AG1® Induces a Favorable Impact on Gut Microbial Structure and Functionality in the Simulator of Human Intestinal Microbial Ecosystem® Model.

Modulation of the human gut microbiome has become an area of interest in the nutraceutical space. We explored the effect of the novel foundational nutrition supplement AG1® on the composition of human microbiota in an in vitro experimental design. Employing the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) model, AG1® underwent digestion, absorption, and subsequent colonic microenvironment simulation under physiologically relevant conditions in healthy human fecal inocula. Following 48 h of colonic simulation, the gut microbiota were described using shallow shotgun, whole genome sequencing. Metagenomic data were used to describe changes in community structure (alpha diversity, beta diversity, and changes in specific taxa) and community function (functional heterogeneity and changes in specific bacterial metabolic pathways). Results showed no significant change in alpha diversity, but a significant effect of treatment and donor and an interaction between the treatment and donor effect on structural heterogeneity likely stemming from the differential enrichment of eight bacterial taxa. Similar findings were observed for community functional heterogeneity likely stemming from the enrichment of 20 metabolic pathways characterized in the gene ontology term database. It is logical to conclude that an acute dose of AG1 has significant effects on gut microbial composition that may translate into favorable effects in humans.

Food Microstructure and Fat Content Affect Growth Morphology, Growth Kinetics, and Preferred Phase for Cell Growth of Listeria monocytogenes in Fish-Based Model Systems.

Food microstructure significantly affects microbial growth dynamics, but knowledge concerning the exact influencing mechanisms at a microscopic scale is limited. The food microstructural influence on Listeria monocytogenes (green fluorescent protein strain) growth at 10°C in fish-based food model systems was investigated by confocal laser scanning microscopy. The model systems had different microstructures, i.e., liquid, xanthan (high-viscosity liquid), aqueous gel, and emulsion and gelled emulsion systems varying in fat content. Bacteria grew as single cells, small aggregates, and microcolonies of different sizes (based on colony radii [size I, 1.5 to 5.0 µm; size II, 5.0 to 10.0 µm; size III, 10.0 to 15.0 µm; and size IV, ≥15 µm]). In the liquid, small aggregates and size I microcolonies were predominantly present, while size II and III microcolonies were predominant in the xanthan and aqueous gel. Cells in the emulsions and gelled emulsions grew in the aqueous phase and on the fat-water interface. A microbial adhesion to solvent assay demonstrated limited bacterial nonpolar solvent affinities, implying that this behavior was probably not caused by cell surface hydrophobicity. In systems containing 1 and 5% fat, the largest cell volume was mainly represented by size I and II microcolonies, while at 10 and 20% fat a few size IV microcolonies comprised nearly the total cell volume. Microscopic results (concerning, e.g., growth morphology, microcolony size, intercolony distances, and the preferred phase for growth) were related to previously obtained macroscopic growth dynamics in the model systems for an L. monocytogenes strain cocktail, leading to more substantiated explanations for the influence of food microstructural aspects on lag phase duration and growth rate.IMPORTANCEListeria monocytogenes is one of the most hazardous foodborne pathogens due to the high fatality rate of the disease (i.e., listeriosis). In this study, the growth behavior of L. monocytogenes was investigated at a microscopic scale in food model systems that mimic processed fish products (e.g., fish paté and fish soup), and the results were related to macroscopic growth parameters. Many studies have previously focused on the food microstructural influence on microbial growth. The novelty of this work lies in (i) the microscopic investigation of products with a complex composition and/or structure using confocal laser scanning microscopy and (ii) the direct link to the macroscopic level. Growth behavior (i.e., concerning bacterial growth morphology and preferred phase for growth) was more complex than assumed in common macroscopic studies. Consequently, the effectiveness of industrial antimicrobial food preservation technologies (e.g., thermal processing) might be overestimated for certain products, which may have critical food safety implications.

Co-Supplementation of Baobab Fiber and Arabic Gum Synergistically Modulates the In Vitro Human Gut Microbiome Revealing Complementary and Promising Prebiotic Properties.

Arabic gum, a high molecular weight heteropolysaccharide, is a promising prebiotic candidate as its fermentation occurs more distally in the colon, which is the region where most chronic colonic diseases originate. Baobab fiber could be complementary due to its relatively simple structure, facilitating breakdown in the proximal colon. Therefore, the current study aimed to gain insight into how the human gut microbiota was affected in response to long-term baobab fiber and Arabic gum supplementation when tested individually or as a combination of both, allowing the identification of potential complementary and/or synergetic effects. The validated Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), an in vitro gut model simulating the entire human gastrointestinal tract, was used. The microbial metabolic activity was examined, and quantitative 16S-targeted Illumina sequencing was used to monitor the gut microbial composition. Moreover, the effect on the gut microbial metabolome was quantitatively analyzed. Repeated administration of baobab fiber, Arabic gum, and their combination had a significant effect on the metabolic activity, diversity index, and community composition of the microbiome present in the simulated proximal and distal colon with specific impacts on Bifidobacteriaceae and Faecalibacterium prausnitzii. Despite the lower dosage strategy (2.5 g/day), co-supplementation of both compounds resulted in some specific synergistic prebiotic effects, including a biological activity throughout the entire colon, SCFA synthesis including a synergy on propionate, specifically increasing abundance of Akkermansiaceae and Christensenellaceae in the distal colon region, and enhancing levels of spermidine and other metabolites of interest (such as serotonin and ProBetaine).

Survival of Probiotic Bacterial Cells in the Upper Gastrointestinal Tract and the Effect of the Surviving Population on the Colonic Microbial Community Activity and Composition.

Many health-promoting effects have been attributed to the intake of probiotic cells. However, it is important that probiotic cells arrive at the site of their activity in a viable state in order to exert their beneficial effects. Careful selection of the appropriate probiotic formulation is therefore required as mainly the type of probiotic species/strain and the administration strategy may affect survival of the probiotic cells during the upper gastrointestinal (GIT) passage. Therefore, the current study implemented Simulator of the Human Microbial Ecosystem (SHIME®) technology to investigate the efficacy of different commercially available probiotic formulations on the survival and culturability of probiotic bacteria during upper GIT passage. Moreover, Colon-on-a-Plate (CoaP™) technology was applied to assess the effect of the surviving probiotic bacteria on the gut microbial community (activity and composition) of three human donors. Significantly greater survival and culturability rates were reported for the delayed-release capsule formulation (>50%) as compared to the powder, liquid, and standard capsule formulations (<1%) (p < 0.05), indicating that the delayed-release capsule was most efficacious in delivering live bacteria cells. Indeed, administration of the delayed-release capsule probiotic digest resulted in enhanced production of SCFAs and shifted gut microbial community composition towards beneficial bacterial species. These results thus indicate that careful selection of the appropriate probiotic formulation and administration strategy is crucial to deliver probiotic cells in a viable state at the site of their activity (distal ileum and colon).

AG1®, a Novel Synbiotic, Maintains Gut Barrier Function following Inflammatory Challenge in a Caco-2/THP1-Blue™ Co-Culture Model.

Nutritional interventions to reduce gastrointestinal (GI) permeability are of significant interest to physically active adults and those experiencing chronic health conditions. This in vitro study was designed to assess the impact of AG1, a novel synbiotic, on GI permeability following an inflammatory challenge. Interventions [AG1 (vitamins/minerals, pre-/probiotics, and phytonutrients) and control (control medium)] were fed separately into a human GI tract model (stomach, small intestine, and colon). In the colonic phase, the GI contents were combined with fecal inocula from three healthy human donors. GI permeability was evaluated with transepithelial electrical resistance (TEER) in a Caco-2 (apical)/THP1-Blue™ (basolateral) co-culture model. The apical side received sodium butyrate (positive control) or Caco-2 complete medium (negative control) during baseline testing. In the 24 h experiment, the apical side received colonic simulation isolates from the GI model, and the basolateral side was treated with Caco-2 complete medium, then 6 h treatment with lipopolysaccharide. TEER was assessed at 0 h and 24 h, and inflammatory markers were measured at 30 h in triplicate. Paired samples t-tests were used to evaluate endpoint mean difference (MD) for AG1 vs. control. TEER was higher for AG1 (mean ± SD: 99.89 ± 1.32%) vs. control (mean ± SD: 92.87 ± 1.22%) following activated THP1-induced damage [MD: 7.0% (p < 0.05)]. AG1 maintained TEER similar to the level of the negative control [-0.1% (p = 0.02)]. No differences in inflammatory markers were observed. These in vitro data suggest that acute supplementation with AG1 might stimulate protective effects on GI permeability. These changes may be driven by SCFA production due to the pre-/probiotic properties of AG1, but more research is needed.

In vitro evaluation of the gastrointestinal delivery of acid-sensitive pancrelipase in a next generation enteric capsule using an exocrine pancreatic insufficiency disease model.

The dissolution characteristics of five capsules (Next Generation Enteric [NGE], Vcaps® Enteric [VCE], VCE DUOCAP® [VCE/VCE] system, Hard Gelatin Capsule [HGC] as negative control, and Creon® 10,000 U as market reference) were evaluated using an in vitro simulation of the stomach and upper intestinal tract with an acidic duodenal incubation (pH 4.5 for the first 10 min, pH 6 for the remaining 17 min) to simulate exocrine pancreatic insufficiency. Caffeine was a marker of capsule dissolution, and tributyrin to butyrate conversion measured pancrelipase activity. All capsules were filled with pancrelipase; the NGE, VCE, VCE/VCE, and HGC capsules also contained 50 mg caffeine. Caffeine was released first from the HGC capsule, followed by the VCE, NGE, and VCE/VCE capsules. Pancrelipase activity followed this trend and demonstrated a similar activity level over time for the NGE, VCE/VCE, and Creon® capsules. The HGC formulation confirmed gastric degradation of unprotected pancrelipase. NGE capsules provided similar protection to the simple fill formulation as observed for the complex formulation of the Creon® capsule in a setting with increased pepsin activity and may hasten the time needed to go from formula development to first-in-human studies for pH sensitive drugs or those requiring small intestine targeting.

Behavior of the Surviving Population of Listeria monocytogenes and Salmonella Typhimurium Biofilms Following a Direct Helium-Based Cold Atmospheric Plasma Treatment.

Although the Cold Atmospheric Plasma (CAP) technology proved promising for inactivation of biofilms present on abiotic food contact surfaces, more research is required to examine the behavior of the CAP surviving biofilm-associated cells. It was therefore examined whether (i) CAP treated (Listeria monocytogenes and Salmonella Typhimurium) biofilm-associated cells were able to further colonize the already established biofilms during a subsequent incubation period and (ii) isolates of the surviving population became less susceptible toward CAP when the number of biofilm development-CAP treatment cycles increased. For this purpose, a direct treatment was applied using a helium-based Dielectric Barrier Discharge electrode configuration. Results indicated that the surviving population was able to further colonize the already established biofilms, since the cell density of the CAP treated + incubated biofilms equaled the initial density of the untreated biofilms. For the L. monocytogenes biofilms, also the total biomass proved to further increase, which might result in an even further increased resistance. The susceptibility of the biofilm-associated cells proved to be influenced by the specific number of CAP treatment cycles, which might potentially result in an overestimation of the CAP treatment efficacy and, consequently, an increased risk of food contamination.

Phosphate Elimination in Emulsified Meat Products: Impact of Protein-Based Ingredients on Quality Characteristics.

The addition of phosphates to meat products improves the emulsifying and gelling properties of meat proteins, in turn enhancing overall product quality. The current market trend towards additive-free products and the health issues related to phosphate challenge the industry to develop phosphate-free meat products. The aim of this study was to evaluate the potential of seven protein-based ingredients (pea, blood plasma, gelatin, soy, whey, egg, and potato) to remediate quality losses of emulsified meat products (cooked sausages) upon phosphate elimination. First, the intrinsic gelling and emulsifying characteristics of the proteins were assessed. Next, the proteins were added to phosphate-free sausages, of which quality characteristics during production (viscoelastic behavior and emulsion stability) and of the final products (texture, cooking loss, and pH) were screened. Blood plasma and soy were superior in phosphate-free cooked sausages, as no significant differences in hardness, cooking yield, or stability were found compared to phosphate-containing sausages. Egg and pea also improved the previously mentioned quality characteristics of phosphate-free sausages, although to a lesser extent. These insights could not entirely be explained based on the intrinsic gelling and emulsifying capacity of the respective proteins. This indicated the importance of a well-defined standardized meat matrix to determine the potential of alternative proteins in meat products.

Inactivation of L. monocytogenes and S. typhimurium Biofilms by Means of an Air-Based Cold Atmospheric Plasma (CAP) System.

Previous (biofilm) inactivation studies using Cold Atmospheric Plasma (CAP) focused on helium (with or without the addition of oxygen) as feeding gas since this proved to result in a stable and uniform plasma. In industry, the use of helium gas is expensive and unsafe for employees. Ambient air is a possible substitute, provided that similar inactivation efficacies can be obtained. In this research, 1 and 7 day-old (single/dual-species) model biofilms containing L. monocytogenes and/or S. typhimurium cells were treated with an air-based Surface Barrier Discharge (SBD) plasma set-up for treatment times between 0 and 30 min. Afterwards, cell densities were quantified via viable plate counts, and predictive models were applied to determine the inactivation kinetics and the efficacy. Finally, the results were compared to previously obtained results using a helium-based SBD and DBD (Dielectric Barrier Discharge) system. This study has demonstrated that the efficacy of the air-based CAP treatment depended on the biofilm and population type, with log-reductions ranging between 1.5 and 2.5 log10(CFU/cm2). The inactivation efficacy was not significantly influenced by the working gas, although the values were generally higher for the air-based system. Finally, this study has demonstrated that the electrode configuration was more important than the working gas composition, with the DBD electrode being the most efficient.

Combined Effect of Cold Atmospheric Plasma and Hydrogen Peroxide Treatment on Mature Listeria monocytogenes and Salmonella Typhimurium Biofilms.

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

Dual-Species Model Biofilm Consisting of Listeria monocytogenes and Salmonella Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP).

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature Listeria monocytogenes and S. Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of L. monocytogenes and S. Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0-30 min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25°C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for S. Typhimurium inactivation when this species was part of the dual-species biofilm, while L. monocytogenes inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.

The Complex Effect of Food Matrix Fat Content on Thermal Inactivation of Listeria monocytogenes: Case Study in Emulsion and Gelled Emulsion Model Systems.

Previous studies on the influence of food matrix fat content on thermal inactivation kinetics of food pathogens have shown contradictory results due to the combined influence of fat content and other factors such as composition. Therefore, thermal inactivation of Listeria monocytogenes at 59, 64, and 69°C was systematically investigated in emulsion and gelled emulsion food model systems with various fat content (1, 5, 10, and 20%), such that the effect of fat content was isolated. Thermal conductivity and rheological properties of the model systems were quantified, as well as the effect of these properties on the thermal load of the model systems. Thermal conductivity was complexly related to fat content, the nature of the food matrix (i.e., viscous or gelled), and temperature. For the emulsions, the consistency index K increased with increasing fat content, while the flow behavior index n followed the opposite trend. For the gelled emulsions, the storage modulus G' was always larger than the loss modulus G″ (i.e., measure of elastic and viscous properties, respectively). The phase angle δ [i.e., arctan (G″/G')] was proportional with fat content, but this relation became more complex at higher temperatures. The thermal load of the model systems was not largely affected by food matrix fat content. Thermal inactivation of L. monocytogenes was investigated by means of the maximum specific inactivation rate k max, log reductions, and sublethal injury (SI). Both for emulsions and gelled emulsions, k max decreased with increasing fat content below approximately 60°C, while a more complex behavior was observed at higher temperatures. In the emulsions, log reductions were considerably lower (i.e., 2-3 log) at 1% fat than in systems with higher fat content. In the gelled emulsions, log reductions generally decreased with increasing fat content. SI decreased with increasing fat content, both in emulsions and gelled emulsions. In conclusion, the inactivation rate (i.e., k max) of L. monocytogenes was affected by a complex relation between food matrix fat content, thermal conductivity, rheological properties, and inactivation temperature. Due to the small scale of the model systems, differences in k max did not directly affect the final log reductions in a similar fashion.

Inactivation of Single Strains of Listeria monocytogenes and Salmonella Typhimurium Planktonic Cells Biofilms With Plasma Activated Liquids.

Recent research has proven the ability of cold atmospheric plasma (CAP) for assuring food safety. A more flexible and transportable alternative is the use of plasma activated liquids (PAL), which are also known to have antimicrobial properties. However, within the context of food safety, little is known on its potential regarding decontamination. This research therefore focusses on identifying the impact of (i) the microbial species and its cell type (planktonic cells or biofilms), (ii) the CAP settings (i.e., gas composition and generation time) and (iii) PAL related factors (treatment time and PAL age) on the technologies efficacy. Cell densities were monitored using the plate counting technique for which the results were analyzed by means of predictive inactivation models. Moreover, the pH and the concentrations of long-lived species (i.e., hydrogen peroxide, nitrite, and nitrate) were measured to characterize the PAL solutions. The results indicated that although the type of pathogen impacted the efficacy of the treatment, mainly the cell mode had an important effect. The presence of oxygen in the operating gas ensured the generation of PAL solutions with a higher antimicrobial activity. Moreover, to ensure a good microbial inactivation, PAL generation times needed to be sufficiently long. Both CAP related factors resulted in a higher amount of long-lived species, enhancing the inactivation. For 30 min. PAL generation using O2, this resulted in log reductions up to 3.9 for biofilms or 5.8 for planktonic cells. However, loss of the PAL activity for stored solutions, together with the frequent appearance of a tailing phase in the inactivation kinetics, hinted at the importance of the short-lived species generated. Different factors, related to (i) the pathogen and its cell mode, (ii) the CAP settings and (iii) PAL related factors, proved to impact the antimicrobial efficacy of the solutions and should be considered with respect to future applications of the PAL technology.