Development of an Affordable, Sustainable and Efficacious Plant-Based Immunomodulatory Food Ingredient Based on Bell Pepper or Carrot RG-I Pectic Polysaccharides.

The prevalence of acute respiratory infections and their impact on quality of life underlies the need for efficacious solutions that are safe, sustainable and economically viable. Polysaccharides in several (traditional) plant extracts have been shown to be immunostimulatory, and some studies suggest beneficial effects against respiratory infections. The aim of this study was to (i) identify the active polysaccharide constituents from affordable and renewable crops (bell pepper and carrot) using activity-guided fractionation, (ii) evaluate in vitro effects on innate immune responses (phagocytosis and cytokine secretion), microbiota modulation and production of short chain fatty acids, followed by (iii) the evaluation of effects of a bell pepper extract enriched for the active component in a human proof of concept study. We identified rhamnogalacturonan-I (RG-I) as the nutricophore responsible for the immunostimulatory activity with substantial structural and functional equivalence between bell pepper (bp) and carrot (c). The in vitro studies showed that bpRG-I and cRG-I comprise similar immune- and microbiota modulatory potential and the human study demonstrated that bpRG-I was well tolerated and enhanced innate immune responsiveness in vivo. This is an important step towards testing the efficacy of RG-I from bpRG-I or cRG-I in an infection trial in humans.

Safety assessment of rhamnogalacturonan-enriched carrot pectin fraction: 90-Day oral toxicity study in rats and in vitro genotoxicity studies.

The dietary fibre product examined is a pectic polysaccharide extract from carrot (Daucus carota), enriched for pectin fragments comprising mainly rhamnogalacturonan-I (RG-I) (abbreviated product name cRG-I). To assess the safety of cRG-I for use as food ingredient, repeated-dose oral toxicity and in vitro genotoxicity studies were conducted. In the subchronic toxicity study (OECD test guideline 408), Wistar Hannover rats received cRG-I at dietary levels (w/w) of 0%, 2.5%, 5% and 10% for 13 weeks. cRG-I induced no adverse effects in this study. The NOAEL was 10% in the diet (equivalent to 6.9 and 7.8 g cRG-I/kg body weight/day in male and female rats, respectively). A package of three in vitro genotoxicity tests (Ames, mouse lymphoma and micronucleus assay in human peripheral blood lymphocytes) was negative for induction of point mutation and chromosome damage. An initial Ames test showed a weak positive response in Salmonella typhimurium strain (TA1537). This response was non-reproducible and attributed to microbial contamination as subsequent tests with an irradiated batch of cRG-I including a repeat Ames test were negative. cRG-I was therefore considered to be non-mutagenic.

Aqueous foams stabilized by chitin nanocrystals.

The aim of the present study was to explore the potential use of chitin nanocrystals, as colloidal rod-like particles, to stabilize aqueous foams. Chitin nanocrystals (ChN) were prepared by acid hydrolysis of crude chitin and foams were generated mainly by sonicating the respective dispersions. The foamability of the chitin nanocrystals was evaluated and the resulting foams were assessed for their stability, in terms of foam volume reduction and serum release patterns, during storage. Additionally, the samples were studied with light scattering and optical microscopy in order to explore the bubble size distribution and morphology of the foam. Nanocrystal concentration and charge density was varied to alter the packing of the crystals at the interface. At low concentrations of ChNs, foams were stable against coalescence and disproportionation for a period of three hours, whereas at higher concentrations, the foams were stable for several days. The enhanced stability of foams prepared with ChNs, compared to surfactant-stabilized foams, can be mainly attributed to the irreversible adsorption of the ChNs at the air-water interface, thereby providing Pickering stabilization. Both foam volume and stability of the foam were increased with an increase in ChNs concentration, and at pH values around the chitin's pKa (pH 7.0). Under these conditions, the ChNs show minimal electrostatic repulsion and therefore a higher packing of the nanocrystals is promoted. Moreover, decreased electrostatic repulsion enhances network formation between the ChNs in the aqueous films, thereby providing additional stability by gel formation. Overall, ChNs were proven to be effective in stabilizing foams, and may be useful in the design of Pickering-stabilized food grade foams.

Effect of soluble polysaccharides addition on rheological properties and microstructure of chitin nanocrystal aqueous dispersions.

Mixtures of chitin nanocrystal aqueous dispersions (at pH 3.0) with soluble polysaccharides of varying molecular features were examined rheologically and microscopically, under different conditions of biopolymer concentration, ionic strength, pH and temperature. The addition of non-adsorbing polysaccharides (guar gum, locust bean gum and xanthan) as well as oppositely charged (κ-carrageenan) to a chitin nanocrystal dispersion, resulted in a network formation and the gel strength increased with the chitin nanocrystal concentration. In contrast, the chitin nanocrystal - chitosan or - pullulan mixed dispersions did not show any network formation (tanδ>1) at the concentration range examined. An increase in ionic strength and pH also resulted in an enhanced elasticity of the chitin nanocrystal-guar gum dispersions. Furthermore, an increase in the elastic modulus, which was irreversible upon cooling, was observed upon heating the chitin nanocrystal-polysaccharide mixed dispersions.

In vitro lipid digestion of chitin nanocrystal stabilized o/w emulsions.

Chitin nanocrystals (ChN) have been shown to form stable Pickering emulsions. These oil-in-water emulsions were compared with conventional milk (whey protein isolate, WPI, and sodium caseinate, SCn) protein-stabilized emulsions in terms of their lipid digestion kinetics using an in vitro enzymatic protocol. The kinetics of fatty acid release were evaluated as well as the change in oil droplet size of the respective emulsions during lipid digestion. The interfacial pressure was measured by addition of the duodenal components using drop tensiometry and the electrical charge of the oil droplets was also assessed, in an attempt to relate the interfacial properties with the stability of the emulsions towards lipolysis. Lipid hydrolysis in the ChN-stabilized emulsion was appreciably slower and the plateau values of the total concentration of fatty acids released were much lower, compared to the WPI- and SCn-stabilized emulsions. Moreover, the ChN-stabilized emulsions were relatively stable to coalescence during lipid digestion, whereas the WPI- and SCn-stabilized emulsions exhibited a significant increase in their droplet size. On the other hand, no major differences were shown among the different emulsion samples in terms of their interfacial properties. The increased stability of the ChN-stabilized emulsions towards lipolysis could be attributed to several underlying mechanisms: (i) strong and irreversible adsorption of the chitin nanocrystals at the interface that might inhibit an extensive displacement of the solid particles by bile salts and lipase, (ii) network formation by the nanocrystals in the bulk (continuous) phase that may reduce lipid digestion kinetics, and (iii) the ability of chitin, and consequently of ChNs, to impair pancreatic lipase activity. The finding that ChNs can be used to impede lipid digestion may have important implications for the design and fabrication of structured emulsions with controlled lipid digestibility that could provide the basis for the development of novel products that may promote satiety, reduce caloric intake and combat obesity.

Metastability of nematic gels made of aqueous chitin nanocrystal dispersions.

Chitin nanocrystal aqueous dispersions were prepared by acid hydrolysis of crude chitin from crab shells. The resulting dispersions were studied with small deformation oscillatory experiments and polarized optical microscopy under different conditions of nanocrystal concentration, ionic strength, pH, and temperature. The chitin nanocrystal dispersions exhibited a nematic gel-like behavior with increasing solids concentration. The appearance of nematic-like structures could be explained by the Onsager theory for parallel alignment of anisotropic particles on entropic terms, while the sol-gel transition could be attributed to associative interactions between the chitin nanocrystals. With increasing ionic strength and pH, such associative interactions were enhanced, because the repulsive forces due to the electrostatic charges were reduced and, thus, stronger gels were formed. Heating of the nanocrystal dispersions led to further increases in the storage modulus (G'), which were irreversible upon cooling; the rate of G' increase (dG'/dt) was dependent on temperature.