16S and 18S rDNA Amplicon Sequencing Analysis of Aesthetically Problematic Microbial Mats on the Walls of the Petralona Cave: The Use of Essential Oils as a Cleaning Method.

The presence of microbial communities on cave walls and speleothems is an issue that requires attention. Traditional cleaning methods using water, brushes, and steam can spread the infection and cause damage to the cave structures, while chemical agents can lead to the formation of toxic compounds and damage the cave walls. Essential oils (EOs) have shown promising results in disrupting the cell membrane of bacteria and affecting their membrane permeability. In this study, we identified the microorganisms forming unwanted microbial communities on the walls and speleothems of Petralona Cave using 16S and 18S rDNA amplicon sequencing approaches and evaluated the efficacy of EOs in reducing the ATP levels of these ecosystems. The samples exhibited a variety of both prokaryotic and eukaryotic microorganisms, including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, the SAR supergroup, Opisthokonta, Excavata, Archaeplastida, and Amoebozoa. These phyla are often found in various habitats, including caves, and contribute to the ecological intricacy of cave ecosystems. In terms of the order and genus taxonomy, the identified biota showed abundances that varied significantly among the samples. Functional predictions were also conducted to estimate the differences in expressed genes among the samples. Oregano EO was found to reduce ATP levels by 87% and 46% for black and green spots, respectively. Consecutive spraying with cinnamon EO further reduced ATP levels, with reductions of 89% for black and 88% for green spots. The application of a mixture solution caused a significant reduction up to 96% in ATP levels of both areas. Our results indicate that EOs could be a promising solution for the treatment of microbial communities on cave walls and speleothems.

Use of risk assessment and predictive microbiology in regulatory science related to the scientific opinions of the EFSA BIOHAZ Panel.

EFSA's Panel on Biological Hazards (BIOHAZ Panel) deals with questions on biological hazards relating to food safety and food-borne diseases. This covers food-borne zoonoses, transmissible spongiform encephalopathies, antimicrobial resistance, food microbiology, food hygiene, animal-by products, and associated waste management issues. The scientific assessments are diverse and frequently the development of new methodological approaches is required to deal with a mandate. Among the many risk factors, product characteristics (pH, water activity etc.), time and temperature of processing and storage along the food supply chain are highly relevant for assessing the biological risks. Therefore, predictive microbiology becomes an essential element of the assessments. Uncertainty analysis is incorporated in all BIOHAZ scientific assessments, to meet the general requirement for transparency. Assessments should clearly and unambiguously state what sources of uncertainty have been identified and their impact on the conclusions of the assessment. Four recent BIOHAZ Scientific Opinions are presented to illustrate the use of predictive modelling and quantitative microbial risk assessment principles in regulatory science. The Scientific Opinion on the guidance on date marking and related food information, gives a general overview on the use of predictive microbiology for shelf-life assessment. The Scientific Opinion on the efficacy and safety of high-pressure processing of food provides an example of inactivation modelling and compliance with performance criteria. The Scientific Opinion on the use of the so-called 'superchilling' technique for the transport of fresh fishery products illustrates the combination of heat transfer and microbial growth modelling. Finally, the Scientific Opinion on the delayed post-mortem inspection in ungulates, shows how variability and uncertainty, were quantitatively embedded in assessing the probability of Salmonella detection on carcasses, via stochastic modelling and expert knowledge elicitation.

Studying the metabolic factors that may impact the growth of co-cultured Listeria monocytogenes strains at low temperature.

The simultaneous presence of more than one strains of Listeria monocytogenes in the same food product may affect the growth capacity of each strain. The present study evaluated the metabolites composition that may potentially influence the growth of individual L. monocytogenes strains in a dual strain composite. Based on previous studies, L. monocytogenes strains, C5 (4b) and 6179 (1/2a) were selected due to the remarkable interaction, which was observed during their co-culture. The selected strains were inoculated (2.0 - 3.0 log CFU/mL) in Tryptic Soy Broth with 0.6% Yeast Extract (TSB-YE) in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic conditions (AC). Their resistance to different antibiotics enabled the selective enumeration of each strain in the co-culture. After reaching stationary phase, single and dual cultures were centrifuged and filtered. The cell-free spent medium (CFSM) was either characterized by Fourier transform infrared (FTIR-ATR) spectrometry or re-inoculated, after the addition of concentrated TSB-YE (for nutrient replenishment), with single and two-strain cultures for the evaluation of growth under the influence of metabolites produced from the same singly and co-cultured strains in the different combinations of strains and CFSM origin (7 °C/AC) (n = 2x3). By the end of storage, singly-cultured C5 and 6179 had reached 9.1 log CFU/mL, while in dual culture, 6179 was affected by the presence of C5 attaining only 6.4 ± 0.8 log CFU/mL. FTIR-ATR spectra of CFSM produced by singly-cultured 6179 and the co-culture were almost identical. Characteristic peaks in FTIR-ATR spectrum of CFSM of singly-cultured C5 at 1741, 1645 and 1223 cm-1 represent functional groups which were not present in the CFSM of the co-culture. These molecules may be located intracellularly or mounted on bacterial cell surface and removed from the supernatant during cell filtration of the co-culture. Both singly- and co-cultured 6179 managed to grow similarly regardless of CFSM origin. Contrarily, both singly- and co-cultured C5 managed to outgrow 6179 in CFSM which contained high concentration of C5 metabolites, while in CFSM produced by singly-cultured 6179, C5 did not grow, suggesting that the produced metabolites of strain 6179 appears to be harmful to strain C5. However, during co-culture, C5 may produce molecules that counteract the inhibitory effect of 6179. The findings shed more light on the mechanism behind the inter-strain interactions of L. monocytogenes indicating that both contact of cells and extracellular metabolites may influence the behavior of the different co-existing strains.

Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

Short-Term Effects of Traditional Greek Meals: Lentils with Lupins, Trahana with Tomato Sauce and Halva with Currants and Dried Figs on Postprandial Glycemic Responses-A Randomized Clinical Trial in Healthy Humans.

Low glycemic index (GI) diets have been associated with decreased chronic disease risk. In a randomized, cross-over study we investigated the GI and glycemic response to three traditional Greek mixed meals: Lentils, Trahana, and Halva. Twelve healthy, fasting individuals received isoglucidic test meals (25 g available carbohydrate) and 25 g glucose reference, in random order. GI was calculated and capillary blood glucose (BG) samples were collected at 0-120 min after meal consumption. Subjective appetite ratings were assessed. All three tested meals provided low GI values. Lentils GI was 27 ± 5, Trahana GI was 42 ± 6, and Halva GI was 52 ± 7 on glucose scale. Peak BG values were lowest for Lentils, followed by Trahana and then by Halva (p for all <0.05). Compared to the reference food, BG concentrations were significantly lower for all meals at all time-points (p for all <0.05). Lentils provided lower glucose concentrations at 30 and 45 min compared to Trahana (p for all <0.05) and at 30, 45, and 60 min compared to Halva (p for all <0.05). BG concentrations did not differ between Trahana and Halva at all time points. No differences were observed for fasting BG, time to peak rise for BG, and subjective appetite ratings. In conclusion, all three mixed meals attenuated postprandial glycemic response in comparison to glucose, which may offer advantages to glycemic control.

Studying the effect of oxygen availability and matrix structure on population density and inter-strain interactions of Listeria monocytogenes in different dairy model systems.

Due to the ubiquitous character of Listeria monocytogenes multiple strains of the pathogen may end up co-existing in/on the same final products and could potentially cause infection during consumption. Such multiple strain contamination may occur in different stages of the food supply chain. The present study evaluated the effect of oxygen availability and matrix structure on inter-strain interactions of L. monocytogenes that may occur at high population levels in/on different dairy model systems. L. monocytogenes strains C5 and ScottA (4b), 6179 (1/2a) and PL25 (1/2b) were selected as resistant to different antibiotics (enabling selective enumeration of each strain in co-culture) and inoculated (2.0-3.0 log CFU/mL, g or cm2) in Ricotta and Camembert broth (1 dairy product: 2 » Ringer solution) and in/on dairy-based structured media (dairy broth supplemented with 0.6 and 1.4% agar), in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic, hypoxic and anoxic conditions. Every experimental treatment was tested with three biological replicates and two technical repeats (n = 3 × 2). The simultaneously presence of different strains of the pathogen in/on the same substrate did not affect neither the duration of the lag phase nor the growth rate of the co-cultured strains. The observed inter-strain interactions were related with the final population reached/decrease during storage and occurred after the "critical" population density of ca. 6.0 log CFU/mL, g or cm2. The phenomenon was more pronounced in/on Ricotta than in/on Camembert-based substrates, indicating that the composition and the available nutrients of the substrate may affect the interactions that expressed as difference in the final population level between singly and co-cultured strains. Under aerobic and hypoxic conditions, most of the observed interactions were more pronounced in dairy-based broths and were mitigated with the addition of agar. The elimination of oxygen resulted in a prolonged lag time, which lasted at least 5 days and no observed interactions by the end of storage, due to low microbial counts. Investigating inter-strain interactions during growth in/on different substrates, which may have undergone temperature abuse during their transport along the supply chain or during storage in household refrigerators, could assist in explaining the mismatch between clinical and food samples during outbreak investigations.

Effects of Spaghetti Differing in Soluble Fiber and Protein Content on Glycemic Responses in Humans: A Randomized Clinical Trial in Healthy Subjects.

This randomized, single blind, cross-over study investigated the glycemic responses to three spaghetti No 7 types differing in dietary protein and soluble fiber content. Fourteen clinically and metabolically healthy, fasting individuals (25 ± 1 years; ten women; BMI 23 ± 1 kg/m2) received isoglucidic test meals (50 g available carbohydrate) and 50 g glucose reference, in random order. GI was calculated using the FAO/WHO method. Capillary blood glucose and salivary insulin samples were collected at 0, 15, 30, 45, 60, and 120 min. Subjective appetite ratings (hunger, fullness, and desire to eat) were assessed by visual analogue scales (VAS, 100 mm) at baseline and 120 min. All three spaghetti types (regular, whole wheat, and high soluble fiber-low carbohydrates) provided low GI values (33, 38, and 41, respectively, on glucose scale) and lower peak glucose values compared to glucose or white bread. No differences were observed between spaghetti No 7 types for fasting glucose, fasting and post-test-meal insulin concentrations, blood pressure (systolic and diastolic), and subjective appetite. Conclusions: all spaghetti No 7 types, regardless of soluble fiber and/or protein content, attenuated postprandial glycemic response, which may offer advantages to glycemic control.

Microbial Ecology of Sheep Milk, Artisanal Feta, and Kefalograviera Cheeses. Part II: Technological, Safety, and Probiotic Attributes of Lactic Acid Bacteria Isolates.

The aim of the present study was to examine 189 LAB strains belonging to the species Enterococcus faecium, E. faecalis, Lactococcus lactis, Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactiplantibacillus pentosus, Latilactobacillus curvatus, Lp. plantarum, Levilactobacillus brevis, and Weissella paramesenteroides isolated form sheep milk, Feta and Kefalograviera cheeses at different ripening stages, for their technological compatibility with dairy products manufacturing, their activities that may compromise safety of the dairy products as well as their capacity to survive in the human gastrointestinal tract. For that purpose, milk acidification and coagulation capacity, caseinolytic, lipolytic, hemolytic, gelatinolytic, and bile salt hydrolase activity, production of exopolysaccharides, antimicrobial compounds, and biogenic amines, as well as acid and bile salt tolerance and antibiotic susceptibility were examined. The faster acidifying strains were Lc. lactis DRD 2658 and P. pentosaceus DRD 2657 that reduced the pH value of skim milk, within 6 h to 5.97 and 5.92, respectively. Strains able to perform weak caseinolysis were detected in all species assessed. On the contrary, lipolytic activity, production of exopolysaccharides, amino acid decarboxylation, hemolytic, gelatinase, and bile salt hydrolase activity were not detected. Variable susceptibility to the antibiotics examined was detected among LAB strains. However, in the majority of the cases, resistance was evident. None of the strains assessed, managed to survive to exposure at pH value 1. On the contrary, 25.9 and 88.9% of the strains survived after exposure at pH values 2 and 3, respectively; the reduction of the population was larger in the first case. The strains survived well after exposure to bile salts. The strain-dependent character of the properties examined was verified. Many strains, belonging to different species, have presented very interesting properties; however, further examination is needed before their potential use as starter or adjunct cultures.

Microbial Ecology of Artisanal Feta and Kefalograviera Cheeses, Part I: Bacterial Community and Its Functional Characteristics with Focus on Lactic Acid Bacteria as Determined by Culture-Dependent Methods and Phenotype Microarrays.

Artisanal cheesemaking is still performed using practices and conditions derived from tradition. Feta and Kefalograviera cheeses are very popular in Greece and have met worldwide commercial success. However, there is a lack of knowledge regarding their lactic acid microecosystem composition and species dynamics during ripening. Thus, the aim of the present study was to assess the microecosystem as well as the autochthonous lactic acid microbiota during the ripening of artisanal Feta and Kefalograviera cheeses. For that purpose, raw sheep's milk intended for cheesemaking, as well as Feta and Kefalograviera cheeses during early and late ripening were analyzed, and the lactic acid microbiota was identified using the classical phenotypic approach, clustering with PCR-RAPD and identification with sequencing of the 16S-rRNA gene, as well as with the Biolog GEN III microplates. In addition, the functional properties of the bacterial community were evaluated using the Biolog EcoPlates, which consists of 31 different carbon sources. In general, concordance between the techniques used was achieved. The most frequently isolated species from raw sheep's milk were Enteroroccus faecium, Lactiplantibacillus plantarum and Pediococcus pentosaceus. The microecosystem of Feta cheese in the early ripening stage was dominated by Lp. plantarum and E. faecium, whereas, in late ripening, the microecosystem was dominated by Weissella paramesenteroides. The microecosystem of Kefalograviera cheese in the early ripening stage was dominated by Levilactobacillus brevis and E. faecium, and in late ripening by W. paramesenteroides and E. faecium. Finally, Carbohydrates was the main carbon source category that metabolized by all microbial communities, but the extent of their utilization was varied. Kefalograviera samples, especially at early ripening, demonstrated higher metabolic activity compared to Feta cheese. However, dominating species within microbial communities of the cheese samples were not significantly different.

In Vitro Virulence Potential, Surface Attachment, and Transcriptional Response of Sublethally Injured Listeria monocytogenes following Exposure to Peracetic Acid.

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes (motB and flaA) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA. Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

Evaluation of antimicrobial activities of plant aqueous extracts against Salmonella Typhimurium and their application to improve safety of pork meat.

Nine odorless laboratory-collected hydro-distilled aqueous extracts (basil, calendula, centrifuged oregano, corn silk, laurel, oregano, rosemary, spearmint, thyme) and one industrial steam-distilled oregano hydrolate acquired as by-products of essential oils purification were screened for their in vitro antimicrobial activity against three Salmonella Typhimurium strains (4/74, FS8, FS115) at 4 and 37 °C. Susceptibility to the extracts was mainly plant- and temperature-dependent, though strain dependent effects were also observed. Industrial oregano hydrolate eliminated strains immediately after inoculation, exhibiting the highest antimicrobial potential. Hydro-distilled extracts eliminated/reduced Salmonella levels during incubation at 4 °C. At 37 °C, oregano, centrifuged oregano, thyme, calendula and basil were bactericidal while spearmint, rosemary and corn silk bacteriostatic. A strain-dependent effect was observed for laurel. The individual or combined effect of marinades and edible coatings prepared of industrial hydrolate and hydro-distilled oregano extracts with or without oregano essential oil (OEO) was tested in pork meat at 4 °C inoculated with FS8 strain. Lower in situ activity was observed compared to in vitro assays. Marinades and edible coatings prepared of industrial oregano hydrolate + OEO were the most efficient in inhibiting pathogen. Marination in oregano extract and subsequent coating with either 50% oregano extract + OEO or water + OEO enhanced the performance of oregano extract. In conclusion, by-products of oregano essential oil purification may be promising alternative antimicrobials to pork meat stored under refrigeration when applied in the context of multiple hurdle approach.

Whole-genome sequence data of the proteolytic and bacteriocin producing strain Enterococcus faecalis PK23 isolated from the traditional Halitzia cheese produced in Cyprus.

Halitzia is a traditional white-brined cheese produced by a limited number of producers in Cyprus. During a survey of the microbiome of a number of different Halitzia samples, we identified a bacterial strain that exhibited enhanced proteolytic activity compared to the other isolates. The strain was further studied, and it was assigned as Enterococcus faecalis PK23. We proceeded with sequencing of its whole genome using Illumina technology. Initial sequencing and assembly produced 116 scaffolds with a length of 3,149,036 bp. Comparison with the available E. faecalis genomes revealed that the strain PK23 exhibited high levels of identity to the genome sequence of E. faecalis isolate 26975_2#180 deposited in GenBank as a single complete contig. From the 116 scaffolds 106 could be aligned to the genome of isolate 26975_2#180 leading to a chromosomal length of 3,132,784 bp with a GC content of 37.3%. From the remaining 10 scaffolds, five showed similarity to plasmid sequences. More specifically, scaffold 54 showed high identity with most part of plasmid pEF1071 of E. faecalis strain BFE 1071, which carries the gene cluster involved in the biosynthesis of enterocins 1071A and 1071B, while scaffold 77 showed high identity with the entire sequence of the unnamed_5 cryptic plasmid of Enterococcus faecium strain PR05720-3. The other three scaffolds were only short parts of larger plasmids. The remaining five scaffolds which could not be related to any plasmid sequence most probably constitute chromosomal sequences present in strain PK23 but absent from isolate 26975_2#180. Their total length was around 2.7 kb, which does not affect the sequence of the PK23 pseudochromosome in a major way. The whole-genome sequence annotation of strain PK23 identified 3161 coding sequences and 62 RNA sequences. The results from the Rapid Annotation using Subsystem Technology (RAST) version 2.0 server indicated the presence of seven putative genes which were related to the subsystem of Protein Degradation. This dataset provides a first overview of the proteolytic and bacteriocin producing properties of E. faecalis PK23. The dataset may also be used in future experiments which could shed light on the adaptation of the strain in the dairy environment and its role in cheese production.

High-quality draft genome sequence data of six Lactiplantibacillus plantarum subsp. argentoratensis strains isolated from various Greek wheat sourdoughs.

Lactiplantibacillus plantarum is a species found in a wide range of foods and other commodities. It can be used as starter or adjunct culture in fermented foods. Herein the annotated high-quality draft genome (scaffolds) of six L. plantarum subsp. argentoratensis strains (LQC 2320, LQC 2422, LQC 2441, LQC 2485, LQC 2516 and LQC 2520) isolated from various Greek wheat sourdoughs is presented. Raw sequence reads were quality checked, assembled into larger contiguous sequences and scaffolds were annotated. The total size of the genomes ranged from 3.13 Mb to 3.49 Mb and the GC content from 45.02% to 45.13%. The total number of coding and non-coding genes were between 3268 and 3723 (3091 to 3492 protein-coding genes, 62 to 107 repeat-region, 54 to 59 tRNAs and 2 to 5 rRNAs, 20 to 30 crispr-repeats, 17 to 26 crispr-spacers and 2 to 4 crispr-arrays). The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers JAEQMR000000000, JAEQMQ000000000, JAEQMP000000000, JAEQMO000000000, JAEQMN000000000 and JAEQMM000000000. The version described in this paper is version JAEQMR010000000, JAEQMQ010000000, JAEQMP010000000, JAEQMO010000000, JAEQMN010000000 and JAEQMM010000000. Raw sequence reads have been submitted in the Sequence Read Archive (SRA) under the BioProject accession number PRJNA689714 (BioSample accession numbers SAMN17215143, SAMN17215144, SAMN17215145, SAMN17215146, SAMN17215147 and SAMN17215148 and SRA accession numbers SRR13357463, SRR13357464, SRR13357465, SRR13357466, SRR13357467, SRR13357468).

Effect οf οxygen availability and pH οn adaptive acid tolerance response of immobilized Listeria monocytogenes in structured growth media.

The aim of the present study was to evaluate the effect of oxygen availability (aerobic, hypoxic and anoxic conditions) and sub-optimal pH (6.2 and 5.5) in a structured medium (10% w/V gelatin) on the growth of two immobilized L. monocytogenes strains (C5, 6179) at 10 °C and their subsequent acid resistance (pH 2.0, e.g., gastric acidity). Anaerobic conditions resulted in lower bacterial population (P < 0.05) (7.8-8.2 log CFU/mL) at the end of storage than aerobic and hypoxic environment (8.5-9.0 log CFU/mL), a phenomenon that was intensified at lower pH (5.5), where no significant growth was observed for anaerobically grown cultures. Prolonged habituation of L. monocytogenes (15 days) at both pH increased its acid tolerance resulting in max. 10 times higher t4D (appx. 60 min). The combined effect though of oxygen availability and suboptimal pH on L. monocytogenes acid resistance was found to vary with the strain. Anoxically grown cultures at pH 5.5 exhibited the lowest tolerance towards lethal acid stress, with countable survivors occurring only until 20 min of exposure at pH 2.0. Elucidating the role of oxygen limiting conditions, often encountered in structured foods, on acid resistance of L. monocytogenes, would assist in assessing the capacity of L. monocytogenes originated from different food-related niches to withstand gastric acidity and possibly initiate infection.

Technological and Safety Attributes of Lactic Acid Bacteria and Yeasts Isolated from Spontaneously Fermented Greek Wheat Sourdoughs.

The aim of the present study was to assess the technological and safety potential of 207 lactic acid bacteria (LAB) and 195 yeast strains isolated from spontaneously fermented Greek wheat sourdoughs. More accurately, the amylolytic, proteolytic, lipolytic, phytase and amino acid decarboxylase activities, along with the production of exopolysaccharides and antimicrobial compounds by the LAB and yeast isolates, were assessed. A well diffusion assay revealed seven proteolytic LAB and eight yeast strains; hydrolysis of tributyrin was evident only in 11 LAB strains. A further Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicated partial hydrolysis of gluten. Lipolysis kinetics over 21 days was applied, exhibiting that lipolytic activity ranged from 6.25 to 65.50 AU/mL. Thirteen LAB inhibited Penicillium olsonii and Aspergillus niger growth and 12 yeast strains inhibited Pe. chrysogenum growth. Twenty-one Lactiplantibacillus plantarum strains exhibited inhibitory activity against Listeria monocytogenes, as well as several sourdough-associated isolates. The structural gene encoding plantaricin 423 was detected in 19 Lcb. plantarum strains, while the structural genes encoding plantaricins NC8, PlnE/F, PlnJ/K, and S were detected in two Lcb. plantarum strains. None of the microbial strains tested exhibited exopolysaccharide (EPS) production, amino acid decarboxylase, amylolytic or phytase activity. The technological and safety potential of the Lcb. plantarum and Wickerhamomyces anomalus strains was highlighted, since some of them exhibited proteolytic, lipolytic, antibacterial and antimould activities.

Εvaluation of oxygen availability on growth and inter-strain interactions of L. monocytogenes in/on liquid, semi-solid and solid laboratory media.

The coexistence and interactions among Listeria monocytogenes strains in combination with the structural characteristics of foods, may influence their growth capacity and thus, the final levels at the time of consumption. In the present study, we aimed to evaluate the effect of oxygen availability in combination with substrate micro-structure on growth and inter-strain interactions of L. monocytogenes. L. monocytogenes strains, selected for resistance to different antibiotics (to enable distinct enumeration), belonging to serotypes 4b (C5, ScottA), 1/2a (6179) and 1/2b (PL25) and were inoculated in liquid (Tryptic Soy Broth supplemented with Yeast Extract - TSB-YE) and solid (TSB-YE supplemented with 0.6% and 1.2% agar) media (2-3 log CFU/mL, g or cm2), single or as two-strain cultures (1:1 strain-ratio). Aerobic conditions (A) were achieved with constant shaking or surface inoculation for liquid and solid media respectively, while static incubation or pour plated media corresponded to hypoxic environment (H). Anoxic conditions (An) were attained by adding 0.1% w/v sodium thioglycolate and paraffin overlay (for solid media). Growth was assessed during storage at 7 °C (n = 3 × 2). Inter-strain interactions were manifested by the difference in the final population between singly and co-cultured strains. Τhe extent of suppression increased with reduction in agar concentration, while the impact of oxygen availability was dependent on strain combination. During co-culture, in liquid and solid media, 6179 was suppressed by C5 by 4.0 (in TSB-YE under H) to 1.8 log units (in solid medium under An), compared to the single culture, which attained population of ca. 9.4 log CFU/mL or g. The growth of 6179 was also inhibited by ScottA by 2.7 and 1.9 log units, in liquid culture under H and An, respectively. Interestingly, in liquid medium under A, H and An, ScottA was suppressed by C5, by 3.3, 2.4 and 2.3 log units, respectively, while in solid media, growth inhibition was less pronounced. Investigating growth interactions in different environments could assist in explaining the dominance of L. monocytogenes certain serotypes.

Application of Enterococcus faecium KE82, an Enterocin A-B-P-Producing Strain, as an Adjunct Culture Enhances Inactivation of Listeria monocytogenes during Traditional Protected Designation of Origin Galotyri Processing.

ABSTRACT: The ability of the enterocin A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during protected designation of origin Galotyri processing was evaluated. Three trials were conducted with artisan cheeses made from traditionally "boiled" (85°C) ewe's milk. The milk was cooled at 42°C and divided in two treatments. A1 milk was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 was inoculated with the basic starter ST1+M78 plus KE82 (step 1). All milks were fermented at 20 to 22°C for 24 h (step 2), and the curds were drained at 12°C for 72 h (step 3) and then salted with 1.5 to 1.8% salt to obtain the fresh Galotyri cheeses (step 4). These fresh cheeses were then ripened at 4°C for 30 days (step 5). Because artificial listerial contamination in the dairy plant was prohibited, samples of A1 and A2 cheese milk (200 mL) or curd (200 g) were collected after steps 1 through 5, inoculated with L. monocytogenes 10 (3 to 4 log CFU/mL or g), incubated at 37, 22, 12, and 4°C for predefined periods, and analyzed for microbial levels and pH. L. monocytogenes levels declined in all cheese curd portions contaminated after steps 2 through 5 (pH 4.36 to 4.84) when stored at 4 or 12°C for 15 days. The final net reductions in Listeria populations were 2.00-, 1.07-, 0.54-, and 0.61-log greater in the A2 than in the A1 curd portions after steps 2, 3, 4, and 5, respectively. In step 1, conducted to simulate the whole cheese milk fermentation process, L. monocytogenes levels declined by 1.47 log CFU/mL more in the A2 than in the A1 milk portions after 72 h at 22°C; however, slight growth (0.6 log CFU/mL) occurred during the first 6 h at 37°C. E. faecium KE82 was compatible with the starter culture and enhanced inactivation of L. monocytogenes during all steps of Galotyri cheese processing. The antilisterial effects of the combined acid and enterocin were the weakest in the fermenting milks, the strongest in the unsalted fermented curds, and declined again in the salted fresh cheeses.

Prior exposure to different combinations of pH and undissociated acetic acid can affect the induced resistance of Salmonella spp. strains in mayonnaise stored under refrigeration and the regulation of acid-resistance related genes.

The innate and inducible resistance of six Salmonella strains (4/74, FS8, FS115, P167807, ATCC 13076, WT) in mayonnaise at 5 °C following adaptation to different pH/undissociated acetic acid (UAA) combinations (15mM/pH5.0, 35mM/pH5.5, 45mM/pH6.0) was investigated. The inherent and acid-induced responses were strain-dependent. Two strains (ATCC 13076, WT), albeit not the most resistant innately, exhibited the most prominent adaptive potential. Limited/no adaptability was observed regarding the rest strains, though being more resistant inherently. The individual effect of pH and UAA adaptation in the phenotypic and transcriptomic profiles of ATCC 13076 and WT was further examined. The type (pH, UAA) and magnitude of stress intensity affected their responses. Variations in the type and magnitude of stress intensity also determined the relative gene expression of four genes (adiA, cadB, rpoS, ompR) implicated in Salmonella acid resistance mechanisms. adiA and cadB were overexpressed following adaptation to some treatments; rpoS and ompR were downregulated following adaptation to 15mM/pH5.0 and 35mM/pH5.5, respectively. Nonetheless, the transcriptomic profiles did not always correlate with the corresponding phenotypes. In conclusion, strain variations in Salmonella are extensive. The ability of the strains to adapt and induce resistant phenotypes and acid resistance-related genes is affected by the type and magnitude of the stress applied during adaptation.

Assessment of Spoilage Potential Posed by Alicyclobacillus spp. in Plant-Based Dairy Beverages Mixed with Fruit Juices during Storage.

ABSTRACT: The scope of the present study was to assess the spoilage potential of different Alicyclobacillus spp. in commercial pasteurized (ambient-stable) plant-based dairy beverages mixed with fruit juices at different inoculation levels and storage temperatures. Different products (coconut and berry [CB]; almond, mango, and passionfruit [AMP]; and oat, strawberry, and banana [OSB]) were inoculated with 10 or 2 × 103 spores per mL of either Alicyclobacillus acidoterrestris, Alicyclobacillus fastidiosus, or Alicyclobacillus acidocaldarius strain composites, whereas noninoculated samples served as controls. Samples inoculated with A. acidoterrestris and A. fastidiosus were stored at 30 and 45°C, whereas A. acidocaldarius storage took place at 50°C for 240 days. Gas composition, Alicyclobacillus spp. populations, total viable counts, pH, water activity, color, and guaiacol off-taste were monitored. CB and AMP supported growth of A. acidoterrestris and A. fastidiosus, reaching populations of 4.0 to 5.0 log CFU/mL. In OSB, populations of A. fastidiosus remained close to the initial inoculation levels during storage at 30°C, whereas at 45°C, the populations declined <1 CFU/mL. A. acidocaldarius growth was supported in CB samples, but not in AMP and OSB samples, reaching ca. 3.0 log CFU/mL at 50°C, regardless of initial inoculum size. Total color change was increased during storage; however, the instrumentally recorded color changes were not macroscopically visible. Spoilage in terms of guaiacol off-taste was identified only in CB and AMP samples inoculated with A. acidoterrestris after 60 days at 30 and 45°C. The increased popularity of these products along with the scarcity of existing literature related to their spoilage by Alicyclobacillus spp., render the contribution of the findings and data of present study critical for assessing the significance of Alicyclobacillus spp. as a potential spoilage hazard in these products and for assisting in the design and implementation of effective mitigation strategies by the beverage industry.