Sterilization of food packaging by UV-C irradiation: Is Aspergillus brasiliensis ATCC 16404 the best target microorganism for industrial bio-validations?

In food industries UV-C irradiation is used to achieve decontamination of some packaging devices, such as plastic caps or laminated foils, and of those smooth surfaces that can be directly irradiated. Since its effectiveness can be checked by microbial validation tests, some ascospore-forming molds (Aspergillus hiratsukae, Talaromyces bacillisporus, Aspergillus montevidensis, and Chaetomium globosum) were compared with one of the target microorganisms actually used in industrial bio-validations (Aspergillus brasiliensis ATCC 16404) to find the species most resistant to UV-C. Tests were carried out with an UV-C lamp (irradiance = 127 µW/cm2; emission peak = 253.7 nm) by inoculating HDPE caps with one or more layers of spores. Inactivation kinetics of each strain were studied and both the corresponding 1D-values and the number of Logarithmic Count Reductions (LCR) achieved were calculated. Our results showed the important role played by the type of inoculum (one or more layers) and by the differences in cell structure (thickness, presence of protective solutes, pigmentation, etc.) of the strains tested. With a single-layer inoculum, Chaetomium globosum showed the highest resistance to UV-C irradiation (1D-value = 100 s). With a multi-layer inoculum, Aspergillus brasiliensis ATCC 16404 was the most resistant fungus (1D-value = 188 s), even if it reached a number of logarithmic reductions that was higher than those of some ascospore-forming mycetes (Aspergillus montevidensis, Talaromyces bacillisporus) tested.

Effect of peracetic acid on ascospore-forming molds and test microorganisms used for bio-validations of sanitizing processes in food plants.

Industrial sterilization of packaging and filling machineries by peracetic acid (PAA) is a widespread practice. In our study we assessed the resistance to PAA of three ascospore-forming molds (Chaetomium globosum ATCC 6205; Talaromyces bacillisporus SSICA 10915; Aspergillus hiratsukae SSICA 3913) compared to that of Aspergillus brasiliensis ATCC 16404 and Bacillus atrophaeus DSM 675, that are currently used as test microorganisms during industrial bio-validations of food packaging and machineries. Tests were carried out at 40 °C using 1,000 mg/l of PAA, with or without a supporting material (aluminium, tin-plate, PET). At all conditions tested, a greater resistance to PAA was registered for C. globosum, followed by T. bacillisporus, A. hiratsukae, A. brasiliensis and B. atrophaeus. D-values of C. globosum varied from 23 to 68 min, whereas T. bacillisporus showed D-values from 83 to 352 s and A. hiratsukae showed D-values from 32 to 65 s. Surprisingly, both test microorganisms (A. brasiliensis and B. atrophaeus) proved less resistant than ascospore-forming molds tested, their D-values being always lower than 30 s. Cells treated without a supporting material proved more resistant than those deposited on plastic or metallic strips, with the exception of tin-plate, where results approaching those obtained without a supporting materials were obtained. Based on the results obtained in this paper, test microorganisms currently used for bio-validations in industrial plants and also heat-resistant strains proved sensibly less resistant to PAA than C. globosum. Therefore, for practical purposes C. globosum should be furtherly studied to understand if its use during bio-validations of sanitizing processes could lead to more performing results.

Sanitization of packaging and machineries in the food industry: Effect of hydrogen peroxide on ascospores and conidia of filamentous fungi.

In the food industry, sterilization of packaging and filling machines by hydrogen peroxide (HP) is a widespread practice. Its effectiveness is usually tested by means of inactivation tests on selected test microorganisms that were any case chosen without taking into account that food products could be also spoiled by microorganisms presumably resistant to HP. For this reason, the aim of this work was to assess the resistance of different ascospore-forming moulds (Talaromyces bacillisporus, Aspergillus hiratsukae, Chaetomium globosum) to HP, in order to find the most resistant to this kind of chemical stress, and to compare their resistance with that registered for other moulds, including test microorganism Aspergillus brasiliensis ATCC 16404. Tests were carried out from 50 to 60 °C on spores or conidia, depending on the strain, either by immersing inoculated strips (aluminium, tin-plate, HDPE, PET) in HP, or by directly inoculating cells in the sanitizing medium. In both tests, T. bacillisporus proved the most resistant strain, followed by A. hiratsukae, C. globosum and A. brasiliensis at all temperatures tested. In test without a supporting material, D values of T. bacillisporus varied from 6 to 23 s. In test with metallic or plastic strips, D values of T. bacillisporus were higher on plastic materials, compared to those obtained on metallic ones up to 53 °C, whereas at higher temperatures D values proved similar. For A. hiratsukae, D values were similar if different materials were compared, except for D50 on aluminium and HDPE, which proved slightly higher (3.1-3.4 s) than those obtained on tin-plate and PET (2.7-2.8 s). Analogously, ascospores of C. globosum behaved in a similar way if different materials were compared, except for D50 values that varied in a wide range (from 2.9 s on tin-plate to 4.0 s on HDPE). A. brasiliensis was rapidly inactivated by the synergistic effect of heat and hydrogen peroxide, so for this strain it was not possible to calculate any D value. Based on the results obtained in this paper, tested ascospore-forming moulds proved to be sensibly more resistant to HP than other heat-sensitive strains tested, their D values always being significantly higher, regardless of the strain considered and the supporting material assessed. Ascospore-forming moulds could be furtherly investigated, as for practical purposes they seemed most suitable as target microorganisms than heat-sensitive microorganisms such as Aspergillus brasiliensis ATCC 16404, their use during bio-validations of sanitizing processes on machineries used for refrigerated products (pH > 4.5) or non-refrigerated acid products (pH ≤ 4.5) leading to more performing results.

Aspergilli with Neosartorya-type ascospores: heat resistance and effect of sugar concentration on growth and spoilage incidence in berry products.

This study focused on four different heat resistant aspergilli: two strains of Aspergillus hiratsukae (≡Neosartorya hiratsukae), one strain of Aspergillus neoglaber (≡Neosartorya glabra), and one strain of Aspergillus thermomutatus (≡Neosartorya pseudofischeri), all isolated from spoiled pasteurized products. Their heat-resistance, the sugar concentration limiting their germination and growth in berry-based media, and a possible relation between the contamination levels of the raw materials used and the spoilage incidence in strawberry jams were assessed. Heat resistance data obtained from thermal death curves showed that the D values of the strains tested ranged between 3.7 and 13.5min at 87°C; 1.5 and 3.5min at 90°C; and 0.3 and 0.4min at 95°C in glucose solution. Similarly, D values ranged between 3.3 and 15.4min at 87°C; 1.3 and 4.3min at 90°C; and 0.3 and 0.6min at 95°C in strawberry-based formulation. For all strains, the corresponding z-values ranged between 5.7 and 8.3°C in glucose solution and from 5.7 to 8.4°C in strawberry formulation. With regard to the limitation of fungal germination and growth in fruit-based media, sucrose concentrations required to avoid growth varied between 45.0 and 55.0% for strawberry medium and between 42.5% and 50.0% for blueberry medium. Spore inactivation was observed below aw 0.88-0.91 for strawberries and aw 0.87-0.90 for blueberries; above 49.7-56.5°Bx for strawberries and 49.6-56.0°Bx for blueberries. The threshold optical refractometric residue proved strain-dependent, but substrate-independent, as for each strain the highest Brix degree value at which germination occurred was the same on both media, despite their different sucrose concentrations. With regard to the relation between contamination of raw materials by heat-resistant mould spores and spoilage incidence on final product, an equation was modelled to estimate the occurrence of fungal spoilage in strawberry jams for low contamination levels (26-46CFU/kg). Although it could not be used as a definitive tool to predict final spoilage in such of products, it could give important practical information to jam producers in preventing spoilage of their products.

Occurrence and ecological distribution of Heat Resistant Moulds Spores (HRMS) in raw materials used by food industry and thermal characterization of two Talaromyces isolates.

In this study, screening of some raw materials used to produce pasteurized products was carried out to determine the occurrence and ecological distribution of heat-resistant fungi. The search for Heat Resistant Mould Spores (HRMS) resulted in the isolation of a limited number of fungal genera: Arthrinium, Aspergillus with either Eurotium-type or Neosartorya-type ascoma, Byssochlamys, Hyphodermella, Monascus, Penicillium, Rasamsonia, Talaromyces and Thermoascus. Sexual aspergilli constituted an overwhelming percentage of the mycobiota, totaling 93.5% of the heat-resistant fungi detected, and being the only fungi to be simultaneously detected in discrete concentrations on almost all matrices found positive for HRMS. Talaromyces spp., Penicillium spp. and Monascus sp. occurred at low percentages (up to 2.1%), though they were the most commonly occurring genera in lemon cells (Talaromyces, Monascus) and blueberries (Penicillium spp.). Among these isolates, two Talaromyces spp. (T. trachyspermus and T. bacillisporus) were tested for heat-resistance in both blueberry and grape juice or in buffered glucose solution, in order to assess their D- and z-values. Data obtained from thermal death curves and statistical elaboration of raw data showed that D-values of T. trachyspermus ranged between 50.0 and 90.9min at 75°C; 13.6 and 20.8min at 78°C; 5.1 and 12.4min at 80°C; 1.6 and 2.6min at 82°C. D values of T. bacillisporus ranged between 44.4 and 60.9min at 82°C; 11.9 and 15.5min at 85°C; 2.7 and 4.1min at 88°C and were equal to 1.2min at 91°C, depending on the medium. The heating times needed for inactivation were comparable to those applied to most heat-resistant species, but significantly lower than those applied to Talaromyces macrosporus or less common ascospore-forming fungal species such as Hamigera avellanea and Thermoascus crustaceus. Therefore, a traditional pasteurization process would be insufficient to avoid potential spoilage problems with T. trachyspermus or T. bacillisporus, even if HRMS contamination of the raw materials processed by food industries is generally low (<100CFU/kg), since the food industry generally tries to achieve five or more log-reduction in their products.

Monitoring the mycobiota of three plants manufacturing Culatello (a typical Italian meat product).

This study reports the composition of the mycobiota growing on the surface of Culatello (a typical Italian meat product) and occurring in the environments of three processing plants. Samples were collected in both winter and summer. A total of 84 culatelli and 14 samples from the plant environment were examined. A total of 331 (from food samples) and 2030 (from air samples) fungal isolates belonging to six genera and 29 species were identified. The substantial correspondence between air- and product-mycobiota in all the manufacturing plants studied seems to indicate a natural selection of those species that have adapted to the thermal-hygrometric conditions to which meat products were subjected. In particular, all sexual Aspergillus spp. with Eurotium-type ascomata, all Scopulariopsis spp. and Sporendonema casei from culatelli exactly matched with those from air samplings, and a prevalence of xerotolerant and xerophilic species belonging to Aspergillus or Penicillium was observed for both culatelli and environments, depending on the plant considered. Aspergillus candidus (16.0%), Penicillium solitum (19.6%), and Aspergillus cristatus (≡ Eurotium cristatum) (17.2%) were the prevalent species in Plants 1, 2, and 3, respectively. Fungal species producing unsightly spots on the casings (Scopulariopsis spp. and Sporendonema casei) were mainly found in the first steps of the aging, but tended to diminish or to change color throughout the process, so ultimately they did not represent a matter of concern. Fungal species potentially producing ochratoxin A (Penicillium nordicum and Aspergillus westerdijkiae) were the least prevalent species collected from a minor number of culatelli, so their presence could be defined as sporadic and did not represent a risk for consumers' health. This study reports the dominance of desirable species over undesirable molds on culatelli, but also highlights the importance of monitoring those meat products where no bacterial starter can degrade mycotoxins and where neither fungal starters nor a skin can inhibit fungal development. The control of the so-called "house mycobiota" in such products should be periodically assessed both in artisanal and industrial plants, since it proved to be fundamental to focus the potential risks connected to consumption of these meat products.

Heat-resistance of Hamigera avellanea and Thermoascus crustaceus isolated from pasteurized acid products.

Products containing sugar or fruit derivatives are usually subjected to a pasteurization process that can anyway be ineffective to kill ascospores from heat-resistant molds. Although the most occurring and economically relevant heat-resistant species belong to Byssochlamys, Neosartorya, Talaromyces, and Eupenicillium genera, an increasing number of uncommon heat-resistant isolates have been recently detected as spoiling microorganisms in such products. Since Hamigera spp. and Thermoascus spp. were those more frequently isolated at SSICA, heat resistance of Hamigera avellanea and Thermoascus crustaceus strains from pasteurized acid products was studied in apple juice, in blueberry and grape juice and in a buffered glucose solution. Data obtained from thermal death curves and statistical elaboration of raw data showed that D values of H. avellanea may vary between 11.11 and 66.67 min at 87°C, between 4.67 and 13.51 at 90°C, and between 0.43 and 1.52 min at 95°C. Similarly, D values of T. crustaceus may vary between 18.52 and 90.91 min at 90°C, between 2.79 and 19.23 at 93°C, and between 1.11 and 2.53 min at 95°C. For both strains studied, the z-values calculated from the decimal reduction time curves did not prove to be significantly influenced by the heating medium, that being 4.35°C, 5.39°C or 5.27°C for H. avellanea and 4.42°C, 3.69°C or 3.37°C for T. crustaceus, respectively in apple juice, in blueberry and grape juice or in the buffered glucose solution. Considering the pasteurization treatments industrially applied to fruit-based foods, the variation of thermal parameters does not seem to be a possible way to avoid product spoilage by these two species and only good practices applied to reduce the original load of heat-resistant fungi can help producers to prevent losses in contaminated finished products, as usually happens for other heat resistant molds.

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents.

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.

Immunoaffinity clean-up and direct fluorescence measurement of aflatoxins B1 and M1 in pig liver: comparison with high-performance liquid chromatography determination.

An improved analytical method for aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) determination in pig liver is described, using an aqueous methanol extraction, an immunoaffinity column clean-up step and a direct fluorometric measurement for toxin detection and quantification. A detection limit of 1.0 microg kg-1 was achieved for AFB1 and AFM1. Mean recoveries of 80.7+/-9.0% for AFB1 spiked at 1.0-9.7 microg kg-1 levels and of 76.7+/-6.6% for AFM1 spiked at 1.0-5.5 microg kg-1 levels were obtained. Recovery data for spiked samples were statistically compared with those obtained by the same extract using classical reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection, showing a significant correlation (p