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Abstract Paecilomyces niveus has long been recognized as an important heat-resistant mold (HRM) causing spoilage in thermally processed apple products. With the recent discovery that P. niveus causes an apple disease called Paecilomyces rot, there is a need to evaluate the possibility that the spores can be introduced via infected fruit and survive thermal processing to contaminate apple products. Previously, research characterizing P. niveus survival and spoilage has been based on simple inoculation of cultured ascospores directly into juice. To determine whether ascospores formed in infected fruit survive through thermal processing with rotating vacuum evaporation (2.5 h, 70 °C), apples infected with P. niveus were used to make apple juice concentrate. Viable P. niveus was quantified in samples taken throughout the apple juice concentrate process: in cider, centrifuged juice, apple solids, and in juice made from concentrate. Additionally, the concentration of the mycotoxin patulin was determined in a subset of samples. Results show that P. niveus survived juice concentration. The thermal process used significantly reduced P. niveus (P = 0.0017); from an average of 5.5 log CFU L−1 in the initial cider made from diseased fruit to an average of 1.5 log CFU L−1 in single strength apple juice diluted from concentrate. Ascospores were observed at a greater abundance than intact asci throughout the concentration process. Patulin concentrations at all stages of apple juice concentration exceeded the United States' regulatory defect action level of 50 ppb. This is the first report that apples infected with P. niveus can survive and remain viable in finished apple products and that patulin is produced in these apples. It raises important questions about the use of damaged and infected fruit as a source of HRM which contribute to spoilage and mycotoxins in apple juice concentrate. Highlights • P. niveus spores in infected apples survive the apple juice concentrate process. • Heat-resistant ascospores survived rotating vacuum evaporation (2.5 h, 70 °C). • Apples infected with Paecilomyces rot contain high levels of patulin. [ABSTRACT FROM AUTHOR]

Abstract Different fungi, including the genera Aspergillus (Neosartorya), Paecilomyces (Byssochlamys) and Talaromyces , produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. The sensitivity of the ascospores to treatments with industrial sanitizers containing chlorine dioxide and iodine (iodophors) has never been assessed before. Ascospores of 4 species of Eurotiales were tested and showed clear variations in sensitivity. The most resilient species, T. macrosporus and Pae. variotii (= B. spectabilis) survive 75, but not 200 ppm chlorine dioxide solution treatments. These species were able to survive 75 ppm iodine solution treatments, but relatively low amounts of ascospores (100–1000 spores) could be inactivated after 16 h of treatment. Inactivated spores did not show any sign of germination after 7 days following treatment on growth medium. As judged by microscopy, iodine inactivation resulted in visibly distorted ascospores. For the interpretation of results, the state of dormancy or activation of ascospores is highly important. Highlights • Ascospores of heat-resistant fungi are inactivated by acidified sodium chlorite, chlorine dioxide and iodine solutions. • There were differences among the heat-resistant mould species used in this study. • The status of dormancy of the ascospores is important for the interpretation of their inactivation by these sanitizers. [ABSTRACT FROM AUTHOR]

Abstract: Species of the genus Byssochlamys, heat-resistant fungi, cause the spoilage and poisoning of heat-processed acidic foods due to the formation of heat-resistant ascospores and the biosynthesis of patulin. Here we evaluated the heat resistance and patulin production of four species of Byssochlamys. Byssochlamys fulva was significantly more heat-resistant than the other species, and Byssochlamys lagunculariae and Byssochlamys nivea synthesized patulin. This is the first report of patulin production by B. lagunculariae. These findings indicate that the four species pose different health risks in food. Thus, we concluded that a method to identify the species of Byssochlamys is necessary. Identification at the species level based on morphology alone is difficult. We developed species-specific primers to identify B. nivea, B. fulva, B. lagunculariae, and Byssochlamys zollerniae using polymerase chain reaction (PCR) to amplify the β-tubulin gene. PCR using the primer sets designed for B. nivea (B. nivea1F/1R) amplified PCR products specific for B. nivea. Similarly, PCR using the B.fulva1F/1R, B.lag1F/1R and B.zol3F/R primer sets produced PCR products specific for B. fulva, B. lagunculariae, and B. zollerniae, respectively. PCR using these four primer sets did not detect other fungi involved in food spoilage and environmental contamination. This identification method is rapid and simple with extremely high specificity. [Copyright &y& Elsevier]

Abstract: Although the production of patulin in apple fruits is mainly by Penicillium expansum, there is no information on the ability of heat resistant moulds that may survive pasteurization to produce this mycotoxin in juice packages during storage and distribution. In this study, the production of patulin by Byssochlamys spp (Byssochlamys nivea FRR 4421, B. nivea ATCC 24008 and Byssochlamys fulva IOC 4518) in cloudy and clarified apple juices packaged in laminated paperboard packages or in polyethylene terephthalate bottles (PET) and stored at both 21°C and 30°C, was investigated. The three Byssochlamys strains were able to produce patulin in both cloudy and clarified apple juices. Overall, the lower the storage temperature, the lower the patulin levels and mycelium dry weight in the apple juices (p<0.05). The greatest variations in pH and °Brix were observed in the juices from which the greatest mycelium dry weights were recovered. The maximum levels of patulin recovered from the juices were ca. 150μg/kg at 21°C and 220μg/kg at 30°C. HPLC-UV, HPCL-DAD and mass spectrometry analyses confirmed the ability of B. fulva IOC 4518 to produce patulin. Due to the heat resistance of B. nivea and B. fulva and their ability to produce patulin either in PET bottles or in laminated paperboard packages, the control of contamination and the incidence of these fungi should be a matter of concern for food safety. Control measures taken by juice industries must also focus on controlling the ascospores of heat resistant moulds. [ABSTRACT FROM AUTHOR]

Abstract: The percentage P (%) of spoiled bottles (n =40) of clarified apple juice due to Byssochlamys fulva, was modeled by using a logistic model: where P max (%) the maximum percentage of spoiled bottles, k (h−1) a slope parameter and τ (h) the time for P = P max /2. Bottles of pasteurized apple juice were inoculated with B. fulva IOC 4518 ascospores for low and high initial loads, 4.8±2.3 ascospores/100mL and 19.3±4.6 ascospores/100mL respectively and incubated at 21°C and 30°C. P max was not significantly different from 100% except for a low initial load at 21°C. Model parameters were estimated with a good accuracy, RMSE in the range 3.89–7.50. Then the model was used to determine the time for 10% bottles spoiled, t 10%. This time was greater at low initial loads, 57.4 and 104h at 30 and 21°C respectively, than at high initial loads 23.9 and 75.1h at 30 and 21°C respectively. This study demonstrated that even at a very low initial contamination, clarified apple juice can be easily spoiled by B. fulva highlighting the importance of controlling critical control steps of fruit juice processing (i.e., fruit washing, juice filtration and pasteurization). [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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Abstract: Nucleotide sequences of the isoepoxydon dehydrogenase gene (idh) for eight strains of Byssochlamys nivea were determined by constructing GenomeWalker libraries. A striking finding was that all eight strains of B. nivea examined had identical nucleotide sequences, including those of the two introns present. The length of intron 2 was nearly three times the size of introns in strains of Penicillium expansum and P. griseofulvum, but intron 1 was comparable in size to the number of nucleotides present in introns 1 and 2 of P. expansum and P. griseofulvum. A high degree of amino acid homology (88%) existed for the idh genes of the strains of B. nivea when compared with sequences of P. expansum and P. griseofulvum. There were many nucleotide differences present, but they did not affect the amino acid sequence because they were present in the third position. The identity of the B. nivea isolates was confirmed by sequencing the ITS/partial LSU (28 S) rDNA genes. Four B. nivea strains were analysed for production of patulin, a mycotoxin found primarily in apple juice and other fruit products. The B. nivea strains produced patulin in amounts comparable to P. expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide. [Copyright &y& Elsevier]

The management of Heat Resistant Moulds (HRMs) is considered a great challenge for the juice fruit industry. Neosartorya , Byssochlamys and Talaromyces are three out of the main genera isolated from fruit juices that show great resistance to heat treatments. Several inactivation parameters can be found in the literature, however all of them were carried out in specific food matrices and using diverse inactivation methods. Thus, this meta-analysis study synthesizes the thermal resistance parameters of the three HRMs by adjusting extended Bigelow-based meta-regression models to data on inactivation experiments conducted in different liquid media. The meta-analytical data, extracted from publications between 1969 and 2017, was composed of decimal reduction time (D), inactivation method, temperature of inactivation, pH, °Brix, age of spores, and type of medium (model, juice, concentrates). Pooled D* values (D at 90 °C, pH 3.5 and 12° Brix) were estimated for B. fulva (1.95 min; 95% CI: 1.21–3.11 min), Talaromyces (4.03 min; 95% CI: 3.43–4.74 min), Neosartorya (0.5.35 min; 95% CI: 4.10–7.08 min), and B. nivea (10.32 min; 95% CI: 5.81–18.4 min). It was found that increasing the soluble solids in concentrates tends to cause a lower decrease in the heat resistance of Neosartorya and Talaromyces than increasing the soluble solids in model liquid or juices (p = 0.001; 0.012). In general, the screw-capped tubes and three neck round inactivation methods render higher D* values (p < 0.05) than the thermal death tubes, the polyethylene bag and the capillary methods. Spores of Talaromyces (overall z pH = 7.56; 95% CI: 5.13–13.5) and Neosartorya (overall z pH = 7.07; 95% CI: 5.04–10.8) appear to be more thermal sensitive to a decrease in medium pH than spores of Byssochlamys (overall z pH = 4.34; 95% CI: 3.20–6.73). The meta-regression models presented in this study can be valuable for estimating pooled inactivation kinetic parameters to be used by the fruit juice industry in the management of thermal processes and in the determination of shelf-life. • Pooled D 90°C * were higher for B. nivea > Neosartorya > Talaromyces > B. fulva. • The addition of preservatives reduced z-values from 8.9–10.1 °C to 6.9–6.8 °C. • Screw-capped tube and three neck round render higher D* values than other methods. • Talaromyces and Neosartorya are more heat sensitive to a drop in pH than Byssochlamys. • Meta-regression models are useful in thermal process design and shelf-life estimation. [ABSTRACT FROM AUTHOR]