microbial Loads, Mycotoxins, and Quality of Durum Wheat from the 2001 Harvest of the Northern Plains Region of the United States

The 2001 durum wheat crop grown in the Northern Plains was surveyed for microbial loads, mycotoxins, and quality. Correlations among these factors were identified. Effects of cleaning, milling, and pasta processing on microbial loads and deoxynivalenol (DON) concentrations were determined. Aerobic plate counts (APCs), mold and yeast counts (MYCs), internal mold infection (IMI), and internal Fusarium infection (IFI) were lowest in grain samples from Montana and highest in grain from northeastern North Dakota. DON and 15-acetyldeoxynivalenol (15-ADON) were not detected in samples from Montana. Nivalenol was not detected in any samples. DON in North Dakota samples ranged from none detected to 23 micrograms/g. 15-ADON was detected in a few North Dakota samples, with a maximum of 0.8 microgram/g. DON positively correlated with APCs, MYCs, IFI, damaged kernels, total defects, U.S. grade number, and tombstone kernel content and negatively correlated with test weight, vitreous kernel content, and kernel weight. APCs, MYCs, and DON concentrations were lower in semolina than whole grain. Processing semolina into spaghetti did not change DON concentrations. APCs for spaghetti were reduced 2.2 to 4.1 logs from those for semolina, whereas MYCs were reduced 0.1 to 1.7 log. Some APCs in durum flour and semolina were higher than certain industry specifications would allow, although other factors were acceptable. However, microbial loads in the spaghetti were all within specifications found in the available literature.

Antifungal Activity Stability of Flaxseed Protein Extract Using Response Surface Methodology

The stability of the antifungal activity of flaxseed (Linum usitatissimum) protein extract was evaluated in this study. Response surface methodology (RSM) using Box-Behnken factorial design was used to evaluate the effects of treatment variables, that is, temperature (50 to 90 degrees C), time (1 to 29 min), and pH (2 to 8), on the residual antifungal activity (RAA) against Penicillium chrysogenum, Fusarium graminearum, Aspergillus flavus, and a Penicillium sp. isolated from moldy noodles. Regression analyses suggested that the linear terms of the temperature and time had significant (P < 0.05) negative effects on the RAA against all test fungi, whereas that of pH had a significant (P < 0.1) positive role on the RAA of all 3 fungi. In addition, the RAA was significantly (P < 0.05) affected by the quadratic terms of time for all fungi, and the quadratic term of temperature played a significant (P < 0.1) role on RAA against F. graminearum. One interaction term (temperature-pH) was found to significantly (P < 0.1) affect the RAA against both Penicillium strains tested. The results indicated that >or= 90% antifungal activity was lost after the protein extracts were heated at 90 degrees C for 8 min except for F. graminearum. At pasteurization condition, >or= 50% activity was retained except for P. chrysogenum. The results also suggested that neutral and alkaline pH favored the antifungal activity stability of the protein extracts. Thus, flaxseed protein might be promising if used as a preservative in foods with neutral or alkaline pH requiring mild heat treatments.

Evaluation of Hot Water and Electron Beam Irradiation for Reducing Fusarium Infection in Malting Barley

The use of Fusarium-infected barley for malting may lead to mycotoxin production and decreased product quality. Physical methods for the treatment of Fusarium-infected barley may prevent these safety and quality defects and allow the use of otherwise good quality barley. Hot water and electron beam irradiation were evaluated for their effectiveness in reducing Fusarium infection while maintaining germinative energy in barley samples. Hot-water treatments involved temperatures of 45, 50. 55, and 60 degrees C and treatment times of 0, 1, 5, 10, and 15 min. Electron beam irradiation involved doses ranging from 0 to 11.4 kGy. Treatment with water at 45 degrees C for 15 min resulted in a reduction in Fusarium infection from 32 to 1% after 15 min, with only a very slight reduction in germination. Treatment with water at 50 degrees C for 1 min resulted in a reduction in Fusarium infection from 32 to 2%, and no effect on germination was observed for up to 5 min of treatment. At higher water temperatures. Fusarium infection was essentially eliminated, but germination was also severely reduced. Electron beam irradiation of Fusarium-infected barley reduced Fusarium infection at doses of >4 kGy, and a slight increase in germination for dry samples was observed with doses of 6 to 8 kGy. Doses of >10 kGy significantly decreased germination. Physical methods may have potential for the treatment of Fusarium-infected malting barley.

Development and Evaluation of an Ozonated Water System for Antimicrobial Treatment of Durum Wheat

Ozonated water is reported to be effective in reducing the microbial load in foods such as fruits, vegetables, and grains. Ozonated water may be an effective alternative to chlorinated water in treating durum wheat before milling. Therefore, durum wheat was washed with ozonated water and analyzed for yeast and mold count (YMC) and aerobic plate count (APC). A system for producing and monitoring ozonated water was developed. The effect of water quality (tap, distilled, and ultra-pure), temperature (7, 15, and 25 degrees C), and pH (2, 4, and 6.5) was evaluated on the following: steady-state dissolved ozone concentration, ozone decay constant, half-life, mass transfer coefficient, equilibrium ozone concentration, and solubility ratio. The study of these parameters was important to attain a stable, high dissolved ozone concentration at the outset of washing and to have information for system improvement and scale-up. A 1% acetic acid solution (pH 2) at 15 degrees C resulted in high dissolved ozone concentration (21.8 mg/L) and long half-life (9.2 min). Subsequently, wheat was washed with 5 wash water types: distilled water, ozonated water (16.5 mg/L), chlorinated water (700 mg/L), acetic acid solution (1%), and acetic acid + ozonated water (1%, 20.5 mg/L). The treated samples were analyzed for YMC and APC. The acetic acid + ozonated water treatment was the most effective, with a reduction of 4.1 and 3.2 log(10) colony forming units/g in YMC and APC, respectively. Though ozonated water was not very effective alone, it was useful in combination with acetic acid.

Quantification of Tri5 gene, expression, and deoxynivalenol production during the malting of barley.

Fusarium can survive, grow, and produce mycotoxins during malting. We evaluated the percentage of barley kernels infected with Fusarium (FI) and deoxynivalenol (DON) concentration in three barley treatments (high-quality, naturally infected, and Fusarium graminearum inoculated barley) during various stages of malting. We also applied real-time polymerase chain reaction (real-time PCR) and real-time reverse transcriptase PCR (real-time RT-PCR) methods to quantify trichothecene-producing (Tri5) DNA concentration and expression, respectively. We observed that FI significantly (P<0.05) increased during the germination stage of malting in all barley treatments. Temperatures of 49°C and higher during kilning reduced the FI in high-quality barley treatments, but for inoculated treatments temperatures in excess of 60°C were needed to reduce FI. The Tri5 DNA concentration ranged from non-detectable to 3.9 ng/50mg, 0.1 to 109.8 ng/50mg and 3.4 to 397.5 ng/50 mg in malted high-quality, inoculated and naturally infected barley treatments respectively. Strong gene expression (Tri5) in naturally infected barley treatments was found during the third day of germination, when compared to high-quality and inoculated barley treatments during malting. Deoxynivalenol was present even at high kilning temperatures, as DON is heat stable. The average DON concentration ranged from non-detectable to 0.1 μg/g, non-detectable to 1.1 μg/g, and 1.5 to 45.9 μg/g during various stages of malting in high-quality, inoculated and infected barley and malt samples respectively. Overall, the last 2 days of germination and initial stages of kilning were peak stages for FI, Tri5 gene production, Tri5 gene expression and DON production.

Multiplex real-time PCR method for detection and quantification of mycotoxigenic fungi belonging to three different genera.

UNLABELLED Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method. PRACTICAL APPLICATION This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.