Effects of recovery, plating, and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries.

Effects of different recovery and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries were studied. Strawberries were spot or dip inoculated with 7 to 8 log CFU per strawberry of each pathogen, air dried for 2 h, and stored for 1, 3, and 7 days at 4 degrees C. The inoculated samples were stomached or washed with phosphate-buffered saline (PBS; pH 7.2) or with modified PBS (pH 8.4). Bacterial levels were determined using a direct selective plating, thin agar layer plating, or membrane-transferring plating (MTP) with tryptic soy agar and sorbital MacConkey agar (E. coli O157:H7) or modified Oxford agar (L. monocytogenes). Under most test conditions, washing with PBS followed by MTP had significantly higher (P < 0.05) recovery for both bacteria compared with other tested methods. Within a 7-day storage period for spot-inoculated strawberries, a stomaching step resulted in an injury of 0.9 to 1.4 log CFU for E. coli O157: H7 and 1.4 to 1.7 log CFU for L. monocytogenes. When a washing step was used instead, this resulted in an injury of only 0.2 to 0.6 log CFU for E. coli O157:H7 and 0.2 to 0.7 log CFU for L. monocytogenes. Both bacteria could survive on strawberry surfaces, but their recovered levels decreased with the increase of storage time at 4 degrees C for both spot and dip inoculation methods. Dip inoculation generally had a lower recovery than spot inoculation. An ideal protocol to recover and enumerate E. coli O157:H7 and L. monocytogenes from strawberries involved shaking and washing samples with 100 ml of PBS for 15 min at 22 degrees C coupled with a MTP enumeration method.

Decontamination of strawberries using batch and continuous chlorine dioxide gas treatments.

Efficacy of chlorine dioxide (ClO2) gas in reducing Escherichia coli O157:H7 and Listeria monocytogenes on strawberries was determined using batch and continuous flow ClO2 gas treatment systems. Effects of continuous ClO2 gas treatment on total aerobic plate count, color, and residual ClO2 and chlorite on strawberries were also evaluated. Strawberries were spot inoculated with 7 to 8 log CFU per strawberry of each pathogen (E. coli O157:H7 and L. monocytogenes), stored for 1 day at 4 degrees C, and treated at 22 degrees C and 90 to 95% relative humidity with 0.2 to 4.0 mg/liter ClO2 gas for 15 or 30 min using a batch treatment system or with 0.6, 1.8, and 3.0 mg/liter for 10 min using a continuous treatment system. Surviving microbial populations were determined using a membrane-transfer plating recovery method. Increased ClO2 gas concentrations resulted in increased log reductions of each pathogen for both the batch and continuous systems. A batch treatment of strawberries with 4 mg/liter ClO2 for 30 min and continuous treatment with 3 mg/liter ClO2 for 10 min achieved greater than a 5-log reduction for both E. coli O157:H7 and L. monocytogenes. After continuous exposure to 3.0 mg/liter ClO2 gas for 10 min followed by 1 week of storage at 4 degrees C, no aerobic microorganisms were detected and the color of the strawberry surface did not change significantly (P > 0.05). Residues of ClO2 and chlorite on strawberries after the treatment were 0.19 +/- 0.33 mg ClO2 per kg and 1.17 +/- 2.02 mg Cl2 per kg, respectively, whereas after 1 week of storage no ClO2 residues were detected and residual chlorite levels were down to 0.07 +/- 0.12 mg Cl2 per kg. These results suggest that ClO2 gas treatment is an effective decontamination technique for improving the safety of strawberries while extending shelf life.

Decontamination of Bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas.

This work examined the efficacy of chlorine dioxide (ClO2) gas for the decontamination of Bacillus thuringiensis spores on paper, wood, epoxy, and plastic surfaces. Spores representing an inoculation level of approximately 6 log colony-forming units (CFU) per surface were treated with 5, 10, 15, 20, 25, or 30 milligrams per liter (mg/L) ClO2 gas for 12 hours under 85-92 percent relative humidity and at 22 +/- 1 degrees C. Under the tested treatment conditions, the highest population of surviving spores was found on the paper surface and the lowest was found on the plastic surface (p < .05). The 5 mg/L ClO2 gas treatment inactivated 2.5, 3.6, 4.0, and 4.9 log spores per surface on paper, wood, epoxy, and plastic surfaces, respectively. A greater than 5-log reduction of spores was achieved on each surface after the 15 mg/L ClO2 gas treatment. The minimum ClO2 gas concentration needed to completely inactivate the inoculated spores was 30 mg/L for paper and wood surfaces, 25 mg/L for epoxy surfaces, and 20 mg/L for plastic surfaces. The results of this study may provide insight into the parameters of effective decontamination procedures for Bacillus spores.

Reduction of listeria monocytogenes on green peppers (Capsicum annuum L.) by gaseous and aqueous chlorine dioxide and water washing and its growth at 7 degrees C.

Reduction of Listeria monocytogenes Scott A on uninjured and injured surfaces of green peppers after 0.3- and 3-mg/ liter gaseous and aqueous ClO2 treatment and water washing for 10 min at 20 degrees C was studied. Growth of the L. monocytogenes untreated or treated with 0.6 mg/liter ClO2 gas for 30 min at 20 degrees C on green peppers also was investigated. A membrane-surface-plating method was used for resuscitation and enumeration of L monocytogenes treated with ClO2. The bacterial viability on pepper surfaces was visualized using confocal laser scanning microscopy (CLSM). Live and dead cells of L. monocytogenes were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively. More than 6 log CFU/5 g L. monocytogenes on uninjured surfaces and about 3.5 log CFU/5 g on injured surfaces were inactivated by both 3-mg/liter and 0.6-mg/liter ClO2 gas treatments. The 3-mg/liter aqueous ClO2 treatment achieved 3.7- and 0.4-log reductions on uninjured and injured surfaces, respectively; whereas, water washing alone showed 1.4- and 0.4-log reductions, respectively. ClO2 gas treatment was the most effective in reducing L. monocytogenes on both uninjured and injured green pepper surfaces, when compared with aqueous ClO2 treatment and water washing. The significant difference (P < 0.05) between log reductions on uninjured and injured surfaces and the results from CLSM analysis suggested that injured surfaces protected more bacteria from sanitation treatments than did uninjured surfaces. Not only could L. monocytogenes grow on green pepper surfaces at 7 degrees C, bacteria that survived the 0.6-mg/liter ClO2 gas treatment also could grow.

Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum L.) by chlorine dioxide gas treatments.

The effects of chlorine dioxide (ClO2) gas concentration (0.1 to 0.5 mg/liter), relative humidity (RH) (55 to 95%), treatment time (7 to 135 min), and temperature (5 to 25 degrees C) on inactivation of Escherichia coli O157:H7 on green peppers were studied using response surface methods. A four-factor, central, composite, rotatable design was used. The microbial log reduction was measured as a response. A direct membrane-surface-plating method with tryptic soy agar and sorbitol MacConkey agar was used to resuscitate and enumerate ClO2-treated E. coli O157:H7 cells. The statistical analysis and the predictive model developed in this study suggest that ClO2 gas concentration, treatment time, RH, and temperature all significantly (P < 0.01) increased the inactivation of E. coli O157:H7. ClO2 gas concentration was the most important factor, whereas temperature was the least significant. The interaction between ClO2 gas concentration and RH indicated a synergistic effect. The predictive model was validated, and it could be used to determine effective ClO2 gas treatments to achieve a 5-log reduction of E. coli O157:H7 on green peppers.

Production of Fumonisin B(inf1) and Moniliformin by Gibberella fujikuroi from Rice from Various Geographic Areas.

Gibberella fujikuroi strains isolated from rice in the United States, Asia, and other geographic areas were tested for sexual fertility with members of mating population D and for production of fumonisin B(inf1) and moniliformin in culture. Of the 59 field strains tested, 32 (54%) were able to cross with tester strains of mating population D, but only a few ascospores were produced in most of these crosses. Thirty-four strains produced more than 10 (mu)g of fumonisin B(inf1) per g, but only three strains produced more than 1000 (mu)g/g. Twenty-five strains produced more than 100 (mu)g of moniliformin per g, and 15 produced more than 1,000 (mu)g/g. Seven field strains produced both fumonisin B(inf1) and moniliformin, but none of these strains produced a high level of fumonisin B(inf1) (>1,000 (mu)g/g). However, a genetic cross between a strain that produced fumonisin B(inf1) but no moniliformin and a strain that produced moniliformin but no fumonisin B(inf1) yielded progeny that produced high levels of both toxins. Strains of G. fujikuroi isolated from rice infected with bakanae disease are similar to strains of mating population D isolated from maize in their ability to produce both fumonisins and moniliformin. This finding suggests a potential for contamination of rice with both fumonisins and moniliformin.

Identity of Fusarium nygamai isolates with long and short microconidial chains from millet, sorghum and soil in Africa.

Several Fusarium species have been found associated with millet and sorghum in Nigeria, Lesotho and Zimbabwe. Amongst these, some isolates were originally identified as short- and long-chained types of F. nygamai. However, there was some question as to the correct identification of the long chained types. This study reclassified some of the isolates with long microconidial chains as F. moniliforme. Morphologically, these strains do not produce chlamydospores like F. nygamai, but produce swollen hyphal cells or resistant hyphae. The isolates in this study were crossed with the mating-type tester strains of Gibberella fujikuroi (F. moniliforme and G. nygamai (F. nygamai). Of the isolates with long chains of microconidia and other characteristics of F. moniliforme, 36% crossed with mating population "A" of G. fujikuroi. Of the isolates with characteristics of F. nygamai, 65% crossed with the testers used to produce the teleomorph of F. nygamai. Mating tests support the separation of the sample population into F. moniliforme and F. nygamai. The results of this study show that genetics can be an aid in resolving some problems in fungal taxonomy.

Processing of nutritious, safe and acceptable foods from CELSS candidate crops.

A controlled ecological life-support system (CELSS) is required to sustain life for long-duration space missions. The challenge is preparing a wide variety of tasty, familiar, and nutritious foods from CELSS candidate crops under space environmental conditions. Conventional food processing technologies will have to be modified to adapt to the space environment. Extrusion is one of the processes being examined as a means of converting raw plant biomass into familiar foods. A nutrition-improved pasta has been developed using cowpea as a replacement for a portion of the durum semolina. A freeze-drying system that simulates the space conditions has also been developed. Other technologies that would fulfill the requirements of a CELSS will also be addressed.

The food-process subsystem for CELSS: a conceptual analysis.

A controlled ecological life support system (CELSS) is required to sustain life for future long-duration space missions. Food processing is an important subsystem component of a CELSS. Factors for designing the food-process subsystem are identified and characterized in this analysis. Interactions of the subsystem with other subsystems in a CELSS are also discussed.

Sexual fertility of forty Fusarium strains from the European Fusarium sambucinum project.

Fungus strains designated as Fusarium sambucinum, F. torulosum, or Fusarium sp. nov. were crossed with MAT1-1 and MAT1-2 tester strains of Gibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated as F. torulosum was fertile with a tester strain of G. pulicaris, suggesting that this is an intraspecies cross and that the strain is G. pulicaris, and, consequently, F. sambucinum rather than F. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are not G. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.

Production of beauvericin by a strain of Fusarium proliferatum isolated from corn fodder for swine.

Beauvericin, a cyclodepsipeptide, was produced by cultures of three strains of Fusarium proliferatum, M-5991, M-6992, and M-6993, grown on cracked corn. M-5991 produced approximately 1,000-mg/kg levels of fumonisins, moniliformin, and beauvericin.

Taxonomy, biology, and clinical aspects of Fusarium species.

There are several taxonomic systems available for identifying Fusarium species. The philosophy used in each taxonomic system is discussed as well as problems encountered in working with Fusarium species in culture. Fusarium species are toxigenic, and the mycotoxins produced by these organisms are often associated with animal and human diseases. The implications for the association of the carcinogens, fumonisins, produced by Fusarium moniliforme and other Fusarium species with human diseases are discussed. Foreign-body-associated fusarial infection such as keratitis in contact lens wearers, onychomycosis, skin infections, and disseminated multiorgan infections are discussed. Disseminated fusarial hyalohyphomycosis has emerged as a significant, usually fatal infection in the immunocompromised host. Successful outcome is determined by the degree of immunosuppression, the extent of the infection, and the presence of a removable focus such as an indwelling central venous catheter. These infections may be clinically suspected on the basis of a constellation of clinical and laboratory findings, which should lead to prompt therapy, probably with one of the newer antifungal agents. Perhaps the use of such agents or the use of colony-stimulating factors may improve the outcome of this devastating infection. However, until new approaches for treatment develop, effective preventive measures are urgently needed.

Conditions and constraints of food processing in space.

NASA: Requirements and constraints of food processing in space include a balanced diet, food variety, stability for storage, hardware weight and volume, plant performance, build-up of microorganisms, and waste processing. Lunar, Martian, and space station environmental conditions include variations in atmosphere, day length, temperature, gravity, magnetic field, and radiation environment. Weightlessness affects fluid behavior, heat transfer, and mass transfer. Concerns about microbial behavior include survival on Martian and lunar surfaces and in enclosed environments. Many present technologies can be adapted to meet space conditions.^ieng

Isolation and characterization of new chlamydosporol related metabolites of Fusarium chlamydosporum and Fusarium tricinctum.

Fusarium chlamydosporum strain T-826 isolated from corn in the USA produced chlamydosporol and two analogs which have been identified by various spectroscopic techniques as: 7,8-dihydro-5-hydroxy-4-methoxy-trans-7,8-dimethyl-2H,5H-pyrano(4, 3-b)pyran-2-one (or isochlamydosporol) and 4-methoxy-5-hydroxymethyl-6-(3-butan-2-ol)-2H-pyran-2-one (or chlamydospordiol). Chlamydosporol (compound a + b) chlamydospordiol (compound c) and isochlamydosporol (compound d) were produced together (up to 6000 micrograms/g) by 3 out of 11 isolates of F. chlamydosporum and by 3 out of 24 isolates of F. tricinctum from various substrates and geographic origin. Three isolates of F. chlamydosporum and one isolate of F. tricinctum produced only chlamydospordiol and 2 isolates of F. tricinctum produced chlamydosporol (a + b), and chlamydospordiol (c).

Fumonisin production and other traits of Fusarium moniliforme strains from maize in northeast Mexico.

Strains of Fusarium moniliforme from maize seed collected in four fields in northeast Mexico were tested for fumonisin production in culture, for sexual compatibility, and for vegetative compatibility by using non-nitrate-utilizing mutants. The test results indicate that a diverse population of fumonisin-producing strains of F. moniliforme (Gibberella fujikuroi) mating population A predominates and that a potential exists for production of fumonisins in Mexican maize.

Soil fertility factors in relation to oesophageal cancer risk areas in Transkei, southern Africa.

Soil samples were collected during certain years for the period 1982-89 from high- and low-risk areas for oesophageal cancer in Transkei, southern Africa. These samples were taken either from cultivated soils under maize monoculture, or from uncultivated soils (1989 only) adjacent to the maize fields. Analyses of mineral elements in the soil samples were performed at two independent laboratories. Furthermore, soil and maize leaf samples, from field trials in a high- and a low-risk area for oesophageal cancer were analysed. The results from this study do not agree with those reported previously for Transkei. Cultivated soils in both high- and low-risk areas were found to be highly fertile. The levels of Mn, Ni, Mg, Ca, K and soil pH were significantly higher, and Al, Fe and organic matter significantly lower in the high-risk compared with the low-risk area. Leaf analysis, although not tested statistically, indicated higher levels of Mn K, Ca and Fe, and lower levels of P, in the high-risk area.

Analysis of naturally occurring mycotoxins in feedstuffs and food.

Aflatoxins, zearalenone, deoxynivalenol, fumonisins, and their respective metabolites require specific procedures for their determination because of their diverse chemistry and occurrence in complex matrices of feedstuffs and foods. Major sources of error in the analysis of these mycotoxins arise from inadequate sampling and inefficient extraction and cleanup procedures. The determinative step in the assay for each of these toxins is sensitive to levels below those that are considered detrimental to humans and animals. Aflatoxins can be determined in grains and animal fluids and tissues by TLC, HPLC, gas chromatography-mass spectrometry (GC-MS), and ELISA procedures. Zearalenone, an estrogenic mycotoxin, can readily be determined in cereal grains and foods by HPLC (50 ng/g) and by TLC (300 ng/g). No incurred levels of zearalenone or its metabolites have been detected in animal tissues destined for human consumption. Deoxynivalenol can be determined in wheat and corn at 300 ng/g by a rapid TLC procedure and at 325 ng/g by a GC method. Although not tested collaboratively, an HPLC procedure and an ELISA screening procedure are capable of detecting deoxynivalenol at low (nanograms/gram) levels in feedstuffs and foods. The recently characterized fumonisins can be detected by TLC, HPLC, and GC-MS at levels below those now considered harmful. Thin-layer chromatography and HPLC (with fluorescence detection of derivatives) procedures can detect fumonisins at approximately 100 ng/g; GC-MS is required for detection at lower levels.

Thermal Resistance and Growth of Bacillus licheniformis and Bacillus subtilis in Tomato Juice.

Bacillus species are common contaminants of soil and can be found with tomatoes and other vegetables and fruits. This research was done to assess the thermal stability of spores of acid tolerant Bacillus licheniformis and Bacillus subtilis in tomato juice. The D values at 90, 95, and 100°C were 29.5, 15.8, and 5.7 min for B. subtilis and 29.9, 12.2, and 5.9 min for B. licheniformis , respectively. The z value for B. subtilis was 14°C and for B. licheniformis was 14.2°C. Aerobically, B. subtilis spores could germinate and outgrow in tomato juice (pH 4.4) within 4 d at 35°C at inoculum levels as low as 1 spore per ml; however, B. licheniformis spores could only germinate and outgrow if levels were 104 spores per mi or higher. Both Bacillus species could raise the pH of tomato juice to above 4.8 when grown aerobically. Neither B. licheniformis nor B. subtilis could germinate and outgrow anaerobically in tomato juice (pH 4.4).