Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity.

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 degrees C in Tris-HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 degrees C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 degrees C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 degrees C only. High pressure up to 600 MPa induced spectral changes which at 20 degrees C were fully reversible upon decompression. At 45 degrees C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 degrees C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.

Antioxidant, Antidiabetic, and Antiobesity Properties, TC7-Cell Cytotoxicity and Uptake of Achyrocline satureioides (Marcela) Conventional and High Pressure-Assisted Extracts.

The growing incidence of non-communicable diseases makes the search for natural sources of bioactive compounds a priority for such disease prevention/control. Achyrocline satureioides ('marcela'), a plant rich in polyphenols and native to Brazil, Uruguay, Paraguay, and Argentina, could be used for this purpose. Data on its antidiabetic/antiobesity properties and cellular uptake of bioactive compounds are lacking. The potentiality of non-thermal technologies such as high-hydrostatic pressure (HP) to enhance polyphenol extraction retains attention. Thus, in the present study aqueous and ethanolic marcela extracts with/without assisted-HP processing were chemically characterized and assessed for their in vitro antioxidant capacity, antidiabetic and antiobesity activities, as well as cellular cytotoxicity and uptake on intestinal cell monolayers (TC7-cells, a clone of Caco-2 cells). Aqueous and ethanolic conventional extracts presented different polyphenolic profiles characterized mainly by phenolic acids or flavonoids, respectively, as stated by reverse phase-high-performance liquid chromatography (RP-HPLC) analyses. In general, ethanolic extracts presented the strongest bioactive properties and HP had none or a negative effect on in vitro bioactivities comparing to conventional extracts. TC7-cell viability and cellular uptake demonstrated in conventional and HP-assisted extracts, highlighted the biological effects of marcela bioactive compounds on TC7-cell monolayers. TC7-cell studies showed no HP-induced cytotoxicity. In sum, marcela extracts have great potential as functional ingredients for the prevention/treatment of chronic diseases such as diabetes.

High pressure-low temperature processing of food proteins.

High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.

Selective transport of staphylococcal enterotoxin A through in vitro generated human M cells.

Staphylococcal enterotoxins are responsible for food poisoning and toxic shock syndrome due to their superantigen activity on T cells. Although their activity necessarily involves passage through the intestinal epithelium, little is known about this critical step. In the present study, we compared the in vitro transport of staphylococcal enterotoxin A through human intestinal absorptive and M cells. We found that the transport of the toxin through M cells was polarized and temperature-sensitive, in contrast with the less efficient transport of the toxin by absorptive cells. These data suggest the involvement of M cells in the intestinal absorption of staphylococcal enterotoxins.

Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics.

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 µM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

UHPH-processed O/W submicron emulsions stabilised with a lipid-based surfactant: physicochemical characteristics and behaviour on in vitro TC7-cell monolayers and ex vivo pig's ear skin.

Submicron O/W emulsions formulated with sesame oil plus a lipid-base surfactant, and with or without retinyl acetate (RAC) as a model hydrophobic biomolecule, were prepared by single-pass homogenisation at ≥ 200 MPa (UHPH) and an initial fluid temperature (Tin) of 24°C. These emulsions were characterised by a monomodal distribution (peak maximum at 260 nm) and a 2-year potential physical stability at ambient temperature. Submicron droplets were investigated in term of (i) physicochemical characteristics (size distribution curves; ζ-potential value), and (ii) impact on TC7-cell monolayers (MTT-assay and cell LDH-leakage). Submicron droplets ± RAC did not affect or increased significantly (p=0.05) TC7-cell metabolic activity after 4-24h of exposure indicating absence of cellular impairment, except when high amounts of droplets were deposed on TC7-cells. Indeed, the lipid-based surfactant deposed alone on TC7-cells at high concentration, induced some significant (p=0.05) cell LDH-leakage, and therefore cell-membrane damage. Cellular uptake experiments revealed a significant (p=0.05) time-dependent internalisation of RAC from submicron droplets, and cellular transformation of RAC into retinol. The turnover of RAC into retinol and therefore RAC bioaccessibility appeared faster for RAC-micelles of similar size-range and prepared at atmospheric pressure with polysorbate 80, than for submicron O/W emulsions. Permeation experiments using pig's ear skin mounted on Franz-type diffusion cells, revealed RAC in dermis-epidermis, in significantly (p=0.05) higher amounts for submicron than coarse pre-emulsions. However, RAC amounts remained low for both emulsion-types and RAC was not detected in the receptor medium of Franz-type diffusion cells.

Interaction of curcumin with phosphocasein micelles processed or not by dynamic high-pressure.

The binding of curcumin to native-like phosphocaseins (PC) dispersed in simulated milk ultrafiltrate at pH 6.6 was assessed by fluorescence spectrophotometry. Curcumin binds to native-like PC micelles with ∼1 binding site per casein molecule, and a binding constant of 0.6-5.6 × 10(4)M(-1). Dynamic high pressure (or ultra-high pressure homogenisation, UHPH) at 200 MPa did not affect the binding parameters of curcumin to processed PC. UHPH-processing of PC dispersions at 300 MPa was followed by a slight but significant (p=0.05) increase in the binding constant of curcumin to processed PC, which may result from the significant UHPH-induced dissociation of initial PC micelles into neo-micelles of smaller sizes, and from the corresponding 1.5-2-fold increase in micelle surface area. PC-curcumin complexes were resistant to pepsin but were degraded by pancreatin, providing the possibility of a spatiotemporally controlled release and protection of bound biomolecules. UHPH-processed PC did not induce TC7-cell damage or major inflammation as assessed by LDH release or IL-8 secretion, respectively, compared with native-like PC. PC micelles could provide a valuable submicron system to vectorise drugs and nutrients.

Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9 degrees C or 20 degrees C: effects on the distribution of minerals and proteins between colloidal and soluble phases.

The effects of high pressure treatments (100-300 MPa; 15 min; 9 degrees C or 20 degrees C) on the distribution of minerals and proteins of raw skim milk (RSM) and of a dispersion of industrial phosphocaseinate (PC) were studied after separation of the micellar and soluble phases by ultracentrifugation (UCF). Whatever the temperature of high pressure treatments, the pressure-induced dissociation of the casein micelles was accompanied by calcium (Ca), phosphorus (P) and casein release from the micelles. The released Ca and P were or became bound to soluble proteins since progressive increases in Ca and P concentrations were observed in the UCF supernatants of RSM and of the PC dispersion but not in the ultrafiltrates from these UCF supernatants (free of soluble proteins). Simultaneously, alpha(S1-), alpha(S2-), beta- and kappa-caseins were progressively released from the micelles, as seen by electrophoretic analysis. The pressure-induced solubilisation of alpha(S1-) and alpha(S2-)caseins, essentially located in the core of the micelles, suggests that high pressure de-stabilized micelles including their internal structure.

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments.

Raw whole milk of high microbial quality (58 degrees C), but markedly decreased above 200 MPa when Tin=24 degrees C (T2>60 degrees C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short (<<1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 degrees C) induced a 2-3 log cycle reduction of the total endogenous milk flora and a 1.5-1.8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2-4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1 approximately 29 degrees C and pressure=200 MPa, increased homogenisation efficiency.