Article

Effect of sugar concentration (°Brix) and storage temperature on the time to visible growth of individual ascospores of six heat-resistant moulds isolated from fruit products

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Abstract

Heat-resistant moulds (HRMs) pose great challenges to processors of fruit-based products due to their thermal resistance and ability to grow across a broad range of conditions. Therefore, the quantification of the effect of inhibitory factors (conditions) on the growth of HRMs is very important to be used to prevent spoilage during shelf-life. This study assessed the minimum temperature and maximum sugar content (degrees Brix) for the growth of six HRMs (Byssochlamys spp. and Neosartorya spp.) previously isolated from fruit products. In addition, the time to form a visible colony (t(v), days) was determined to assess biological variability of individual ascospores within same population. Heat activated ascospores (10 min at 80 degrees C) were spread plated (+/- 100 spores) on acidified Potato Dextrose Agar (aPDA, pH 3.5) plates from which the degrees Brix was adjusted with fructose-glucose (1:1) to levels between 44 and 59 degrees Brix followed by incubation at 30 degrees C. To assess the effect of temperature, inoculated plates of aPDA were incubated at 4, 7, 8, 10, 12 and 14 degrees C. Three replicates (= 3 aPDA plates) were prepared per condition evaluated. The number of visible colonies were counted daily for up to two months. Probability distribution functions were then fitted in @Risk to the cumulative t(v)'s. With regards to cold tolerance, B. nivea was the most cold sensitive as it had the least ability to germinate and form visible colonies at low temperatures (no growth when T <= 10 degrees C). On the other hand, N. hiratsukae was the most cold tolerant, being able to form visible colonies at temperatures >= 7 degrees C. Likewise, B. nivea was the most sensitive to increased degrees Brix values, whilst N. udagawae was able to grow out at the highest degrees Brix evaluated (59 degrees/a(w) = 0.86). The tolerance of potential spoilage HRMs to high sugar levels and their ability to grow under chilled conditions represent a challenge for the microbial stability of high-sugar fruit products. Differences in individual t(v)'s were mostly observed under conditions at the growth/no growth regions. For instance, individual t(v)'s of B. nivea ascospores ranged from 24 to 46 days at 12 degrees C and those of N. udagawae ranged from 20 to 45 days at 59 degrees Brix. The t(v)'s data from each HRM and condition evaluated were then fitted to different statistical distributions (Exponential, Normal, Lognormal, Weibull, Logistic or Pareto) to allow the use of the obtained data in further Quantitative Microbial Spoilage Risk Assessment work for pasteurized fruit products.

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... Also, in QMRA, the risk is characterized by adverse effects on human health (e.g. Daelman et al., 2013), while in the QMSRA, the risk is characterized by spoilage defects such as microbial surface growth, off-flavors and off-odors due to lipolysis, sugar fermentation, and protein hydrolysis as a result of microbial growth (Membré and Dagnas, 2016;Santos et al., 2018a;Santos et al., 2019;Santos et al., 2020). ...
... The model inputs comprise original data collected in fruit puree processing industries and reported in previous studies (i.e. Santos et al., 2018a;Santos et al., 2019;Santos et al., 2020). Additionally, the following two assumptions were considered: (i) there is no post contamination after pasteurization so all ascospores were subjected to a thermal heat treatment, (ii) the raw material (strawberries) was not treated with sanitizers (e.g. ...
... (6)) having as parameter the probability of one ascospore to germinate and present visible growth during storage time. The input time visible growth came from data previously obtained (Santos et al., 2019;Santos et al., 2020). This variable was described as lognormal and normal distributions (Santos et al., 2020) or empirical distributions (discrete when more than two values, uniform when only two values) according to the sample size and study performed. ...
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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.
... B. nivea ascospores are 4-5.5 � 3-4 μm presenting a Samson, R.A., Houbraken, J., Varga, J., Frisvad, J. C., 2009 smooth ornamentation (Fig. 3, photo f) (Samson et al., 2009), while N. fischeri ascospores are longer with 7-8 � 3-4 μm dimensions and presenting convex surfaces bearing anastomosing ridges (reticulate) (Fig. 4, photos G-I) (Samson et al., 2007). These moulds are able to grow as low as 10-12 � C for B. nivea (Panagou et al., 2010;Kotzekidou, 2014;Lane Paixão dos Santos et al., 2020) and 10 � C for N. fischeri (Nielsen et al., 1989;Lane Paixão dos Santos et al., 2020), lower temperatures than bacteria. They are both able to grow under lower water activity (as low as 0.89) than bacteria (Zimmermann et al., 2011), and under reduced oxygen inside fruit packs and carbonated beverages (Sant'Ana et al., 2010). ...
... B. nivea ascospores are 4-5.5 � 3-4 μm presenting a Samson, R.A., Houbraken, J., Varga, J., Frisvad, J. C., 2009 smooth ornamentation (Fig. 3, photo f) (Samson et al., 2009), while N. fischeri ascospores are longer with 7-8 � 3-4 μm dimensions and presenting convex surfaces bearing anastomosing ridges (reticulate) (Fig. 4, photos G-I) (Samson et al., 2007). These moulds are able to grow as low as 10-12 � C for B. nivea (Panagou et al., 2010;Kotzekidou, 2014;Lane Paixão dos Santos et al., 2020) and 10 � C for N. fischeri (Nielsen et al., 1989;Lane Paixão dos Santos et al., 2020), lower temperatures than bacteria. They are both able to grow under lower water activity (as low as 0.89) than bacteria (Zimmermann et al., 2011), and under reduced oxygen inside fruit packs and carbonated beverages (Sant'Ana et al., 2010). ...
... Tremarin et al. (2017) have The Journal of Engineering and Exact Sciences -jCEC 4 studied the influence of soluble solid content and storage temperature on the growth of B. fulva in agar-added apple juices, developing a model to predict the growth of this microorganism in apple juice as a function of these factors. Santos et al. (2020) have investigated the effect of sugar concentration and storage temperature on the time for visible growth (tv) of ascospores of heatresistant moulds, including Byssochlamys spp. The authors have observed that reduction in temperature (14ºC to 7ºC) have a more pronounced effect on the distribution of tv than the effect of sugar content (44 to 59ºBrix). ...
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A wide variety of microorganisms can contaminate food products. Due to low pH, fruits are mostly spoiled by fungi. The growth of fungi on fruits depends on the characteristics of the food and on environmental factors. In this context, the predictive microbiology is one option for studying and designing strategies to control microbial growth on foods. Thus, the aim of this work was to develop a model to predict the growth and the spoilage of low acid fruits contaminated by Byssochlamys fulva spores based on the storage temperature, pH and sugar content of the fruit. The growth behavior of this microorganism in modified potato-dextrose culture medium was studied as a function of temperature (20, 28, 36oC), pH (4, 5, 6) and sugar content (0, 5, 10, 15% m/v). A mathematical model was constructed to quantify the effect of these factors on the growth kinetics of this fungus and on the time to spoil the product, using linear model and Gibson-type model. The growth curves of B. fulva in pulps of mango, melon, papaya and tomato were obtained experimentally from which the time to spoil was determined and compared with the results predict by the mathematical model in the range of 20ºC to 36ºC. The developed model described satisfactorily the time to spoil, showing better results for melon pulp, for which the accuracy of the model was greater than 90%. The proposed model has a great potential to predict the shelf life of those foods contaminated by this fungus, mainly under lower storage temperature conditions.
... Naturally, freshly squeezed vegetable juice has a lower Brix value, between 5 to 12. Concentrates, caused by the thermal evaporation of water, have higher Brix values, between 25 and 60 [63]. Results obtained by Lane Paixão dos Santos et al. [64] confirmed that increasing • Brix lowers the livelihood chances of Neosartorya. Within tested species, N. udagawae was the most resilient and possessed the ability to grow at the highest evaluated • Brix (59 • /aw = 0.86) [65]. ...
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Soil-borne Neosartorya spp. are the highly resilient sexual reproductive stage (teleomorph) of Aspergillus spp. Fungi of this genus are relevant components of root-associated microbial community, but they can also excrete mycotoxins and exhibit great resistance to high temperatures. Their ascospores easily transfer between soil and crops; thus, Neosartorya poses a danger to horticulture and food production, especially to the postharvest quality of fruits and vegetables. The spores are known to cause spoilage, mainly in raw fruit produce, juices, and pulps, despite undergoing pasteurization. However, these fungi can also participate in carbon transformation and sequestration, as well as plant protection in drought conditions. Many species have been identified and included in the genus, and yet some of them create taxonomical controversy due to their high similarity. This also contributes to Neosartorya spp. being easily mistaken for its anamorph, resulting in uncertain data within many studies. The review discusses also the factors shaping Neosartorya spp.'s resistance to temperature, preservatives, chemicals, and natural plant extracts, as well as presenting novel solutions to problems created by its resilient nature.
... It may also serve as the flavouring agent. The concentration of around 60-65°Brix renders the concentrates safe from bacterial attacks; however, the presence of resistant spores and moulds can be a potential health risk (dos Santos et al., 2020b). A brief discussion of the safety concerns associated with the concentrates is given in section 5. ...
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The demand for pre‐processed fruits has grown rapidly in recent years as a result of consumer attention towards fresh, healthy and convenient foods. Most of the fruits are available seasonally and they are perishable, so it is necessary to have a continuous supply of the fruits to avoid the blockages in the fruit processing industry. The review is written in the purview of this issue, and it presents pre‐processed fruits as a sustainable solution. The present review is part II of the series of articles on pre‐processed fruits as the raw material for various food industries. The fruits covered here are mango, pineapple, pear, olive and papaya. The article continues to emphasise the critical processing and storage condition for these fruits ranked higher in terms of their production quantity and economic value. The fruits are critically reviewed for their processing, storage and safety aspects along with key research findings of different processing techniques. The important safety aspects associated with such processed commodities are also discussed.
... On one hand, fruit soluble solids content (SSC) is composed of sugars, organic acids, and inorganic salts, which comprehensively represents the taste information of apple fruits and can be simply and fast obtained by spectroscopy (Fan et al. 2016;Dos Santos et al. 2020). SSC directly affects the apple grading level and the popularity of consumers, and thus widely adopted for flavor quality detection of apple fruit (Xu et al. 2019). ...
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... Because of biological variability within a population of spores, different statistical distributions (Exponential, Normal, Lognormal, Weibull, Logistic or Pareto) of t v can be applied. It was reported that at 53 Brix, 0.883 a w , the 95th percentile t v was four fold the 5th percentile for Byssochlamys fulva [22 ]. ...
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The objective of this mini-review is to guide microbiologists through the selection and implementation of suitable predictive models for specific mold growth applications. Most food microbiologists including mycologists are not familiar with modelling techniques while people involved in modelling are unaware of mold specificities. As compared to bacteria, the development of mold is characterized by distinct features, such as the ability to form mycelium and to produce asexual conidia and sexual spores for some species. Therefore, it is necessary to determine the relevant biological responses and select the most significant factors that impact these responses, in addition to attaining proven predictive capacity.
... On the other hand, increasing temperature to 30°C will potentially increase the risk of outgrowth of A. fischerianus. Despite no visible growth being observed in this study at 10°C (after at most 90 days), previous investigation showed that A. fischerianus is able to grow at refrigerated temperatures (≥10°C) when the other growth determining factors are optimal (Santos et al., 2020). Accordingly, Tremarim et al. (2015) assessed the growth kinetics of A. fischerianus in diluted apple juice (a w 0.99) from 10°C to 30°C and observed lag times ranging from 19 days, at the lowest temperature, to less than one day, when the temperature was optimum for growth (30°C). ...
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The raw water quality and associations between the factors considered as threats to water safety were studied in 20 groundwater supplies in central Finland in 2002-2004. Faecal contaminations indicated by the appearance of Escherichia coli or intestinal enterococci were present in five small community water supplies, all these managed by local water cooperatives. Elevated concentrations of nutrients in raw water were linked with the presence of faecal bacteria. The presence of on-site technical hazards to water safety, such as inadequate well construction and maintenance enabling surface water to enter into the well and the insufficient depth of protective soil layers above the groundwater table, showed the vulnerability of the quality of groundwater used for drinking purposes. To minimize the risk of waterborne illnesses, the vulnerable water supplies need to be identified and appropriate prevention measures such as disinfection should be applied.
Article
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The safety of drinking water is evaluated by the results obtained from faecal indicators during the stipulated controls fixed by the legislation. However, drinking-water related illness outbreaks are still occurring worldwide. The failures that lead to these outbreaks are relatively common and typically involve preceding heavy rain and inadequate disinfection processes. The role that classical faecal indicators have played in the protection of public health is reviewed and the turning points expected for the future explored. The legislation for protecting the quality of drinking water in Europe is under revision, and the planned modifications include an update of current indicators and methods as well as the introduction of Water Safety Plans (WSPs), in line with WHO recommendations. The principles of the WSP approach and the advances signified by the introduction of these preventive measures in the future improvement of drinking water quality are presented. The expected impact that climate change will have in the quality of drinking water is also critically evaluated.
Article
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Since publication of the 3rd Edition of the World Health Organisation (WHO) Drinking Water Quality guidelines, global adoption of water safety plans (WSPs) has been gathering momentum. Most guidance lists managerial commitment and 'buy-in' as critical to the success of WSP implementation; yet the detail on how to generate it is lacking. This commentary discusses aspects of managerial commitment to WSPs. We argue that the public health motivator should be clearer and a paramount objective and not lost among other, albeit legitimate, drivers such as political or regulatory pressures and financial efficiency.
Article
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This geographical study aimed to show natural or water-processing-related factors of faecal contamination incidents (FCIs) of drinking water in continental France. We defined a FCI as the occurrence of at least 20 colony-forming Escherichia coli or enterococci among all the 100 mL samples collected for regulatory purpose within one day from a given drinking water supply zone (SZ). We explored correlations between the standardized number of FCIs per département (N_Pols) and various indicators related to weather, land cover, topography, geology and water management for three SZ size sub-classes. In 2003-2004, 2,739 FCIs occurred in SZs supplying fewer than 2,000 people, mainly with simply disinfected groundwater. N_Pols correlates with four covariates: (1) precipitation; (2) the extension of the karst outcrops; (3) the extent of disinfection; and (4) catchment protection. One hundred millimetres of yearly excess in precipitation increases the pollution risk by 28-37%, depending on the sub-class. A 10% extension of the karst areas, a 10% increase of unprotected resources, or of SZs with no disinfection, could entail a higher risk of FCI by about 10%. The correlations are reproducible over the three sub-classes and corroborate expert appraisals. These results encourage the ongoing effort to generalize disinfection and catchment protection.
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To determine thermal resistance, the effect of pasteurization temperature variations (c. 2 degrees C) in a continuous system in the number of decimal reductions (n) of a Byssochlamys strain in clarified apple juice (CAJ). Thermal destruction kinetics of Byssochlamys fulva IOC 4518 in thermal death tubes were determined at 85 degrees , 90 degrees , 92 degrees and 95 degrees C by using Weibull distribution frequency model. Three processes with different heating and holding temperatures (A: 94 degrees , 92 degrees C; B: 95 degrees , 93 degrees C; C: 96 degrees , 94 degrees C, respectively) were performed in a continuous system. Process time was 30 s. delta (time of first decimal reduction) values were: 42.98, 8.10, 3.62 and 1.81 min. Variable n ranged from 0.16 to >4.78 for process B (equivalent to industrial). Variable n (0.95-2.66 log CFU ml(-1)) were obtained in CAJ bottles processed under condition B, while process A resulted in total heat-resistant mould (HRM) survival and process C in total HRM destruction. This study demonstrates that small variations in temperature during the CAJ pasteurization could result in elimination or survival of HRM due to its nonlogarithmic behaviour. This was the first study to use Weibull frequency method to model inactivation of HRM in fruit juices. Temperature variations could culminate in the presence of HRM in pasteurized juices even when low counts (<10 spores per 100 ml) were present in the raw materials.
Article
Heat-resistant strains of Byssochlamys fulva, B. nivea, and other heat-resistant fungi were isolated from vineyard soil, grapes, grape-processing lines, and waste pomace. They are known to remain in grape juice occasionally and to grow in grape juice products. Ascospores of these fungi have a D value (decimal reduction time) of about 10 min at 190 F (88 C), but in the presence of 90 μliters of SO2 per liter (normally added to the juice) the D value was cut in half. Filtration through a commercial diatomaceous filter aid (also a common processing step) entrapped all but about 0.001% of experimentally added spores. Thus, heat in the presence of SO2 and filtration together can reduce the population of these spores by several orders of magnitude. Growth was also prevented by benzoate or sorbate in low concentrations. Oxygen must be reduced to extremely low levels before lack of oxygen limits growth.
Article
Heat-resistant moulds (HRMs) are well known for their ability to survive pasteurization and spoil high-acid food products, which is of great concern for processors of fruit-based products worldwide. Whilst the majority of the studies on HRMs over the last decades have addressed their inactivation, few data are currently available regarding their contamination levels in fruit and fruit-based products. Thus, this study aimed to quantify and identify heat-resistant fungal ascospores from samples collected throughout the processing of pasteurized high-acid fruit products. In addition, an assessment on the effect of processing on the contamination levels of HRMs in these products was carried out. A total of 332 samples from 111 batches were analyzed from three processing plants (=three processing lines): strawberry puree (n = 88, Belgium), concentrated orange juice (n = 90, Brazil) and apple puree (n = 154, the Netherlands). HRMs were detected in 96.4% (107/111) of the batches and 59.3% (197/332) of the analyzed samples. HRMs were present in 90.9% of the samples from the strawberry puree processing line (1–215 ascospores/100 g), 46.7% of the samples from the orange juice processing line (1–200 ascospores/100 g) and 48.7% of samples from the apple puree processing line (1–84 ascospores/100 g). Despite the high occurrence, the majority (76.8%, 255/332) of the samples were either not contaminated or presented low levels of HRMs (<10 ascospores/100 g). For both strawberry puree and concentrated orange juice, processing had no statistically significant effect on the levels of HRMs (p > 0.05). On the contrary, a significant reduction (p < 0.05) in HRMs levels was observed during the processing of apple puree. Twelve species were identified belonging to four genera - Byssochlamys, Aspergillus with Neosartorya-type ascospores, Talaromyces and Rasamsonia. N. fumigata (23.6%), N. fischeri (19.1%) and B. nivea (5.5%) were the predominant species in pasteurized products. The quantitative data (contamination levels of HRMs) were fitted to exponential distributions and will ultimately be included as input to spoilage risk assessment models which would allow better control of the spoilage of heat treated fruit products caused by heat-resistant moulds.
Article
The major aims of this study were to assess inter- and intra-species variability of heat resistant moulds (HRMs), Byssochlamys fulva and Byssochlamys nivea, with regards to (i) heat resistance and (ii) effect of heat treatment intensity on subsequent outgrowth. Four-week-old ascospores were suspended in buffered glucose solution (13° Brix, pH 3.5) and heat treated in a thermal cycler adjusted at 85 °C, 90 °C and 93 °C. Two variants of the Weibull model were fitted to the survival data and the following inactivation parameters estimated: b (inactivation rate, min⁻¹), n (curve shape) and δ (the time taken for first decimal reduction, min). In addition to the assessment of heat resistance, outgrowth of Byssochlamys sp. from ascospores heated at 70 °C, 75 °C, 80 °C, 85 °C and 90 °C for 10 min and at 93 °C for 30 and 70 s was determined at 22 °C for up to 30 days. The Baranyi and Roberts model was fitted to the growth data to estimate the radial growth rates (μmax, mm.day⁻¹) and lag times (λ, days). Inter-species variability and significant differences (p < 0.05) were observed for both inactivation and growth estimated parameters among B. fulva and B. nivea strains. The effect of heat treatment intensity on outgrowth of B. fulva strains was more apparent at the most intense heat treatment evaluated (90 °C/10 min), which was also the condition in which greater dispersion of the estimated kinetic parameters was observed. On the other hand, B. nivea strains were more affected by heating, resulting in greater variability of growth parameters estimated at different heating intensities and in very long lag phases (up to 25 days). The results show that inter- and intra-species variability in the kinetic parameters of Byssochlamys sp. needs to be taken into account for more accurate spoilage prediction. Furthermore, the effect of thermal treatments on subsequent outgrowth from ascospores should be explored in combination with other relevant factors such as °Brix and oxygen to develop thermal processes and storage conditions which can prevent the growth of HRMs and spoilage of heat treated food products.
Article
Effective risk management helps ensure safe drinking water and protect public health. Even in high-income countries, risk management sometimes fails and waterborne disease, including outbreaks, occur. To help reduce waterborne disease, the WHO Guidelines for Drinking Water Quality recommend water safety plans (WSPs), a systematic preventive risk management strategy applied from catchment to consumer. Since the introduction of WSPs, international guidelines, national and state legislation, and local practices have facilitated their implementation. While various high-income OECD countries have documented successes in improving drinking water safety through implementing WSPs, others have little experience. This review synthesizes the elements of the enabling environment that promoted the implementation of WSPs in high-income countries. We show that guidelines, regulations, tools and resources, public health support, and context-specific evidence of the feasibility and benefits of WSPs are elements of the enabling environment that encourage adoption and implementation of WSPs in high-income countries. These findings contribute to understanding the ways in which to increase the uptake and extent of WSPs throughout high-income countries to help improve public health.
Article
Spoilage of thermally processed products by heat-resistant molds (HRM) is a widespread problem for food and beverage industries. These molds can survive not only the temperatures applied during pasteurization but also the high pressure used during high-pressure processing (HPP), causing numerous incidents of spoilage and significant economic losses. The ability of HRM to survive these treatments is due to the formation of highly heat-resistant and pressure-resistant spores called ascospores. Ascospores are widely distributed in the soil, particularly in cultivated soils in which fruits and vegetables are grown. They have been isolated from ingredients, packaging and the processing environment. Prevention of spoilage of heat-processed products by HRM would require reduction or elimination of soil contamination and ascospores from the ingredients, packaging and processing environment. The suppliers of the ingredients and packaging as well as the beverage manufacturers must have programs in place to reduce contamination of these products by HRM ascospores. This review provides an insight into the recent advances on the assessment of HRM contamination and spoilage prevention and highlights the need for further research in this field.
Article
Access to safe water is essential for public health and is one of the most important prerequisites for good living and safe food production. Many studies have shown that non-compliance with drinking water quality standards in small water supply systems is much higher than in large systems. Nevertheless, people served by small water supply systems have the right to the same level of health protection. Actions are therefore needed to improve the situation. The objective of the present study was to carry out a baseline analysis of the situation in the Nordic region and provide recommendations for governmental policy and actions. Data were gathered on number of water supplies, population served, compliance with regulations and waterborne disease outbreaks from various sources in the Nordic countries. The collected data showed that there are about 12500 regulated water supplies, 9400 of which serve fewer than 500 persons. The number of unregulated and poorly regulated supplies is unknown, but it can be roughly estimated that these serve 10% of the Nordic population on a permanent basis or 2.6 million people. However, this does not tell the whole story as many of the very small water supplies serve transient populations, summerhouse dwellers and tourist sites, with many more users. Non-compliance regarding microbes is much higher in the small supplies. The population weighted average fecal contamination incidence rate in the Nordic region is eleven times higher in the smaller supplies than in the large ones, 0.76% and 0.07%, respectively. Registered waterborne disease outbreaks were also more frequent in the small supplies than in the large ones.
Article
The objective of this study was to estimate the growth rate, lag time and the times for the appearance of visible mycelium (tv) of six fungal species (Paecilomyces variotii, Penicillium paneum, Penicillium polonicum, Penicillium spinulosum, Penicillium citrinum and Aspergillus sydowii). The growth parameters were determined at 20 °C, 25 °C and 30 °C and in two culture media (18% Dichloran Glycerol Agar - DG-18 and Dichloran Rose Bengal Chloramphenicol Agar – DRBC) aiming the selection of most appropriate strains for use in challenge tests and for the development of predictive models. Seventy-two growth curves were obtained after measuring the diameter of the colonies for 14 days at 20 °C, 25 °C and 30 °C. Values of growth rate between 1.4 and 11.1 (day⁻¹) were obtained in this study. Faster growth rates (> 7 day⁻¹) were observed for Pa. variotii (LMQA-001) and P. paneum (LMQA-002) at 25 °C. The lag times varied between 0.7 and 4.2 days, with 50% of the values being as short as 0.7−1.4 day. The variability between the species was analyzed and coefficients of variation (CV) of 44.2% and 41% for the growth rate were observed at 30 °C. At 20 °C and 25 °C, CV varied from 28.3% to 32.9%, respectively. Given these results, Pa. variotii and P. paneum could be considered for further use in microbiological challenge tests aiming to assess the robustness of bakery products formulations.
Article
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 13.5 min at 87 °C; 1.5 and 3.5 min at 90 °C; and 0.3 and 0.4 min at 95 °C in glucose solution. Similarly, D values ranged between 3.3 and 15.4 min at 87 °C; 1.3 and 4.3 min at 90 °C; and 0.3 and 0.6 min 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 (26–46 CFU/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.
Article
A quantitative risk assessment model of yogurt spoilage by Aspergillus niger was developed based on a stochastic modeling approach for mycelium growth by taking into account the important sources of variability such as time-temperature conditions during the different stages of chill chain and individual spore behavior. Input parameters were fitted to the appropriate distributions and A. niger colony's diameter at each stage of the chill chain was estimated using Monte Carlo simulation. By combining the output of the growth model with the fungus prevalence, that can be estimated by the industry using challenge tests, the risk of spoilage translated to number of yogurt cups in which a visible mycelium of A. niger is being formed at the time of consumption was assessed. The risk assessment output showed that for a batch of 100,000 cups in which the percentage of contaminated cups with A. niger was 1% the predicted numbers (median (5 th , 95 th percentiles)) of the cups with a visible mycelium at consumption time were 8 (5, 14). For higher percentages of 3, 5 and 10 the predicted numbers (median (5 th , 95 th percentiles)) of the spoiled cups at consumption time were estimated to be 24 (16, 35), 39 (29, 52) and 80 (64, 94), respectively. The developed model can lead to a more effective risk-based quality management of yogurt and support the decision making in yogurt production.
Article
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 90.9 min at 75 °C; 13.6 and 20.8 min at 78 °C; 5.1 and 12.4 min at 80 °C; 1.6 and 2.6 min at 82 °C. D values of T. bacillisporus ranged between 44.4 and 60.9 min at 82 °C; 11.9 and 15.5 min at 85 °C; 2.7 and 4.1 min at 88 °C and were equal to 1.2 min 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 (< 100 CFU/kg), since the food industry generally tries to achieve five or more log-reduction in their products.
Article
Assuring sufficient quality of drinking water is of great importance for public wellbeing and prosperity. Nations have developed regulatory system with the aim of providing drinking water of sufficient quality and to minimize the risk of contamination of the water supply in the first place. In this study the chemical quality of Icelandic drinking water was evaluated by systematically analyzing results from audit monitoring where 53 parameters were assessed for 345 samples from 79 aquifers, serving 74 water supply systems. Compliance to the Icelandic Drinking Water Regulation (IDWR) was evaluated with regard to parametric values, minimum requirement of sampling, and limit of detection. Water quality compliance was divided according to health-related chemicals and indicators, and analyzed according to size. Samples from few individual locations were benchmarked against natural background levels (NBLs) in order to identify potential pollution sources. The results show that drinking compliance was 99.97% in health-related chemicals and 99.44% in indicator parameters indicating that Icelandic groundwater abstracted for drinking water supply is generally of high quality with no expected health risks. In 10 water supply systems, of the 74 tested, there was an indication of anthropogenic chemical pollution, either at the source or in the network, and in another 6 water supplies there was a need to improve the water intake to prevent surface water intrusion. Benchmarking against the NBLs proved to be useful in tracing potential pollution sources, providing a useful tool for identifying pollution at an early stage.
Article
For over 30 years, predictive microbiology focused on food-pathogenic bacteria. The objective of predictive mycology is to understand and to predict the development of fungi in food and raw materials, the inactivation of fungal spores in the food industry, and the accumulation of secondary metabolites, such as mycotoxins, throughout the food chain. The number of studies dedicated to food spoilage fungi has increased a lot these recent years. Most of these studies were concerned with the effect of environmental factors on fungal growth, but the major issues were not addressed. Methods are not unique, and may depend on the objective of the study. Through the examples of fungal starters, food spoilage fungi, and mycotoxin accumulation, the more relevant factors, biological responses and predictive models are detailed.
Article
Heat-resistant fungi are often factors causing spoilage of heat-processed products. Contamination of agricultural raw materials is often a result of their contact with soil. Materials contaminated by spores of heat-resistant fungi can be a risk to consumers' health because of toxic metabolites (mycotoxins) produced by these microorganisms. Due to resistance of fungi to high temperatures they are able to survive industry pasteurization process. Therefore, the only way to prevent the growth of these microorganisms in the product is suitable selection of material by conducting tests for the presence of heat-resistant fungi. The use of traditional culture methods is long and, therefore, does not apply in the selection of raw materials for production. However, time is a critical factor in assessing the acceptance or rejection of a given batch of raw material, due to the necessity of processing it fresh, which is very important especially in the case of fruit.
Book
The first and second editions of Fungi and Food Spoilage established a reputation as the foremost book on foodborne fungi. This completely revised and updated third edition is an invaluable reference for food microbiologists investigating fungal spoilage and sources of mycotoxin contamination in foods. The introductory chapters of the book deal with the ecology of food spoilage and give an overview of how food processing, packaging and storage affect fungal growth. Subsequent chapters cover the fundamentals of classifying and naming fungi and current methods for isolation and enumeration, including general and special purpose media, incubation conditions, etc. The major part of the book provides keys, descriptions and illustrations of all yeasts and moulds commonly encountered in foods. Characteristics of the species, including their ecology and potential for mycotoxin production, are also included. The broad and practical nature of the coverage will appeal to microbiologists, mycologists and biotechnologists in the food industry, academic, research and public health institutions. Dr John Pitt and Dr Ailsa Hocking are both Honorary Research Fellows at CSIRO Food Science Australia, North Ryde, NSW, Australia. © Springer Science+Business Media, LLC 2009. All rights reserved.
Article
This paper explores the fate and transport of microbial contamination in a cold climate and coarse aquifers. A confirmed norovirus outbreak in a small rural water supply in the late summer of 2004, which is estimated to have infected over 100 people, is used as a case study. A septic system, 80 m upstream of the water intake, is considered to have contaminated drinking water. Water samples tested were negative for coliform and strongly positive for norovirus. Modeling predicts that a 4.8-log10 removal was possible in the 8 m thick vadose zone, while only a 0.7-log10 and 2.7-log10 removal in the aquifer for viruses and E. coli, respectively. The model results support that the 80 m setback distance was inadequate and roughly 900 m aquifer transport distance was needed to achieve 9-log10 viral removal. Sensitivity analysis showed that the most influential parameters on model transport removal rate are grain size diameter and groundwater velocity, temperature and acidity. The results demonstrate a need for systematic evaluation of septic systems in rural areas in lesser studied coarse strata at low temperatures, thereby strengthening data used for regulatory requirements for more confident determination on safe setback distances. Keywords: norovirus outbreak; septic system; groundwater; microbial transport, coarse soil.
Article
Spores are an integral part of the life cycle of the gross majority of fungi. Their morphology and the mode of formation are both highly variable among the fungi, as is their resistance to stressors. The main aim for spores is to be dispersed, both in space, by various mechanisms or in time, by an extended period of dormancy. Some fungal ascospores belong to the most stress-resistant eukaryotic cells described to date. Stabilization is a process in which biomolecules and complexes thereof are protected by different types of molecules against heat, drought, or other molecules. This review discusses the most important compounds that are known to protect fungal spores and also addresses the biophysics of cell protection. It further covers the phenomena of dormancy, breaking of dormancy, and early germination. Germination is the transition from a dormant cell toward a vegetative cell and includes a number of specific changes. Finally, the applied aspects of spore biology are discussed.
Article
Pressure transients in drinking water pipelines (i.e., surges) may cause hydraulic pressure gradients, resulting in the potential for intrusion of pathogens present in the external environment into the distribution system. The objectives of this study were to determine the occurrence of indicator microorganisms and pathogens in the vicinity of potable water pipelines and assess the potential for intrusion attributable to transient distribution system pressure changes. As part of an earlier study (Kirmeyer et al, 2001), soil and water samples were collected at sites immediately exterior to drinking water pipelines at eight locations in six states. Samples were then tested for occurrence of total and fecal coliforms, Clostridium perfringens, Bacillus subtilis, coliphage, and enteric viruses. Indicator microorganisms and enteric viruses were detected in more than 50% of the samples examined. Monitoring of pressure transients at a large distribution system indicated that pressure transients occurred frequently, although negative pressures were detected on only one occasion. The results of this study suggest that during negative- or low-pressure events, microorganisms may enter the treated drinking water through pipeline leaks.
Article
In order to assess the effect of the inoculum size on the time to visible growth for Penicillium chrysogenum, the correlation described by González et al. (González, H.H.L., Resnik, S.L., Vaamonde, G., 1987. Influence of inoculum size on growth rate and lag phase of fungi isolate from Argentine corn. International Journal of Food Microbiology 4, 111-117) was compared to the model introduced by Gougouli et al. (Gougouli, M., Kalantzi, K., Beletsiotis, E., Koutsoumanis, K.P., 2011. Development and application of predictive models for fungal growth as tools to improve quality control in yogurt production. Food Microbiology 28, 1453-1462). Based on the regression coefficient, the latter model performed better than the former one to fit the data obtained for P. chrysogenum grown on Potato Dextrose Agar at 25°C. Inoculum sizes in the range 10(1)-10(5) spores were tested at 0.930, 0.950, 0.970, and 0.995 aw. By extrapolation of the straight line, the model of Gougouli et al. (2011) provided accurate estimations of the time to visible growth for a single spore inoculum, tvg (N=1). In order to avoid experiments at reduced water activities, the influence of water activity on the model parameters, and on the ratio tvg (N=1) over the germination time was assessed.
Article
Unlabelled: Germination is the process by which a spore is transformed from a dormant state of metabolic activity to one of high activity. Effects of different fruit components, aeration and cold storage at 5°C on the germination of ascospores of Neosartorya fischeri after heat treatment were investigated by measuring rate and percentage germination and outgrowth of fungal biomass. Some activated spores reverted back to dormancy after 12 and 24 h of storage at 5°C. Ascospores germinated easily in acetate buffers. Germination of ascospores was retarded in medium containing citric acid and sugars. Ascospores cultured under low aeration showed no signs of germination. Different food components and growth conditions have varying effects on germination of ascospores. Significance and impact of the study: The findings of this study can be used in the fruit industry to predict the effect of some fruit components on the germination of heat-resistant ascospores. The impact of cold storage on ascospores, which may be activated by heat schedules used to manufacture some types of fruit products, will help in determining the best storage conditions for enhanced shelf life to be adopted immediately after heat treatment. These findings also help to determine for how long a fruit juice is safe at room temperature after production before mycotoxin synthesis by heat-resistant fungi sets in.
Article
The relation between germination time and lag time of mycelium growth of individual spores was studied by combining microscopic and macroscopic techniques. The radial growth of a large number (100-200) of Penicillium expansum and Aspergillus niger mycelia originating from single spores was monitored macroscopically at isothermal conditions ranging from 0 to 30°C and 10 to 41.5°C, respectively. The radial growth curve for each mycelium was fitted to a linear model for the estimation of mycelium lag time. The results showed that the lag time varied significantly among single spores. The cumulative frequency distributions of the lag times were fitted to the modified Gompertz model and compared with the respective distributions for the germination time, which were obtained microscopically. The distributions of the measured mycelium lag time were found to be similar to the germination time distributions under the same conditions but shifted in time with the lag times showing a significant delay compared to germination times. A numerical comparison was also performed based on the distribution parameters λ(m) and λ(g), which indicate the time required from the spores to start the germination process and the completion of the lag phase, respectively. The relative differences %(λ(m)-λ(g))/λ(m) were not found to be significantly affected by temperatures tested with mean values of 72.5±5.1 and 60.7±2.1 for P. expansum for A. niger, respectively. In order to investigate the source of the above difference, a time-lapse microscopy method was developed providing videos with the behavior of single fungal spore from germination until mycelium formation. The distances of the apexes of the first germ tubes that emerged from the swollen spore were measured in each frame of the videos and these data were expressed as a function of time. The results showed that in the early hyphal development, the measured radii appear to increase exponentially, until a certain time, where growth becomes linear. The two phases of hyphal development can explain the difference between germination and lag time. Since the lag time is estimated from the extrapolation of the regression line of the linear part of the graph only, its value is significantly higher than the germination time, t(G). The relation of germination and lag time was further investigated by comparing their temperature dependence using the Cardinal Model with Inflection. The estimated values of the cardinal parameters (T(min), T(opt), and T(max)) for 1/λ(g) were found to be very close to the respective values for 1/λ(m), indicating similar temperature dependence between them.
Article
The effects of potassium sorbate, sodium benzoate, SO2 and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice were determined. Growth at 21, 30 and 37°C over a 25-day incubation period was significantly retarded by 75 ppm SO2, 150 ppm potassium sorbate and 500 ppm sodium benzoate. Biomass accumulated to approximately 500 mg/100 ml in control samples of apple juice. Patulin was produced in the highest concentrations at 21°C after 20 days incubation. After reaching a maximum concentration at 30 and 37°C, a rapid decline in patulin content was observed. Patulin production was also observed at 12°C. On the basis of concentration, SO2 had the most significant effect on the rate of biomass and patulin production by B. nivea followed by potassium sorbate and sodium benzoate, respectively.
Article
Behaviour of Byssochlamys nivea Westling ascospores in fruit products supplemented with sucrose was determined. Growth and ascospore production occurred at 30° in juice with water activity as low as 0.90. Sucrose protected ascospores against death in fruit products stored at 7° and −30° and in grape juice heated at 75°. Colony development was retarded or inhibited on grape juice agar to which more than 30 g sucrose per 100 ml had been added.
Article
The effect of storage temperature (0-40 °C) and inoculum size (10¹-10⁵ spores) on the mycelium growth kinetics of 12 fungal species on yogurt were monitored. A cardinal model with inflection (CMI) was used to describe the effect of temperature on the growth rate (μ) and the lag time (λ) of each isolate. Significant differences on the temperature dependence of the mycelium growth between the tested species were observed. Depending on the strain, the estimated minimum, optimum and maximum temperature parameters for μ (T(min), T(opt), T(max)) ranged from -7.6 to 9.6, 19.5 to 37.8 and 29.8 to 46.9 °C, respectively. Only λ was found to be affected by the inoculum size and a linear relation between Ln (λ) and Log (inoculum size) was revealed. The inoculum level did not influence the values of T(min), T(opt) and T(max) for λ. Based on the above observations, the combined effect of inoculum size and temperature on λ was modeled using a modified CMI. The parameter λ(opt) (λ at optimum conditions) was expressed as a function of the inoculum size. Validation studies showed a good performance of the developed models. The application scheme of the models for improving fungi control in yogurt productions is discussed.
Article
Predictive models can be a tool to develop strategies to prevent mould development and consequently mycotoxin production. The aims of this work were to assess the impact of a) high/low levels of inoculum and b) optimal/suboptimal environmental conditions on fungal responses based on both kinetic and probabilistic models. Different levels of spore suspensions of Aspergillus carbonarius and Penicillium expansum were prepared and inoculated centrally with a needlepoint load on malt extract agar (MEA) with 50 replicates. While optimum conditions led to a colony diameter increase which followed Baranyi's function, suboptimal conditions led to different grow functions. In general, growth rate (mu) and lag phase (lambda) were normally distributed. Specifically, the growth rate (mu) showed similar distributions under optimal growth conditions, regardless of the inoculum level, while suboptimal a(w) and temperature conditions led to higher kurtosis distributions, mainly when the inoculum levels were low. Regarding lambda, more skewed distributions were observed, mainly when the inoculum levels were low. Probability models were not much affected by the inoculum size. Lower probabilities of growth were in general predicted under marginal conditions at a given time for both strains. The slopes of the probability curves were smaller under suboptimal growth conditions due to wider distributions. Results showed that a low inoculum level and suboptimal conditions lead to high variability of the estimated growth parameters and growth probability.
Article
Appropriate implementation of WSPs offers an important opportunity to engage in and promote preventative risk management within water utilities. To ensure success, the whole organization, especially executive management, need to be advocates. Illustrated by two case studies, we discuss the influence of organizational culture on buy-in and commitment to public health protection and WSPs. Despite an internal desire to undertake risk management, some aspects of organizational culture prevented these from reaching full potential. Enabling cultural features included: camaraderie; competition; proactive, involved leaders; community focus; customer service mentality; transparency; accountability; competent workforce; empowerment; appreciation of successes, and a continual improvement culture. Blocking features included: poor communication; inflexibility; complacency; lack of awareness, interest or reward and coercion. We urge water utilities to consider the influence of organizational culture on the success and sustainability of WSP adoption, and better understand how effective leadership can mould culture to support implementation.
Article
The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 °C) and aw levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) (Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 °C, 46.4 °C and 32.1 °C for B. fulva and 10.5 °C, 43.2 °C and 32.1 °C for B. nivea. The respective values for aw were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 aw regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 °C irrespective of aw. Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4-4.2% false positive and 2.1-3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.
Article
More than 50% of the outbreaks of waterborne disease in the United States are due to the consumption of contaminated groundwater. An estimated 65% of the cases in these outbreaks are caused by enteric viruses. Little, however, is known about the persistence of viruses in groundwater. The purpose of this study was to determine whether measurable chemical and physical factors correlate with virus survival in groundwater. Groundwater samples were obtained from 11 sites throughout the United States. Water temperature was measured at the time of collection. Several physical and chemical characteristics, including pH, nitrates, turbidity, and hardness, were determined for each sample. Separate water samples were inoculated with each of three viruses (poliovirus 1, echovirus 1, and MS-2 coliphage) and incubated at the in situ groundwater temperature; selected samples were also incubated at other temperatures. Assays were performed at predetermined intervals over a 30-day period to determine the number of infective viruses remaining. Multiple regression analysis revealed that temperature was the only variable significantly correlated with the decay rates of all three viruses. No significant differences were found among the decay rates of the three viruses, an indication that MS-2 coliphage might be used as a model of animal virus survival in groundwater.
Article
Spoilage of pasteurized and canned fruit and fruit products caused by heat-resistant molds have been reported repeatedly in recent years. Species most commonly implicated in fruit and fruit product disintegration are Byssochlamys fulva, Byssochlamys nivea, Neosartorya fischeri, Talaromyces flavus, and Eupenicillium brefeldianum. These organisms are saprophytic rather than parasitic and usually contaminate fruits on or near the ground. They can survive heat treatments used for fruit processing and can grow and spoil the products during storage at room temperature, which results in great economic losses. Mold heat resistance is attributed to the formation of sexual spores, ascospores. Ascospores have a wide range of heat resistance, depending on species, strain, age of organism, heating medium, pH, presence of sugars, fats, and acids in heating medium, growth conditions, etc. The mechanism(s) of thermoresistance are not clear; probably some very stable compound(s) critical to germination and outgrowth are present in the heat-resistant ascospores. Besides spoilage, the heat-resistant molds produce a number of toxic secondary metabolites, such as byssotoxin A; byssochlamic acid; the carcinogen, patulin, the tremorgenic substances, fumitremorgin A and C, and verruculogen; fischerin, which caused fatal peritonitis in mice; and eupenifeldin, a compound possessing cytotoxicity as well as in vivo antitumor activity. Growth of heat-resistant fungi can be controlled by lowering the water activity, adding sulfur dioxide, sorbate, or benzoate; washing of fruits in hypochlorite solution before heat treatment reduces the number of ascospores and makes the heat destruction more successful. More research is needed to elucidate the mechanism(s) of thermoresistance and develop new methods for the complete inactivation of resistant ascospores.