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Relating water holding capacity of ovalbumin gels to aggregate structure

To increase understanding of water holding in heat-set ovalbumin gels, the relation between aggregate structure (both in solution and in gels) and gel coarseness or gel stiffness is studied. The aggregate morphology obtained by preparation of ovalbumin gels between pH 5.8 and 6.8 differs, as shown by a combination of light scattering, confocal microscopy, electron microscopy and neutron scattering techniques. It is shown that with decreasing pH larger aggregates are formed that are more compact, indicated by the fractal dimension that increased from 1.9 to 2.6. Furthermore, larger, more compact aggregates at pH 5.8 as compared to pH 6.8 resulted in more coarse gels with a less merged microstructures. In addition, a lower gel stiffness is observed at lower pH. The combination of a higher coarseness and lower gel stiffness explains both the lower total water holding and the easier exudation of water from the gel upon applied forces.

The contribution of time-dependent stress relaxation in protein gels to the recoverable energy that is used as a tool to describe food texture

The recoverable energy (RE), defined as the ratio of the work exerted on a test specimen during compression and recovered upon subsequent decompression, has been shown to correlate to sensory profiling of protein-based food products. Understanding the mechanism determining the time-dependency of RE is primordial. This work aims to identify the protein-specific impact on the recoverable energy by stress dissipation via relaxation of (micro)structural rearrangements within protein gels. To this end, caseinate and gelatin gels are studied for their response to time-dependent mechanical deformation as they are known to develop structurally distinct network morphologies. This work shows that in gelatin gels no significant stress relaxation occurs on the seconds timescale, and consequently no time-dependency of the amount of energy stored in this material is observed. In caseinate gels, however, the energy dissipation via relaxation processes does contribute significantly to the time-dependency of reversible stored energy in the network. This can explain the obtained RE as a function of applied deformation at slow deformation rates. At faster deformation, an additional contribution to the dissipated energy is apparent, that increases with the deformation rate, which might point to the role of energy dissipation related to friction of the serum entrapped by the protein-network. This work shows that engineering strategies focused on controlling viscous flow in protein gels could be more effective to dictate the ability to elastically store energy in protein gels than routes that direct proteinspecific aggregation and/or network-assembly.

The effect of polysaccharides on the ability of whey protein gels to either store or dissipate energy upon mechanical deformation

The addition of polysaccharides to proteins during gel formation can alter the mechanical and textural properties of the resultant gels. However, the effect of addition of different polymers on mechanical properties of whey protein (WP) gels including their ability to elastically store energy, often measured in terms of the recoverable energy (RE), or dissipate energy, has not been fully reported. In this paper heatinduced WP gels containing high (HM) or low (LM) methylated negatively charged pectin or the neutral pullulan were prepared to study how the addition of polysaccharides to WP affects the mechanical properties of the formed gels. These gels were subjected to uniaxial compression and mechanical properties, including RE, were evaluated. The addition of pullulan to WP did not enhance the RE, whereas an increase in LM pectin resulted in higher RE. For gels containing HM pectin, the presence of the polymer induced an initial decrease of the RE. Nevertheless, RE increased with further increase in pectin concentration. These findings indicate that the addition of polysaccharides to whey proteins during gel formation results in changes in the RE but to different extent for different polymers. The results from this study suggest that the addition of polysaccharides to WP can be used as a tool to modulate the ability of whey protein gels to elastically store energy upon mechanical deformation.

Activation energy of the disruption of gel networks in relation to elastically stored energy in fine-stranded ovalbumin gels

The aim of this study was to relate the activation energy of the disruption of ovalbumin networks to elastically stored energy (i.e. recoverable energy, RE) obtained from mechanical deformation tests. To this end, heat-set ovalbumin gels were prepared at a fixed volume fraction and pH, but varying incubation temperatures. The activation energy required to disrupt the gels was derived from the Arrhenius equation. Increasing incubation temperature from 65 to 95 C during gel formation resulted in a gradual increase in the activation energy up to a factor of ~8. Gels obtained at or just below the protein denaturation temperature of around 75 C had significantly lower recoverable energy (RE). These latter gels also had lower fracture stress and strain. At incubation temperatures above 70 C RE was constant around 75%, although a steady increase in activation energy was observed. This demonstrates that storing energy in a protein network is not directly related to the interactions that make up the network. A combination of electron microscopy, water holding, and stress relaxation experiments were performed to study the different energy dissipation modes. It was shown that different dissipation modes for various gels were comparable, and this explains why the RE was similar, with the exception of gels prepared at lower incubation temperatures where (micro) fracture events could have occurred that lowered the RE. These results suggest that RE is not a network characteristic related to microstructural or smaller length scale interactions, but the result of various material-related energy dissipation mechanisms.

Interactions in protein mixtures. Part I : Second virial coefficients from osmometry

The interaction of proteins (b-lactoglobulin, Bovine Serum Albumin (BSA), gelatins and whey protein isolate (WPI)) in solution was quantified by measuring their second virial coefficient using membrane osmometry. At neutral pH below 20e40 mM ionic strength, electrostatic repulsion dominated the interaction. At higher ionic strength, BSA, WPI and whey protein aggregates (WPA) were well approximated as hard spheres. On the other hand b-lactoglobulin behaves as an adhesive hard sphere for which the stickiness parameter t (known from the Baxter model for sticky hard spheres) and depth and width of the adhesive part of the interaction potential were calculated.

Interactions in protein mixtures. Part II: A virial approach to predict phase behavior

A virial theory was used to relate molecular interactions (in terms of second virial coefficients, B0) and molecular size ratios to liquideliquid phase separation. Application of the virial theory to binary hard sphere mixtures (additive and non-additive) confirmed the applicability of this simple approach towards predicting phase behavior based on two-particle interactions. Experimentally, second cross virial coefficients were obtained for dextran/gelatin, whey protein isolate (WPI)/gelatin mixtures and whey protein aggregate (WPA)/gelatin mixtures using membrane osmometry at varying ionic strength. From this, solvent conditions where interactions between proteins are dominated by electrostatics and solvent conditions where interactions are dominated by hard body interactions could be determined. Using experimentally obtained second virial coefficients, the liquideliquid phase separation for gelatin/dextran mixtures was successfully predicted. Second cross virial coefficients for gelatin/whey protein isolate and for gelatin/whey protein aggregate could be related to the absence of phase separation in these mixtures. This could be related to a similar size of the proteins and their non-additive behavior at conditions where they mainly interact via hard body interactions.

Microstructure and rheology of globular protein gels in the presence of gelatin

The microstructure and rheological response of globular protein gels (whey protein isolate (WPI) and soy protein isolate (SPI)) in the presence of gelatin (type A, type B and hydrolyzed type A) was investigated. Microstructural information was obtained using a combination of confocal laser scanning microscopy (CLSM) and spin echo small-angle neutron scattering (SESANS). Addition of gelatin led to a coarsening of the globular protein gel structure and to a reduction in storage modulus of globular protein gels. The presence of hydrolyzed gelatin on the other hand did not induce these changes in the globular protein gels. Results were obtained at conditions where proteins only interact via hard body interactions. For these conditions it was concluded that the ratio of molecular sizes is the most important determinant for the occurrence or absence of rheological and microstructural changes in globular protein gels prepared in the presence of gelatin.

Calcium binding restores gel formation of succinylated gelatin and reduces brittleness with preservation of the elastically stored energy

To better tailor gelatins for textural characteristics in (food) gels, their interactions are destabilized by introduction of electrostatic repulsions and creation of affinity sites for calcium to “lock” intermolecular interactions. For that purpose gelatins with various degrees of succinylation are obtained. Extensive succinylation hampers helix formation and gel strength is slightly reduced. At high degrees of succinylation the helix propensity, gelling/melting temperatures, concomitant transition enthalpy, and gel strength become calcium-sensitive, and relatively low calcium concentrations largely restore these properties. Although succinylation has a major impact on the brittleness of the gels formed and the addition of calcium makes the material less brittle compared to nonmodified gelatin, the modification has no impact on the energy balance in the gel, where all energy applied is elastically stored in the material. This is explained by the unaffected stress relaxation by the network and high water-holding capacity related to the small mesh sizes in the gels.

Water holding of soy protein gels is set by coarseness, modulated by calcium binding, rather than gel stiffness

This work aims to differentiate between the contributions to water holding (WH) by gel microstructure and network stiffness of soy protein (SP) gels. SP were succinylated to increase calcium binding affinity, and the presence of different calcium salts were used to generate gels with different morphologies while keeping ionic strength and protein concentration constant. It was found that not gel stiffness, but coarseness (gel microstructure inhomogeneity) is more dominant in setting the WH ability. A higher energy dissipation of applied stress onto the protein network was related to inability of a gel network to retain water.

Permeability of gels is set by the impulse applied on the gel

To better understand sensory perception of foods, water exudation studies on protein-based gels are of a high importance. It was aimed to study the interplay of gel coarseness and gel stiffness on water holding (WH) and water flow kinetics from the gel once force is applied onto the material. Ovalbumin heat-set gels were used as a model system, where protein volume fraction was kept constant and ionic strength was varied to obtain a range of different gel morphologies and stiffness. WH of gels was measured both as a function of time and force applied. From experimental data (i) an effective gel permeability coefficient and (ii) an effective water flux coefficient were obtained and related to gel coarseness and stiffness. Gel coarseness determined maximum amount of water removed from the gel at defined conditions, where lower (0.1 mm) and upper ( 0.4 mm) limiting scales for water removal were identified. Gel stiffness is the major determinant for water removal kinetics from the gel. The combination of gel coarseness and gel stiffness showed a cooperative effect on gel WH. The insights can be exploited in product development to predict and tune oral perception properties of (new) products.

Characterizing length scales that determine the mechanical behavior of gels from crosslinked casein micelles

Mechanical behavior of a protein gel plays a large role in sensory properties. Despite the large amount of research on caseins, the origin of the mechanical behavior is not well understood yet. To determine the length scales that are relevant for the mechanical behavior of casein gels, casein micelles were crosslinked with increasing amount of transglutaminase followed by acidification to form gels. The gel heterogeneity, observed with confocal microscopy, electron microscopy and light scattering showed a gradual decrease on a micrometer length scale with increasing crosslinking. Such gradual change as a function of crosslinking was also observed in the elastic modulus and the Young’s modulus of the gels. Furthermore, particle size both prior to gelation and in the gel decreased with increasing crosslinking. Casein micelle stiffness (determined by atomic force microscopy) showed a maximum and the amount of water entrapped by the gel particles and their aggregates in the gel (determined by neutron scattering) showed a minimum with increasing crosslinking. These extrema coincides with the extrema observed in kinetics of gel formation and in gel breakdown properties. It was concluded that the elasticity of the gel originates on the length scale of the casein micelle (a few hundred nanometer), while fracture properties are determined at a smaller length scale, by the structure within the casein micelle.

Electrophoretic behavior in relation to the structural integrity of codfish parvalbumin upon heat treatment

This work evaluates the impact of heat processing of parvalbumin, a major fish allergen, on the consequences for quantitative analysis of this protein embedded in different matrices during heating (either isolated, in an aqueous extract, or in whole fillets) to assess potential health risks. It is shown that oligomerization of parvalbumin does occur, but only upon heat treatment above 80 °C. This coincides with the ability of the isolated protein to refold up to this temperature in a fully reversible way, as demonstrated by circular dichroism analysis. In autoclaved samples a disintegration of the protein structure is observed. The situation becomes different when parvalbumin is embedded in a matrix with other constituents, as in fish extracts or whole fillets. The electrophoretic analysis of parvalbumin (SDS-PAGE and immunoblotting) is largely determined by complexation with other proteins resulting in insoluble materials caused by the partial unfolding of the parvalbumin at elevated temperatures. This effect is more strongly observed for cod fish extract, compared to whole cod fillets, as in the latter situation the integrity of the tissue hampers this interprotein complexation. Moreover, it is shown by ELISA analysis of heat-treated samples that using blotting procedures where disintegration of complexes may be promoted, restoring some of the IgG-binding propensity, may provide false outcomes. It was concluded that antibody binding to parvalbumin is dominated by the potential to form heatinduced complexes with other proteins. The possibly less-soluble or extractable character of these complexes may provide confusing information regarding potential health risks of fish and fish protein-containing food composites when such heat-treated samples are analyzed by immunochemical assays.

Quantitative analysis of the network structure that underlines the transitioning in mechanical responses of pea protein gels

The objective of this study was to analyze quantitatively the network structure that underlines the transitioning in the mechanical responses of heat-induced pea protein gels. To achieve this, gels were prepared from pea proteins at varying pHs from 3.0 to 4.2 at a fixed 100 mg/mL protein concentration. Gels were also prepared by varying the protein concentration from 100 to 150 mg/mL at a fixed pH 3.0. Mechanical deformation properties of the gels were determined. An increase in protein concentration at a fixed pH resulted in an increase in fracture stress and Young's modulus. Variation of the pH at a fixed protein concentration resulted in transitioning in mechanical responses such as fracture stress, fracture strain, and the recoverable energy. The network structures were visualized by the use of confocal laser scanning and scanning electron microscopy, and the characteristic length scales of these structures were quantitatively analyzed in terms of the pair correlation function. Variation of the protein concentration at a fixed pH did not significantly alter the microstructure of the gels, whereas variation of the pH at a fixed protein concentration resulted in significant changes in the gel structure. Structural transitioning was shown to occur around pH 3.7. The findings from this study show transitioning in rheological responses of pea protein gels occur as a result of structural changes. The results from this study offer opportunities to broaden the application of pea proteins in food products, as products with desirable rheological (textural) and structural properties can be designed from pea proteins.

Protein aggregates may differ in water entrapment but are comparable in water confinement

Aggregate size and density are related to gel morphology. In the context of the water distribution in complex food systems, in this study, it was aimed to investigate whether protein aggregates varying in size and density differ in entrapped and confined water. Heat-set soy protein aggregates (1%, v/v) prepared in the presence of 3.5 mM divalent salts increased in size and decreased in apparent density following the salt type order MgSO4, MgCl2, CaSO4, and CaCl2. In the absence of applied (centrifugal) forces, larger and less dense aggregates entrap more water. When force is applied from larger and more deformable aggregates, more water can be displaced. Entrapped water of ∼8−13 g of water/g of protein is associated with (pelleted) aggregates, of which approximately 4.5−8.5 g of water/g of protein is not constrained in exchangeability with the solvent. The amount of confined water within aggregates was found to be independent of the aggregate density and accounted for ∼3.5 g of water/g of protein. Confined water in aggregates is hindered in its diffusion because of physical structure constraints and, therefore, not directly exchangeable with the solvent. These insights in the protein aggregate size and deformability in relation to water entrapment and confinement could be used to tune water holding on larger length scales when force is applied.

Relation between gel stiffness and water holding for coarse and fine-stranded protein gels

The sensory perception of foods is directly related to gel morphology. The aim of this research was to investigate the relationship between gel water holding and stiffness for gels with a different morphology. Whey protein gels were prepared by varying ionic strength to create fine and coarse-stranded gels. These gels were characterized for their coarseness, stiffness and their water holding capacity. Fine gels were referred to typical coarseness length scale smaller than 0.1 mm and coarse gels were referred to coarseness length scale larger than 0.1 mm.Water holding was measured both as a function of time and of applied pressure. Increased gel coarseness length scale in both fine and coarse gels resulted in a larger extent of network deformation at a certain applied force. For fine gels, the coarseness length scale of the gel was shown to determine water removal. In the case of coarse gels, coarseness length scale and stiffness had a counteracting effect, but coarseness length scale was still dominant. These results show that the tuning of coarseness length scale of protein networks independent of stiffness or, the other way round, provides a tool to set the water holding capacity in food gels.

Ligation of pork skin gelatin with glucose moieties affects the junction zones in gelled networks

This study aims to investigate the role of the ligation of steric moieties on the formation of junction zones during network formation of gelatin gels. The molecular conformational propensities, heat stability and mechanical properties of gradually chemically modified pork skin gelatin have been evaluated. To this end, glucose moieties are ligated to lysine residues on the protein using the Maillard reaction. It is shown that ligation of small sugar moieties only marginally impairs with triple helix formation and does not interfere with the mechanism of network formation. The Young's modulus (gel stiffness) and the fracture properties decrease with increasing degree of modification, but not the ability to reversibly store energy in the network. It is suggested that Maillardation affects the strandestrand interaction energy, but not necessarily the number of junction zones. A retained recoverable energy while the fracture properties can be affected allows industry to tailor food products in terms of their expected sensory performance.

Short-chain fatty acids protect against high-fat-induced obesity via a PPARgamma-dependent switch from lipogenesis to fatty-acid oxidation

Short-chain fatty acids (SCFAs) are the main products of dietary fiber fermentation and are believed to drive the fiber-related prevention of the metabolic syndrome. Here we show that dietary SCFAs induce a peroxisome proliferator-activated receptor (PPAR) γ-dependent switch from lipid synthesis to utilization. Dietary SCFA supplementation prevented and reversed high-fat diet-induced metabolic abnormalities in mice by decreasing PPARγ expression and activity. This increased the expression of mitochondrial uncoupling protein 2 and raised the AMP/ATP ratio, thereby stimulating oxidative metabolism in liver and adipose tissue via AMP-activated protein kinase. The SCFA-induced reduction in body weight and stimulation of insulin sensitivity were absent in mice with adipose-specific disruption of PPARγ. Similarly, SCFA-induced reduction of hepatic steatosis was absent in mice lacking hepatic PPARγ. These results demonstrate that adipose and hepatic PPARγ are critical mediators of the beneficial effects of SCFA on the metabolic syndrome, with clearly distinct and complementary roles. Our findings indicate that SCFAs may be used therapeutically as cheap and selective PPARγ modulators.

Gut colonization with methanobrevibacter smithii is associated with childhood weight development

Objective: To prospectively investigate the presence and counts of archaea in feces of 472 children in association with weight development from 6 to 10 years of age. Methods: Within the KOALA Birth Cohort Study, a single fecal sample from each child was analyzed by quantitative polymerase chain reaction to quantify archaea (Methanobrevibacter smithii, Methanosphera stadtmanae). Anthropometric outcomes (overweight [body mass index {BMI} ≥ 85th percentile], age- and sex-standardized BMI, weight, and height z-scores) were repeatedly measured at ages (mean ± SD) of 6.2 ± 0.5, 6.8 ± 0.5, 7.8 ± 0.5, and 8.8 ± 0.5 years. Generalized estimating equation was used for statistical analysis while controlling for confounders. Results: Methanobrevibacter smithii colonization was associated with an increased risk of overweight (adjusted odds ratio [OR] = 2.69; 95% confidence interval [CI] 0.96-7.54) from 6 to 10 years of age. Children with high levels (>7 log10 copies/g feces) of this archaeon were at highest risk for overweight (OR = 3.27; 95% CI 1.09-9.83). Moreover, M. smithii colonization was associated with higher weight z-scores (adj. β 0.18; 95% CI 0.00-0.36), but not with height. For BMI z-scores, the interaction (P = 0.008) between M. smithii and age was statistically significant, implying children colonized with M. smithii had increasing BMI z-scores with age. Conclusions: Presence and higher counts of M. smithii in the gut of children are associated with higher weight z-scores, higher BMI z-scores, and overweight.

Hepatic uptake of rectally administered butyrate prevents an increase in systemic butyrate concentrations in humans

BACKGROUND:Short-chain fatty acids (SCFAs), fermentation products of undigested fibers, are considered beneficial for colonic health. High plasma concentrations are potentially harmful; therefore, information about systemic SCFA clearance is needed before therapeutic use of prebiotics or colonic SCFA administration. OBJECTIVE:The aim of this study was to investigate the effect of rectal butyrate administration on SCFA interorgan exchange. METHODS:Twelve patients (7 men; age: 66.4 ± 2.0 y; BMI 24.5 ± 1.4 kg/m2) undergoing upper abdominal surgery participated in this randomized placebo-controlled trial. During surgery, 1 group received a butyrate enema (100 mmol sodium butyrate/L; 60 mL; n = 7), and the other group a placebo (140 mmol 0.9% NaCl/L; 60 mL; n = 5). Before and 5, 15, and 30 min after administration, plasma samples were taken from the radial artery, hepatic vein, and portal vein. SCFA concentrations were analyzed, and fluxes from portal-drained viscera, liver, and splanchnic area were calculated and used for the calculation of the incremental area under the curve (iAUC) over a 30-min period. RESULTS:Rectal butyrate administration led to higher portal butyrate concentrations at 5 min compared with placebo (92.2 ± 27.0 μmol/L vs. 14.3 ± 3.4 μmol/L, respectively; P < 0.01). iAUCs of gut release (282.8 ± 133.8 μmol/kg BW · 0.5 h) and liver uptake (-293.7 ± 136.0 μmol/kg BW · 0.5 h) of butyrate were greater in the butyrate-treated group compared with placebo [-16.6 ± 13.4 μmol/kg BW · 0.5 h (gut release) and 16.0 ± 13.8 μmol/kg BW · 0.5 h (liver uptake); P = 0.01 and P < 0.05, respectively]. As a result, splanchnic butyrate release did not differ between groups. CONCLUSION: After colonic butyrate administration, splanchnic butyrate release was prevented in patients undergoing upper abdominal surgery. These observations imply that therapeutic colonic SCFA administration at this dose is safe. The trial was registered at clinicaltrials.gov as NCT02271802.

Proton magnetic resonance spectroscopy reveals increased hepatic lipid content after a single high-fat meal with no additional modulation by added protein

Background: Fat accumulation in nonadipose tissue is linked to insulin resistance and metabolic diseases. Earlier studies have shown that hepatic lipid accumulation can occur after 4 d of a high-fat diet in humans, and this fat accumulation can be blunted by the ingestion of additional proteins. Objectives: In this study, we explored whether a single high-fat meal increased the lipid content in liver and skeletal muscle as measured by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) and whether the addition of protein can modulate the postprandial ectopic lipid storage. Design: Intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) concentrations were determined by using ¹H-MRS before and 3 and 5 h after a high-fat with added protein meal (61.5% of energy from fat) or a high-fat without added protein meal (mean ± SEM: 51.1 ± 7.9 g of protein; 191.9 ± 9.9 kcal added) in a randomized crossover study. IHL and IMCL concentrations were converted to absolute concentrations (g/kg wet weight) by using water as an internal reference. Results: Nine lean, healthy subjects [6 men and 3 women; mean (±SD) age: 22.7 ± 3.0 y; mean body mass index (in kg/m²): 21.8 ± 1.8] were included in this study. IHL concentrations increased ∼20% (P < 0.01) at 3 h after the meal and did not further increase after 5 h. In contrast, IMCL concentrations were not altered during the postprandial period (P = 0.74). The addition of protein to a single high-fat meal did not change the postprandial accumulation of fat in the liver (P = 0.93) or skeletal muscle (P = 0.84). Conclusions: In this study, we showed that a single energy-dense, high-fat meal induced net lipid accumulation in the liver, which was detected by using in vivo ¹H-MRS. This noninvasive approach might bring new opportunities to study postprandial hepatic lipid dynamics. The addition of protein did not change the ectopic lipid retention after a single high-fat meal.

Effects of high-fat feeding on ectopic fat storage and postprandial lipid metabolism in mouse offspring

Objective: Parental high-fat feeding was proposed to negatively impact metabolic health in offspring. Here, the ectopic fat storage in heart and liver in offspring was investigated, and the effects on mitochon- drial function, de novo lipogenesis, and postprandial lipid metabolism were explored in detail. Methods: Male and female mice received either a high-fat (HF) or standard chow (LF) diet during mating, gestation and lactation. All offspring animals received the HF diet. Results: Abdominal visceral adipose tissue tended to be higher in HF/HF mice. Cardiac lipid content was also higher in the HF/HF mice (LF/HF vs. HF/HF: 1.03% 6 0.08% vs. 1.33% 6 0.07% of water signal, P 5 0.01). In contrast, hepatic lipid content tended to be lower in HF/HF mice compared to LF/HF mice. A severely disturbed postprandial lipid clearance was revealed in HF/HF mice by the results from the tri- glyceride (TG) tolerance tests (LF/HF vs. HF/HF: 6,753 6 2,213 vs. 14,367 6 1,978 mmol l21min21,P 5 0.01) and 13 C-fatty acid retention test (LF/HF vs. HF/HF: 2.73% 6 0.85% vs. 0.89% 6 0.26% retention from bolus, P 5 0.04), which may underlie the lower hepatic lipid content. Conclusions: Here it is shown that HF diet negatively impacts postprandial TG clearance in offspring and results in an overall metabolic unfavorable phenotype and ectopic lipid deposition in the heart and in visceral storage sites.

Protein intake and lean body mass preservation during energy intake restriction in overweight older adults

Background:  Dietary-induced weight loss is generally accompanied by a decline in skeletal muscle mass. The loss of muscle mass leads to a decline in muscle strength and impairs physical performance. A high dietary protein intake has been suggested to allow muscle mass preservation during energy intake restriction. Objective: To investigate the impact of increasing dietary protein intake on lean body mass, strength and physical performance during 12 weeks of energy intake restriction in overweight older adults. Design: Sixty-one overweight and obese men and women (63±5 years) were randomly assigned to either a high protein diet (HP; 1.7 g kg−1 per day; n=31) or normal protein diet (NP; 0.9 g kg−1 per day; n=30) during a 12-week 25% energy intake restriction. During this controlled dietary intervention, 90% of the diet was provided by the university. At baseline and after the intervention, body weight, lean body mass (dual-energy X-ray absorptiometry), leg strength (1-repetition maximum), physical performance (Short Physical Performance Battery, 400 m) and habitual physical activity (actigraph) were assessed. Results: Body weight declined in both groups with no differences between the HP and NP groups (−8.9±2.9 versus −9.1±3.4 kg, respectively;P=0.584). Lean body mass declined by 1.8±2.2 and 2.1±1.4 kg, respectively, with no significant differences between groups (P=0.213). Leg strength had decreased during the intervention by 8.8±14.0 and 8.9±12.8 kg, with no differences between groups (P=0.689). Physical performance as measured by 400 m walking speed improved in both groups, with no differences between groups (P=0.219). Conclusions: Increasing protein intake above habitual intake levels (0.9 g kg−1 per day) does not preserve lean body mass, strength or physical performance during prolonged energy intake restriction in overweight older adults.

Post-prandial muscle protein synthesis: “You are what you just ate”

Background: Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults. Objective: To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein. Design: 12 healthy young males ingested 20 g intrinsically [1-13C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-2H5]-phenylalanine, L-[ring-2H2]-tyrosine, and L-[1-13C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment. Results: 55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min-1·100 mL leg volume-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h-1 based upon the muscle protein bound L-[ring-2H5]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-13C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE). Conclusion: Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Short-term muscle disuse lowers myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion

Disuse leads to rapid loss of skeletal muscle mass and function. It has been hypothesized that short successive periods of muscle disuse throughout the lifespan play an important role in the development of sarcopenia. The physiological mechanisms underlying short-term muscle disuse atrophy remain to be elucidated. We assessed the impact of 5 days of muscle disuse on postabsorptive and postprandial myofibrillar protein synthesis rates in humans. Twelve healthy young (22 ± 1 yr) men underwent a 5-day period of one-legged knee immobilization (full leg cast). Quadriceps cross-sectional area (CSA) of both legs was assessed before and after immobilization. Continuous infusions of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine were combined with the ingestion of a 25-g bolus of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled dietary protein to assess myofibrillar muscle protein fractional synthetic rates in the immobilized and nonimmobilized control leg. Immobilization led to a 3.9 ± 0.6% decrease in quadriceps muscle CSA of the immobilized leg. Based on the l-[ring-(2)H5]phenylalanine tracer, immobilization reduced postabsorptive myofibrillar protein synthesis rates by 41 ± 13% (0.015 ± 0.002 vs. 0.032 ± 0.005%/h, P < 0.01) and postprandial myofibrillar protein synthesis rates by 53 ± 4% (0.020 ± 0.002 vs. 0.044 ± 0.003%/h, P < 0.01). Comparable results were found using the l-[1-(13)C]leucine tracer. Following protein ingestion, myofibrillar protein bound l-[1-(13)C]phenylalanine enrichments were 53 ± 18% lower in the immobilized compared with the control leg (0.007 ± 0.002 and 0.015 ± 0.002 mole% excess, respectively, P < 0.05). We conclude that 5 days of muscle disuse substantially lowers postabsorptive myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

There are no nonresponders to resistance-type exercise training in older men and women

Objective To assess the proposed prevalence of unresponsiveness of older men and women to augment lean body mass, muscle fiber size, muscle strength, and/or physical function following prolonged resistance-type exercise training. Design/Setting/Participants A retrospective analysis of the adaptive response to 12 (n = 110) and 24 (n = 85) weeks of supervised resistance-type exercise training in older (>65 years) men and women. Measurements Lean body mass (DXA), type I and type II muscle fiber size (biopsy), leg strength (1-RM on leg press and leg extension), and physical function (chair-rise time) were assessed at baseline, and after 12 and 24 weeks of resistance-type exercise training. Results Lean body mass increased by 0.9 ± 0.1 kg (range: −3.3 to +5.4 kg; P < .001) from 0 to 12 weeks of training. From 0 to 24 weeks, lean body mass increased by 1.1 ± 0.2 kg (range: −1.8 to +9.2 kg; P < .001). Type I and II muscle fiber size increased by 324 ± 137 μm2 (range: −4458 to +3386 μm2; P = .021), and 701 ± 137 μm2 (range: −4041 to +3904 μm2; P < .001) from 0 to 12 weeks. From 0 to 24 weeks, type I and II muscle fiber size increased by 360 ± 157 μm2 (range: −3531 to +3426 μm2; P = .026) and 779 ± 161 μm2 (range: −2728 to +3815 μm2; P < .001). The 1-RM strength on the leg press and leg extension increased by 33 ± 2 kg (range: −36 to +87 kg; P < .001) and 20 ± 1 kg (range: −22 to +56 kg; P < .001) from 0 to 12 weeks. From 0 to 24 weeks, leg press and leg extension 1-RM increased by 50 ± 3 kg (range: −28 to +145 kg; P < .001) and 29 ± 2 kg (range: −19 to +60 kg; P < .001). Chair-rise time decreased by 1.3 ± 0.4 seconds (range: +21.6 to −12.5 seconds; P = .003) from 0 to 12 weeks. From 0 to 24 weeks, chair-rise time decreased by 2.3 ± 0.4 seconds (range: +10.5 to −23.0 seconds; P < .001). Nonresponsiveness was not apparent in any subject, as a positive adaptive response on at least one training outcome was apparent in every subject. Conclusions A large heterogeneity was apparent in the adaptive response to prolonged resistance-type exercise training when changes in lean body mass, muscle fiber size, strength, and physical function were assessed in older men and women. The level of responsiveness was strongly affected by the duration of the exercise intervention, with more positive responses following more prolonged exercise training. We conclude that there are no nonresponders to the benefits of resistance-type exercise training on lean body mass, fiber size, strength, or function in the older population. Consequently, resistance-type exercise should be promoted without restriction to support healthy aging in the older population.

Impact of the Macronutrient Composition of a Nutritional Supplement on Muscle Protein Synthesis Rates in Older Men: A Randomized, Double Blind, Controlled Trial

GEEN TIFN AFFILIATIE Context: An impaired muscle protein synthetic response to feeding likely contributes to muscle loss with aging. There are few data available on the effect of the macronutrient composition of clinical supplements on the postprandial muscle protein synthetic response in older subjects. Objective: The objective of the study was to determine the impact of the macronutrient composition of a nutritional supplement on the postprandial muscle protein synthetic response in older men. Methods: A total of 45 nonsarcopenic older men (aged 69 ± 1 y; body mass index 25.7 ± 0.3 kg/m(2)) were randomly assigned to ingest 21 g of leucine-enriched whey protein with carbohydrate (9 g) and fat (3 g) (Pro-En), an isonitrogenous amount of 21 g of leucine-enriched whey protein without carbohydrate and fat (Pro), or an isocaloric mixture (628 kJ) containing carbohydrate and fat only (En). Stable isotope tracer methodology was applied to assess the basal as well as the postprandial muscle protein synthesis rates in the three groups. Results: Ingestion of protein in the Pro-En and Pro groups significantly increased muscle protein synthesis rates when compared with the basal rates (from 0.032 ± 0.003%/h to 0.05%/h 3 ± 0.004%/h and 0.040%/h ± 0.003%/h to 0.049%/h ± 0.003%/h, respectively; P < .05), whereas ingestion of carbohydrate and fat did not increase muscle protein synthesis rates in the En group (from 0.039%/h ± 0.004%/h to 0.040%/h ± 0.003%/h; P = .60). Despite the greater postprandial rise in circulating insulin concentration in the Pro-En group, no significant differences were observed in postprandial muscle protein synthesis rates between the Pro-En and Pro groups (P = .32). Postprandial muscle protein synthesis rates were higher in the Pro-En vs En group (P = .01). Conclusion: The ingestion of a nutritional supplement containing 21 g of leucine-enriched whey protein significantly raises muscle protein synthesis rates in nonsarcopenic older men, but coingestion of carbohydrate and fat does not modulate the postprandial muscle protein synthetic response to protein ingestion in older men.

Capsaicin-induced satiety is associated with gastrointestinal distress but not with release of satiety hormones

Background: Capsaicin, which is the major pungent principle in chili peppers, is able to induce satiety and reduce caloric intake. The exact mechanism behind this satiating effect is still unknown. We hypothesized that capsaicin induces satiety through the release of gastrointestinal peptides, such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), from enteroendocrine cells in the small intestine. OBJECTIVE: We investigate the effects of an intraduodenal capsaicin infusion (1.5 mg pure capsaicin) in healthy volunteers on hunger, satiety, and gastrointestinal symptoms and the release of GLP-1 and PYY. DESIGN: Thirteen participants (7 women) [mean ± SEM age: 21.5 ± 0.6 y; body mass index (in kg/m(2)): 22.8 ± 0.6] participated in this single-blind, randomized, placebo-controlled crossover study with 2 different treatments. During test days, an intraduodenal infusion of either capsaicin or a placebo (physiologic saline) was performed with the use of a nasoduodenal catheter over a period of 30 min. Visual analog scale scores were used to measure hunger, satiety, and gastrointestinal symptoms. Blood samples were drawn at regular intervals for GLP-1 and PYY. Gallbladder volumes were measured with the use of real-time ultrasonography. RESULTS: The intraduodenal capsaicin infusion significantly increased satiety (P-treatment effect < 0.05) but also resulted in an increase in the gastrointestinal symptoms pain (P-treatment × time interaction < 0.0005), burning sensation (P-treatment × time interaction < 0.0001), nausea (P-treatment × time interaction < 0.05), and bloating (P-treatment × time interaction < 0.001) compared with the effects of the placebo infusion. Satiety scores had a positive correlation with all gastrointestinal symptoms. No differences in GLP-1 and PYY concentrations and gallbladder volumes were observed after the capsaicin infusion compared with after the placebo infusion. CONCLUSIONS: An intraduodenal infusion of capsaicin significantly increases satiety but does not affect plasma concentrations of GLP-1 and PYY. Rather, the effect on satiety seems related to gastrointestinal stress as shown by the associations with pain, burning sensation, nausea, and bloating scores. This trial was registered at clinicaltrials.gov as NCT01667523.

Cholecystokinin regulates satiation independently of the abdominal vagal nerve in a pig model of total subdiaphragmatic vagotomy

The vagal nerve and gut hormones CCK and GLP-1 play important roles in the control of food intake. However, it is not clear to what extent CCK and GLP-1 increase satiation by stimulating receptors located on abdominal vagal nerve endings or via receptors located elsewhere. This study aimed to further explore the relative contribution of the abdominal vagal nerve in mediating the satiating effects of endogenous CCK and GLP-1. Total subdiaphragmatic vagotomy or sham operation was combined with administration of CCK1 and GLP-1 receptor antagonists devazepide and exendin (9–39) in 12 pigs, applying an unbalanced Latin Square within-subject design. Furthermore, effects of vagotomy on preprandial and postprandial acetaminophen absorption, glucose, insulin, GLP-1 and CCK plasma concentrations were investigated. Ad libitum liquid meal intake (mean ± SEM) was similar in sham and vagotomized pigs (4180 ± 435 and 3760 ± 810 g/meal). Intake increased by about 20% after blockade of CCK1 receptors, independently of the abdominal vagal nerve. Food intake did not increase after blockade of GLP-1 receptors. Blockade of CCK1 and GLP-1 receptors increased circulating CCK and GLP-1 concentrations in sham pigs only, suggesting the existence of a vagal reflex mechanism in the regulation of plasma CCK1 and GLP-1 concentrations. Vagotomy decreased acetaminophen absorption and changed glucose, insulin, CCK and GLP-1 concentrations indicating a delay in gastricemptying. Our data showthat at liquid feeding, satiation is decreased effectively by pharmacological blockade of CCK1 receptors. We conclude that regulation of liquid meal intake appears to be primarily regulated by CCK1 receptors not located on abdominal vagal nerve endings.

Ileal brake activation: Macronutrient-specific effects on eating behavior?

Background: Activation of the ileal brake, by infusing lipid directly into the distal part of the small intestine, alters gastrointestinal (GI) motility and inhibits food intake. The ileal brake effect on eating behavior of the other macronutrients is currently unknown. Objective: The objective of this study was to investigate the effects of ileal infusion of sucrose and casein on food intake, release of GI peptides, gastric emptying rate and small-bowel transit time with safflower oil as positive control. Design: This randomized, single-blind, crossover study was performed in 13 healthy subjects (6 male; mean age 26.4 ± 2.9 years; mean body mass index 22.8 ± 0.4 kgm− 2) who were intubated with a naso–ileal catheter. Thirty minutes after the intake of a standardized breakfast, participants received an ileal infusion, containing control ((C) saline), safflower oil ((HL) 51.7 kcal), low-dose casein ((LP) 17.2 kcal) or high-dose casein ((HP) 51.7 kcal), low-dose sucrose ((LC) 17.2 kcal) and high-dose sucrose ((HC) 51.7 kcal), over a period of 90 min. Food intake was determined during an ad libitum meal. Visual analogue score questionnaires for hunger and satiety and blood samples were collected at regular intervals. Results: Ileal infusion of lipid, protein and carbohydrate resulted in a significant reduction in food intake compared with control (HL: 464.3 ± 90.7 kcal, Po0.001; HP: 458.0 ± 78.6 kcal, Po0.005; HC: 399.0 ± 57.0 kcal, Po0.0001 vs control: 586.7 ± 70.2 kcal, Po0.001, respectively). A reduction in energy intake was still apparent when the caloric amount of infused nutrients was added to the amount eaten during the ad libitum meal. Secretion of cholecystokinin and peptide YY but not of glucagon-like peptide-1 (7–36) was increased during ileal perfusion of fat, carbohydrates and protein. During ileal perfusion of all macronutrients, a delay in gastric emptying and intestinal transit was observed, but differences were not significant compared with control. Conclusion: Apart from lipids, also sucrose and casein reduce food intake on ileal infusion, thereby activating the ileal brake. In addition to food intake, also satiety and GI peptide secretion were affected.

Intraduodenal infusion of a combination of tastants decreases food intake in humans

Background: Taste receptors are expressed not only in taste buds but also in the gastrointestinal tract. It has been hypothesized that these receptors may play a role in satiety and food intake. Objective: This study investigated the effect of intraduodenal tastant infusions (bitter, sweet, and umami) on food intake, hunger and fullness, gastrointestinal symptoms, and gastrointestinal peptide release. Design: Fifteen healthy volunteers [6 male; mean ± SEM age: 23.9 ± 2.0 y; mean ± SEM body mass index (in kg/m(2)): 22.4 ± 0.3] received 5 treatments in a double-blind, randomized, placebo-controlled crossover design. Test days started with the insertion of a nasoduodenal catheter followed by a standardized liquid breakfast. Participants received an intraduodenal infusion 150 min after breakfast, containing quinine (bitter), rebaudioside A (sweet), monosodium glutamate (umami), a combination of the 3 tastants, or placebo (tap water) over a period of 60 min. Food intake was measured during an ad libitum meal, and visual analog scales were used to monitor gastrointestinal complaints and hunger and fullness scores. Blood samples were drawn at regular intervals for cholecystokinin, glucagon-like peptide 1 (GLP-1), and peptide YY (PYY) analysis. Results: Infusion of the combination of tastants substantially decreased food intake (422 ± 97 compared with 486 ± 104 kcal for placebo, P < 0.05), whereas both a combination of tastants and umami decreased hunger scores compared with placebo. No change in cholecystokinin, GLP-1, or PYY concentrations was observed during the infusions. Intraduodenal infusions of the tastants did not result in gastrointestinal symptoms. Conclusions: Intraduodenal infusion of umami and a combination of tastants inhibits feelings of hunger, but only the latter also reduces food intake. However, these alterations were not accompanied by changes in the plasma concentrations of the gut-derived peptides cholecystokinin, GLP-1, or PYY. This trial was registered at clinicaltrials.gov as NCT01956838.

Cholecystokinin regulates satiation independently of the abdominal vagal nerve in a pig model of total subdiaphragmatic vagotomy

The vagal nerve and gut hormones CCK and GLP-1 play important roles in the control of food intake. However, it is not clear to what extent CCK and GLP-1 increase satiation by stimulating receptors located on abdominal vagal nerve endings or via receptors located elsewhere. This study aimed to further explore the relative contribution of the abdominal vagal nerve in mediating the satiating effects of endogenous CCK and GLP-1. Total subdiaphragmatic vagotomy or sham operation was combined with administration of CCK1 and GLP-1 receptor antagonists devazepide and exendin (9-39) in 12 pigs, applying an unbalanced Latin Square within-subject design. Furthermore, effects of vagotomy on preprandial and postprandial acetaminophen absorption, glucose, insulin, GLP-1 and CCK plasma concentrations were investigated. Ad libitum liquid meal intake (mean±SEM) was similar in sham and vagotomized pigs (4180±435 and 3760±810 g/meal). Intake increased by about 20% after blockade of CCK1 receptors, independently of the abdominal vagal nerve. Food intake did not increase after blockade of GLP-1 receptors. Blockade of CCK1 and GLP-1 receptors increased circulating CCK and GLP-1 concentrations in sham pigs only, suggesting the existence of a vagal reflex mechanism in the regulation of plasma CCK1 and GLP-1 concentrations. Vagotomy decreased acetaminophen absorption and changed glucose, insulin, CCK and GLP-1 concentrations indicating a delay in gastric emptying. Our data show that at liquid feeding, satiation is decreased effectively by pharmacological blockade of CCK1 receptors. We conclude that regulation of liquid meal intake appears to be primarily regulated by CCK1 receptors not located on abdominal vagal nerve endings.

Dynamic texture perception and oral processing of semi-solid food gels: Part 2: Impact of breakdown behaviour on bolus properties and dynamic texture perception

Food texture perception depends on food structure and oral processing behaviour. The aim of this study was to explain dynamic texture perception of emulsion-filled, semi-solid gels by properties of the boli formed during three stages of oral processing. Texture perception of emulsion-filled gels varying in fracture stress and strain was found to be a dynamic process. Specific texture attributes were perceived as dominant sensations in the beginning (firm), middle (moist, refreshing, elastic, sticky) and end of oral processing (grainy, melting, creamy). In the beginning of oral processing mechanical properties of the boli, such as first penetration peak force and flowability, were correlated to sensory firmness. In the middle of oral processing, correlations between boli properties and texture perception were more complex. Perception of moist and refreshing was related to mechanical properties of the boli, such as flowability, rather than to the amount of saliva incorporated into the boli. Perception of elastic and sticky was related to the mechanical bolus properties resilience and adhesiveness. In the end of oral processing, emulsion-filled gels were perceived either as creamy or grainy. Gels perceived as creamy revealed a high bolus flowability while gels perceived as grainy consisted of boli with a high number of broken down particles. We conclude that bolus formation and changes in the properties of the bolus underlay the changes in texture perception during oral processing.

Evidence for ball-bearing mechanism of microparticulated whey protein as fat replacer in liquid and semi-sold multi-component model foods

The current understanding of the mechanisms underlying the fat mimicking properties of fat replacers, such as microparticulated whey protein (MWP), is limited. MWP is known to provide fat-related mouthfeel in specific foods, such as yoghurts and cheeses. Tribological and rheological properties are well known to contribute to the perception of fat related sensory attributes. This study investigated the tribological and rheological properties of MWP in liquid and semi-solid model foods to reveal the mechanisms underlying the fat mimicking properties of MWP. In liquids, addition of MWP reduced the friction coefficient effectively. After scaling out the impact of viscosity on lubrication, results provide strong evidence that the reduction of friction of MWP in liquids is mainly due to ball-bearing lubrication. In semi-solid gels, addition of MWP also decreased the friction, but to a smaller extent compared to liquid model foods. The mechanism underlying the lubrication behavior of multi-component semi-solid foods that contain fat droplets, emulsifier, fat replacers such as MWP embedded in a gel matrix is complex.We conclude that different components affect the lubrication properties of the composite food through different mechanisms. Fat droplets reduce friction due to the formation of a fat film following a plate-out mechanism. We suggest that emulsifiers influence the formation of a fat or emulsifier film, whereas MWP is suggested to reduce friction due to a ball-bearing mechanism. In addition, the influence of bulk viscosity, gel fracture behavior, and the interactions between the MWP and its surrounding matrix and emulsifiers, need to be accounted for.

Uncoupling the impact of fracture properties and composition on sensory perception of emulsion-filled gels

The aim of the study is to investigate the effect of fracture properties and composition of emulsion-filled gels on dynamic texture perception. Twelve emulsion-filled gels varying in fracture stress (High/Low) and strain (High/Low) were prepared from three binary gel mixtures. Mechanical properties, syneresis, friction properties, microstructure, melting behavior, oral breakdown and texture perception of the gels were determined. Gels varying in composition but exhibiting similar fracture properties were obtained. Serum release, melting in mouth and friction varied between gels differing in composition. Fracture properties and melting of gels impacted oral breakdown. Fracture properties impacted perception of texture attributes at first bite and during chew down. Melting and syneresis impacted chew down perception of gels. We conclude that fracture stress mainly impacted texture perception at first bite, whereas fracture strain impacted perception of chew down texture attributes with high fracture strain gels being perceived creamy. The composition of gels impacted properties such as melting and serum release, which accounted for high variations in perception of moistness and creaminess between samples.

Dynamic texture perception, oral processing behaviour and bolus properties of emulsion-filled gels with and without contrasting mechanical properties

Many highly palatable foods are composed of multiple components which can have considerably different mechanical properties leading to contrasting texture sensations. The aim of this study was to better understand the impact of contrasting mechanical properties in semi-solid gels on oral processing behaviour and dynamic texture perception. Four reference emulsion-filled gels without mechanical contrast were prepared using agar (1 or 2 wt%) or gelatine (2.5 or 5.5% wt). Six emulsion-filled gels with contrasting mechanical properties were obtained by combining two different gel layers. Agar reference gels displaying low fracture strain produced boli with many small particles and were perceived as grainy. Gelatine reference gels displaying high fracture strain produced boli with few large particles which melted in mouth and were perceived as creamy. Reference gels with large fracture stress were masticated for long times with a high chewing muscle activity and perceived as firm and grainy. Bolus properties, oral processing behaviour and dynamic sensory perception of the 6 contrasting gels were compared to the 4 reference gels using Principal Component Analysis. The presence of an agar layer in contrasting gels dominated bolus properties which contained many small particles and did not mix readily in mouth. The temporal sensory profiles and sensory trajectories of contrasting gels fell between the temporal sensory profiles and sensory trajectories of the two gel layers which they were composed of. We conclude that distinct features of dynamic texture perception in emulsion-filled gels with mechanical contrast are perceived separately in mouth.

Influence of stimulus properties and sensory task instructions on oral processing behavior of liquid stimuli

This study determined the influence of texture properties and sensory task instructions on oral processing behavior of liquid stimuli. Oral processing of one sip of water, skimmed milk, thickened skimmed milk, cream and cream with poppy seeds was quantified using Surface Electromyography. Oral behavior was determined while subjects performed or did not perform sensory evaluation. Oral clearance time and time after the second swallow differed between liquid stimuli displaying large differences in texture properties, probably due to subjects attempting to remove oral coatings from the tongue surface during the oral clearance phase. Time to swallow decreased and total muscle activity increased when liquid stimuli were orally processed without performing a sensory evaluation. No differences in oral processing behavior of liquid stimuli were observed between different types of sensory evaluations (assessment of different sensory attributes). We suggest that some texture attributes of liquid foods might be perceived without requiring additional oral processing. We conclude that performing sensory evaluation influences oral processing behavior of liquid stimuli compared to not performing sensory evaluation.

Dynamic texture perception and oral processing of semi-solid food gels: Part 1: Comparison between QDA, progressive profiing and TDS

Texture perception of food is a dynamic phenomenon depending on food properties and oral processing. Several sensory techniques enable to measure texture perception over time. The aim of this study was to compare quantitative descriptive analysis (QDA), temporal dominance of sensation (TDS) and progressive profiling in the assessment of dynamic texture of emulsion filled gels varying in fracture stress (low/ high), fracture strain (low/high) and oil release (oil droplets bound/unbound to the gel matrix). The QDA results revealed that the variation of mechanical properties led to significant differences in texture properties perceived at first bite (firmness and brittleness). Texture attributes perceived at later stages of mastication showed significant differences between gels depending on the first bite properties e.g. soft gels were perceived as more melting. Progressive profiling showed that creaminess increased over eating time while firmness decreased. TDS results were in agreement with the other methods and additionally conveyed information on the succession of perceived attributes over time. The TDS sensory trajectories demonstrated that for all gels dynamic perception evolved in a similar fashion but samples with a high or low fracture strain differed at the end of oral processing. We conclude that texture perception of semi-solid gels is dynamic and can be measured by either of the three sensory methods. The mechanical properties of the gels influence the perception of texture attributes at first bite and at later stages of mastication. QDA, TDS and progressive profiling gave matching and complementary results in the assessment of dynamic sensory texture.

Eating behaviour explains differences between individuals in dynamic texture perception of sausages

Texture perception of foods has been demonstrated to be influenced by age, dental health and oral processing behaviour. Eating duration is a significant factor contributing to and determining food oral processing behaviour. The influence of eating duration on dynamic texture perception, oral processing behaviour and properties of the food bolus have not been investigated extensively. The aims of this study are (i) to determine the influence of naturally preferred eating duration on dynamic texture perception of sausages and (ii) to explain differences in dynamic texture perception between short and long duration eaters by chewing behaviour and bolus properties. Two groups of subjects were selected based on their natural eating duration for a controlled portion size of two sausages. The group of ‘‘long duration eaters’’ (n = 11) took on average twice as long to consume a piece of sausage compared to the group of ‘‘short duration eaters’’ (n = 12). Independent of eating duration, short and long eating duration subjects chewed sausages with the same chewing frequency (p = 0.57) and muscle effort rate (p = 0.15) during oral processing. Total muscle effort and total number of chews were significantly higher (p < 0.05 for both) for long duration eaters mainly due to the longer eating time compared to short duration eaters. Bolus properties showed that short duration eaters did not break down the boli as much as long duration eaters resulting in fewer (p < 0.001) and larger (p < 0.05) sausage bolus fragments, firmer (p < 0.001) and less adhesive (p < 0.001) boli with lower fat content (p < 0.05) and less saliva incorporation (p < 0.001) at swallow compared to the bolus properties of long duration eaters. These differences in bolus properties influenced dynamic texture perception of the sausages as the bolus of short duration eaters revealed different properties than the bolus of long duration eaters. Temporal dominance of sensations (TDS) showed that short and long duration eaters perceived the same sausage similarly in the early stages of oral processing, but started to perceive the texture of the same sausage differently from the middle of oral processing towards the end. We conclude that short duration eaters did not compensate for their shorter eating duration by chewing more efficiently but were comfortable swallowing a less broken down bolus than long duration eaters. Moreover, we conclude that differences in eating behaviour between subjects can lead to differences in bolus properties of sausages causing differences in dynamic texture perception of the same sausage.

Fat droplet characteristics affect rheological, tribological and sensory properties of food gels

This work aims to investigate the effect of fat droplet characteristics in emulsion-filled gels on their dynamic rheological, tribological and microstructure properties during breakdown, and their sensory perceptions. Fat droplet characteristics investigated were the interaction of the fat droplet with the gel matrix (modulated by using different emulsifiers to yield droplets being either bound or unbound to the matrix) and the solid fat content (SFC, varying from 4% to 48%). Fat content was varied from 0% to 20%. Elastic modulus and fracture properties of these gels (determined under uni-axial compression) were affected by dropletematrix interaction, fat content, and SFC. A mouth-mimicking tribometer connected to a CLSM was used to determine tribological properties (friction) and microstructural evolution (fat coalescence) of gels under shear. Gels with unbound droplets led to more coalescence (than bound) and increased fat content also led to more coalescence. The observed increase in fat coalescence related to a decrease in friction, which was also related to an enhancement of the perception of fat-related sensory attributes (determined by quantitative descriptive sensory analysis). The effects of unbound droplets and higher fat content on increasing coalescence and decreasing friction were further enhanced by increasing SFC. Having found that decrease in friction and increase in coalescence relates to an enhancement of perception of fat-related attributes, one would expect that increasing SFC would further enhance the perception of fat-related attributes. This was not found. We attribute this to the fact that our systems are gels that have complicated breakdown behavior.

Properties of oil/water emulsions affecting the deposition, clearance, and after-feel sensory perception of oral coatings

The aims of this study were to investigate the influence of (i) protein type, (ii) protein content 32 and (iii) viscosity of o/w emulsions on the deposition and clearance of oral oil coatings and 33 after-feel perception. Oil fraction (moil/cm2tongue) and after-feel perception differed 34 considerably between emulsions which do not flocculate under in mouth conditions (Na-35 caseinate) and emulsions which flocculate under in mouth conditions (lysozyme). The 36 irreversible flocculation of lysozyme stabilized emulsions caused slower oil clearance from 37 the tongue surface compared to emulsions stabilized with Na-caseinate. Protein content had a 38 negative relation with oil fraction for lysozyme stabilized emulsions and no relation with oil 39 fraction for Na-caseinate stabilized emulsions immediately after expectoration. Viscosity 40 differences of the o/w emulsions did not affect oil deposition and clearance, although the 41 presence of xanthan as thickener decreased deposition of oil on tongue compared to emulsion 42 without xanthan. We conclude that after-feel perception of o/w emulsions is complex and 43 depends on the deposited oil fraction, the behavior of proteins in mouth and thickeners.

Just noticeable differences and Weber fraction or oral thickness perception of model beverages

Rheological properties of beverages contribute considerably to texture perception. When developing new beverages, it is important to have knowledge on the smallest differences of viscosity which a consumer can discriminate. Thickness is the sensory attribute most commonly used to describe the viscosity of beverages. The aim of this study was to determine the Just Noticeable Differences (JNDs) of oral thickness perception and the Weber fraction (K) of Newtonian model stimuli (maltodextrin solutions). JNDs were determined using the method of constant stimuli with 5 reference stimuli ranging in viscosity from 10 to 100 mPas. JNDs increased with increasing viscosity of the reference stimulus. The Weber fraction (K) for oral thickness perception of model beverages was K = 0.26 for the studied viscosity range. The Weber fraction for oral thickness perception is comparable to Weber fractions reported in literature for perception of kinesthetic food firmness and spreadability, creaminess, sourness, and bitterness perception. This demonstrates that the human sensitivity towards oral discrimination of thickness of liquid stimuli is comparable to the human sensitivity towards discrimination of specific texture properties and specific taste stimuli.

Formation, clearance and mouthfeel perception of oral coatings formed by emulsion-filled gels

Four emulsion-filled gelatin gels varying in fat content (5 and 15%) and type of emulsifier (whey protein isolate: fat droplets bound to matrix; Tween 20: fat droplets unbound to matrix) were studied. We investigated (1) the formation and clearance dynamics of fat deposition on the tongue using in vivo fluorescence during oral processing; (2) influence of fat droplet characteristics on fat deposition on tongue and fatty mouthfeel; and (3) effect of follow-up consumption (water or gelatin gel) on the removal of fat deposition on the tongue. We conclude that fat fraction deposited on tongue and fatty perception increased with increasing mastication time, and decreased after expectoration with increasing clearance time. Fat fraction deposited on tongue and fatty perception are higher in gels with unbound droplets compared to bound droplets, as well as in gels with 15% fat compared to 5% fat.Water removed deposited fat from the tongue faster than gelatin gel.

Formation dynamics of oral oil coatings and their effect on subsequent sweetness perception of liquid stimuli

Knowledge of the formation of oral coatings and their influence on subsequent taste perception is necessary to understand possible taste-masking effects by oil coatings. This study investigated (a) the dynamics of the formation of oral oil coatings formed by o/w emulsions and (b) the effect of oral oil coatings on subsequent sweetness perception of sucrose solutions. In vivo fluorescence was used to quantitate the oil fraction deposited on the tongue after oral processing of oil-in-water emulsions for different times. A trained panel evaluated sweetness perception of sucrose solutions after orally processing the emulsions. The oil fraction reached its maximum value within the first 3 s of oral processing. The oil fraction did not significantly affect subsequent sweetness perception of sucrose solutions. It is suggested that the oil droplets deposited on the tongue did not form a hydrophobic barrier that is sufficient to reduce the accessibility of sucrose to taste buds.

Bolus matters: Impact of food oral breakdown on dynamic texture perception

link to thesis

 

Background and aims:
Texture is an important, yet complex, quality attribute of food. Food structure and properties can be linked to texture perception during the first bite. However, the perception of attributes during chew down is more difficult to explain, as food requires being broken down to be swallowed safely. Food oral processing, which is a recent discipline connecting food science to the physiology of the eating process, is considered to be the key for understanding dynamic food texture perception. The aim of this thesis is to understand the link between food properties and texture perception by investigating oral food breakdown, in simple model foods.
Methods:
Gels were used as a model for soft solid foods. Several properties of the gels were controlled by modifying the composition of gels, including fracture stress and fracture strain, oil droplets binding to the gels matrix, melting, serum release and mechanical contrast. The texture perception of the gels was measured using several sensory methods. Qualitative descriptive analysis (QDA), progressive profiling and temporal dominance of sensations (TDS) were compared in the assessment of dynamic texture perception. In order to link gel properties to texture perception, the oral processing of gels was measured through analyses on the gel bolus and measurements of chewing behaviour. Gel boli were expectorated at various stages of oral processing and were analysed for gel fragments size and number, mechanical properties and saliva incorporation. These analyses were used to quantify the degree of breakdown of gels and to relate bolus properties to changes in texture perception. Chewing behaviour was measured using Electromyography (EMG) to understand the role of oral processing behaviour in bolus formation and dynamic texture perception.
Results:
Dynamic texture perception of gels could be measured by QDA, progressive profiling and TDS, which were complementary methods. Fracture properties of gels could predict the perception of first bite texture attributes. Fracture stress and fracture strain were correlated to first bite firmness and brittleness respectively. During chew down, the link between gel properties and texture perception became less clear. Nonetheless, fracture properties and other gels properties, such as melting and serum release, related to chew down perception. Bolus properties depended on gel properties, but related better to chew down texture perception than gel properties. Mainly changes in mechanical properties and fragmentation of the bolus could explain the perception of complex texture attributes, such as creaminess and graininess respectively. Chewing behaviour depended on products properties. In addition, chewing behaviour impacted the formation of the bolus and could result in differences in dynamic texture perception between groups of individuals.
Conclusions:
The oral breakdown of food is a valuable input to understand the perception of complex chew down texture attributes. Such an input could be used to design foods with a desired texture sensory profile for reformulation of foods fitting in a healthier diet or foods for target consumer groups.

Oral coatings: A study on the formation, clearance and perception

link to thesis

Oral coatings are residues of food and beverages that coat the oral mucosa after consumption. Oral coatings are one of the factors influencing lubrication properties in mouth, taste and aroma perception. Although it is known that oral coatings can influence sensory perception, the understanding of the chemical composition and physical properties of oral coatings in relation to sensory perception is limited. The aim of this thesis is to understand which factors influence the composition, formation and clearance of oral coatings and their sensory perception.

Method

This thesis consisted of studies of oil coatings deposited on the tongue formed by oil droplets of oil/water (o/w) emulsions and protein coatings formed by the aqueous phase of o/w emulsions. The effect of oil content, protein content, protein in-mouth behavior and presence of thickener on the formation and clearance dynamics of oral coatings was investigated. A calibration method for in vivo fluorescence measurements to represent conditions occurring in mouth was developed to quantify oil oral coatings. Protein content of oral coatings was quantified using cotton swabs to collect protein oral coatings and subsequently quantifying protein concentration of the coating (mass protein/mass coating) with the Lowry method. Progressive sensory profiling was used to assess sensory perception of coatings over time. The effect of oral coatings on subsequent sweetness perception was studied by first coating the tongues of participants with o/w emulsions, and subsequently providing sucrose solutions and evaluating sweetness intensity.

Results

Oral coatings are formed within the first seconds of contact with the oral mucosa. These fast dynamics were observed for formation of oil coatings by liquid o/w emulsions and semi-solid emulsion-filled gels as well as for formation of protein coatings by the aqueous phase of o/w emulsions. Oil coatings consisted of individual oil droplets deposited on the tongue surface rather than a continuous oil film. Increasing oil or protein content in the stimuli increased the oil or protein content deposited on the tongue. Proteins which display different in mouth behavior (proteins flocculating vs. proteins not flocculating with salivary biopolymers) deposit similarly on the tongue. Presence of xanthan gum decreased the amount of oil and protein deposited on the tongue.

This suggests that the formation dynamics of coatings is dependent on the availability of the ingredients to deposit between the voids of the papillae. In general, clearance of oil/fat coatings followed a similar tendency for all stimuli studied in this thesis. Most of the coating (> 60%) is cleared from the tongue in the first 45s. Exception occurred when oil coatings were formed by o/w emulsions stabilized by lysozyme. Perception of oral coatings depends on the amount of oil and protein deposited on the tongue, the type of protein used (proteins which flocculate lead to high astringency) and the presence of thickener. Oral coatings formed by o/w emulsions do not influence subsequent sweetness perception of sucrose solutions.

Conclusions

Several factors were identified which affect after-feel and after-taste of oral oil and protein coatings, such as protein and oil content, protein type and lubrication in-mouth. Oral coatings formed by o/w emulsions do not influence subsequent sweetness perception since the amount of oil deposited on the tongue is not sufficient to form a hydrophobic barrier to limit the accessibility of sucrose to the taste buds

Impact of growth conditions and role of sigB on L. monocytogenes fitness in single and mixed biofilms cultured with L. plantarum WCFS1

The role of sigB, a major transcriptional regulator of stress response genes,was assessed in formation of single and mixed species biofilms of Listeria monocytogenes EGD-e and Lactobacillus plantarum WCFS1 as secondary species at 20 °C and 30 °C using differentmediumcompositions (nutrient-richmediumwith andwithout supplementary manganese, glucose and salt). L. monocytogenes showed significant biofilm development at both temperatures and in all media tested although less biofilm was formedwhen glucosewas supplemented only. The contribution of L.monocytogenes to themixed species biofilm declined especially at higher temperature in glucose-richmedium in the absence and presence ofmanganese, due to lactic acid formationwith concomitant decrease in culture pH belowthe pHmin of L. monocytogenes. Using an in-frame sigB deletion mutant and a complementationmutant we showed that sigB contributed to survival under these acid stress conditions. Notably, the additional presence of salt protected L. monocytogenes in the acidic mixed species biofilms resulting in an increase of around 2–3 log10 cfu/ml and this phenomenon showed to be sigB-dependent.

Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates

Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry.

Bacillus cereus ATCC 14579 RpoN (Sigma 54) is a pleiotropic regulator of growth, carbohydrate metabolism, motility, biofilm formation and toxin production

Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.

Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores

Background: Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. Results: The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned foodmatrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. Conclusion: This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complexfood matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies.

Comparative analysis of biofilm formation by Bacillus cereus reference strains and undomesticated food isolates and the effect of free iron

Biofilm formation of Bacillus cereus reference strains ATCC 14579 and ATCC 10987 and 21 undomesticated food isolates was studied on polystyrene and stainless steel as contact surfaces. For all strains, the biofilm forming capacity was significantly enhanced when in contact with stainless steel (SS) as a surface as compared to polystyrene (PS). For a selection of strains, the total CFU and spore counts in biofilms were determined and showed a good correlation between CFU counts and total biomass of these biofilms. Sporulation was favoured in the biofilm over the planktonic state. To substantiate whether iron availability could affect B. cereus biofilm formation, the free iron availability was varied in BHI by either the addition of FeCl3 or by depletion of iron with the scavenger 2,2-Bipyridine. Addition of iron resulted in increased air-liquid interface biofilm on polystyrene but not on SS for strain ATCC 10987, while the presence of Bipyridine reduced biofilm formation for both materials. Biofilm formation was restored when excess FeCl3 was added in combination with the scavenger. Further validation of the iron effect for all 23 strains in microtiter plate showed that fourteen strains (including ATCC10987) formed a biofilm on PS. For eight of these strains biofilm formation was enhanced in the presence of added iron and for eleven strains it was reduced when free iron was scavenged. Our results show that stainless steel as a contact material provides more favourable conditions for B. cereus biofilm formation and maturation compared to polystyrene. This effect could possibly be linked to iron availability as we show that free iron availability affects B. cereus biofilm formation.

Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties

The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of timetemperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply.

Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores

Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH5.5) meat-based BHI. Rice, broccoli and BHI pH5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1min at 95°C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH7.4 was more pronounced than on rice, broccoli and BHI pH5.5 conceivably reflecting that conditions in BHI pH7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, pH of BHI and the effect of food matrix highlighting the impact of different (model) food recovery media on outgrowth efficiency and heterogeneity of non-heat-treated and heat-damaged B. cereus spores.

Bacillus thermoamylovorans spores with very-high-level heat resistance germinate poorly in rich medium despite the presence of ger clusters but efficiently upon exposure to calcium-dipicolinic acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12 °C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available.

T-Rex: Transcriptome analysis webserver for RNA-seq Expression data

Background: Transcriptomics analyses of bacteria (and other organisms) provide global as well as detailed information on gene expression levels and, consequently, on other processes in the cell. RNA sequencing (RNA-seq) has over the past few years become the most accurate method for global transcriptome measurements and for the identification of novel RNAs. This development has been accompanied by advances in the bioinformatics methods, tools and software packages that deal with the analysis of the large data sets resulting from RNA-seq efforts. Results: Based on years of experience in analyzing transcriptome data, we developed a user-friendly webserver that performs the statistical analysis on the gene expression values generated by RNA-seq. It also provides the user with a whole range of data plots. We benchmarked our RNA-seq pipeline, T-REx, using a case study of CodY mutants of Bacillus subtilis and show that it could easily and automatically reproduce the statistical analysis of the cognate publication. Furthermore, by mining the correlation matrices, k-means clusters and heatmaps generated by T-REx we observed interesting gene-behavior and identified sub-groups in the CodY regulon. Conclusion: T-REx is a parameter-free statistical analysis pipeline for RNA-seq gene expression data that is dedicated for use by biologists and bioinformaticians alike. The tables and figures produced by T-REx are in most cases sufficient to accurately mine the statistical results. In addition to the stand-alone version, we offer a user-friendly webserver that only needs basic input ( http://genome2d.molgenrug.nl)

Quantifying variability on thermal resistance of Listeria monocytogenes

Knowledge of the impact of strain variability and growth history on thermal resistance is needed to provide a realistic prediction and an adequate design of thermal treatments. In the present study, apart from quantifying strain variability on thermal resistance of Listeria monocytogenes, also biological variability and experimental variability were determined to prioritize their importance. Experimental variability was defined as the repeatability of parallel experimental replicates and biological variability was defined as the reproducibility of biologically independent reproductions. Furthermore, the effect of growth history was quantified. The thermal inactivation curves of 20 L. monocytogenes strains were fitted using the modified Weibull model, resulting in total 360 Dvalue estimates. The D-value ranged from 9 to 30 min at 55 °C; from 0.6 to 4 min at 60 °C; and from 0.08 to 0.6 min at 65 °C. The estimated z-values of all strains ranged from 4.4 to 5.7 °C. The strain variability was ten times higher than the experimental variability and four times higher than the biological variability. Furthermore, the effect of growth history on thermal resistance variability was not significantly different from that of strain variability and was mainly determined by the growth phase.

Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU

The dynamic response of microorganisms to environmental conditions depends on the behavior of individual cells within the population. Adverse environments can select for stable stress resistant subpopulations. In this study, we aimed to get more insight in the diversity within Listeria monocytogenes LO28 populations, and the genetic basis for the increased resistance of stable resistant fractions isolated after acid exposure. Phenotypic cluster analysis of 23 variants resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation, and virulence indicators. A higher glutamate decarboxylase activity correlated with increased acid resistance. Whole genome sequencing revealed mutations in rpsU, encoding ribosomal protein S21 in the largest phenotypic cluster, while mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and high hydrostatic pressure resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants. The finding that acid stress selects for rpsU variants provides potential insights in the mechanisms underlying population diversity of L. monocytogenes.

Performance and resistance of Listeria monocytogenes wild type and multiple stress resistant variants in mixed culture biofilms with Lactobacillus plantarum

Population diversity and the ability to adapt to changing environments allow Listeria monocytogenes to grow and survive under a wide range of environmental conditions. In this study, we aimed to evaluate the performance of a set of acid resistant L. monocytogenes variants in mixed-species biofilms with Lactobacillus plantarum as well as their benzalkonium chloride (BAC) resistance in these biofilms. L. monocytogenes LO28 wild type and acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Homolactic fermentation of glucose by L. plantarum created an acidic environment with pH values below the growth boundary of L. monocytogenes. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, indicating differences in adaptive behaviour between the variants. Two variants that showed a high level of acid adaptation when grown in liquid cultures, showed also better performance in mixed species biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. It can be concluded that acid resistant variants of L. monocytogenes show diversity in their adaptation to acidic conditions and their capacity to survive in mixed cultures and biofilms with L. plantarum.

Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein

Listeria monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress resistant variants. The presence of genetic variants allows for growth and survival of the population under a wide range of environmental conditions with specific fitness and robustness parameters considered a trade-off. A variant may have an advantage under one condition, while this might be a disadvantage under other conditions. This study aims to evaluate the impact of the presence of stress resistant variants and their corresponding trade-offs on population composition under different environmental conditions. A set of robustness and fitness parameters of WT and variants was obtained and used to model their behaviour under combined mild stress conditions and in a simulated food chain. Predictions of performance were validated in single cultures by plate counts and in mixtures by qPCR in which WT and an rpsU deletion variant were distinguished by specific primers. Growth predictions for WT and rpsU deletion variant were matching the experimental data generally well. Validation of performance in the simulated food chain confirmed the trend of higher fitness and lower robustness for the WT compared to the rpsU variant. This study provides insights into the conditions which can select for stress resistant variants in industrial settings and discusses their potential persistence in food processing environments.

Quantifying strain variability in modeling growth of Listeria monocytogenes

Prediction of microbial growth kinetics can differ from the actual behavior of the target microorganisms. In the present study, the impact of strain variability on maximum specific growth rate (μmax) (h−1) was quantified using twenty Listeria monocytogenes strains. The μmax was determined as function of four different variables, namely pH, water activity (aw)/NaCl concentration [NaCl], undissociated lactic acid concentration ([HA]), and temperature (T). The strain variability was compared to biological and experimental variabilities to determine their importance. The experiment was done in duplicate at the same time to quantify experimental variability and reproduced at least twice on different experimental days to quantify biological (reproduction) variability. For all variables, experimental variability was clearly lower than biological variability and strain variability; and remarkably, biological variabilitywas similar to strain variability. Strain variability in cardinal growth parameters, namely pHmin, [NaCl]max, [HA]max, and Tmin was further investigated by fitting secondary growth models to the μmax data, including amodified secondary pHmodel. The fitting results showed that L. monocytogenes had an average pHmin of 4.5 (5–95% prediction interval (PI) 4.4–4.7), [NaCl]max of 2.0mM(PI 1.8–2.1), [HA]max of 5.1mM (PI 4.2–5.9), and Tmin of −2.2 °C (PI (−3.3)–(−1.1)). The strain variability in cardinal growth parameters was benchmarked to available literature data, showing that the effect of strain variability explained around 1/3 or less of the variability found in literature. The cardinal growth parameters and their prediction intervals were used as input to illustrate the effect of strain variability on the growth of L. monocytogenes in food products with various characteristics, resulting in 2–4 log CFU/ml(g) difference in growth prediction between the most and least robust strains, depending on the type of food product. This underlined the importance to obtain quantitative knowledge on variability factors to realistically predict the microbial growth kinetics.

Metallic taste in cancer patients treated with chemotherapy

Background: Metallic taste is a taste alteration frequently reported by cancer patients treated with hemotherapy. Attention to this side effect of chemotherapy is limited. This review addresses the definition, assessment methods, prevalence, duration, etiology, and management strategies of metallic taste in chemotherapy treated cancer patients. Methods: Literature search for metallic taste and chemotherapy was performed in PubMed up to September 2014, resulting in 184 articles of which 13 articles fulfilled the inclusion criteria: English publications addressing metallic taste in cancer patients treated with FDA-approved chemotherapy. An additional search in Google Scholar, in related articles of both search engines, and subsequent in the reference lists, resulted in 13 additional articles included in this review. Cancer patient forums were visited to explore management strategies. Findings: Prevalence of metallic taste ranged from 9.7% to 78% among patients with various cancers, chemotherapy treatments, and treatment phases. No studies have been performed to investigate the influence of metallic taste on dietary intake, body weight, and quality of life. Several management strategies can be recommended for cancer patients: using plastic utensils, eating cold or frozen foods, adding strong herbs, spices, sweetener or acid to foods, eating sweet and sour foods, using ‘miracle fruit’ supplements, and rinsing with chelating agents. Interpretation: Although metallic taste is a frequent side effect of chemotherapy and a much discussed topic on cancer patient forums, literature regarding metallic taste among chemotherapy treated cancer patients is scarce. More awareness for this side effect can improve the support for these patients.

Neural processing of basic tastes in healthy young and older adults – an fMRI study

Ageing affects taste perception as shown in psychophysical studies, however, underlying structural and functional mechanisms of these changes are still largely unknown. To investigate the neurobiology of age-related differences associated with processing of basic tastes, we measured brain activation (i.e. fMRI-BOLD activity) during tasting of four increasing concentrations of sweet, sour, salty, and bitter tastes in young (average 23 years of age) and older (average 65 years of age) adults. The current study highlighted age-related differences in taste perception at the different higher order brain areas of the taste pathway. We found that the taste information delivered to the brain in young and older adults was not different, as illustrated by the absence of age effects in NTS and VPM activity. Our results indicate that multisensory integration changes with age; older adults showed less brain activation to integrate both taste and somatosensory information. Furthermore, older adults directed less attention to the taste stimulus; therefore attention had to be reallocated by the older individuals in order to perceive the tastes. In addition, we considered that the observed age-related differences in brain activation between taste concentrations in the amygdala reflect its involvement in processing both concentration and pleasantness of taste. Finally, we state the importance of homeostatic mechanisms in understanding the taste quality specificity in age related differences in taste perception.

Functional specialization of the human insula during taste perception

The primary gustatory area is located in the insular cortex. Although the insular cortex has been the topic of multiple parcellation studies, its functional specialization regarding taste processing received relatively little attention. Studies investigating the brain response to taste suggested that the insular cortex is involved in processing multiple characteristics of a taste stimulus, such as its quality, intensity, and pleasantness. In the current functional magnetic resonance study, younger and older adult male subjects were exposed to four basic tastes in five increasing concentrations.Weapplied a data-driven analysis to obtain insular response maps,which showed that the insular cortex processes the presence of taste, its corresponding pleasantness, as well as its concentration. More specifically, the left and right insular cortices are differentially engaged in processing the aforementioned taste characteristics: representations of the presence of a taste stimulus as well as its corresponding pleasantness dominate in the left insular cortex, whereas taste concentration processing dominates in the right insular cortex. These results were similar across both age groups. Our results fit well within previous cytoarchitectural studies and show insular lateralization in processing different aspects of taste stimuli in men.

Metallic taste in cancer patients treated with chemotherapy

Background: Metallic taste is a taste alteration frequently reported by cancer patients treated with chemotherapy. Attention to this side effect of chemotherapy is limited. This review addresses the definition, assessment methods, prevalence, duration, etiology, and management strategies of metallic taste in chemotherapy treated cancer patients. Methods: Literature search for metallic taste and chemotherapy was performed in PubMed up to September 2014, resulting in 184 articles of which 13 articles fulfilled the inclusion criteria: English publications addressing metallic taste in cancer patients treated with FDA-approved chemotherapy. An additional search in Google Scholar, in related articles of both search engines, and subsequent in the reference lists, resulted in 13 additional articles included in this review. Cancer patient forums were visited to explore management strategies. Findings: Prevalence of metallic taste ranged from 9.7% to 78% among patients with various cancers, chemotherapy treatments, and treatment phases. No studies have been performed to investigate the influence of metallic taste on dietary intake, body weight, and quality of life. Several management strategies can be recommended for cancer patients: using plastic utensils, eating cold or frozen foods, adding strong herbs, spices, sweetener or acid to foods, eating sweet and sour foods, using ‘miracle fruit’ supplements, and rinsing with chelating agents. Interpretation: Although metallic taste is a frequent side effect of chemotherapy and a much discussed topic on cancer patient forums, literature regarding metallic taste among chemotherapy treated cancer patients is scarce. More awareness for this side effect can improve the support for these patients.

What reported food-evoked emotions may add: A model to predict consumer food choice

Food-evoked emotions provide information that goes beyond the information from traditional hedonic ratings. The objectives of our study were: (i) to investigate how intrinsic (sensory) and extrinsic (packaging) cues affect consumers’ emotional responses to foods, and (ii) to explore whether emotional responses to these cues combined with liking, predict actual food choice. Participants (n = 103) rated emotional responses to seven products under a blind taste, a package and a package and taste condition using the EsSense Profile™. During the blind taste condition participants also scored liking of the products. Test products were breakfast drinks and desserts. Food choice was measured in two different breakfast sessions reflecting a different choice context. In one choice context, products were presented blind to taste, after which participants chose one out of the seven foods to consume for breakfast. In the other choice context, participants based their choice on the package of the seven foods without tasting them. Results showed that emotions evoked by food products could be organised in a two-dimensional space, representing a valence (pleasantness) and an activation/arousal dimension. Specific emotional profiles generated for products differed across the blind taste, package and the package and taste condition, meaning that intrinsic and extrinsic product properties elicit in part different emotions. Liking and valence together had the strongest predictive value for product choice based on the product’s taste. The combination of liking, valence and arousal had the strongest predictive value for package-based choice. In conclusion, food-evoked emotions add predictive value to solely liking ratings, and may guide consumers’ product choice behaviour.

Learning to (dis)like: The effect of evaluative conditioning with tastes and faces on odor valence assessed by implicit and explicit measurements

Evaluative conditioning may be an important mechanism for learning food preferences and aversions; however, in both real life and experimental settings it has not been consistently successful. The current study aimed to gain more insight into which underlying factors may contribute to a successful outcome of olfactory evaluative conditioning. Two groups of 18 participants came in on three consecutive days, and were repeatedly exposed to four novel, neutral odors (CS) coupled to varying disliked, neutral, liked, or no stimuli (taste and/or pictures, US), following a 50% reinforcement schedule, leading to 40 odor presentations per session. Liking ratings, as well as changes in the autonomic nervous system were assessed before, during and after conditioning. We were able to induce negative, but not positive, affective changes by pairing neutral odorswith tastes and pictures differing in valence. Negative as well as multimodal stimuli appear to be more potent US, since they may be considered more salient. Lastly, results of the current study imply that heart rate is responsive to changes in valence of olfactory stimuli, and perhaps even more sensitive than explicit ratings of liking.

 

Affective and cognitive drivers of food choice

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The aim of this thesis was to test if food-evoked emotional and cognitive associations explain and predict food choice better than sensory liking per se. Hereby we focused on the sensory and packaging product properties. In addition, we investigated the link between sensory properties and emotional responses to foods; and the influence of the context appropriateness on choice. Methods: We conducted a series of product profiling experiments of test products (breakfast drinks) with regular consumers. Participants rated emotional responses and liking to a set of tasted test products, and subsequently, after an interval of one week, participants’ actual choice was observed, after again tasting the series of product samples (presented blind) to choose from. In the following study we took the same measures, but now included the products packaging. Thus, participants rated emotional responses also to the product’s package and they chose one product after viewing the packages of all test products (without tasting). Two dessert products were included in the product set to assess the impact of eating occasion appropriateness. The test products were also evaluated by a trained panel on sensory characteristics using descriptive analysis. In the last study, we assessed cognitive terms (emotional and functional words) participants associate with sensory attributes and the products’ package. And, participants rated liking and chose, after an interval of one week, a product based on the products’ packages.
Results: The measured emotional responses could be decomposed in two dimensions, i.e. valence (pleasant to unpleasant) vs. arousal (calm to excitement). The combination of emotion valence and liking scores predicted individual choice based on the products taste for over 50% of all participants and was a better predictor of choice than liking scores alone. The combination of liking, valence and also arousal resulted in the best prediction for package-based choice with correct predicted individual choices for 41% of all participants. Furthermore, we demonstrated that the match, between the cognitive associations to the products sensory and packaging cues, was positively related to choice. However, liking ratings outperformed the product-packagematch  in predicting individual product choice. In particular, expected liking (based on the product’s package) predicted 25% more individual choices correct than the product-packagematch. Furthermore, we demonstrated that a product was more likely to be chosen when the package provided context appropriate information (i.e. breakfast context for breakfast drinks). Lastly, we found that texture-related attributes were drivers of positive emotions and that specific taste-related attributes were drivers of specific arousal emotions. Conclusion:Emotional and cognitive responses to foods are relevant drivers of choice behaviour. Foodevoked emotional responses predicted choice consistently better than liking scores alone. However, the combination of liking scores and emotions was the best predictor of food choice based on the product’s taste and packaging. Hence, emotions may explain and guide consumers’ choice behaviour. Furthermore, product profiles, based on cognitive product associations, seem to be related to choice behaviour; but it is still unclear what their contribution is in predicting choice based on liking per se. In addition, it was shown that appropriateness also influences package-based choice. Lastly, links between sensory and emotional profiling were identified which offer a possible application of the findings on food-evoked emotions in product development.

Global reverse supply chain redesign for household plastic waste under the emission trading scheme

With increasing global resource scarcity, waste becomes a resource that can be managed globally. A reverse supply chain network for waste recycling needs to process all the waste with minimum costs and environmental impact. As re-processing of waste is one of the major sources of pollution in the recycling processes, a mechanism is needed to control and reduce the emission impact in the re-processing as a key to facilitate the globalized reverse supply chain and avoid spreading pollutants overseas. Emission Trading Schemes (ETS) can function as policy instruments for controlling emissions. The ETS introduces a trade-off between the economic efficiency and the environmental impacts. ETS has been implemented in Europe and is developing rapidly in China too. The aim of the research is to re-design a reverse supply chain from a global angle based on a case study conducted on household plastic waste distributed from Europe to China. Emission trading restrictions are set on the processing plants in both Europe and China. We modeled a network optimization problem using integer programing approach, allowing the reallocation of intermediate processing plants under emission trading restrictions. Optimization results show that global relocation of re-processors leads to both a reduction of total costs and total transportation emission. ETS applied to re-processors further helps to reduce emissions from both reprocessing and transportation sectors. Carbon cap should be carefully set in order to be effective. With a given carbon cap, the model also shows the effective carbon price range. These results give an insight into the feasibility of building a global reverse supply chain for household plastic waste recycling and demonstrate the impact of ETS on the network design.

 

Fast cooling of (non)-nucleated virgin and recycled poly(propylenes): Effect of processing conditions on structural and mechanical properties

In this study, the effect of processing parameters, i.e., the cooling rate and pressure, on the structureproperty relationships for nucleated and non-nucleated i-PP from virgin and recycled sources was investigated. Special attentionwas given to the brittle-to-ductile transition of nucleated i-PP from plastic packaging waste. Differential fast scanning calorimetry and dilatometry were used to mimic industrial process conditions. From the calorimetric experiments, it was observed that under ambient pressure the mesomorphic phase was formed upon fast cooling, which was confirmed by WAXD analysis. The dilatometry results showed that by applying pressure also g-phase crystals are formed. Nucleated samples showed an increased tendency for the g-phase formation and a decreased tendency for mesomorphic phase formation. Up to now, recycled i-PP showed a brittle behavior, but this study showed that by applying a sufficiently high cooling rate, the yield stress can be reduced and a stabilization of the deformation can be obtained leading to a ductile behavior for recycled i-PP.

 

Mechanical recycling of plastic packaging waste

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As a result of an increasing world population and increasing prosperity, the need for plastics has been growing exponentially in the last decades. The majority of the plastics used are derived from depleting oil sources. In order to fulfill the future need for plastics, the plastics consumption should decrease, alternative feedstocks should be sought, and/or plastics should be recycled. The latter is the subject of this thesis. The largest plastics market is packaging and accounts for approx. 40 % of the total plastics production. Packaging materials are short-lived and are usually discarded within one year, in contrast to plastics used in e.g. automotive applications. The recycling of plastic packaging waste is therefore essential for a sustainable society. However, most of the plastic packaging waste ends up at the consumer and has to be recovered. The so-called post-consumer plastic packaging waste is an extremely complex waste stream and consists of a potpourri of plastic products, types, and grades, all in contact with a different product, of which residuals may still adhere to the plastic. These fillers act as contaminants and limit further applicability of waste plastics in recycled products. The objective of this research is twofold: 1) to identify typical contaminants in sorted plastic packaging waste and 2) to investigate their influence on each individual step of the reprocessing chain. This chain consists out of several steps: collection, sorting, cleaning, and normal polymer processing. Moreover, adjustments of the process parameters are done to improve the mechanical properties of the recyclate, making it suitable for use in more demanding applications. This project is tackled by considering the reprocessing chain of plastic packaging waste. The results obtained for waste materials are compared to model systems of virgin materials. This thesis strongly focuses on isotactic  polypropylene (i-PP), which is a versatile polymer and one of the main constituents of the packaging waste stream