Conformational Changes of Whey and Pea Proteins upon Emulsification Approached by Front-Surface Fluorescence.

Proteins are widely used to stabilize emulsions, and plant proteins have raised increasing interest for this purpose. The interfacial and emulsifying properties of proteins depend largely on their molecular properties. We used fluorescence spectroscopy to characterize the conformation of food proteins from different biological origins (dairy or pea) and transformation processes (commercial or lab-made isolates) in solution and at the oil-water interface. The fourth derivative of fluorescence spectra provided insights in the local environment of tryptophan (Trp) residues and thus in the protein structure. In emulsions, whey proteins adsorbed with their Trp-rich region at the oil-water interface. Proteins in the commercial pea isolate were present as soluble aggregates, and no changes in the local environment of the Trp residues were detected upon emulsification, suggesting that these structures adsorb without conformational changes. The lab-purified pea proteins were less aggregated and a Trp-free region of the vicilin adsorbed at the oil-water interface.

Mechanisms Governing the Transfer of Pure and Plant Matrix Carotenoids Toward Emulsified Triglycerides.

SCOPE: The study aims to assess the role of factors assumed to be involved in the transfer of carotenoids from plant matrices to dietary emulsions in the upper digestive tract. METHODS AND RESULTS: Transfer is first measured as a function of time of pure ß-carotene (ßC), lutein (LUT), and lycopene (LYC) to triglyceride (TG) droplets dispersed in water. Then the transfer to TG droplets stabilized with either bovine serum albumin (BSA), phospholipids (PL), or both is measured. Finally, transfer of tomato and spinach puree carotenoids to these emulsions is measured. The maximal transfer efficiency of the pure carotenoids to uncoated emulsions is very efficient, ranging from 59% to 77%. However, it is dramatically impaired, ranging from 0.5% to 31% (p < 0.05), when emulsions are stabilized by the emulsifiers. Conversely, when LUT, and to a less extent ßC, but not LYC, is provided by the vegetable purees, its maximal transfer efficiency is significantly higher for the coated emulsions than for the uncoated one. CONCLUSIONS: Emulsifiers can dramatically impair the transfer of pure carotenoids to emulsion TG while they can facilitate the transfer of carotenoids from plant matrices. This suggests that specific interactions between plant matrix compounds and emulsifiers can enhance the transfer efficiency of carotenoids.

The initial characteristics of marine oil emulsions and the composition of the media inflect lipid oxidation during in vitro gastrointestinal digestion.

This work aimed at evaluating the effect of simulated digestive fluids, interface and lipid droplet sizes on the oxidation of oil-in-water emulsions containing long chain n-3 fatty acyls. Emulsions stabilised by a protein or by phosphatidyl-choline/Tween 80 were submitted to gastro-intestinal in vitro conditions in presence of metmyoglobin. The gastric phase was characterised by a decrease of tocopherol amounts and moderate O2 uptake and aldehyde formation. Oxidation developed further during the intestinal phase, with tocopherols tending to zero, oxygen uptake and production of aldehydes at potentially toxic concentrations. The simulated digestive fluids reduced oxygen uptake and MDA formation only during the intestinal step of the phospholipid-stabilised emulsion. Quantitative losses of PUFA (e.g. EPA, DHA) were less than 10% even significant at the end of the digestion.

n-3 PUFA added to high-fat diets affect differently adiposity and inflammation when carried by phospholipids or triacylglycerols in mice.

BACKGROUND: Dietary intake of n-3 polyunsaturated fatty acids (PUFA) is primarily recognized to protect against cardiovascular diseases, cognitive dysfunctions and the onset of obesity and associated metabolic disorders. However, some of their properties such as bioavailability can depend on their chemical carriers. The objective of our study was to test the hypothesis that the nature of n-3 PUFA carrier results in different metabolic effects related to adiposity, oxidative stress and inflammation. METHODS: 4 groups of C57BL/6 mice were fed for 8 weeks low fat (LF) diet or high-fat (HF, 20%) diets. Two groups of high-fat diets were supplemented with long-chain n-3 PUFA either incorporated in the form of phospholipids (HF-ω3PL) or triacylglycerols (HF-ω3TG). RESULTS: Both HF-ω3PL and HF-ω3TG diets reduced the plasma concentrations of (i) inflammatory markers such as monocyte chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6), (ii) leptin and (iii) 4-hydroxy-2-nonenal (4-HNE), a marker of n-6 PUFA-derived oxidative stress compared with the control HF diet. Moreover, in both HF-ω3PL and HF-ω3TG groups, MCP-1 and IL-6 gene expressions were decreased in epididymal adipose tissue and the mRNA level of gastrointestinal glutathione peroxidase GPx2, an antioxidant enzyme, was decreased in the jejunum compared with the control HF diet. The type of n-3 PUFA carrier affected other outcomes. The phospholipid form of n-3 PUFA increased the level of tocopherols in epididymal adipose tissue compared with HF-ω3TG and resulted in smaller adipocytes than the two others HF groups. Adipocytes in the HF-ω3PL and LF groups were similar in size distribution. CONCLUSION: Supplementation of mice diet with long-chain n-3 PUFA during long-term consumption of high-fat diets had the same lowering effects on inflammation regardless of triacyglycerol or phospholipid carrier, whereas the location of these fatty acids on a PL carrier had a major effect on decreasing the size of adipocytes that was not observed with the triacyglycerol carrier. Altogether, these results would support the development functional foods containing LC n-3 PUFA in the form of PL in order to prevent some deleterious outcomes associated with the development of obesity.

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells.

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

Gastric conditions control both the evolution of the organization of protein-stabilized emulsions and the kinetic of lipolysis during in vitro digestion.

During digestion, lipids undergo modifications of their colloidal and molecular structures, which depend on the digestive conditions and the composition of the digestive juices. The aim of this work was to evaluate whether gastric pH and pepsin modulate the colloidal evolution and the bioacessibility of fatty acids of an oil-in-water emulsion stabilized by a protein during in vitro digestion. The fate of BSA-stabilized rapeseed oil-in-water emulsion during gastric phase at pH 2.5 or 4.0 with or without pepsin and its consequences on intestinal lipolysis was measured in the simulated gastric and duodenal conditions. The pH had limited impact but pepsin favoured flocculation and coalescence of the droplets, modulating the early stage of lipolysis but not its final extent.

Modifications of interfacial proteins in oil-in-water emulsions prior to and during lipid oxidation.

Lipid oxidation is a major cause for the degradation of biological systems and foods, but the intricate relationship between lipid oxidation and protein modifications in these complex multiphase systems remains unclear. The objective of this work was to have a spatial and temporal insight of the modifications undergone by the interfacial or the unadsorbed proteins in oil-in-water emulsions during lipid oxidation. Tryptophan fluorescence and oxygen uptake were monitored simultaneously during incubation in different conditions of protein-stabilized oil-in-water emulsions. Kinetic parameters demonstrated that protein modifications, highlighted by decrease of protein fluorescence, occurred as an early event in the sequence of the reactions. They concerned more specifically the proteins adsorbed at the oil/water interface. The reactions led in a latter stage to protein aggregation, carbonylation, and loss of protein solubility.

Effect of lateral heterogeneity in mixed surfactant-stabilized interfaces on the oxidation of unsaturated lipids in oil-in-water emulsions.

The development of lipid oxidation in oil-in-water (O/W) emulsions is widely influenced by the properties of the interfacial layer, which separates the oil and water phases. In this work, the effect of the structure of the interface on the oxidative stability of surfactant stabilized O/W emulsions was investigated. Emulsions were prepared with either single Tween 20 or Tween 20/co-surfactant mixtures in limiting amounts. The co-surfactants, Span 20 and monolauroyl glycerol have the same hydrophobic tail as Tween 20 but differ by the size and composition of their polar headgroup. Metal-initiated lipid oxidation, monitored through the measurement of oxygen uptake, formation of conjugated dienes and volatile compounds, developed more rapidly in the emulsions stabilized by the surfactant mixture than in the single Tween 20-stabilized emulsion. The reconstitution of Tween 20/co-surfactant films at the air-water interface and their surface-pressure isotherms highlighted that, contrary to single Tween 20 molecules, Tween 20/co-surfactant mixtures exhibited an heterogeneous distribution within the interfacial layer, offering probably easier access of water-soluble pro-oxidants to the oil phase. These observations provide direct information about the link between the homogeneity of the interface layer and the oxidative stability of emulsions.

Contribution of the interfacial layer to the protection of emulsified lipids against oxidation.

The oxidative stability of oil-in-water (O/W) emulsions is highly dependent on the type of emulsifier. The purpose of this work was to investigate the specific role of the adsorbed emulsifiers on lipid oxidation of O/W emulsions. Emulsions of similar droplet size distribution stabilized by minimum amounts of proteins or surfactants were oxidized at 25 °C in the presence of equimolar iron-EDTA complex. The pH and the amount of emulsifier in the aqueous phase were also varied to investigate the role of the droplet charge and the emulsifier in the aqueous phase. Oxygen uptake, conjugated dienes (CD), and volatile compound formation demonstrated that the protein-stabilized interfaces are less efficient at protecting emulsified lipids against oxidation than surfactant-stabilized interfaces. The antioxidant effect of unadsorbed proteins was also confirmed.

Quantification of unadsorbed protein and surfactant emulsifiers in oil-in-water emulsions.

Unadsorbed emulsifiers affect the physical and chemical behaviour of oil-in-water (O/W) emulsions. A simple methodology to quantify unadsorbed emulsifiers in the aqueous phase of O/W emulsions has been developed. Emulsions were centrifuged and filtered to separate the aqueous phase from the oil droplets and the concentration of unadsorbed emulsifiers in the aqueous phase determined. The quantification of unadsorbed surfactants based on the direct transesterification of their fatty acids was validated for Tween 20, Tween 80, citric acid ester (Citrem), Span 20 and monolauroyl glycerol. To determine unadsorbed proteins, results obtained with Folin-Ciocalteu reagent or UV-spectrophotometry were compared on emulsions stabilized by ß-lactoglobulin (BLG), ß-casein (BCN) or bovine serum albumin (BSA). The first method gave more accurate results especially during aging of emulsions in oxidative conditions. The whole methodology was applied to emulsions stabilized with single or mixed emulsifiers. This approach enables optimization of emulsion formulations and could be useful to follow changes in the levels of unadsorbed emulsifiers during physical or chemical aging processes.

Meat processing and colon carcinogenesis: cooked, nitrite-treated, and oxidized high-heme cured meat promotes mucin-depleted foci in rats.

Processed meat intake is associated with colorectal cancer risk, but no experimental study supports the epidemiologic evidence. To study the effect of meat processing on carcinogenesis promotion, we first did a 14-day study with 16 models of cured meat. Studied factors, in a 2 x 2 x 2 x 2 design, were muscle color (a proxy for heme level), processing temperature, added nitrite, and packaging. Fischer 344 rats were fed these 16 diets, and we evaluated fecal and urinary fat oxidation and cytotoxicity, three biomarkers of heme-induced carcinogenesis promotion. A principal component analysis allowed for selection of four cured meats for inclusion into a promotion study. These selected diets were given for 100 days to rats pretreated with 1,2-dimethylhydrazine. Colons were scored for preneoplastic lesions: aberrant crypt foci (ACF) and mucin-depleted foci (MDF). Cured meat diets significantly increased the number of ACF/colon compared with a no-meat control diet (P = 0.002). Only the cooked nitrite-treated and oxidized high-heme meat significantly increased the fecal level of apparent total N-nitroso compounds (ATNC) and the number of MDF per colon compared with the no-meat control diet (P < 0.05). This nitrite-treated and oxidized cured meat specifically increased the MDF number compared with similar nonnitrite-treated meat (P = 0.03) and with similar nonoxidized meat (P = 0.004). Thus, a model cured meat, similar to ham stored aerobically, increased the number of preneoplastic lesions, which suggests colon carcinogenesis promotion. Nitrite treatment and oxidation increased this promoting effect, which was linked with increased fecal ATNC level. This study could lead to process modifications to make nonpromoting processed meat.

Chemical modeling of heme-induced lipid oxidation in gastric conditions and inhibition by dietary polyphenols.

The gastric tract may be the first site exposed to diet-related oxidative stress. After food intake, dietary iron such as (met)myoglobin, the pigment of meat, oxygen, and polyunsaturated lipids come into close contact. The main goal of this work is the in vitro investigation of lipid oxidation taking place in the gastric compartment and its inhibition by dietary polyphenols. Oil-in-water emulsions stabilized either by bovine serum albumin (BSA) or egg yolk phospholipids (PL) were designed to model the gastric content. The metmyoglobin-initiated lipid oxidation led to the accumulation of lipid-derived conjugated dienes and volatile aldehydes. These reactions were faster in the BSA model than in the PL model, highlighting the influence of the interfacial composition. Quercetin, rutin, (+)-catechin, caffeic acid, and chlorogenic acid proved to be better inhibitors than alpha-tocopherol and ascorbic acid. Emulsions as models of the gastric environment are valuable tools to study the stability of macro- and micronutrients.

Oxidative stability of bovine serum albumin- and sodium caseinate-stabilized emulsions depends on metal availability.

Proteins often stabilize food emulsions and are also able to promote or delay lipid oxidation in complex systems. The purpose of this work was to investigate the relationship between metal ion availability and oxidative stability of oil-in-water emulsions stabilized by bovine serum albumin (BSA) or sodium caseinate (NaCas). Emulsions with similar and stable droplet size distributions were prepared with stripped sunflower oil (30 vol %) and protein solutions (20 g L(-)(1); pH = 6.5). In the absence of the water-soluble metal chelator EDTA, oxygen uptake, conjugated dienes, and volatile compounds developed faster in NaCas-stabilized emulsions than in those prepared with BSA. This effect is attributed to the chelating properties of NaCas and to electrostatic interactions that attract some metal ions at the interface where they could initiate lipid oxidation. When EDTA (100 muM) was present, oxidation was delayed to a greater extent in emulsions made with NaCas than in BSA stabilized emulsions. These conditions probably enabled NaCas to exert free-radical-scavenging activity.

The nature of the apolar phase influences the structure of the protein emulsifier in oil-in-water emulsions stabilized by bovine serum albumin. A front-surface fluorescence study.

Proteins are widely used as emulsifiers in food emulsions. Model emulsions, designed to study emulsifying properties of proteins and their conformation at the interfaces often contain a hydrocarbon as apolar phase instead of natural triglycerides as found in food products. Yet, some results indicate that the protein conformation at the interface depends on the nature of the apolar phase. Front-surface fluorescence spectroscopy was used to evidence differences in the structure of bovine serum albumin (BSA) adsorbed at the interface of emulsions prepared with different apolar phases: an hydrocarbon (n-dodecane), a synthetic medium-chain triglyceride (miglyol) and a natural vegetable oil (sunflower oil). Emulsions had similar size distributions of oil droplets. Front-surface fluorescence emission spectra of tryptophanyl residues of the protein (Trp) in emulsions, creams and serums varied as a function of the nature of hydrophobic phase. In emulsions and creams, wavelength of the maximum fluorescence intensities was blue-shifted as compared to the BSA solution. The shift was larger in creams than in emulsions and in samples containing dodecane than with the other apolar phases. Fourth derivative spectra of emulsions and creams exhibited two peaks assigned, respectively, to Trp located in hydrophilic and hydrophobic environments. The peaks were slightly red-shifted in the presence of sunflower oil as compared to miglyol and dodecane and the relative intensity of the "hydrophobic peak" was higher in dodecane. The effects were greater in creams than in emulsions. Fluorescence intensity of Trp was the highest in the serums of emulsions prepared with dodecane as compared to serums issued from sunflower oil and miglyol emulsions. Thus, proportion of adsorbed protein was lower in dodecane emulsions than with the other apolar phases. These results evidence that the mean environment of Trp was more hydrophobic in emulsions and creams than in solutions due to a displacement of some of the Trp of the protein to a more hydrophobic environment. Dodecane had the greatest impact on Trp environment (more hydrophobic) followed by miglyol and then by sunflower oil. This is likely due to differences in the conformation of the protein at the hydrocarbon-water interface as compared to the triacylglycerol-water ones. In addition, sunflower oil provoked a large decrease of Trp fluorescence intensity in emulsions and creams as compared to miglyol or dodecane. This could be due to contaminant quenchers in the oil or to interactions of the unsaturated fatty chains with the protein inducing quenching of the Trp. These observations should be related to the physical properties of the apolar phase and its molecular organization and interactions with the protein at the interface.

2,2-diphenyl-1-picrylhydrazyl (DPPH*) test demonstrates antiradical activity of Dorstenia psilurus and Dorstenia ciliata plant extracts.

Plants of the Dorstenia genus, used in traditional medicine and as food ingredient in Africa, are rich in polyphenolic compounds which can be involved in prevention of disease and food spoilage through their antioxidant activity. The antiradical activities of extracts of Dorstenia psilurus. D. ciliata and a phenolic compound (6-prenylapigenin) from D. ciliata were evaluated with the 2,2-diphenyl-1-picrylhydrazyl (DPPH*) test. D. psilurus chloroform and D. ciliata ethyl acetate extracts were shown to exhibit antiradical activities, with slow kinetic action. The efficient concentrations at different kinetic times "EC50,t" for the D. ciliata extract: 699, 479, 311 and 251 g/kg after 30, 60, 120 and 180 min, respectively, were always much lower than for the D. psilurus extract: 2341, 2312, 1672 and 1281 g/kg at the same times. The antiradical activity of 6-prenylapigenin was weak compared to the extracts. These results suggest the antioxidant activities of Dorstenia extracts and might help to understand their traditional use. We propose "EC50, t", calculated on the curves obtained with DPPH test, as a parameter to screen and compare antiradical activities of plant extracts with slow kinetic behaviour.