Potential of multiparametric characterization of foodstuffs by nuclear magnetic resonance to better predict microbial behavior.

Numerous predictive microbiology models have been proposed to describe bacterial population behaviors in foodstuffs. These models depict the growth kinetics of particular bacterial strains based on key physico-chemical parameters of food matrices and their storage temperature. In this context, there is a prominent issue to accurately characterize these parameters, notably pH, water activity (aw ), and NaCl and organic acid concentrations. Usually, all these product features are determined using one destructive analysis per parameter at macroscale (>5 g). Such approach prevents an overall view of these characteristics on a single sample. Besides, it does not take into account the intra-product microlocal variability of these parameters within foods. Nuclear magnetic resonance (NMR) is a versatile non-invasive spectroscopic technique. Experiments can be recorded successively on a same collected sample without damaging it. In this work, we designed a dedicated NMR approach to characterize the microenvironment of foods using 10-mg samples. The multiparametric mesoscopic-scale approach was validated on four food matrices: a smear soft cheese, cooked peeled shrimps, cold-smoked salmon, and smoked ham. Its implementation in situ on salmon fillets enabled to observe the intra-product heterogeneity and to highlight the impact of process on the spatial distribution of pH, NaCl, and organic acids. This analytical development and its successful application can help address the shortcomings of monoparametric methods traditionally used for predictive microbiology purposes.

A microscopy-based approach for determining growth probability and lag time of individual bacterial cells.

The development of relevant predictive models for single-cell lag time and growth probability near growth limits is of critical importance for predicting pathogen behavior in foods. The classical methods for data acquisition in this field are based on turbidity measurements of culture media in microplate wells inoculated with approximately one bacterial cell per well. Yet, these methods are labour intensive and would benefit from higher throughput. In this study, we developed a quantitative experimental method using automated microscopy to determine the single-cell growth probability and lag time. The developed method consists of the use of direct cell observation with phase-contrast microscopy equipped with a 100× objective and a high-resolution device camera. The method is not a time-lapse method but is based on the observation of high numbers of colonies for a given time. Automation of image acquisition and image analysis was used to reach a high throughput. The single-cell growth probabilities and lag times of four strains of Listeria monocytogenes were determined at 4 °C. The microscopic method was shown to be a promising method for the determination of individual lag times and growth probability at the single-cell level.

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

Identification of markers of thermal processing ("roasting") in aqueous extracts of Coffea arabica L. seeds through NMR fingerprinting and chemometrics.

Roasting of Coffea arabica L. seeds gives rise to chemical reactions that produce more than 800 compounds, some being responsible for the desired organoleptic properties for which the beverage called "coffee" is known. In the industry, the "roasting profile," that is, the times and temperatures applied, is key to influence the composition of roasted coffee beans and the flavour of the beverage made from them. The impact of roasting on the chemical composition of coffee has been the subject of numerous studies, including by nuclear magnetic resonance (NMR) spectroscopy. However, the roasting equipment and profiles applied in these studies are often far from real industrial conditions. In this work, the effects of two critical technological parameters of the roasting process, namely, the "development time" (the period of time after the "first crack," a characteristic noise due to seed disruption) and the final roasting temperature on coffee extracts, were investigated. Seeds were roasted at pilot scale according to 13 industrial roasting profiles and extracted in D2 O. The extracts were analysed by 1 H NMR experiments. The NMR spectra were compared using (a) quantitative analysis of main signals by successive orders of magnitude and (b) chemometric tools (principal component analysis, partial least squares and sparse-orthogonal partial least squares analysis). This allowed to identify compounds, which may serve as markers of roasting and showed that changes in chemical composition can be detected even for slight change in final temperature (~1°C) or in total roasting time (~25 s).

Combined microplate-ABTS and HPLC-ABTS analysis of tomato and pepper extracts reveals synergetic and antagonist effects of their lipophilic antioxidative components.

The antioxidant capacity of 9 pure lipophilic compounds was examined by microplate-ABTS and HPLC-ABTS, using similar experimental conditions. Results obtained showed that HPLC-ABTS method can be used for a rapid determination of individual antioxidant capacity of compounds in standard solutions or complex mixtures. The application of both methods to real lipophilic extracts from tomato (Solanum lycopersicum L.), green and red peppers (Capsicum annuum) reveals possible interactions between antioxidants. Thus, synthetic mixtures of two compounds identified in tomato and peppers were measured using microplate-ABTS and HPLC-ABTS. Synergistic effects were observed between (ß-carotene-capsanthin) (1:9) and (1:1), (α-tocopherol-capsanthin) (1:9), (lutein-lycopene) (9:1) and (capsanthin-δ-tocopherol) (9:1). On the contrary, antagonistic effects were observed for (lutein-δ-tocopherol) and (α-tocopherol-δ-tocopherol). The interactions observed with two-compound mixtures are not systematically observed in the natural lipophilic extracts from tomato, green and red peppers, probably since extracts are more complex and are susceptible to cause interferences.

Reducing Salt in Raw Pork Sausages Increases Spoilage and Correlates with Reduced Bacterial Diversity.

UNLABELLED: Raw sausages are perishable foodstuffs; reducing their salt content raises questions about a possible increased spoilage of these products. In this study, we evaluated the influence of salt reduction (from 2.0% to 1.5% [wt/wt]), in combination with two types of packaging (modified atmosphere [50% mix of CO2-N2] and vacuum packaging), on the onset of spoilage and on the diversity of spoilage-associated bacteria. After 21 days of storage at 8°C, spoilage was easily observed, characterized by noticeable graying of the products and the production of gas and off-odors defined as rancid, sulfurous, or sour. At least one of these types of spoilage occurred in each sample, and the global spoilage intensity was more pronounced in samples stored under modified atmosphere than under vacuum packaging and in samples with the lower salt content. Metagenetic 16S rRNA pyrosequencing revealed that vacuum-packaged samples contained a higher total bacterial richness (n = 69 operational taxonomic units [OTUs]) than samples under the other packaging condition (n = 46 OTUs). The core community was composed of 6 OTUs (Lactobacillus sakei, Lactococcus piscium, Carnobacterium divergens, Carnobacterium maltaromaticum, Serratia proteamaculans, and Brochothrix thermosphacta), whereas 13 OTUs taxonomically assigned to the Enterobacteriaceae, Enterococcaceae, and Leuconostocaceae families comprised a less-abundant subpopulation. This subdominant community was significantly more abundant when 2.0% salt and vacuum packaging were used, and this correlated with a lower degree of spoilage. Our results demonstrate that salt reduction, particularly when it is combined with CO2-enriched packaging, promotes faster spoilage of raw sausages by lowering the overall bacterial diversity (both richness and evenness). IMPORTANCE: Our study takes place in the context of raw meat product manufacturing and is linked to a requirement for salt reduction. Health guidelines are calling for a reduction in dietary salt intake. However, salt has been used for a very long time as a hurdle technology, and salt reduction in meat products raises the question of spoilage and waste of food. The study was conceived to assess the role of sodium chloride reduction in meat products, both at the level of spoilage development and at the level of bacterial diversity, using 16S rRNA amplicon sequencing and raw pork sausage as a meat model.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

TiO2 photocatalysis damages lipids and proteins in Escherichia coli.

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O2·(-)), inhibited this effect by half, showing us that O2·(-) radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO2, respectively, which is accounted for by the cytotoxic effect of TiO2. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO2 increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O2·(-) on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics.

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.

Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance.

BACKGROUND: Lactic acid bacteria are commonly marketed as probiotics based on their putative or proven health-promoting effects. These effects are known to be strain specific but the underlying molecular mechanisms remain poorly understood. Therefore, unravelling the determinants behind probiotic features is of particular interest since it would help select strains that stand the best chance of success in clinical trials. Bile tolerance is one of the most crucial properties as it determines the ability of bacteria to survive in the small intestine, and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study was to investigate the natural protein diversity within the Lactobacillus plantarum species with relation to bile tolerance, using comparative proteomics. RESULTS: Bile tolerance properties of nine L. plantarum strains were studied in vitro. Three of them presenting different bile tolerance levels were selected for comparative proteomic analysis: L. plantarum 299 V (resistant), L. plantarum LC 804 (intermediate) and L. plantarum LC 56 (sensitive). Qualitative and quantitative differences in proteomes were analyzed using two-dimensional electrophoresis (2-DE), tryptic digestion, liquid chromatography-mass spectrometry analysis and database search for protein identification. Among the proteins correlated with differences in the 2-DE patterns of the bacterial strains, 15 have previously been reported to be involved in bile tolerance processes. The effect of a bile exposure on these patterns was investigated, which led to the identification of six proteins that may be key in the bile salt response and adaptation in L. plantarum: two glutathione reductases involved in protection against oxidative injury caused by bile salts, a cyclopropane-fatty-acyl-phospholipid synthase implicated in maintenance of cell envelope integrity, a bile salt hydrolase, an ABC transporter and a F0F1-ATP synthase which participate in the active removal of bile-related stress factors. CONCLUSIONS: These results showed that comparative proteomic analysis can help understand the differential bacterial properties of lactobacilli. In the field of probiotic studies, characteristic proteomic profiles can be identified for individual properties that may serve as bacterial biomarkers for the preliminary selection of strains with the best probiotic potential.