Lebanese fermented goat milk products: From tradition to meta-omics.

Ambriss, Serdaleh and Labneh El Darff are traditional Lebanese products made from fermented goat's milk. A questionnaire completed by 50 producers of these products showed that they are prepared by periodic percolation either by milk or by Laban in amphora or goat skins during the lactation season. Production is carried out on a small scale and in a limited number of production units, often by elderly people, resulting in a real risk of disappearance of these products and loss of the corresponding microbial resources. In this study, 34 samples from 18 producers were characterized by culture-dependent and -independent analyses. The results obtained from these two methods were radically different, the latter revealing in Ambriss and Serdaleh the co-dominance of Lactobacillus kefiranofaciens, a fastidious-growing species, and Lactococcus lactis in a viable but not culturable state. Overall, their composition is reminiscent of kefir grains. Phylogenomic and functional analyses of the genomes of the key species Lb. kefiranofaciens have revealed differences from those found in kefir, particularly in their polysaccharide genes, which may explain the absence of grains. However, Labneh El Darff displayed a dominance of Lactobacillus delbrueckii, probably due to the addition of Laban. In addition, the study identified several zoonotic pathogens, including Streptococcus parasuis, which dominated in one sample. Metagenome-Assembled Genome (MAG) analysis indicated that this pathogen acquired lactose utilization genes through horizontal gene transfer. The contamination of the herd with Mycoplasmopsis agalactiae in the Chouf region was also revealed by MAG analysis of the Serdaleh samples. Antibiotic resistance genes were detected in most of the samples, particularly in the Serdaleh ones, where the dominant L. lactis strains possessed a plasmid with a multi-resistance island. Finally, this study paves the way for further analyses to shed light on the resilience of these ecosystems established in amphora or in goatskins and to improve hygiene practices for milk production.

[Glassy cell carcinoma of the uterine cervix: An aggressive type of cancer]. / Glassy cell carcinoma du col de l'utérus : une maladie tumorale agressive.

INTRODUCTION: Cervical cancer is the twelfth most frequent cancer in women in France. Glassy cell carcinoma is a rare histological entity, rapidly aggressive, associated with a poor prognosis. CASE REPORT: A 30-year-old woman was admitted in an internal medicine department for polyarthralgia with high grade fever, evolving for 3 weeks. There was an inflammatory syndrome. The 18-FDG-PET-scan showed inflammatory lymph nodes as well as disseminated osteolytic lesions, and a primitive pelvic tumor. A 3cm tumor of the cervix was found during the gynaecologic examination. Histological analysis elicited a high-index mitotic carcinoma, glassy cell carcinoma type. Despite chemotherapy, the outcome was poor, with early death occurring after three months of follow-up. CONCLUSION: The glassy cell carcinoma of the cervix should be considered as an aetiology of bone metastases in young female patients.

Perinatal grief following neonatal comfort care for lethal fetal condition.

BACKGROUND: The objective of the study was to assess perinatal grief experienced after continuing pregnancy and comfort care in women diagnosed with lethal fetal condition compared with termination of pregnancy for fetal anomaly (TOPFA). METHODS: This was a retrospective observational study which included women who chose to continue their pregnancy after the diagnosis of lethal fetal condition with comfort care support at birth at the Prenatal Diagnosis Center of Rennes Hospital from January 2007 to January 2017. Women were matched with controls who underwent TOPFA for the same type of fetal anomaly, gestational age at diagnosis and year. Women were evaluated by a questionnaire including the Perinatal Grief Scale. RESULTS: There were 28 patients in the continuing pregnancy group matched with 56 patients in the TOPFA group. Interval between fetal loss and completion of questionnaire was 6±3 years. Perinatal grief score was similar at 61±22 vs 58±18 (p = 0.729) in the continuing pregnancy and TOPFA groups, respectively. Women in the TOPFA group expressed more guilt. The cesarean-section rate in the continuing pregnancy group was 25%. CONCLUSION: Perinatal grief experienced by women opting for continuing pregnancy and comfort care after diagnosis of a potentially lethal fetal anomaly is not more severe than for those choosing TOPFA.

Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus.

Filamentous fungi have a complex morphology that induces fermentation process development issues, as a consequence of viscosity increase and diffusion limitations. In order to better understand the relationship between viscosity changes and fungus morphology during fermentations of Trichoderma reesei, an accurate image analysis method has been developed to provide quantitative and representative data for morphological analysis. This method consisted of a new algorithm called FACE that allowed sharp images to be created at all positions, segmentation of fungus, and morphological analysis using skeleton and topological approaches. It was applied and validated by characterizing samples of an industrial strain of Trichoderma reesei that had or had not been exposed to an extreme shear stress. This method allowed many morphological characteristics to be identified, among which nine relevant criteria were extracted, regarding the impact of shear stress on the fungus and on the viscosity of the fermentation medium.

Development of a conceptual framework of holistic risk assessment - Landfill as a particular type of contaminated land.

Landfills can be regarded as a particular type of contaminated land that has a potential to directly and indirectly pollute all of the four main spheres of the environment which are the lithosphere, atmosphere, hydrosphere and eventually adversely impact the biosphere. Therefore, environmental risk assessment of a landfill has to be more integrated and holistic by virtue of its nature of being a multidimensional pollutant source. Despite this, although various risk assessment approaches have been adopted for landfill waste disposal sites, there are still wide-ranging knowledge gaps and limitations which need to be addressed. One important knowledge gap and limitation of current risk assessment approaches is the inability to fully identify, categorise and aggregate all individual risks from all combinations of hazards, pathways and targets/receptors (e.g. water, air, soil and biota) in connection to a certain landfill leachate and yet at any stage of the landfill cycle. So such an approach is required that could not only integrate all possible characteristics of varying scenarios but also contain the ability to establish an overall risk picture, irrespective of the lifecycle stage of the landfill (e.g. planning stage/pre-operation, in-operation or post-operation/closed). One such approach to address the wide-breadth of landfill impact risks is by developing a more holistic risk assessment methodology, whose conceptual framework is presented in this paper for landfill leachate in a whole-system format. This conceptual framework does not only draw together various constituting factors and sub-factors of risk assessment in a logical sequence and categorical order, but also indicates the "what, why, when and how" outputs of and inputs to these factors and sub-factors can be useful. The framework is designed to identify and quantify a range of risks associated with all stages of the landfill lifecycle, and yet in a more streamlined, logical, categorical and integrated format, offering a more standardised and unified whole-system approach.

Osmotic stress affects the stability of freeze-dried Lactobacillus buchneri R1102 as a result of intracellular betaine accumulation and membrane characteristics.

AIMS: To help cells to better resist the stressful conditions associated with the freeze-drying process during starter production, we investigated the effect of various osmotic conditions on growth, survival and acidification activity of Lactobacillus buchneri R1102, after freeze-drying and during storage for 3 months at 25°C. METHODS AND RESULTS: High survival rates during freeze-drying, but not during storage, were obtained when 0·1 mol l(-1) KCl was added at the beginning of fermentation, without any change in membrane properties and betaine accumulation. This condition made it possible to maintain a high acidification rate throughout the process. In contrast, the addition of 0·6 mol l(-1) KCl concentrations at the beginning of fermentation led to a high survival rate during storage that was related to high intracellular betaine levels, low membrane fluidity and high cycC19:0 concentrations. However, these modifications induced the degradation of acidification activity during storage. When a moderate stress was applied by combining 0·1 mol l(-1) KCl at the beginning and 0·6 mol l(-1) KCl at the end of fermentation, betaine accumulated in the cells without any membrane alteration, allowing them to maintain high acidification activity and survival rate during storage. CONCLUSION: Specific osmotic conditions during fermentation induced intracellular betaine accumulation and modifications of membrane character-istics, thus affecting stress resistance of Lact. buchneri R1102. A slight osmotic stress made it possible to maintain a high acidification activity, whereas a high osmotic stress at the end of fermentation led to the preservation of cell survival during freeze-dried storage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that the survival and preservation of acidification activity of freeze-dried Lact. buchneri R1102 during starter production can be improved by using appropriate osmotic conditions.

Salivary enzymes and exhaled air affect Streptococcus salivarius growth and physiological state in complemented artificial saliva.

To better understand the phenomena governing the establishment of the oral bacterium Streptococcus salivarius in the mouth, the effect of some environmental factors has been studied in complemented artificial saliva, under oral pH and temperature conditions. Three salivary enzymes at physiological concentrations were tested: peroxidase, lysozyme and amylase, as well as injection of exhaled air. Injection of air containing 5% CO2 and 16% O2 induced a deleterious effect on S. salivarius K12, mainly by increasing redox potential. Addition of lysozyme slightly affected the physiological state of S. salivarius by altering membrane integrity. In contrast, peroxidase was not detrimental as it made it possible to decrease the redox potential. The addition of amylase reduced the specific growth rate of S. salivarius by formation of a complex with amylase and mucins, but led to high final biomass, as a result of enzymatic degradation of some nutrients. Finally, this work demonstrated that salivary enzymes had a slight impact on S. salivarius behaviour. It can thus be concluded that this bacterium was well adapted to in-mouth conditions, as it was able to resist certain salivary enzymes, even if tolerance to expired air was affected, as a result of an increased redox potential.

Characterization of Streptococcus salivarius growth and maintenance in artificial saliva.

AIMS: To help gain a better understanding of factors influencing the establishment within the oral cavity of Streptococcus salivarius K12, a commensal oral bacterium, we characterized its behaviour in artificial saliva. METHODS AND RESULTS: Streptococcus salivarius K12 was grown in artificial saliva complemented with a representative meal, under oral pH and temperature conditions. Exponential growth phase was characterized by a high specific growth rate (2.8 h(-1)). During maintenance phase, an uncoupling between growth and lactic acid production occurred, which allowed maintaining viability (95%), intracellular pH (6.6) and membrane polarisation (95%), and thus proton motive force. However, in late stationary phase, viability (64%) and vitality were degraded as a result of lower synthesis of energetic and glycogen-related proteins as compared to a richer medium. CONCLUSIONS: Streptococcus salivarius was able to rapidly grow in complemented artificial saliva. Nevertheless, a degradation of its physiological state was observed in late-stationary phase. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates, for the first time, that artificial saliva was a convenient medium that permitted Strep. salivarius to grow in oral conditions (physico-chemical environment, addition of meals) but not to maintain cellular viability and vitality in starvation conditions.

Lipid analysis of Neochloris oleoabundans by liquid state NMR.

This study is an evaluation of liquid state NMR as a tool for analyzing the lipid composition of algal cultures used for biodiesel production. To demonstrate the viability of this approach, (13)C NMR was used to analyze the lipid composition of intact cells of the algal species, Neochloris oleoabundans (UTEX #1185). Two cultures were used in this study. One culture was "healthy" and grown in conventional media, whereas the other culture was "nitrogen-starved" and grown in media that lacked nitrate. Triglyceride was determined to be present in both cultures by comparing the algal NMR spectra with published chemical shifts for a wide range of lipids and with a spectrum obtained from a triglyceride standard (glyceryl trioleate). In addition, it is shown that (1) the signal-to-noise ratio of the approximately 29.5 ppm methylene peak is indicative of the lipid content and (2) the nitrogen-starved culture contained a greater lipid content than the healthy culture, as expected. Furthermore, the nitrogen-starved culture produced spectra that primarily contained the characteristic peaks of triglyceride (at approximately 61.8 and approximately 68.9 ppm), whereas the healthy culture produced spectra that contained several additional peaks in the glycerol region, likely resulting from the presence of monoglyceride and diglyceride. Finally, potential interferences are evaluated (including the analysis of phospholipids via (31)P NMR) to assess the specificity of the acquired spectra to triglyceride. These results indicate that NMR is a useful diagnostic tool for selectively identifying lipids in algae, with particular relevance to biodiesel production.

Micro-Raman spectroscopy of algae: composition analysis and fluorescence background behavior.

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo-bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood.

Acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus induces physiological responses at membrane and cytosolic levels that improves cryotolerance.

AIMS: This work aimed at clarifying the physiological responses of Lactobacillus delbrueckii subsp. bulgaricus CFL1 cells after exposure to acidification at the end of fermentation, in relation to their cryotolerance. METHODS AND RESULTS: Cells acidified at the end of the fermentation (pH 5.25 for 30 min) had their cryotolerance improved as compared to the reference condition (pH 6.0). By analyzing the cytosolic proteome, it was established that changes occurred in the synthesis of 21 proteins, involved in energy metabolism, nucleotide and protein synthesis and stress response. Acidification also induced a slight decrease in unsaturated to saturated and cyclic to saturated membrane fatty acid ratios. CONCLUSIONS: Lactobacillus bulgaricus CFL1 was able to develop a combined physiological response at both membrane and cytosolic levels. This acid adaptation was referred as a cross-protection phenomenon as it allowed the cells to become more tolerant to cold stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increased knowledge concerning the physiological mechanisms that explained the cross-protection by acid adaptation. It may be useful for improving cryotolerance of lactic acid bacteria, either in cells banks or in an industrial context.

Long-term flow rates and biomat zone hydrology in soil columns receiving septic tank effluent.

Soil absorption systems (SAS) are used commonly to treat and disperse septic tank effluent (STE). SAS can hydraulically fail as a result of the low permeable biomat zone that develops on the infiltrative surface. The objectives of this experiment were to compare the hydraulic properties of biomats grown in soils of different textures, to investigate the long-term acceptance rates (LTAR) from prolonged application of STE, and to assess if soils were of major importance in determining LTAR. The STE was applied to repacked sand, Oxisol and Vertisol soil columns over a period of 16 months, at equivalent hydraulic loading rates of 50, 35 and 8L/m(2)/d, respectively. Infiltration rates, soil matric potentials, and biomat hydraulic properties were measured either directly from the soil columns or calculated using established soil physics theory. Biomats 1 to 2 cm thick developed in all soils columns with hydraulic resistances of 27 to 39 d. These biomats reduced a 4 order of magnitude variation in saturated hydraulic conductivity (K(s)) between the soils to a one order of magnitude variation in LTAR. A relationship between biomat resistance and organic loading rate was observed in all soils. Saturated hydraulic conductivity influenced the rate and extent of biomat development. However, once the biomat was established, the LTAR was governed by the resistance of the biomat and the sub-biomat soil unsaturated flow regime induced by the biomat. Results show that whilst initial soil K(s) is likely to be important in the establishment of the biomat zone in a trench, LTAR is determined by the biomat resistance and the unsaturated soil hydraulic conductivity, not the K(s) of a soil. The results call into question the commonly used approach of basing the LTAR, and ultimately trench length in SAS, on the initial K(s) of soils.

Fermentation pH and temperature influence the cryotolerance of Lactobacillus acidophilus RD758.

The effects of 3 fermentation temperatures (30, 37, and 42 degrees C) and 3 fermentation pH (4.5, 5, and 6) on the cryotolerance of Lactobacillus acidophilus RD758 were studied in relation to their fatty acid composition. Cryotolerance was defined as the ability of the cells to recover their acidification activity after freezing and frozen storage at -20 degrees C. Better cryotolerance was obtained in cells grown at 30 degrees C or at pH 5; these cells showed no loss in acidification activity during freezing and a low rate of loss in acidification activity during frozen storage. On the other hand, cells grown at 42 degrees C or at pH 4.5 displayed poor cryotolerance. The membrane fatty acid composition was analyzed and related to the cryotolerance using principal component analysis. The improved cryotolerance observed during the freezing step was associated with a high ratio of unsaturated to saturated fatty acids, a low C18:0 content, and high C16:0 and cyclic C19:0 relative concentrations. High resistance during frozen storage was related to a high cycC19:0 concentration. Finally, the low cryotolerance observed after fermentation at pH 4.5 was explained by a low C18:2 content.

Improvement of the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing by natural selection.

Lactic acid bacteria are often produced as frozen or freeze-dried cultures that can be used for the direct inoculation of milk in cheese and fermented milk production processes. The objective of this study was to investigate whether the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing could be improved by natural selection. Three parallel cultures of strain CFL1 were propagated for 30 cycles in which each cycle involved three serial transfers through milk, one freezing step, and one thawing step. The concentration in viable cells after thawing as well as the acidifying activity of the thawed cultures increased dramatically throughout the experiment. This may be explained by the random appearance of better-adapted mutants that can outcompete the other genotypes. However, after 30 cycles of subcultivation, freezing, and thawing, all the cultures contained subpopulations having different survival rates to freezing. Our results show that serial transfer culture experiments may be used to improve technological properties of lactic acid bacteria. Furthermore, investigation of the mutations that are responsible for an increased cryotolerance may help to define new targets for improving the resistance of lactic acid bacteria to several stresses.

Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition.

The resistance to freezing and frozen storage of Streptococcus thermophilus was related to the fatty acid composition of the cell membrane. The effects of four experimental factors were investigated on the fatty acid concentrations and on the recovery of acidification activity of S. thermophilus stored at -20 degrees C by using a complete experimental design: incorporating oleic acid in the culture medium, fermentation pH, addition of glycerol as cryoprotective agent and duration of storage. The acidification activity decreased during the freezing and the frozen storage of S. thermophilus. The storage time slightly enhanced the unsaturated fatty acid concentrations. The addition of glycerol did not modify the fatty acid composition but increased the resistance to frozen storage. The addition of oleic acid and the decrease of the fermentation pH enhanced the ratio unsaturated:saturated fatty acids and improved the recovery of the acidification activity. These results indicate that the resistance to frozen storage was closely related to the membrane fatty acid composition. We interpreted this as an adaptation of S. thermophilus to the addition of oleic acid and the unfavorable growth conditions that corresponded to a low fermentation pH.

Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage.

Thermophilic lactic acid bacteria exhibit different survival rates during freezing and frozen storage, depending on the processing conditions. We used a Plackett and Burman experimental design to study the effects of 13 experimental factors, at two levels, on the resistance of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to freezing and frozen storage. The resistance was evaluated by quantifying the decrease of acidification activity during freezing and throughout 8 weeks of storage. Acidification activity after freezing and frozen storage was affected by 12 experimental factors. Only the thawing temperature did not show any significant effect. S. thermophilus was more resistant than L. bulgaricus and the cryoprotective effect of glycerol during freezing and storage was confirmed. The temperature and duration of the cryoprotection step influenced acidification activity following the freezing step: the lower the temperature and the shorter the duration, the higher the activity. Acidification activity after storage was affected by several experimental factors involved in the fermentation stage: use of NaOH instead of NH4OH for pH control, addition of Tween 80 in the culture medium, and faster cooling led to better cryotolerance. Resistance to freezing and frozen storage was improved by using a high freezing rate and a low storage temperature. Finally, this study revealed that the conditions under which lactic acid bacteria are prepared should be well controlled to improve their preservation and to limit the variability between batches and between species.

Method of quantifying the loss of acidification activity of lactic acid starters during freezing and frozen storage.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (delta tm) between the tm values of the concentrated cell suspension before and after freezing. During storage at -20 degrees C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9-14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.

Modelling the interactions between Lactobacillus curvatus and Enterobacter cloacae. I. Individual growth kinetics.

Mixed cultures of Lactobacillus curvatus and Enterobacter cloacae were chosen as a model system to quantitatively study microbial interactions involved in food spoilage and food preservation. In this paper models were developed to predict the individual behaviour of L. curvatus and E. cloacae in pure suspension cultures as a function of the glucose and lactate concentration and the pH. In a second paper these models will be used to predict the moment of interaction in mixed suspension cultures and the cell concentrations at this moment. The effect of pH on the maximum specific growth rate could be described by a parabolic equation for L. curvatus and E. cloacae. E. cloacae is clearly more sensitive to a pH decrease than L. curvatus, as may be concluded from the theoretical minimum pH for growth of 5.6 for E. cloacae and 4.3 for L. curvatus. For both organisms no effect of glucose on the maximum specific growth rate could be detected and Monod kinetics with a low Monod constant was assumed. For L. curvatus the effect of lactate on the maximum specific growth rate at different initial pH values could be described by a linear relationship. For E. cloacae a slight effect of lactate on the maximum specific growth rate could only be detected at pH 7. However, this effect was negligible compared to the effect of the pH. For both organisms the lag time was modelled by the inverse of the specific growth rate. It can be concluded that, with regard to interactions between L. curvatus and E. cloacae, the pH is likely to be the most important factor in the case where L. curvatus is the dominant organism. Substrate limitation is likely to become important in the case where E. cloacae is the dominant organism or for media with a high buffer capacity.

Modelling the interactions between Lactobacillus curvatus and Enterobacter cloacae. II. Mixed cultures and shelf life predictions.

The modelling approach presented in this study can be used to predict when interactions between microorganisms in homogenous systems occur. It was tested for the interaction between Lactobacillus curvatus and Enterobacter cloacae. In this binary system, L. curvatus produces lactic acid which decreases the pH in the system. The pH decrease was found to be the main limiting factor of growth of both E. cloacae and L. curvatus. This resulted in E. cloacae reaching its final concentration earlier when compared to its growth in pure culture. The models consisted of a set of first order ordinary differential equations describing the growth, consumption and production rates of both microorganisms. The parameters for these equations were obtained from pure culture studies and from literature. These equations were solved using a combination of analytical and numerical methods. The prediction of growth in mixed culture using parameters from pure culture experiments and literature were close to the experimental data. Both model predictions and experimental validation indicated that interaction occurs when the concentration of L. curvatus reaches 10(8) cfu/ml. At that moment in time, the pH had decreased to inhibiting levels. These concentrations of microorganisms (10(8) cfu/ml) do occur in fermented products where interactions obviously are important. In nonfermented foods however, this level of microorganisms indicate that spoilage has occurred or is about to start. Microbial interactions can therefore be neglected when predicting shelf life or safety of food products in most cases.

An analysis of the Hooper Visual Organization Test with neurological patients.

An item analysis of the Hooper Visual Organization Test (VOT) was performed using the test protocols of 320 German-speaking neurological patients with a mean age of 51.3 years. All patients presented some form of cerebral pathology. The sample yielded a mean test score of 18.8 (SD = 6.2). Estimates of internal consistency of the total scale yielded acceptable results (>.88). Although the VOT was judged to be a useful instrument for assessing visuoperceptual and visuospatial-organizational dysfunction, item characteristics of the test are not satisfactory. In particular, the item ranking does not correspond to the order of difficulty. Moreover, in their current form, scoring rules of a number of items seem to be arbitrary and not founded on empirical data. A revision of the VOT is proposed to improve its psychometric properties.