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

1241 Journal of Food Protection, Vol. 66, No. 7, 2003, Pages 1241 1246 Copyright q, International Association for Food Protection Evaluation of Hot Water and Electron Beam Irradiation for Reducing Fusarium Infection in Malting Barley BALASUBRAHMANYAM KOTTAPALLI, 1 CHARLENE E. WOLF-HALL, 2 * PAUL SCHWARZ, 1 JURGEN SCHWARZ, 1 AND JAMES GILLESPIE 1 1 Department of Cereal and Food Sciences and 2 Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, North Dakota 58105, USA MS 02-343: Received 26 September 2002/Accepted 21 January 2003 ABSTRACT 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 608C 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 458C 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 508C 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 signi cantly decreased germination. Physical methods may have potential for the treatment of Fusarium-infected malting barley. Different microorganisms can contaminate barley from maturation to the storage stage (10). In recent years, fungi that cause a serious plant disease known as Fusarium head blight (FHB) in barley have become increasingly persistent (16). Mycotoxins may occur in FHB-infected grain (19, 21, 27), and the consumption of these mycotoxins may lead to health problems for humans and animals (2). There is signi cant concern in the malting and brewing industry about the use of FHB-infected grain for malting (18). Researchers have shown that the growth of Fusarium during the malting process not only resulted in mycotoxin production but also affected the germinative capacity and malting characteristics of barley (10, 24). Fusarium graminearum and Fusarium poae, commonly found in FHBinfected grain, have been reported to reduce kernel plumpness, to increase wort soluble nitrogen and free amino nitrogen, and to affect wort color (25). Fusarium mycotoxins such as deoxynivalenol (DON) and diacetoxyscripenol (DAS) have been reported to affect the malting process by reducing alpha-amylase activity and alpha-amino nitrogen levels in worts and may also have a negative impact on yeast growth and fermentation (23, 28). Another problem associated with FHB-infected grain, and of signi cant concern to the brewing industry, is the gushing of beer (17). Beattie et al. (3) explained that because of the various effects caused by the FHB-infected grain, maltsters might * Author for correspondence. Tel: 701-231-6387; Fax: 701-231-6536; E-mail: charlene.hall@ndsu.nodak.edu. not accept infected grain or may purchase it only at a reduced price. This situation has had a negative impact on the barley growers, the malting industry, and the barleyproducing regions of the United States (15). Grain containing.2.0 ppm of DON is not accepted for malting, and some maltsters do not accept grain containing any detectable DON (3). As a result of the various problems caused by FHB, it has become very dif cult for regional barley growers to pro tably market even mildly FHB-infected grain for malting. Our objective was to evaluate nonchemical treatments (hot-water and electron beam irradiation treatments) for their effects on Fusarium infection and germinative energy in barley. Such treatments may allow maltsters to use mildly FHB-infected barley for malting with a low risk of mycotoxin production and product quality defects. However, to be acceptable, treatments must not signi cantly damage the germinative energy of the barley. MATERIALS AND METHODS Experimental design and statistical analysis. This project included three sets of experiments. The rst set of experiments evaluated the effects of electron beam irradiation on Fusarium infection (FI) and germinative energy (GE) in three barley samples. The second set of experiments evaluated the effects of hot water on FI and GE in an FHB-infected barley sample. The third set of experiments evaluated the effect of electron beam irradiation on FI and GE in the same barley sample used for the hot-water experiments. The GE analyses for the second and third experiments included GE values obtained after 3 and 5 days of germi-

1242 KOTTAPALLI ET AL. J. Food Prot., Vol. 66, No. 7 nation, while the GE analysis for the rst experiment included only values obtained after 3 days. A randomized complete block design was used to test the treatment differences with respect to Fusarium infection for all experiments. For the rst experiment, a randomized complete block design was used for the analysis of GE. For the second and third experiments, in which GE was determined after 3 and 5 days, a split-plot design was used. The results of FI and GE were analyzed by analysis of variance and a general linear model procedure to compare differences between treatments. The level of signi cance used was 5%. Barley. Three barley samples from the 1999 barley harvest in North Dakota were used for the rst experiment. The rst sample, designated sound barley (cultivar Robust) contained no detectable DON. The second sample was an FHB-infected barley sample (a composite of the cultivars Minnbrite and Robust) containing 8 mg of DON per g. To create the third sample, the FHBinfected barley was steeped by placing 100-g portions of barley in 400 ml of water and soaking the barley for 10 h with 6 min of aeration per h; after each 10 h of soaking, the barley was subjected to a 2-h air rest during which the water was changed. The total soaking time was 30 h. The combined steeped sample was then stored at 48C until it was irradiated (;24 h). For the second and third sets of experiments, the barley used was an FHB-infected Robust cultivar from the 2000 harvest in North Dakota. This barley had a DON concentration of 3 mg/g. All DON levels were determined by gas chromatography (26). Hot-water treatments. Barley seeds (200 per treatment) were randomly selected, counted, and placed in cheesecloth bags. The samples were presoaked, since fungal propagules within the seed may become metabolically activated upon imbibition and thus may theoretically become more sensitive to heat damage when treated with hot water. For presoaking, the samples were placed in 1-liter beakers containing 700 ml of sterile distilled water, and the beakers were placed in a refrigerated bath maintained at 168C. A coiled pipe containing holes was immersed into each beaker for aeration of the seeds. Aeration with compressed air was carried out to provide adequate dissolved oxygen for the germ. The soaking period was 4 h, and samples were aerated for 5 min every hour. After soaking, the bags were removed from the beakers and the water was drained. The presoaked barley seed bags were then immersed in a water bath that was maintained at the desired treatment temperature. The treatments were carried out at four different water temperatures (45, 50, 55, and 608C) for different treatment times (0, 5, 10, and 15 min). After they were treated, bags were removed from the water bath. The bags were opened in a laminar ow hood, and the treated samples along with the presoaked control sample were then analyzed. Treatments were repeated three times. Electron beam irradiation treatments. Irradiation treatments with an electron beam were carried out at the Iowa State University Linear Accelerator Facility (Thomson CSF Linac, Saint Aubin, France). Samples of barley (200 g each) were placed in sterile plastic 720-ml Whirl-Pak stomacher bags. The bags had indicator strips or dosimeters (Bruker Instruments Inc., Billerica, Mass.) attached to them. Dosimeters from the chamber were analyzed to verify actual doses with a dosimeter analyzer (Bruker Analytische Messtechnik Karlsruhe, Germany). The doses used ranged from 2.3 to 11.3 kgy for the rst experiment and from 2.4 to 11.4 kgy for the third experiment. The treatments were repeated three times. After the samples had been irradiated, they were returned within 24 h to the lab at North Dakota State University in Fargo for analysis. FI. One hundred barley seeds were aseptically transferred into standard-sized petri dishes (5 seeds per petri dish) containing half-strength acidi ed potato dextrose agar (22). The petri dishes were then incubated under ambient lighting for 5 days at room temperature. After 5 days, the molds that grew on the seeds were identi ed to the genus level and counted. The number of seeds colonized by Fusarium was represented as a percentage. The FI values for control samples were always determined at the time individual experiments were conducted, because Fusarium viability in barley is known to decrease with storage time (3). GE. GE was determined by the method of the American Society of Brewing Chemists (1). One hundred barley kernels of each sample to be tested were placed in petri dishes containing Whatman no. 4 lter paper saturated with sterile distilled water. All dishes were placed in an environmental chamber (Caron Products and Services, Inc., Marietta, Ohio). The chamber was maintained at 168C and at 100% relative humidity. Chitted (sprouted) kernels were counted and removed after 24, 48, and 72 h to avoid excessive moisture uptake by the early-germinating kernels. For the second and third set of experiments, testing was also carried out after 120 h (5 days). The total percentages of kernels germinated after 72 h and after 120 h were recorded and designated 3- day GE and 5-day GE, respectively. RESULTS AND DISCUSSION Electron beam irradiation treatments. The results of the rst experiment, in which sound, FHB-infected, and steeped FHB-infected barley samples were treated with electron beam irradiation, are shown in Figures 1 and 2. The FI value for the steeped FHB-infected sample was signi cantly (P, 0.05) higher than that for the dry FHBinfected barley. This result is likely due to cross-contamination with Fusarium propagules during steeping. For the sound sample and the steeped FHB infected sample, FI values decreased signi cantly (P, 0.05) at doses of.2.4 kgy. For the dry FHB-infected sample, a dose of.4.3 kgy was required to achieve a signi cant reduction in FI. These results are similar to previously reported results for the gamma irradiation (20) of dry barley with an initial FI of ca. 19%. The more extensive FI reduction for the steeped barley may be due to fungal propagules being more sensitive to ionizing radiation after imbibition. Thus, increasing the moisture content of the barley seeds may improve the effectiveness of ionizing radiation in reducing Fusarium levels. However, it is apparent from Figure 2 that the germinative capacity of the higher-moisture barley was much more sensitive to ionizing radiation as well. The GE in the sound barley sample decreased signi - cantly (P, 0.05) with a dose of 6.9 kgy, indicating that the germ may be affected at higher doses. For the FHBinfected barley, there were slight but signi cant decreases in GE at 4.6 and 6.9 kgy; however, the GE for barley treated at 8.6 kgy was not signi cantly different from that for untreated barley (control). Ramakrishna et al. (20) reported a similar phenomenon for gamma-irradiated FHBinfected barley, while Yen et al. (29) reported that gamma irradiation doses of 0.3 to 4.8 kgy stimulated germination in maize kernels. Gilbert and Tekauz (11) demonstrated that

J. Food Prot., Vol. 66, No. 7 TREATING FUSARIUM IN MALTING BARLEY 1243 FIGURE 1. Effects of electron beam irradiation on FI in sound barley, FHB-infected barley, and steeped FHB-infected barley. Error bars indicate standard errors for three data points. FIGURE 2. Effects of electron beam radiation on FI and 3-day GE in an FHBinfected barley sample. Error bars indicate FIGURE 3. Effects of electron beam irradiation on 3-day GE in sound barley, FHB-infected barley, and steeped FHB-infected barley. Error bars indicate standard errors for three data points. FHB infection could affect germination in grain (11), and thus the increase in GE at 8.6 kgy could possibly be attributed to the concurrent decrease in FI. Electron beam irradiation treatments were repeated with a sample of the barley used for the hot-water experiments. The results of these irradiation treatments are summarized in Figure 3. Signi cant (P, 0.05) reductions in FI occurred at doses of.2.3 kgy. There was no detectable FI after treatment at.6.9 kgy and only 3% FI after treatment at 4.7 kgy, results consistent with the trend observed by Ramakrishna et al. (20) for gamma irradiation. Signi - cant (P, 0.05) reductions in 3- and 5-day GE started at doses of.4.7 kgy. These effects were similar to those observed in the rst set of experiments with different barley

1244 KOTTAPALLI ET AL. J. Food Prot., Vol. 66, No. 7 FIGURE 4. Effects of 458C water on FI and on 3- and 5-day GE in a presoaked FHB-infected barley sample. Error bars indicate FIGURE 5. Effects of 508C water on FI and on 3- and 5-day GE in a presoaked FHB-infected barley sample. Error bars indicate FIGURE 6. Effects of 558C water on FI and on 3- and 5-day GE in a presoaked FHB-infected barley sample. Error bars indicate FIGURE 7. Effects of 608C water on FI and on 3- and 5-day GE in a presoaked FHB-infected barley sample. Error bars indicate samples. The 5-day GE values were signi cantly (P, 0.05) higher than the 3-day GE values, with differences increasing as the dose increased, indicating that some metabolic injury that was not re ected in the 3-day GE value alone had occurred. Although there appeared to be some recovery after the two additional days, this loss in vigor may not be acceptable to maltsters. Hot-water treatments. Barley was presoaked for 4 h at 168C prior to hot-water treatment to provide time for imbition and thereby increase the susceptibility of the Fusarium. However, for the untreated control barley, an unexpected decrease in GE from 86.5% when the barley was dry to 73% following the presoaking process was observed. The reason for this result is not clear, but this decrease could have been due to water sensitivity in the grain. Water sensitivity is the failure of grain to germinate in excess water and is attributed to competition of the embryo with microorganisms for oxygen (14). Presoaking was also observed to increase the mean FI level from 28 to 32%. This result, again, was likely due to cross-contamination of kernels during soaking. Figures 4 through 7 show the effects of hot-water treatments on FI and on 3- and 5-day GE over time. Signi cant (P, 0.05) decreases in FI occurred within 1 min at all temperatures. The complete absence of FI was observed for samples treated at 55 and 608C for 10 and 5 min, respectively. Treatment at 458C decreased the FI level from 32 to 1% after 15 min. There was no signi cant (P, 0.05) reduction in 5-day GE even after 15 min. However, there was a slight but statistically signi cant decrease in 3-day GE (from 70% at 0 h to 64%) after 10 min at 458C. A dramatic reduction in FI (from 32 to 2%) was observed after only 1 min at 508C, but treatment with 508C for up to 5 min water had no apparent effect on 3- or 5- day GE. However, at 55 and 608C, the 3- and 5-day GE signi cantly (P, 0.05) decreased within 1 min. Hot-water treatment for Fusarium infections in maize seeds has been studied by Eredey et al. (7, 8). The trends found by these investigators for reductions in FI and germination as temperatures and times increased were similar to those observed in the present study. Dry heat has also been shown to be effective in reducing FI in barley while maintaining germination (4). However, the requirement for treatment for 5 to 21 days at 60 to 808C seems impractical

J. Food Prot., Vol. 66, No. 7 TREATING FUSARIUM IN MALTING BARLEY 1245 for large-scale operations and calls into question potential effects on other malt quality parameters. Both hot water and electron beam irradiation show potential as treatments for reducing levels of FI without damaging germination in mildly FHB-infected malting barley. These treatments could be a more acceptable than the use of inhibitory chemicals. Residues and undesirable reaction products in malt are a concern with chemical treatment, since they have the potential to affect both the quality of the malt and yeast fermentation performance during brewing. Sanitizing chemicals such as sodium hypochlorite may even damage seed germination and are not effective in eliminating internal microbial infections (5). Irradiation may result in oxidative chemical reactions in the grain; however, the effects these reaction products would have on quality needs to be determined. Delincee (6) indicated that total lipids (constituent in cereals that may be most affected by ionizing radiation) were not signi cantly changed in cereals with treatment at doses of up to 10 kgy. An advantage of irradiation is that a process could be developed to treat grain either prior to or after storage. Treating the grain prior to storage may also help in the maintenance of quality by eliminating insect infestations. Ionizing radiation doses of.2 kgy would theoretically eliminate insects and many non spore-forming bacteria (9). Hanis et al. (13) demonstrated reductions in overall micro- ora from approximately 6.5 to 4 log units in various cereal meals treated at 1 kgy, and only 1 to 10 CFU/g of Enterococcus and Clostridium species survived at 10 kgy. Altering the microbial ora could also potentially affect malt quality and safety and will require further research. Halasz et al. (12) observed an increase in mycotoxin production by two strains of Fusarium graminearum in grain cultures treated at 1 and 3 kgy with gamma irradiation. The use of starter cultures containing desirable microorganisms that can outcompete or inhibit undesirable ora during malting may be an additional measure that can be taken to improve both the safety and the quality of treated barley. One advantage of the hot-water treatments is that DON is water soluble, and some preformed toxin that may be present in mildly FHB-infected barley could be washed out of the grain. Further research is needed to optimize the time and temperature under scaled-up conditions. Additional studies are under way to further evaluate these treatments for barley samples from different cultivars and for different levels of infection. These additional studies will also determine the effects of these treatments on malt quality and evaluate the mycotoxigenicity of surviving Fusarium. 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