Inactivation Of Ascospores Research Articles

Modeling the Thermal Inactivation of Ascospores from Heat-Resistant Molds in Pineapple Juice and Evaluating Disinfection Efficiency of Sodium Hypochlorite and Chlorine Dioxide

The contamination and spoilage of heat-treated fruit juices by heat-resistant mold ascospores present significant challenges to the food industry. Understanding effective strategies to mitigate this contamination is vital for ensuring the shelf-life and microbial safety of heat-treated fruit juices. This study investigated the thermal resistance of ascospores from different heat-resistant mold species, including Aspergillus laciniosus, A. chevalieri, A. denticulatus, A. siamensis, Hamigera pallida, and Talaromyces macrosporus, isolated from pineapple and sugarcane field soils. Ascospores inactivation kinetics in pineapple juice under heat treatment (75–97 °C) were analyzed using log-linear and Weibull models. Among these species, A. laciniosus displayed the highest heat resistance (δ-value: 104.59 min at 85 °C), while A. siamensis exhibited the lowest (δ-value: 3.39 min at 80 °C). Furthermore, A. laciniosus, the most heat-resistant species, showed notable tolerance to sanitizers. The most effective inactivation was achieved using 1.0% (w/v) sodium hypochlorite for 15 min. Chlorine dioxide, however, was generally ineffective and even activated dormant ascospores in some cases. The combination of hot water (65 °C for 5 min) with sanitizer increased ascospore reduction in most species but did not achieve the 3-log reduction required by the European Standard N13697. This study revealed a correlation between ascospore resistance to heat and chlorine dioxide, offering significant findings for practical inactivation strategies.

Development Control and Inactivation of Byssochlamys nivea Ascospores by Hyperbaric Storage at Room Temperature.

This study tested hyperbaric storage (25-150 MPa, for 30 days) at room-temperature (HS/RT, 18-23 °C) in order to control the development of Byssochlamys nivea ascospores in apple juice. In order to mimic commercially pasteurized juice contaminated with ascospores, thermal pasteurization (70 and 80 °C for 30 s) and nonthermal high pressure pasteurization (600 MPa for 3 min at 17 °C, HPP) took place, and the juice was afterwards placed under HS/RT conditions. Control samples were also placed in atmospheric pressure (AP) conditions at RT and were refrigerated (4 °C). The results showed that HS/RT, in samples without a pasteurization step and those pasteurized at 70 °C/30 s, was able to inhibit ascospore development, contrarily to samples at AP/RT and refrigeration. HS/RT for samples pasteurized at 80 °C/30 s evidenced ascospore inactivation, especially at 150 MPa, wherein an overall reduction of at least 4.73 log units of ascospores was observed to below detection limits (1.00 Log CFU/mL); meanwhile, for HPP samples, especially at 75 and 150 MPa, an overall reduction of 3 log units (to below quantification limits, 2.00 Log CFU/mL) was observed. Phase-contrast microscopy revealed that the ascospores do not complete the germination process under HS/RT, hence avoiding hyphae formation, which is important for food safety since mycotoxin development occurs only after hyphae formation. These findings suggest that HS/RT is a safe food preservation methodology, as it prevents ascospore development and inactivates them following commercial-like thermal or nonthermal HPP pasteurization, preventing mycotoxin production and enhancing ascospore inactivation.

Quantitative microbial spoilage risk assessment (QMSRA) of pasteurized strawberry purees by Aspergillus fischeri (teleomorph Neosartorya fischeri)

Aspergillus fischeri ascospores are known as potential spoilage microorganisms of pasteurized fruit products due to their high incidence in fruits, the ability to survive pasteurization and to grow in acidic conditions. This study aimed to develop a quantitative microbial spoilage risk assessment (QMSRA) model approach to estimate the spoilage risk of packaged strawberry purees due to A. fischeri under various scenarios regarding product formulation, processing and storage conditions. The development of the risk assessment comprised three steps: (1) initial contamination level of raw material by ascospores (N0), (2) inactivation of ascospores during thermal processing (Np) and (3) determination of the number of ascospores which are able to survive thermal processing and develop visible mycelia (D = 2 mm) during storage (Nf). Data of visible growth (tv, days) comprised distributions previously obtained as function of water activity (aw) (0.860–0.985), oxygen (0–21%), temperature (8–30 °C) and pasteurization (95–105 °C/15 s). The simulations were performed in triplicate with 100,000 iterations using the software R. The outcome “spoilage risk” was defined as the probability of having at least one ascospore (Nf) capable of forming visible colonies in 100 g-pack strawberry puree within the typical use-by dates. Overall, high probabilities of spoilage were estimated for purees pasteurized at milder treatments at 85 °C/15–60 s (67%) and 90 °C/15–60 s (≥40%) stored at ambient temperature (22 °C). The spoilage risk was only effectively reduced (0.02%) by increasing pasteurization conditions to 95 °C for at least 45 s. Moreover, the microbial stability of such purees, i.e., spoilage risk <0.001% (=less than 1 spoilage pack out of 105 produced units) was predicted to occur for purees treated at 100 °C/15 s or stored at chilled conditions (≤8 °C) or at strict anaerobic conditions or produced as concentrates (aw ≤ 0.860). Based on the outcomes obtained, a set of specifications for Heat-Resistant Moulds (HRMs) in raw material and pasteurized purees aimed to be used as an ingredient was suggested. Furthermore, the results can be used to support risk management decisions in identifying and quantifying the impact of possible interventions during formulation, processing and storage conditions of fruit purees to effectively reduce this risk.

Study of thermal stability of ascospores Aspergillus (Neosartorya) fischeri depending on the concentration of soluble solids in apple juice

Kinetic studies of the thermal inactivation of test crop spores are necessary to develop optimal heat treatment regimes for fruit juices. The purpose of the work is to study the dynamics of changes in the thermal stability parameters DT and z depending on changes in the soluble solids content in canned fruit products using the example of certain types of apple juice products with a pH of 3.80. The regularity of thermal inactivation of ascospores of the mesophilic mold Aspergillus fischeri in concentrated apple juice (JAC) with a soluble dry matter (RSV) content of 70%, in restored apple juice with RSV – 11.2%, and in restored apple juice with pulp with RSV – 16% was studied. The parameters of thermal stability were determined by the capillary method at temperatures of 80, 85, 90, and 95 °C. It was experimentally established that the heat resistance of A. fischeri spores in clarified apple juice was DT 95 °С = 0.16 min, and the parameter value z = 6.76 °C, in apple juice with pulp parameters: DT 95 °C = 0.24 min, z – 7.12 °C, in YaKS – DT 95 °C = 0.39 min, and z – 7.8 °C. The dynamics of thermal stability parameters D and z of A. fischeri mold fungus spores (test cultures) versus RSV concentration of juice products was established. The research results showed that with an increase in the concentration of RSV, the thermal stability of spores increases exponentially. The rate of increase in thermal stability decreases with increasing concentration of RSV. Since the concentration of RSV affects the rheological properties of the product (viscosity), this leads to a change in the kinetics of heating in products with convection heat transfer. Therefore, an increase in the concentration of RSV should inevitably lead not only to an increase in the thermal stability of spores of microorganisms, but also to a shift in the region of optimal modes of heat treatment of products toward an increase in the thermal load to ensure regulatory requirements for microbiological safety.

Inactivation of ascospores of Talaromyces macrosporus and Neosartorya spinosa by UV-C, UHPH and their combination in clarified apple juice

This work addresses the use of a thin-film short wave ultraviolet radiation (UVC) reactor and its combination with ultra-high pressure homogenization (UHPH) for the inactivation of Talaromyces macrosporus (CBS 130.89) and Neosartorya spinosa (CBS 586.90), fungal species that can cause food spoilage in products made of fruits. Ascospores of these microorganisms were inoculated in phosphate buffer solution (PBS) and clarified apple juice at a concentration between 5 and 6 Log10 spores/mL before treatments. Ascospores of both microorganisms were not detected in PBS after UV-C treatments even at the lowest assayed dose (1.8 J/mL), but in apple juice T. macrosporus showed to be significantly more UV-C resistant than N. spinosa. A UV-C dose of 21.5 J/mL in a single pass achieved decimal reductions of 2.15 Log10 and 5.4 Log10 respectively. Decimal reductions of both strains significantly increased in many cases when the same UV-C dose was applied in 2 or 3 passes throughout the UV-C reactor achieving T. macrosporus a maximum reduction of 3.88 Log10 after a UV-C dose of 21.5 J/mL applied in 3 passes. UHPH treatments at 100 and 200 MPa were ineffective in inactivating ascospores of both microorganisms, However, UHPH left ascospores more vulnerable to UV-C resulting in a synergistic effect achieving a maximum reduction of 3.6 Log10 on T. macrosporus after a combined treatment of UHPH at 200 MPa and a single pass of UV-C at 21.5 J/mL. The use of scanning electron microscopy revealed that only UHPH caused small changes in the cell wall structure of ascospores, but that might have left cells better exposed to UV-C.

Resistance of Byssochlamys nivea and Neosartorya fischeri mould spores of different age to high pressure thermal processing and thermosonication

Byssochlamys nivea and Neosartorya fischeri are heat resistant moulds that spoil acid foods and can produce mycotoxins. This study investigated the efficacy of 600 MPa high pressure thermal processing (HPTP) and thermosonication (TS, 24 kHz, 0.33 W/mL) at 75 °C for the inactivation of 4–12 week old ascospores of B. nivea in strawberry puree and N. fischeri in apple juice. Spore resistance increased with increasing age for both species, with differences in the extent of inactivation of up to 2 log between spore age groups. HPTP was more effective than TS for spore inactivation. The 12 week B. nivea and N. fischeri spores had similar resistance to HPTP. TS caused increase in spore numbers, which was more pronounced for N. fischeri. The kinetics of mould ascospore inactivation by HPTP and TS were well described by Weibull and Lorentzian models, respectively. The Weibull's scale factor b decreased with spore age for N. fischeri, and the Lorentzian parameter b also decreased with spore age for both moulds, indicating more resistance. The results demonstrated that mould spore age is an issue for fruit product manufacturers, since spores contaminating fruits can be very old, exhibiting higher resistance.

Ultrasound assisted thermal pasteurization of beers with different alcohol levels: Inactivation of Saccharomyces cerevisiae ascospores

The industrial production of beer ends with a process of pasteurization. This study investigated the ultrasound assisted thermal pasteurization of beer or thermosonication (TS), aiming the inactivation of Saccharomyces cerevisiae ascospores, the most resistant form of the yeast. The efficacy of 30 s TS in batch and continuous operation mode at 60, 65, and 70 °C was studied. After that the ascospore inactivation in beers was modelled and TS pasteurization conditions recommended. Lastly, the inactivation of S. cerevisiae ascospores in beer by TS vs. thermal processing at 55 °C was compared. Ultrasound alone and continuous TS operation were not enough to achieve the minimum beer pasteurization requirement aiming at S. cerevisiae spore inactivation. The TS survival curves were fitted with a Weibull model. TS at 50 °C-1.9 min and TS at 55 °C-26 s were enough for pasteurization, as opposed to 55°C-38 min thermal process. The results are helpful for designing appropriate thermosonication conditions to pasteurize beer with different alcohol contents and other beverages.

Nonthermal pasteurization of beer by high pressure processing: modelling the inactivation of saccharomyces cerevisiae ascospores in different alcohol beers

ABSTRACTThe industrial production of beer ends with a process of thermal pasteurization. In this research, the nonthermal pasteurization of beer by high pressure processing (HPP) was carried out. First, the effect of alcohol content on Saccharomyces cerevisiae ascospore inactivation at 400 MPa was studied. The number of ascospores in 0.0%, 4.8%, and 7.0% alc/vol beers for 10 min processing time decreased by 3.1, 4.9, and ≥ 6.0 log, respectively. The Weibull model fitted the ascospore inactivation by HPP in 0.0%, 4.8%, and 7.0% alc/vol beers. At 400 MPa, 7.2 s could ensure the minimum pasteurization of beers and for 600 MPa 5 s were enough for ≥ 7 log reductions. The overall flavour of HPP vs. untreated beers was evaluated for a lager and an ale, with no significant differences between the untreated and HPP beers. Thus, nonthermal HPP is a feasible technology to pasteurize beer with different alcohol contents without heat.

High pressure processing and thermosonication of beer: Comparing the energy requirements and Saccharomyces cerevisiae ascospores inactivation with thermal processing and modeling

In this research, pasteurization of beer by nonthermal high pressure processing (HPP) and thermosonication (TS) were compared with thermal pasteurization. The inactivation of Saccharomyces cerevisiae ascospores in beer was studied and modelled for HPP at 200, 300 and 400 MPa, and for TS at 50, 55 and 60 °C with an acoustic energy density of 16.15 W/mL. The energy requirements for equivalent ascospore inactivation by HPP, TS, and thermal processes were compared. For the same processing time, ascospore inactivation was greatest with nonthermal HPP, followed by 60 °C TS, then 60 °C thermal processing. Nonlinear survival curves which could be described by the Weibull model were observed for both HPP and TS. To achieve a 2.5 log reduction in ascospores, HPP required 77 kJ/L compared with 189 kJ/L for thermal processing and 2612 kJ/L for TS. HPP and thermosonication may be alternatives to thermal beer pasteurization, achieving greater log reductions in S. cerevisiae ascospores with shorter processing times (TS and HPP) or less energy (HPP).

Inactivation of Byssochlamys nivea ascospores in strawberry puree by high pressure, power ultrasound and thermal processing

Byssochlamys nivea is a mold that can spoil processed fruit products and produce mycotoxins. In this work, high pressure processing (HPP, 600MPa) and power ultrasound (24kHz, 0.33W/mL; TS) in combination with 75°C for the inactivation of four week old B. nivea ascospores in strawberry puree for up to 30min was investigated and compared with 75°C thermal processing alone. TS and thermal processing can activate the mold ascospores, but HPP–75°C resulted in 2.0 log reductions after a 20min process. For a 10min process, HPP–75°C was better than 85°C alone in reducing B. nivea spores (1.4 vs. 0.2 log reduction), demonstrating that a lower temperature in combination with HPP is more effective for spore inactivation than heat alone at a higher temperature. The ascospore inactivation by HPP–thermal, TS and thermal processing was studied at different temperatures and modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature in spore inactivation, alone or combined with other physical processes. The Weibull model described the spore inactivation by 600MPa HPP–thermal (38, 50, 60, 75°C) and thermal (85, 90°C) processing, whereas the Lorentzian model was more appropriate for TS treatment (65, 70, 75°C). The models obtained provide a useful tool to design and predict pasteurization processes targeting B. nivea ascospores.

Modeling the inactivation of Neosartorya fischeri ascospores in apple juice by high pressure, power ultrasound and thermal processing

Neosartorya fischeri is a mould that spoils acid foods and can produce mycotoxins. In this work, the efficacy of high pressure processing (HPP, 600 MPa) and power ultrasound (24 kHz, 0.33 W/mL) in combination with 75 °C for the inactivation of four week old N. fischeri ascospores in apple juice was investigated and compared with 75 °C thermal processing alone. The HPP-75 °C process was the most effective technique for inactivating N. fischeri spores, resulting in 3.3 log reductions after 10 min vs. no inactivation for thermosonication (TS) and thermal processing. Unexpectedly, activation shoulders were observed during the TS process. Then, the effect of different temperatures on the ascospore inactivation in apple juice by HPP-thermal, TS and thermal processing was investigated, and the log survivors vs. time were modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature for the spore inactivation, alone or combined with other processes. The Weibull model described the spore inactivation better by 600 MPa HPP-thermal (50, 60, 75 °C) and thermal (85, 90 °C), whereas Lorentzian was more appropriate for the TS treatment (65, 70, 75 °C). In conclusion, HPP is the best food preservation technology due to higher spore inactivation in apple juice at the same temperature.

Ascospore inactivation and germination by high pressure processing is affected by ascospore age

Abstract The effects of age on high pressure resistance of the ascospores of heat resistant moulds Byssochlamys fulva, B. nivea, Neosartorya fischeri and N. spinosa were determined. Ascospores were harvested from cultures grown for 3–15 weeks at 30 °C on malt extract agar. Following filtration and determination of concentration, the ascospores were subjected to high pressure processing (HPP) at 600 MPa for 10 min in 0.1 M citrate phosphate buffer (pH 4 and 6) and mango puree (pH 5). The results supported our hypothesis that age (maturity) affects high pressure resistance of ascospores of heat resistant moulds. A reduction of log10 2.5 cfu mL− 1 was achieved for three week old ascospores of B. nivea whereas for nine week old ascospores only a half log reduction was achieved. Similar results were observed for B. nivea and N. fischeri. The HPP treatment caused activation of ascospores of N. spinosa, with older ascospores showing increased activation. Industrial relevance The observation of activation of some ascospores by HPP, indicates that HPP alone is insufficient for elimination of these problematic spoilage microorganisms. HPP would need to be combined with other hurdles in order to produce high quality pressure-treated shelf-stable fruit products.

Influence of activation and germination on high pressure inactivation of ascospores of the mould Eurotiumrepens

We investigated heat activation and germination of Eurotium repens ascospores to follow high pressure inactivation. Activation energy and entropy values strengthen the idea of protein denaturation as the underlying mechanism of heat activation. Preceding activation, germination or a combination of both affected high pressure inactivation in different ways. Activation followed immediately by high pressure treatment led to the most efficient improvement in inactivation. However, a pause after activation caused a partial re-establishment of the spores’ stability and less efficient high pressure inactivation. Germination stabilized the spores against high pressure. A combined treatment of activation and germination led to an initially fast inactivation, but compared to high pressure treatment of only activated spores the time course of inactivation was slowed down.

Heat resistance of Monascus ruber ascospores isolated from thermally processed green olives of the Conservolea variety

A heat-resistant fungus identified as Monascus ruber was isolated from thermally processed green olives of the Conservolea variety. Thermal death parameters of the ascospores were determined using Baranyi's primary predictive model in phosphate buffer (pH 7.0), citrate buffer (pH 4.0) and brine (pH 3.8) at two different NaCl levels (5.6% and 10.5%). The decimal reduction times ( D values) at 70, 75 and 80 °C in phosphate buffer (pH 7.0) were 37.31, 7.14 and 1.66 min, respectively, and the calculated z value was 7.4 °C. In citrate buffer (pH 4.0), the relative D values were 38.61, 7.83 and 2.10 min and the z value was 7.9 °C. In brine, the D 70 °C, D 75 °C and D 80 °C values increased from 46.08, 4.91 and 0.88 min to 72.99, 7.98 and 1.04 min, respectively, as the level of NaCl in brine increased from 5.6% to 10.5%, indicating a protective effect in ascospore inactivation. This effect was more pronounced at lower heating temperatures. It was concluded that a thermal process of F 80 °C=5 min could be considered as an effective thermal processing treatment. Results from this research could assist the table olive industry in designing an effective pasteurization plan to eliminate the fungus in thermally processed table olives.

Modulation of thermal resistance of ascospores of Neosartorya fischeri by acidulants and preservatives in mango and grape juice

Minimal thermal processing is desirable for near natural organoleptic and nutritional qualities of fruit based products. In the present investigation, the effect of heat (85°C) in combination with acidulants or common preservatives on inactivation of ascospores of Neosartorya fischeri, a heat resistant mould isolated from grapes, has been studied in mango and grape juice. The ascospores were found to survive for >300 min of heating at 70, 75 and 80°C in these fruit juices and complete inactivation required 120 min of heating at 85°C. The synergistic effect of heat and organic acids or preservatives in fruit juices was noticed. The thermal death rate (1/k85°C) values did not vary much in the presence of lactic (20), malic (20) and citric (19) acids, but tartaric acid showed least inactivation effect (1/k85°C=54 min) in mango juice. The 1/k85°Cvalues for ascospores of N. fischeri in mango juice containing 0·1% of potassium sorbate or sodium benzoate or combination of both at 0·05% were found to be 44, 35 and 29 min respectively. These values were respectively, 32, 13 and 14 min in grape juice. Nearly 50 and 67% of the heating time was reduced by the use of potassium sorbate and sodium benzoate (0·05% each) in mango and grape juice to inactivate 3 log number of ascospores of N. fischeri. These results may be useful in thermal processing of fruit juices.

Thermal Death Rate of Ascospores of Neosartorya fischeri ATCC 200957 in the Presence of Organic Acids and Preservatives in Fruit Juices

Heat-resistant molds, including Neosartorya fischeri, are known to spoil thermally processed fruit products. The control measures required for such problems must not cause an appreciable loss of the organoleptic qualities of the final products. In the present study we determined the thermal death rates of ascospores of N. fischeri ATCC 200957 in fruit juices containing organic acids and preservatives. The ascospores were able to survive for more than 6 h of heating at 75°C, 5 h at 80°C, and 3 to 4 h at 85°C in mango or grape juice. Of the four organic acids tested, citric acid exhibited the maximal destruction of ascospores in mango juice at 85°C (l/k = 27.22 min), and tartaric acid the least (1/k = 61.73 min). The effect of common preservatives on the thermal death rates of ascospores at 85°C in mango and grape juices was studied. Almost similar effects on thermal inactivation of ascospores were noted when potassium sorbate (l/k = 29.38 min) or sodium benzoate (l/k = 27.64 min) or the combination of both (l/k = 27.53 min) was used in mango juice. In grape juice, potassium sorbate (l/k = 25.07 min) was more effective than sodium benzoate (l/k = 50.08 min) or the combination of both (l/k = 40.79 min) in inactivation of ascospores of the mold. The thermal death rate (l/k) values in mango and grape juices in the absence of any preservative were 63.51 and 69.27 min respectively.

Heat resistance of ascospores of Byssochlamys nivea in milk and cream

Byssochlamys nivea strains of rather varying origin used for heat inactivation experiments were cultured for 28 days at 30°C on malt extract agar, since under these conditions the highest degree of heat resistance of the ascospores was observed. Inactivation was performed in steel capillary tubes to obtain reproducible results under experimental conditions comparable to those prevailing in practice. An inactivation temperature of 92°C proved to be most practical. Decimal reduction times for the individual strains at this temperature in Ringer's solution varied between 1.3 and 2.4 s. In the temperature range studied, inactivation of ascospores in UHT milk (1.5% w/w fat content) and cream (10% w/w fat content) has not been found to differ significantly from that in Ringer's solution. Homogenization of milk as applied in practice did not affect heat inactivation of ascospores. Assuming the most unfavorable conditions (50 ascospores/l and using the most heat-resistant strain) the following relations between the level of infected 500 g packages were calculated: 1 of 10 6 packs infected; 24 s at 92°C; 1 of 10 3 packs infected; 16.5 s at 92°C; 1 of 10 2 packs infected, 14 s at 92°C.

Heat resistance of ascospores of Neosartorya fischeri as affected by sporulation and heating medium

Ascospores of three strains of Neosartorya fischeri grown on three different sporulation media were evaluated for heat resistance in apple juice, grape juice and 0.1 M potassium phosphate buffer (pH 7.0). The type of sporulation medium did not affect heat resistance of ascospores. Ascospores (approx. 10 6/ml, initial viable population) of all three strains survived heat treatment at 84° C for 120 min. The rate of thermal inactivation of ascospores was lower in apple juice than in grape juice and phosphate buffer. Fumaric acid (1 and 2%) at pH 2.5, 3.0 and 3.5 greatly increased the rate of heat inactivation of ascospores. At similar concentrations and pH values, acetic, citric and tartaric acids resulted in moderate increases in inactivation rates. Malic acid had little influence on rates of inactivation. Rates of inactivation increased as the pH of heating media containing fumaric, citric, tartaric and acidic acids decreased. Thus, the pH and the type, percentage and molarity of organic acid in the heating medium act synergistically with heat to inactivate ascospores of N. fischeri.