Substantial differences between organ and muscle specific tracer incorporation rates in a lactaying dairy cow

We aimed to produce intrinsically L-[1-13C]phenylalanine labeled milk and beef for subsequent use in human nutrition research. The collection of the various organ tissues after slaughter allowed for us to gain insight into the dynamics of tissue protein turnover in vivo in a lactating dairy cow. One lactating dairy cow received a constant infusion of L- [1-13C]phenylalanine (450 mmol/min) for 96 h. Plasma and milk were collected prior to, during, and after the stable isotope infusion. Twenty-four hours after cessation of the infusion the cow was slaughtered. The meat and samples of the various organ tissues (liver, heart, lung, udder, kidney, rumen, small intestine, and colon) were collected and stored. Approximately 210 kg of intrinsically labeled beef (bone and fat free) with an average L-[1-13C]phenylalanine enrichment of 1.860.1 mole percent excess (MPE) was obtained. The various organ tissues differed substantially in L-[1-13C]phenylalanine enrichments in the tissue protein bound pool, the highest enrichment levels were achieved in the kidney (11.7 MPE) and the lowest enrichment levels in the skeletal muscle tissue protein of the cow (between 1.5–2.4 MPE). The estimated protein synthesis rates of the various organ tissues should be regarded as underestimates, particularly for the organs with the higher turnover rates and high secretory activity, due to the lengthened (96 h) measurement period necessary for the production of the intrinsically labeled beef. Our data demonstrates that there are relatively small differences in L- [1-13C]phenylalanine enrichments between the various meat cuts, but substantial higher enrichment values are observed in the various organ tissues. We conclude that protein turnover rates of various organs are much higher when compared to skeletal muscle protein turnover rates in large lactating ruminants.

Microstructure, texture and oral processing: New ways to reduce sugar and salt in foods

Food oral processing as the bridge between food texture, microstructure and sensory perception has gained enormous interest in the last decade. This review provides an overview of the role of the microstructure of soft- and semi-solid foods in food oral processing and sensory perception. Phase separated mixed protein– polysaccharide gels and emulsion-filled gels are described as suitable model foods to investigate food oral processing systematically. Special attention is given to the sensory perception of texture, taste and interactions thereof. Several approaches to reduce the salt and sugar content of semi- and soft-solid foods without compromising taste are reviewed. These reduction approaches are based on an understanding of food oral processing in relation to the microstructure of the foods and its breakdown.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) sporeforming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Live-cell imaging tool optimization to study gene expression levels and snamics in single cells of Bacillus cereus

Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium Bacillus cereus specifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of single B. cereus cells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression in B. cereus. With a case study concerning cotD expression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show that cotD is not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

Isolation and quantification of highly acid resistant variants of Listeria monocytogenes

Heterogeneity in stress response of bacteria is one of the biggest challenges posed by minimal processing, which aims at finding the balance between microbiologically stable foods while maintaining the characteristics of fresh products. In this study, exposure of Listeria monocytogenes LO28 to acid stress, which can be encountered in the food processing environment as well as in the human body upon ingestion, led to inactivation kinetics showing considerable tailing, which was described by a biphasic inactivation model. Stable acid resistant variants of L. monocytogenes LO28 were isolated after exposure of late-exponential phase cells to pH3.5 for 90min. The resulting 23 stable resistant isolates could be divided in three groups: (a) highly increased acid resistance (<1log10 reduction, n=16), (b) slightly increased acid resistance (1-3log10 reduction, n=6), and (c) one isolate showing a variable acid stress response. The highly acid resistant group showed increased resistance to the tested pH range of 2.5 to 3.5 in both late-exponential and stationary phase. Increased acid resistance showed to be significantly correlated to reduced growth rate. The Weibull model was reparameterized, resulting in improved parameter estimation, and was used to estimate the inactivation kinetics at mild pH. Studying the growth boundaries of the wild type and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress. This study shows that acid exposure of late-exponential phase cells reveals the presence of acid resistant subpopulations and that there is a phenotypic diversity amongst them. The occurrence of heterogeneity and stress resistant subpopulations may lead to a higher number of surviving microorganisms than expected. Also, stress resistant subpopulations can become part of the domestic flora in a food production line. The currently isolated acid resistant variants are a new group of stress resistant variants and underline the importance of gaining more insight in the mechanisms underlying this heterogeneity and increased resistance.

Physiological and psychosocial age-related changes associated with reduced food intake in older persons

Dietary intake changes during the course of aging. Normally an increase in food intake is observed around 55 years of age, which is followed by a reduction in food intake in individuals over 65 years of age. This reduction in dietary intake results in lowered levels of body fat and body weight, a phenomenon known as anorexia of aging. Anorexia of aging has a variety of consequences, including a decline in functional status, impaired muscle function, decreased bone mass, micronutrient deficiencies, reduced cognitive functions, increased hospital admission and even premature death. Several changes during lifetime have been implicated to play a role in the reduction in food intake and the development of anorexia of aging. These changes are both physiological, involving peripheral hormones, senses and central brain regulation and non-physiological, with differences in psychological and social factors. In the present review, we will focus on age-related changes in physiological and especially non-physiological factors, that play a role in the age-related changes in food intake and in the etiology of anorexia of aging. At the end we conclude with suggestions for future nutritional research to gain greater understanding of the development of anorexia of aging which could lead to earlier detection and better prevention.


Multimodal network design for sustainable household plastic recycling

Purpose – This research studies a plastic recycling system from a reverse logistics angle and investigates the potential benefits of a multimodality strategy to the network design of plastic recycling. This research aims to quantify the impact of multimodality on the network, to provide decision support for the design of more sustainable plastic recycling networks in the future. Design/methodology/approach – A MILP model is developed to assess different plastic waste collection, treatment and transportation scenarios. Comprehensive costs of the network are considered, including emission costs. A baseline scenario represents the optimized current situation while other scenarios allow multimodality options (barge and train) to be applied. Findings – Results show that transportation cost contributes to about 7 percent of the total cost and multimodality can bring a reduction of almost 20 percent in transportation costs (CO2-eq emissions included). In our illustrative case with two plastic separation methods, the post-separation channel benefits more from a multimodality strategy than the source-separation channel. This relates to the locations and availability of intermediate facilities and the quantity of waste transported on each route. Originality/value – This study applies a reverse logistics network model to design a plastic recycling network with special structures and incorporates a multimodality strategy to improve sustainability. Emission costs (carbon emission equivalents times carbon tax) are added to the total cost of the network to be optimized.


The quest for probiotic effector molecules – Unraveling strain specificity at the molecular level

Pharmaceutical agents are widely applied for the treatment of gastrointestinal (and systemic) disorders and their role as modulators of host cell responses is relatively well characterized. By contrast, we are only beginning to understand the molecular mechanisms by which health-promoting, probiotic bacteria act as host cell modulators. The last decade has seen a rapid development of the genomics field for the widely applied probiotic genus Lactobacillus, and nowadays dozens of full genome sequences are available, as well as sophisticated post genomic and genetic engineering tools. This development has enabled comparative (functional) genomics approaches to identify the bacterial effector molecules involved in molecular communication with the host system that may underlie the probiotic effects observed. These efforts can also be complemented with dedicated mutagenesis approaches to eliminate or alter these effector molecules, followed by assessment of the host interaction consequences thereof, allowing the elucidation of the molecular mechanisms involved in probiotic health effects. Many of these approaches have pinpointed that the Lactobacillus cell envelope contains several effector molecules that are pivotal in the direct signaling capacity of these bacteria that underlies their immunomodulatory effects, including lipoteichoic acid, peptidoglycan, and (glyco)proteins. Moreover, the cell envelope contains several compounds such as wall teichoic acid and capsular polysaccharides that may not be involved in direct signaling to the host cell, but still affect signaling through shielding of other bacterial effector molecules. Initial structural studies revealed subtle strain- and species-specific biochemical differences in the canonical cell envelope compounds that are involved in these host interactions. These biochemical variations include the degree and positioning of d-alanyl and glycosyl substitution in lipoteichoic acids, and acetylation of peptidoglycan. Furthermore, specific peptides derived from peptidoglycan and envelope associated (glyco)proteins were recently identified as potent immunomodulators. The latter findings are exciting in the light of the possibility of more pharmacological application of these bioactive probiotic molecules, and especially cost-effective production and targeted delivery of bioactive peptides seems to emerge as a feasible strategy to harness this knowledge.



The structure of an alternative wall teichoic acid produced by a Lactobacillus plantarum WCFS1 mutant contains a 1,5-linked poly(ribitol phosphate) backbone with 2-α-D-glucosyl substitutions

A tagF1-tagF2 deletion mutant of Lactobacillus plantarum lacks poly(glycerol phosphate) polymerase activity required for glycerol-type wall teichoic acid (WTA) biosynthesis. The mutant activates an alternative genetic locus, tarIJKL, encoding the enzymes for nucleotide activation and incorporation of ribitol in the WTA backbone polymer. This alternative ribitol-type WTA backbone and its repeating unit were isolated and characterized by HPAEC, UPLC-MS, NMR spectroscopy, and MALDI-TOF MS, using synthetic molecules as references. The structure was established as 1,5-linked poly(ribitol phosphate) which was substituted at the C-2 hydroxyl group of the ribitol residue with α-D-glucosyl at a frequency of 28%.



Cell surface-associated compounds of probiotic lactobacilli sustain the strain-specificity dogma

Probiotic lactobacilli can positively impact on the health status of targeted (diseased) populations but efficacy depends strongly on the strain employed and the molecular basis for this phenomenon is poorly understood. This review discusses the current state-of-the-art in the field of molecular probiotic-host interactions, focusing on subtle strain-specific differences in the biochemical characteristics of cell surface-associated probiotic ligands and the consequences thereof for the immune responses elicited. This research is bound to enhance our understanding of strain-specificity in relation to probiotic functionality and will allow molecular science-based design of screening and characterization assays targeted to improved selection of probiotic candidate strains. Moreover, identified bioactive effector molecules could be isolated or produced for administration in a more pharmacological regime.



A comprehensive metatranscriptome analysis pipeline and its validation using human small intestine microbiota datasets

Background: Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy. Results: The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems. Conclusions: A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.



Recent developments in basophil research: Do basophils initiate and perpetuate Th2 responses?

Basophils account for only 0.1-1% of all peripheral blood leukocytes. They were considered to be a redundant cell type for a long time. However, several findings show a non-redundant role for basophils in type 2 T-helper cell (Th2) immune responses in helminth infections, allergy and autoimmunity. Both immunoglobulin-E-dependent and -independent pathways have been described to contribute to basophil activation. In addition, several recent studies reported that basophils can function as antigen-presenting cells and are important in the initiation of Th2 immune responses. However, there are also conflicting studies that do not corroborate the importance of basophils in Th2 immune responses. This review discusses the role of basophils in Th2 immune responses in view of these recent findings.


Profile of volatile organic compounds in exhaled breath changes as a result of gluten free diet

In the present longitudinal study, we followed volatile organic compounds (VOCs) excreted in exhaled breath of 20 healthy individuals over time, while adhering to a gluten-free diet for 4 weeks prior to adherence to a normal diet. We used gas chromatography coupled with mass spectrometry (TD-GC-tof-MS) in combination with chemometric analysis to detect an array of VOCs in exhaled breath. Multivariate analysis was applied to extract the maximal information from the obtained data. Dietary intake was assessed to verify adherence to the diet and to get insight into macronutrient intake during the intervention period. A set of 12 volatile compounds distinguished the samples obtained during the gluten-free diet from those obtained during a normal diet. Seven compounds could be chemically identified (2-butanol, octane, 2-propyl-1pentanol, nonanal, dihydro-4-methyl-2(3H)-furanone, nonanoic acid and dodecanal) and speculated on a possible origin. Our findings suggest that a gluten-free dietary period had a reversible impact on participants' excreted metabolites visible in their breath. Several explanations are proposed of influencing metabolic status through dietary interventions. Although the exact origin of the discriminating compounds is not yet known, the main goal of this paper was to share a new potential use of exhaled air analysis and might become a useful tool in fields of nutrition and metabolism.

Moisture diffusivity in food materials

This paper investigates whether moisture diffusion can be predicted for food materials. We focus especially on mixtures of glucose homopolymers and water. The predictions are based on three theories: (1) the Darken relation, linking the mutual diffusivity to the self diffusivities, (2) the generalised Stokes–Einstein relation for the solute self diffusivity, and (3) the free volume theory for water self diffusivity. Using literature data obtained for the whole class of glucose homopolymer, we show that these theories predict the moisture diffusivity for the whole range of volume fractions, from zero to one, and a broad range of temperatures. Furthermore, we show that the theories equally holds for other hydrophilic biopolymers one finds in food. In the concentrated regime, all experimental data collapse to a single curve. This universal behaviour arises because these biopolymers form a hydrogen bonded network, where water molecules move via rearrangement of the free volume.



A paradigm shift in drying of food materials via free-volume concepts

We give an overview of the prediction of thermodynamics of food materials, and the kinetics of water transport in them using universal theories based on free volume concepts. These material properties are highly relevant to the prediction of food drying. These presented theories are shown to hold for a large class of polysaccharides and proteins. These different food materials apparently follow the soft matter paradigm that materials’ behavior at length scales larger than the molecular scale are dominantly determined by physical characteristics rather than their chemical details. We pose that for food and other bio-materials hydrogen-bonding is largely determining their physical behavior, as in drying.



Oil bodies: An insight in their microstructure – maize germ vs sunflower seeds

Storage triacylglycerols in oleaginous seeds are surrounded by a layer that consists of phospholipids and proteins, mainly oleosins. These entities are intracellular organelles, known as oil bodies. It is often reported that they have a spherical shape, but imaging using cryo-SEM analysis showed that they are rather elastic and their shape depends on their surrounding environment. In this research we have shown that in maize germ, which has a relatively low moisture content, the oil body geometry depends on the available space they have. On the other hand, oil bodies in sunflower seeds, which contain double amounts of water, appear with an almost spherical shape. Oil bodies can be extracted from oleaginous seeds using an aqueous alkaline extraction, which leads to a stable natural oil-in-water emulsion. As no additional energy is required, this method can be considered as sustainable and may find a lot of potential uses in industry. Extraneous co-extracted proteins most likely form a second layer around the oil body surface, which protects the oil bodies from coalescence, even at high oil concentration. The extraneous proteins of maize germ oil body emulsions could be removed by applying aqueous washing steps, but not in the case of sunflower seed oil bodies.


Microbe–microbe interactions in mixed culture food fermentations

Most known natural and industrial food fermentation processes are driven by either simple or complex communities of microorganisms. Obviously, these fermenting microbes will not only interact with the fermentable substrate but also with each other. These microbe–microbe interactions are complex but thought to be crucial for obtaining the desired product characteristics. Microbial interactions are mediated through a variety of molecular and physiological mechanisms. Examples of interaction mechanisms which have an impact on the outcome of food fermentation processes will be discussed. Finally, the technological and scientific challenges associated with the production and propagation of complex mixed starter cultures are briefly addressed. Research on the composition and functionality of complex microbial consortia is gaining momentum and will open new avenues for controlling and improving food fermentation processes, and developing new applications for mixed cultures.

Availability of public goods shapes the evolution of competing metabolic strategies

Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.