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.

Effect of storage temperature, water activity, oxygen headspace concentration and pasteurization intensity on the time to growth of Aspergillus fischerianus (teleomorph Neosartorya fischeri).

This study aims to assess, by means of a full factorial design, the effect of storage temperature (10-30 °C), water activity (aw, 0.87-0.89), headspace oxygen (O2) level (0.15-0.80%) and pasteurization intensity (95 °C-105 °C/15sec) on the time to visible growth (tv, days) of Aspergillus fischerianus on acidified Potato Dextrose Agar (aPDA, pH 3.6) for up to 90 days. Moreover, in order to validate the results obtained on aPDA, 12 conditions were selected and assessed in concentrate strawberry-puree based medium. Overall, storage temperature had the greatest effect on the tv of A. fischerianus on the evaluated conditions. At 10 °C, no visible growth was observed over the 90 day incubation period, whilst visible mycelia (diameter ≥ 2 mm) were present in 37% and 89% of the conditions at 22 °C and 30 °C, respectively. Pasteurization intensity had only a minor effect on the outgrowth of A. fischerianus. Growth inhibition was observed when aw was reduced to 0.870 ± 0.005 in combination with very low headspace O2 levels (0.15% ± 0.10) in both, aPDA and concentrate strawberry-based media, regardless of the incubation temperature and heat pasteurization intensity. Overall, longer tv's were required when incubation was done at 22 °C compared to 30 °C. Ultimately, the effect of O2 (0.05 and 1%) and pasteurization intensity (95 °C and 105 °C/15sec) were evaluated on totally 22 fruit purees (un-concentrates and concentrates) over a 60 day storage period. None of the concentrates purees (aw ≤0.860) evaluated in this study supported the growth of A. fischerianus. On the other hand, A. fischerianus growth inhibition was only observed when the O2 levels were ≤0.05% on un-concentrates fruit purees (aw ≥ 0.980) stored at ambient temperature (22 °C). Combination of multiple stress factors effectively inhibited growth of A. fischerianus. In general, storage of fruit purees at low temperatures (<10 °C) or distribution in the form of concentrates can be considered as important strategies to prevent the growth of spoilage associated heat-resistant moulds.

Assessment of minimum oxygen concentrations for the growth of heat-resistant moulds.

This study evaluated the effect of both gaseous and dissolved oxygen (O2) concentration (0 - 21%) on the growth of six heat-resistant moulds (HRMs) (Neosartorya and Byssochlamys spp.) previously isolated from high-acid fruit products. The study was performed in acidified potato dextrose agar (aPDA) with all six HRMs and with B. fulva and N. fischeri in strawberry, apple and orange juice-based media. At ≥ 0.15% O2, visible growth of the HRMs occurred within 3-6 days. Complete inhibition on aPDA did not occur even at very low levels of dissolved O2 (ca. 0.01% O2). With the exception of B. fulva, decrease of the O2 concentration to ≤0.03% resulted in significantly (p < 0.05) longer times to visible growth. The growth of N. laciniosa, N. fischeri, B. nivea and B. fulva was inhibited for 30 days when they were incubated under strict anaerobic conditions. As in aPDA, B. fulva and N. fischeri grew in the three fruit-based media at O2 concentrations ≥0.15%. Significantly slower (p < 0.05) growth was observed for N. fischeri in orange juice medium. Strategies to inhibit the growth of HRMs should therefore not be based entirely on establishing low headspace O2 levels. With this in mind, the effect of low O2 concentrations (<1%) should be studied in combination with other factors (hurdles) such as antioxidants, organic acids, sugars (aw), storage temperature and pasteurization intensity, in order to predict the growth inhibition of the HRMs.

Occurrence, distribution and contamination levels of heat-resistant moulds throughout the processing of pasteurized high-acid fruit products.

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.

Inter- and intra-species variability in heat resistance and the effect of heat treatment intensity on subsequent growth of Byssochlamys fulva and Byssochlamys nivea.

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.

Effect of Initial Headspace O2 Level on the Growth and Volatile Metabolite Production of Leuconostoc Mesenteriodes and the Microbial and Sensorial Quality of Modified Atmosphere Packaged Par-Fried French Fries.

This study evaluated the effect of residual O2 level (0% to 5%) on microbial growth and volatile metabolite production on par-fried French fries packaged in a modified atmosphere with 60% CO2 (rest N2 ) at 4 °C. The results obtained showed that the initial headspace (IH) O2 level had an effect on growth of Leuconostoc mesenteroides on French fry simulation agar, whereby growth was slightly faster under 5% O2 . In terms of quantity, ethanol, 2-methyl-1-propanol, and dimethyl disulphide were the most significant volatile metabolites produced by L. mesenteroides. The production of ethanol by L. mesenteroides was highest on simulation agar packaged under low IH O2 levels (0% to 1%), indicating that the fermentative metabolism was induced under these conditions. In agreement with the results observed on the simulation medium, growth of native lactic acid bacteria was faster under an IH O2 level of 5%. In addition, ethanol, 2-methyl-1-propanol, and dimethyl disulphide were also quantitatively the most important volatile metabolites. However, in contrast, greater quantities of ethanol and dimethyl disulphide were produced on par-fried French fries packaged under 5% O2 . This was attributed to the limited growth of the native flora on the par-fried French fries under residual O2 levels of 0% and 1%. Although some significant differences (P < 0.05) occurred between the French fries packaged in 0%, 1%, and 5 % residual O2 during storage, all products were considered to be acceptable for consumption. The results of this study can be used to optimize the shelf-life of packaged chill stored potato products.

Decontamination of Pangasius fish (Pangasius hypophthalmus) with chlorine or peracetic acid in the laboratory and in a Vietnamese processing company.

This study evaluated the decontamination of Pangasius fillets in chlorine or peracetic acid treated wash water. First, the decontamination efficacy of the washing step with chlorinated water applied by a Vietnamese processing company during trimming of Pangasius fillets was evaluated and used as the basis for the experiments performed on a laboratory scale. As chlorine was only added at the beginning of the batch and used continuously without renewal for 239min; a rapid increase of the bacterial counts and a fast decrease of chlorine in the wash water were found. This could be explained by the rapid accumulation of organic matter (ca. 400mg O2/L of COD after only 24min). Secondly, for the experiments performed on a laboratory scale, a single batch approach (one batch of wash water for treating a fillet) was used. Chlorine and PAA were evaluated at 10, 20, 50 and 150ppm at contact times of 10, 20 and 240s. Washing with chlorine and PAA wash water resulted in a reduction of Escherichia coli on Pangasius fish which ranged from 0-1.0 and 0.4-1.4logCFU/g, respectively while less to no reduction of total psychrotrophic counts, lactic acid bacteria and coliforms on Pangasius fish was observed. However, in comparison to PAA, chlorine was lost rapidly. As an example, 53-83% of chlorine and 15-17% of PAA were lost after washing for 40s (COD=238.2±66.3mg O2/L). Peracetic acid can therefore be an alternative sanitizer. However, its higher cost will have to be taken into consideration. Where (cheaper) chlorine is used, the processors have to pay close attention to the residual chlorine level, pH and COD level during treatment for optimal efficacy.

Microbial ecology of Vietnamese Tra fish (Pangasius hypophthalmus) fillets during processing.

There are numerous factors that can have an impact on the microbial ecology and quality of frozen Pangasius hypophthalmus fillets during processing in Vietnam. The presence of spoilage bacteria along the processing line can shorten the shelf-life of thawed frozen fish products. Therefore, the spoilage microbiota throughout the processing chain of two companies (BC: large scale factory, chlorine-based process, BW: large scale factory, water-based process and SC: small scale factory, chlorine-based process) was identified by culture-dependent techniques and 16S rRNA gene sequencing. The microbiological counts were observed to be insignificantly different (p>0.05) between BC and BW. Surprisingly, chlorine treated fillets from the SC line were revealed to have significantly higher microbial counts than potable water treated fillets at BW line. This was determined to be a result of temperature abuse during processing at SC, with temperatures even greater than 10 °C being recorded from skinning onwards. On the contrary, the microbiota related to spoilage for BC and BW lines was determined by 16S rRNA gene sequencing to be more diverse than that on the SC line. A total of 174 isolates, 20 genera and 38 species were identified along the processing chains. The genera Aeromonas, Acinetobacter, Lactococcus and Enterococcus were prevalent at various processing steps on all the processing lines evaluated. A diverse range of isolates belonging to the Enterobacteriaceae such as Providencia, Shigella, Klebsiella, Enterobacter and Wautersiella were isolated from fillets sampled on the SC line whereas Serratia was only observed on fillets sampled on the BC and BW lines. The results can be used to improve Good Manufacturing Practices for processed Pangasius fillets and to select effective measures to prolong the shelf-life of thawed Vietnamese Pangasius fillets products.

Effect of high oxygen and high carbon dioxide atmosphere packaging on the microbial spoilage and shelf-life of fresh-cut honeydew melon.

This study evaluated the potential of modified atmospheres (MAs) combining high oxygen (O2) and high carbon dioxide (CO2) levels to extend the shelf-life of fresh-cut honeydew melon. Firstly, the effect of MA on the growth and volatile organic metabolite production of Candida sake, Leuconostoc mesenteroides and Leuconostoc gelidum, which had all been previously isolated from spoiled commercial fresh-cut honeydew melon, was evaluated separately on honeydew melon agar at 7 °C. Additionally, the effect of selected MAs on the microbial, physico-chemical and sensory quality of commercial fresh-cut honeydew melon cubes was evaluated at 7 °C. The results showed that MAs with high O2 and high CO2 levels greatly retarded the growth, CO2 and volatile metabolite production (i.e. ethanol, 2-methyl-1-butanol, ethyl acetate, phenylacetic acid, nonanal) of C. sake on honeydew melon agar; especially MAs consisting of 50% O2+50% CO2 and 70% O2+30% CO2. In contrast, the MAs evaluated only had a minor effect on the growth and volatile metabolite production of L. mesenteroides and L. gelidum. Overall, the effect of MAs on colour, juice leakage, juiciness, and firmness of fresh-cut honeydew melon was small during storage. Sensory preference was generally for fresh-cut honeydew melon cubes packaged in MA of 50% O2+50% CO2. These were still acceptable on day five of storage and had appreciably lower populations of yeasts and lactic acid bacteria, lower quantities of volatile organic compounds, but slightly stronger colour oxidation compared to honeydew melon that was packaged in air. Additionally, most of the samples packed in air had blown by day five due to the large quantity of CO2 production during storage. Therefore, 50% O2+50% CO2 is a potential MA solution for extending the shelf-life of fresh-cut honeydew melon.

Effect of O(2)-CO(2) enriched atmospheres on microbiological growth and volatile metabolite production in packaged cooked peeled gray shrimp (Crangon crangon).

This study evaluated the effect of modified atmospheres (MAs) with different O(2) concentrations on microbial growth and volatile metabolite production in gray shrimp (Crangon crangon) during storage at 4 °C. Eight MAs were evaluated in total. Four of the MAs evaluated were without CO(2): 0/0/100, 0/10/90, 0/30/70, 0/50/50 (% CO(2)/O(2)/N(2)) whilst the other four MAs all contained 50% CO(2): 50/0/50, 50/10/40, 50/30/20, 50/50/0 (% CO(2)/O(2)/N(2)). Volatile spoilage metabolites were identified by thermal desorption GC-MS and quantified during storage by selective ion flow tube mass spectrometry (SIFT-MS). In comparison to microbial growth observed with an atmosphere of 100% N(2), microbial growth was stimulated by the addition of O(2) in the MAP in the absence of CO(2.) Under these conditions the total psychrotrophic counts exceeded 7 log cfug(-1) after just 3 days of storage. However, in the presence of 50% CO(2) the total psychrotrophic count exceeded 7 log cfug(-1) after 5 days of storage. The combination of 50% CO(2) and 50% O(2) significantly inhibited microbial growth. For this MA condition, a diminishing effect on the production of metabolites was also observed, especially for amines and sulfur compounds, which constituted the major fraction of components causing the offensive odor.

Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled-stored food products at the end of their shelf-life.

The major objective of this study was to determine the role of psychrotrophic lactic acid bacteria (LAB) in spoilage-associated phenomena at the end of the shelf-life of 86 various packaged (air, vacuum, modified-atmosphere) chilled-stored retail food products. The current microbiological standards, which are largely based on the total viable mesophilic counts lack discriminatory capacity to detect psychrotrophic LAB. A comparison between the total viable counts on plates incubated at 30 °C (representing the mesophiles) and at 22 °C (indicating the psychrotrophs) for 86 food samples covering a wide range - ready-to-eat vegetable salads, fresh raw meat, cooked meat products and composite food - showed that a consistent underestimation of the microbial load occurs when the total aerobic mesophilic counts are used as a shelf-life parameter. In 38% of the samples, the psychrotrophic counts had significantly higher values (+0.5-3 log CFU/g) than the corresponding total aerobic mesophilic counts. A total of 154 lactic acid bacteria, which were unable to proliferate at 30 °C were isolated. In addition, a further 43 with a poor recovery at this temperature were also isolated. This study highlights the potential fallacy of the total aerobic mesophilic count as a reference shelf-life parameter for chilled food products as it can often underestimate the contamination levels at the end of the shelf-life.

Exposure of infants to fumonisins in maize-based complementary foods in rural Tanzania.

Feeding children with maize may expose them to fumonisins (FBs). This study assessed FB exposure for infants consuming maize in Tanzania by modeling maize consumption data (kg/kg body weight (bw)/day) with previously collected total FB contamination (microg/kg) patterns for sorted and unsorted maize harvested in 2005 and 2006. Consumption was estimated by twice conducting a 24 h dietary recall for 254 infants. The exposure assessment was performed with the @RISK analysis software. Of the infants, 89% consumed maize from 2.37 to 158 g/person/day (mean; 43 g/person/day +/- 28). Based on the contamination for sorted maize; in 2005, the percentage of infants with FB exposures above the provisional maximum tolerable daily intake (PMTDI) of 2 microg/kg (bw) (26% (95% confidence interval (CI); 23-30)) was significantly higher than the level of 3% (90% CI; 2-12) in 2006. Pooling the datasets for sorted maize from the two seasons resulted in a seemingly more representative risk (10% (95% CI; 6-17)) of exceeding the PMTDI. However, infants who might have consumed unsorted maize would still be at a significantly higher risk (24% (95% CI; 15-34)) of exceeding the PMTDI. Sorting and other good maize management practices should be advocated to farmers in order to minimize FB exposure in rural areas.

Modeling the effect of temperature on the growth rate and lag phase of Penicillium expansum in apples.

The objective of the present study was to develop validated models that describe the effect of storage temperature on the growth rate and lag phase of six Penicillium expansum strains. The growth of the selected strains was therefore studied on Apple Puree Agar Medium (APAM) at 30, 25, 16, 10, 4 and 2 degrees C. Growth rates and lag phases were estimated using linear regression. Several secondary models were evaluated and for the growth rate, a modification of the extended Ratkowsky model was selected. Regarding the lag phase, the Arrhenius-Davey model provided the best adjustment to the observed data. Model validation was performed in two steps. Firstly, the developed models were validated on APAM. The obtained bias factors (Bf) ranged from 0.91 to 1.14 and the accuracy factors (Af) were <1.2 for the validation performed on APAM, indicating that the models were good predictors of the true mean colony growth rate and lag phase. Afterwards, an external validation was carried out in apples. For the growth rate, Bf ranged from 0.64 to 0.81 and Af<1.39, indicating conservative predictions. On the contrary for the lag phase, a clear deviation was observed between predictions and observed values on apples (0.351.6). These results highlight that the use of simulation or synthetic media for the development of predictive models for the lag phase of moulds can lead to inadequate predictions and that a validation on the real food matrix is necessary. Application of the developed models is possible in the framework of Quantitative Risk Assessment to develop control strategies against blue mould rot in apple and enables the inclusion of strain variability. However, possible underestimation of the lag phase should be taken into account.

Growth kinetics of cultures from single spores of Aspergillus flavus and Fusarium verticillioides on yellow dent corn meal.

A dilution protocol originally developed for the isolation of single bacterial cells was modified to suite the specificities of fungal growth. The modified protocol was used to study the growth kinetics of single spores of Aspergillus flavus and Fusarium verticillioides on yellow dent corn meal. Both a(w) and temperature significantly influenced the distributions of the colony growth rates and lag phases and the rate at which individual spores of both isolates completed the lag period. An interaction between a(w) and temperature was noted on the spread of the distributions of these growth parameters. The histograms of the single spore colony growth rates and lag phases generally became wider the more compromising the conditions for growth became, indicating a greater variation in growth ability at these conditions. The rate at which the single spores passed through the lag phases generally decreased with decrease in temperature and/or a(w), with an interaction again noted between these two factors on the rate. These results show the potential range and variability in growth of individual fungal spores at the lowest inoculum level possible.

Influence of experimental parameters on the fluorescence response and recovery of the high-performance liquid chromatography analysis of fumonisin B1.

The effect of mobile phase pH, column temperature, extraction method and derivatisation time on the fluorescence response and recovery of fumonisin B1 in corn was investigated. Column temperature and mobile phase pH were negatively and positively correlated with the fluorescence response, respectively. Use of an Ultraturrax blender for extraction resulted in higher FB1 recoveries compared to a rotary shaker. In contrast to other reports, maximum fluorescence response occurred after a derivatisation time of 8 min. These results reflect on the absolute requirement for standardisation of the aforementioned parameters.

Effect of water activity and temperature on growth and the relationship between fumonisin production and the radial growth of Fusarium verticillioides and Fusarium proliferatum on corn.

The two major fumonisin-producing Fusarium species are Fusarium verticillioides and Fusarium proliferatum. The growth and fumonisin production of these two isolates on corn was studied at water activities (a(w)) between 0.860 and 0.975 and at temperatures between 15 and 30 degrees C. Growth rates (g, mm/day) were obtained by linear regression during the linear phase of growth. In general, growth rates for both isolates increased significantly (P < 0.05) with increases in a(w) and temperature. Both fumonisin production and radial growth (mycelial development) for both isolates increased with a(w) at all temperatures investigated, but the effect of temperature on this relationship was not obvious. The effect of temperature on fumonisin production at high a(w) values optimal for growth was only marginal, whereas at lower a(w) values the effect of temperature was more pronounced, with more fumonisin production occurring at temperatures not optimal for growth. The optimum temperature for fumonisin production was between 15 and 25 degrees C. For F. proliferatum, the optimum temperature for growth at all a(w) values, 30 degrees C, resulted in the poorest fumonisin production. For both isolates, the slowest initial rate of fumonisin production was at 15 degrees C, the temperature at which the slowest growth rates were obtained.