Inactivation of Vegetative and Sporulated Bacteria by Dry Gaseous Ozone

Inactivation by gaseous ozone of different types of microorganisms is successfully achieved provided, as is well known, the gaseous phase is strongly humidified. The inactivation mechanisms and species involved in this process are, however, not yet clearly identified. To gain insight, we considered exposure of bacterial spores to dry rather than humidified ozone, a less complex chemical environment. In contrast to most of the published literature, it is shown that, under strict dry ozone conditions, bacterial spores can be inactivated, but to a degree that is largely dependent on the spore type and substrate material. In this case, the O₃ molecule is determined to be responsible for the inactivation process through its diffusion into and oxidative action within the spore, as no outer erosion of the spore is detected. With humidified ozone, a higher inactivation efficiency is observed that is most probably related, in part, to the swelling of the spore, which facilitates the diffusion of oxidative species within it and up to the core; besides O₃, these oxidative agents stem from the interaction of O₃ with H₂O, which in the end leads to a heavily damaged spore structure, in contrast to dry-ozone exposure where the spore integrity is maintained.     

Microbiological quality of Brazilian UHT milk: Identification and spoilage potential of spore‐forming bacteria

Counts of aerobic mesophilic micro‐organisms and aerobic mesophilic spore‐forming bacteria were determined in 91 ultra‐high‐temperature (UHT) commercial Brazilian samples. Forty‐six spore‐forming bacteria were identified and characterised in terms of their spoilage potential. Among the 20 brands evaluated, 45% had counts of aerobic mesophilic micro‐organisms higher than 1.0 × 102 cfu/mL. The sporulated bacteria were identified as Bacillus subtilis/amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus megaterium, and 31% and 33% showed proteolytic and lipolytic activity, respectively. Our findings indicate that there is a potential risk of UHT milk samples becoming spoiled during their commercial shelf life.     

Evaluation of Process Control Alternatives for the Inactivation of Escherichia coli,MS2 Bacteriophage,and Bacillus subtilis Spores during Wastewater Ozonation

Dissolved ozone concentration integrated over time (CT) is a reliable indicator of disinfection efficacy in drinking water treatment. However, ozone CT may not be measurable in some wastewater ozone applications. In this study, alternative process control parameters, specifically ozone to total organic carbon (O₃:TOC) ratio, differential UV₂₅₄ absorbance (ΔUV₂₅₄), and differential total fluorescence (ΔTF), were correlated with the inactivation of Escherichia coli, the bacteriophage MS2, and Bacillus subtilis spores in five secondary wastewater effluents. CT values greater than 9 mg-min/L were generally required for measurable inactivation of B. subtilis spores, and CT values of 1 and 2 mg-min/L consistently achieved greater than 6- and 5-log inactivation of MS2 and E. coli, respectively. The O₃:TOC, ΔUV_254, and ΔTF correlations for MS2 and B. subtilis were useful for predicting inactivation, while those of E. coli were characterized by greater variability. [Supplementary materials are available for this article. Go to the publisher's online edition of Ozone: Science & Engineering for the following free supplemental resources: additional figures and data tables.]     

Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage

Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n = 467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100 °C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125 °C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.     

Isolation and species diversity of arbuscular mycorrhizal fungi in the rhizosphere of <i>Puccinellia tenuiflora</i> of Songnen saline-alkaline grassland,China

Salinization has led to the deterioration of the ecological environment, affected the growth of plants, and hindered the development of agriculture and forestry. Arbuscular mycorrhizal (AM) fungi, as important soil microorganisms, play significant physiological and ecological roles in promoting plant nutrient absorption and improving soil structure. Puccinellia tenuiflora (Turcz.) Scribn. et Merr. in Songnen saline-alkaline grassland was selected as the research object to observe AM fungal colonization of the roots and explore the species and diversity of AM fungi in symbiotic association with P. tenuiflora. This study showed that AM fungi colonized in P. tenuiflora roots and formed a typical Arum–type mycorrhizal structure. A significant correlation was observed between vesicular abundance and the colonization intensity of mycorrhiza. Isolation and identification revealed 40 species of AM fungi in the rhizosphere of P. tenuiflora, belonging to 14 genera, of which two species could not be identified. The richness of the genus Glomus was the highest, accounting for 30% of the total species. Funneliformis mosseae and Rhizophagus intraradices were isolated from all the samples and were the species with the widest distribution in the rhizosphere of P. tenuiflora. Correlation analysis showed that pH only had a significant impact on the distribution of a few species, such as Glomus pustulatum, Diversispora spurca, Glomus aggregatum, Rhizophagus clarum, and Acaulospora foveata. The present study provides a theoretical basis for further exploring the resources of AM fungi in saline-alkaline soil.     

Shift of pH-Value During Thermal Treatments in Buffer Solutions and Selected Foods

The pH value is one of the most important process parameters during thermal treatments, regardless if the medium is a simple buffer solution or a complex food matrix. When the temperature increases after an initial measurement of the pH at ambient temperature (25°C), a significant pH shift could occur, which could produce incomparable results in different buffer solutions or lead to side reactions during food preservation. Consequently, a measurement cell was constructed to record online the pH-value and temperature up to 130°C. By applying the Nernst equation, it was possible to exclude the temperature-dependent influence of the pH glass electrode on the total pH value. The pH shift was measured over a wide temperature range (ΔT 20–130°C) in the most commonly used buffer solutions and some selected food matrices. The ΔpH of certain buffer solutions, namely TRIS and ACES, showed a significant pH decrease of??2.01 ± 0.08 (ΔT 20–130°C) and??1.27 ± 0.1 (ΔT 20–130°C), respectively, whereas the pH of PBS buffer solution was nearly independent of temperature. The ΔpH decrease recorded in milk (?0.89 ± 0.6, ΔT 20–130°C) as well as commercial and self-made baby food (?0.56 ± 0.05, ΔT 20–130°C) is of special interest for the food industry to get a deeper insight in occurring reactions during thermal preservation processes.     

The survivability of Bacillus anthracis (Sterne strain) in processed liquid eggs

In this study, we investigated the survival and inactivation kinetics of a surrogate strain of Bacillus anthracis (Sterne strain) in whole egg (WE), egg white (EW), sugared egg yolk (YSU), and salted egg yolk (YSA) at low (−20, 0, and 5 °C), moderate (15, 20, 25, 30, 35, and 40 °C), and high storage temperatures (45, 50, 55, and 60 °C). Outgrowth of the spores was measured as lag phase duration (LPD). Replication of vegetative cells was measured in terms of growth rate (GR) and maximum population density (MPD). Spore inactivation was recorded as inactivation rate and percent reduction in viable count. In general, spore viability decreased at low and high temperatures and increased at moderate temperatures. At 0 and 5 °C, a 60–100% reduction in spore viability was seen within 2–3 weeks in WE and YSU, 0–30% in YSA, and 50–100% in EW. At −20 °C, however, no drop in spore titer was observed in YSU and EW but a 20% drop in titer was seen in YSA and 50% in WE within 2–3 weeks. At high temperatures, WE, EW, and YSA produced a 20–50% drop in the spore titer within 1–4 h whereas YSU showed 100% inactivation within 0.75 h. At moderate storage temperatures, as the temperature increased from 15 to 40 °C, LPD decreased from 13.5 to 0.75 h and MPD reached 0.27–2.2 × 10⁹ CFU/ml in YSU and WE, respectively. Markedly lower growth was observed in YSA (LPD = 24–270 h, MPD = 9 × 10⁵ CFU/ml) and spores were inactivated completely within 1–6 h in EW. The survivability and inactivation data of B. anthracis in liquid egg products reported in this investigation will be helpful in developing risk assessment models on food biosecurity.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

Flow cytometric assessment of Bacillus spore response to high pressure and heat

The physiological response of Bacillus licheniformis spores to high pressure and thermal inactivation in sodium citrate buffer and nutrient broth was investigated using multiparameter flow cytometry. Spores were treated by heat-only at 121 °C, by high pressure at 150 MPa (37 °C), or by a combined high pressure and heat treatment at 600 MPa and 77 °C, and then dual stained with the fluorescent dyes SYTO 16 and propidium iodide (PI). For pressure treated spores, but not heat-only treated spores, four distinct sub-populations were detected by flow cytometry, and for these we suggest a three step model of inactivation involving a germination step following hydrolysis of the spore cortex, an unknown step, and finally an inactivation step with physical compromise of the spore's inner membrane.

Industrial relevance

This preliminary study offers a simple and fast flow cytometric method for the rapid assessment of the physiological state of bacterial spores following high pressure and thermal processing. An improved understanding of the mechanisms of spore inactivation will aid in the food safety assessment of pressure assisted thermal sterilisation in particular, and also assist in the commercialisation of these processes facilitating adoption by industry.     

Origin of bacterial spores contaminating foods

Bacterial spores (= endospores) are common contaminants in foods. Sources of contamination in the food chain may include soil, faeces, animal feeds and food ingredients and processing chain themselves. Sporulation may occur in very diverse environments. The environment of sporulation has a strong influence on spore properties relevant for food quality and safety.