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.

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.

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.

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.

Differential Modulation of Listeria monocytogenes Fitness, In Vitro Virulence, and Transcription of Virulence-Associated Genes in Response to the Presence of Different Microorganisms.

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCEListeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.

Using the gamma concept in modelling fungal growth: A case study on brioche-type products.

Fungi are common spoilers of intermediate moisture foods such as bakery products. Brioche are bakery products prone to fungal spoilage due to their pH (5.8-6.2) and water activity (aw) (0.82-0.84). The aims of the present study were: (i) the identification of fungal species occurring in brioche products, (ii) the in vitro assessment of their growth potential, and (iii) the development of a validated growth model following the gamma concept. A total of 102 fungal strains were isolated, with Penicillium sp., Cladosporium sp., and Aspergillus sp. being the main genera, representing 90% of the isolates. Given the isolation frequency, any potential fungal prevalence throughout the bakery processs and/or the results of in vitro assessment of fungal growth potential under conditions mimicking brioche (pH, aw, temperature), Aspergillus flavus, Aspergillus fumigatus, and Penicillium sp. were selected for the development of the gamma model. According to in vitro validation, the model successfully predicted fungal growth, while on in situ experiments, the intrinsic parameters (aw and/or level of used preservative) of brioche in combination with packaging conditions (modified atmosphere) did not allow fungal growth.

Modeling transfer of Escherichia coli O157:H7 and Listeria monocytogenes during preparation of fresh-cut salads: impact of cutting and shredding practices.

Cutting and shredding of leafy vegetables increases the risk of cross contamination in household settings. The distribution of Escherichia coli O157:H7 and Listeria monocytogenes transfer rates (Tr) between cutting knives and lettuce leaves was investigated and a semi-mechanistic model describing the bacterial transfer during consecutive cuts of leafy vegetables was developed. For both pathogens the distribution of log10Trs from lettuce to knife was towards low values. Conversely log10Trs from knife to lettuce ranged from -2.1 to -0.1 for E. coli O157:H7 and -2.0 to 0 for L. monocytogenes, and indicated a more variable phenomenon. Regarding consecutive cuts, a rapid initial transfer was followed by an asymptotic tail at low populations moving to lettuce or residing on knife. E. coli O157:H7 was transferred at slower rates than L. monocytogenes. These trends were sufficiently described by the transfer-model, with RMSE values of 0.426-0.613 and 0.531-0.908 for L. monocytogenes and E. coli O157:H7, respectively. The model showed good performance in validation trials but underestimated bacterial transfer during extrapolation experiments. The results of the study can provide information regarding cross contamination events in a common household. The constructed model could be a useful tool for the risk-assessment during preparation of leafy-green salads.

Organic acids for control of Salmonella in different feed materials.

BACKGROUND: Salmonella control in animal feed is important in order to protect animal and public health. Organic acids is one of the control measures used for treatment of Salmonella contaminated feed or feed ingredients. In the present study, the efficacy of formic acid (FA) and different blends of FA, propionic acid (PA) and sodium formate (SF) was investigated. Four Salmonella strains isolated from feed were assayed for their acid tolerance. Also, the effect of lower temperatures (5°C and 15°C) compared to room temperature was investigated in rape seed and soybean meal. RESULTS: The efficacy of acid treatments varied significantly between different feed materials. The strongest reduction was seen in pelleted and compound mash feed (2.5 log10 reduction) followed by rapeseed meal (1 log10 reduction) after 5 days exposure. However, in soybean meal the acid effects were limited (less than 0.5 log10 reduction) even after several weeks' exposure. In all experiments the survival curves showed a concave shape, with a fast initial death phase followed by reduction at a slower rate during the remaining time of the experiment.No difference in Salmonella reduction was observed between FA and a blend of FA and PA, whereas a commercial blend of FA and SF (Amasil) was slightly more efficacious (0.5-1 log10 reduction) than a blend of FA and PA (Luprocid) in compound mash feed. The Salmonella Infantis strain was found to be the most acid tolerant strain followed by, S. Putten, S. Senftenberg and S. Typhimurium. The tolerance of the S. Infantis strain compared with the S. Typhimurium strain was statistically significant (p<0.05). The lethal effect of FA on the S. Typhimurium strain and the S. Infantis strain was lower at 5°C and 15°C compared to room temperatures. CONCLUSIONS: Acid treatment of Salmonella in feed is a matter of reducing the number of viable bacterial cells rather than eliminating the organism. Recommendations on the use of acids for controlling Salmonella in feed should take into account the relative efficacy of acid treatment in different feed materials, the variation in acid tolerance between different Salmonella strains, and the treatment temperature.

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.

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.

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.

Quorum sensing in the context of food microbiology.

Food spoilage may be defined as a process that renders a product undesirable or unacceptable for consumption and is the outcome of the biochemical activity of a microbial community that eventually dominates according to the prevailing ecological determinants. Although limited information are reported, this activity has been attributed to quorum sensing (QS). Consequently, the potential role of cell-to-cell communication in food spoilage and food safety should be more extensively elucidated. Such information would be helpful in designing approaches for manipulating these communication systems, thereby reducing or preventing, for instance, spoilage reactions or even controlling the expression of virulence factors. Due to the many reports in the literature on the fundamental features of QS, e.g., chemistry and definitions of QS compounds, in this minireview, we only allude to the types and chemistry of QS signaling molecules per se and to the (bioassay-based) methods of their detection and quantification, avoiding extensive documentation. Conversely, we attempt to provide insights into (i) the role of QS in food spoilage, (ii) the factors that may quench the activity of QS in foods and review the potential QS inhibitors that might "mislead" the bacterial coordination of spoilage activities and thus may be used as biopreservatives, and (iii) the future experimental approaches that need to be undertaken in order to explore the "gray" or "black" areas of QS, increase our understanding of how QS affects microbial behavior in foods, and assist in finding answers as to how we can exploit QS for the benefit of food preservation and food safety.

Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction.

Staphylococcus aureus strains producing the bacteriophage-encoded staphylococcal enterotoxin A (SEA) were divided into two groups, high- and low-SEA-producing strains, based on the amount of SEA produced. After growth under favorable conditions in batch cultures, 10 of the 21 strains tested produced more than 1,000 ng/ml SEA, and 9 strains produced less than 10 ng/ml SEA; two enterotoxigenic strains, MRSA252 and Newman, produced intermediate levels of SEA (around 450 ng/ml). The differences in the production of SEA were found to be associated with the expression level of sea and whether the strains hosted the sea(1) or sea(2) version. Furthermore, differences in nucleotide sequence in the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was correlated with the capacity for a massive increase in SEA levels by prophage induction as demonstrated using mitomycin C (MC). This was also confirmed by the occurrence of additional sea expression, presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. Remarkably, the SEA level was increased up to 10-fold in some strains due to prophage induction. The low-SEA-producing group and the high-SEA-producing subgroup lacking phage-activated sea transcription showed no increase in SEA formation after the addition of MC. This study demonstrates that sea expression in enterotoxigenic strains is correlated with the clonal lineage of sea-carrying phages. The high-SEA-producing group, in particular the prophage-inducible sea(1) group, may be more relevant to staphylococcal food poisoning than the low-SEA-producing group, harboring mainly sea(2).

Bayesian inference for quantifying Listeria monocytogenes prevalence and concentration in minced pork meat from presence/absence microbiological testing.

The purpose of this work was to estimate the prevalence and concentration of Listeria monocytogenes in minced pork meat by the application of a Bayesian modeling approach. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media. Presence of the pathogen was confirmed through biochemical and molecular tests. Independent experiments (n = 10) for validation purposes were performed. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. Sensitivity and specificity varied depending on the culture method. L. monocytogenes concentration was estimated at 14-17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = -2.17%). The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained.

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.

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.

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 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.

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.

Development of a model describing the effect of temperature, water activity and (gel) structure on growth and ochratoxin A production by Aspergillus carbonarius in vitro and evaluation in food matrices of different viscosity.

The present study aimed: (i) to develop models for the combined effect of water activity (0.99, 0.94 and 0.90), microstructure expressed as 0, 5, 10 and 20% w/v gelatin, and temperature (15, 20 and 25 °C), on growth and OTA production rates by Aspergillus carbonarius; and (ii) to evaluate the performance of the developed models on food matrices (jelly, custard and marmalade) of different viscosity at pH 5.5. The square root of biomass increase rate (fungal growth rate) and OTA production rate were determined by the Baranyi model and were further modeled as a function of temperature, gelatin concentration and a(w) by applying polynomial models. Time for visible growth and the upper asymptote of the OTA production curve were also determined by the Baranyi model. Increase in gelatin concentration resulted in a significant delay in all parameters describing fungal growth and OTA production rates, at all temperatures and a(w). The effect of microstructure on fungal growth and OTA production rates was less evident at stress conditions of a(w) and temperature. Detection time for visible fungal growth was markedly influenced by a(w) and temperature. Coefficients of determination were 0.899 and 0.887 for the models predicting the square root (√µ(max)) of growth and OTA production rate, respectively. Predictions of growth rate agreed well with the recorded data of custard and marmalade, while observations of OTA production rate indicated low agreement with model predictions, in all food matrices except for marmalade. The present findings may provide a basis for reliable assessment of the risk of fungal growth and OTA production in foods of different structural and rheological properties.