Inactivation of conidia from three Penicillium spp. isolated from fruit juices by conventional and alternative mild preservation technologies and disinfection treatments.

Fungi are able to grow on diverse food products and contribute to food spoilage worldwide causing food loss. Consumers prefer freshly squeezed fruit juices, however, the shelf life of these juices is limited due to outgrowth of yeast and fungi. The shelf life of pulsed electric field (PEF) treated juice can be extended from 8 days up to a few weeks before spoilage by moulds becomes apparent. Conidia produced by three Penicillium ssp. (Penicillium expansum, Penicillium buchwaldii and Penicillium bialowiezense), previously isolated from spoiled PEF treated fruit juice and smoothie, were characterized for resistance towards selected mild physical processing techniques in orange juice and toward sanitizers on surfaces. The results show that Penicillium spp. conidia are susceptible to mild heat, high pressure pasteurization (HPP), PEF, cold atmospheric plasma (CAP), UV, and chemical sanitizers chlorine dioxide and hypochlorite albeit with different susceptibility. Treatment with mild heat, HPP, PEF, or chlorine dioxide reduced conidia by more than 5 log. For hypochlorite, UV, and CAP the reduction was between 1 and 3 log. Together, this study provides data for the development of intervention strategies to eliminate spoilage mould conidia in fruit juices.

Characterization of four Paenibacillus species isolated from pasteurized, chilled ready-to-eat meals.

Food spoilage is often caused by microorganisms. The predominant spoilage microorganisms of pasteurized, chilled ready-to-eat (RTE) mixed rice-vegetable meals stored at 7°C were isolated and determined as Paenibacillus species. These sporeforming psychrotrophic bacteria are well adapted to grow in the starch-rich environment of pasteurized and chilled meals. Growth of the Paenibacillus isolates appeared to be delayed by decreased (<7°C) temperature or chilled temperature (7°C) combined with decreased pH (<5), increased sodium chloride (>5.5%, corresponding with an aw<0.934), or decreased aw (<0.931; using sucrose). To gain insight in the effect of the pasteurization processing of the meal on spore inactivation, heat-inactivation kinetics were determined and D-values were calculated. According to these kinetics, pasteurization up to 90°C, necessary for inactivation of vegetative spoilage microorganisms and pathogens, does not significantly contribute to the inactivation of Paenibacillus spores in the meals. Furthermore, outgrowth of pasteurized spores was determined in the mixed rice-vegetable meal at several temperatures; P. terrae FBR-61 and P. pabuli FBR-75 isolates did not substantially increase in numbers during storage at 2°C, but had a significant increase within a month of storage at 4°C or within several days at 22°C. Overall, this work shows the importance of Paenibacillus species as spoilage microorganisms of pasteurized, chilled RTE meals and that the meals' matrix, processing conditions, and storage temperature are important hurdles to control microbial meal spoilage.

Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores.

Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity.

Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation.

BACKGROUND: To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. RESULTS: While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33 ± 0.02 h(-1) and 0.34 ± 0.03 h(-1), respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. CONCLUSIONS: Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium.

Genotypic adaptations associated with prolonged persistence of Lactobacillus plantarum in the murine digestive tract.

Probiotic bacteria harbor effector molecules that confer health benefits, but also adaptation factors that enable them to persist in the gastrointestinal tract of the consumer. To study these adaptation factors, an antibiotic-resistant derivative of the probiotic model organism Lactobacillus plantarum WCFS1 was repeatedly exposed to the mouse digestive tract by three consecutive rounds of (re)feeding of the longest persisting colonies. This exposure to the murine intestine allowed the isolation of intestine-adapted derivatives of the original strain that displayed prolonged digestive tract residence time. Re-sequencing of the genomes of these adapted derivatives revealed single nucleotide polymorphisms as well as a single nucleotide insertion in comparison with the genome of the original WCFS1 strain. Detailed in silico analysis of the identified genomic modifications pinpointed that alterations in the coding regions of genes encoding cell envelope associated functions and energy metabolism appeared to be beneficial for the gastrointestinal tract survival of L. plantarum WCFS1. This work demonstrates the feasibility of experimental evolution for the enhancement of the gastrointestinal residence time of probiotic strains, while full-genome resequencing of the adapted isolates provided clues towards the bacterial functions involved. Enhanced gastrointestinal residence is industrially relevant because it enhances the efficacy of the delivery of viable probiotics in situ.

Congruent strain specific intestinal persistence of Lactobacillus plantarum in an intestine-mimicking in vitro system and in human volunteers.

BACKGROUND: An important trait of probiotics is their capability to reach their intestinal target sites alive to optimally exert their beneficial effects. Assessment of this trait in intestine-mimicking in vitro model systems has revealed differential survival of individual strains of a species. However, data on the in situ persistence characteristics of individual or mixtures of strains of the same species in the gastrointestinal tract of healthy human volunteers have not been reported to date. METHODOLOGY/PRINCIPAL FINDINGS: The GI-tract survival of individual L. plantarum strains was determined using an intestine mimicking model system, revealing substantial inter-strain differences. The obtained data were correlated to genomic diversity of the strains using comparative genome hybridization (CGH) datasets, but this approach failed to discover specific genetic loci that explain the observed differences between the strains. Moreover, we developed a next-generation sequencing-based method that targets a variable intergenic region, and employed this method to assess the in vivo GI-tract persistence of different L. plantarum strains when administered in mixtures to healthy human volunteers. Remarkable consistency of the strain-specific persistence curves were observed between individual volunteers, which also correlated significantly with the GI-tract survival predicted on basis of the in vitro assay. CONCLUSION: The survival of individual L. plantarum strains in the GI-tract could not be correlated to the absence or presence of specific genes compared to the reference strain L. plantarum WCFS1. Nevertheless, in vivo persistence analysis in the human GI-tract confirmed the strain-specific persistence, which appeared to be remarkably similar in different healthy volunteers. Moreover, the relative strain-specific persistence in vivo appeared to be accurately and significantly predicted by their relative survival in the intestine-mimicking in vitro assay, supporting the use of this assay for screening of strain-specific GI persistence.

Transcriptomes reveal genetic signatures underlying physiological variations imposed by different fermentation conditions in Lactobacillus plantarum.

Lactic acid bacteria (LAB) are utilized widely for the fermentation of foods. In the current post-genomic era, tools have been developed that explore genetic diversity among LAB strains aiming to link these variations to differential phenotypes observed in the strains investigated. However, these genotype-phenotype matching approaches fail to assess the role of conserved genes in the determination of physiological characteristics of cultures by environmental conditions. This manuscript describes a complementary approach in which Lactobacillus plantarum WCFS1 was fermented under a variety of conditions that differ in temperature, pH, as well as NaCl, amino acid, and O(2) levels. Samples derived from these fermentations were analyzed by full-genome transcriptomics, paralleled by the assessment of physiological characteristics, e.g., maximum growth rate, yield, and organic acid profiles. A data-storage and -mining suite designated FermDB was constructed and exploited to identify correlations between fermentation conditions and industrially relevant physiological characteristics of L. plantarum, as well as the associated transcriptome signatures. Finally, integration of the specific fermentation variables with the transcriptomes enabled the reconstruction of the gene-regulatory networks involved. The fermentation-genomics platform presented here is a valuable complementary approach to earlier described genotype-phenotype matching strategies which allows the identification of transcriptome signatures underlying physiological variations imposed by different fermentation conditions.

Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers.

BACKGROUND: Lactic acid bacteria (LAB) are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. METHODOLOGY/PRINCIPAL FINDINGS: In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI) survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log) and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na(+)/H(+) antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. CONCLUSION: Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-)robustness, which is important for our molecular understanding of GI-tract survival and could facilitate the design of culture conditions aimed to enhance probiotic culture robustness.

Short- and long-term adaptation to ethanol stress and its cross-protective consequences in Lactobacillus plantarum.

This paper describes the molecular responses of Lactobacillus plantarum WCFS1 toward ethanol exposure. Global transcriptome profiling using DNA microarrays demonstrated adaptation of the microorganism to the presence of 8% ethanol over short (10-min and 30-min) and long (24-h) time intervals. A total of 57 genes were differentially expressed at all time points. Expression levels of an additional 859 and 873 genes were modulated after 30 min and 24 h of exposure to the solvent, respectively. Ethanol exposure led to induced expression of genes involved in citrate metabolism and cell envelope architecture, as well as canonical stress response pathways controlled by the central stress regulators HrcA and CtsR. Correspondingly, cells grown for 24 h in medium containing 8% ethanol exhibited higher levels of citrate consumption and modified cell membrane fatty acid composition and showed invaginating septa compared with cells grown in liquid medium without ethanol. In addition, these physiological changes resulted in cross-protection against high temperatures but not against several other stresses tested. To evaluate the role of HrcA and CtsR in ethanol tolerance, ctsR and hrcA gene deletion mutants were constructed. The growth rate of the L. plantarum ΔctsR::cat strain was impaired in de Man-Rogosa-Sharpe (MRS) medium containing 8% ethanol, whereas growth of the L. plantarum ΔhrcA::cat and ΔctsR ΔhrcA::cat mutants was indistinguishable from that of wild-type cells. Overall, these results suggest that the induction of CtsR class III stress responses provides cross-protection against heat stress.