Stability of food proteins at high pressure conditions

High pressure (HP) is particularly suited to study protein folding/unfolding, revealing subtle structural rearrangements not accessible by other types of denaturation. HP also has many industrial-scale advantages over heat treatments, including “greener” processing and preservation of nutritional values, colors, and flavors of foods. We have combined in situ HP with small-angle (X-ray and neutron) scattering (SAS) and spectrophotometry to follow the structure in solution of proteins of interest for the food industry. SAS is an essential technique for obtaining structural, but low-resolution, information about proteins, when conventional high-resolution structural biology methods are not possible. I will illustrate this approach with two studies on proteins of food interest: (i) bovine β-lactoglobulin (BLG), a whey protein with a high propensity to bind to various bioactive molecules. We probed by SANS1 and absorbance the effects on pressure stability and reversibility of BLG of the binding of retinol (vitamin A), resveratrol (polyphenol), and biliverdin (linear tetrapyrrole chromophore) to different sites on the protein2, 3. (ii) C-phycocyanin (CPC), a phycobiliprotein from cyanobacteria, to which tetrapyrrole chromophores are covalently attached and which can be used as a natural blue dye in the food industry. We studied by SAXS and absorbance HP-induced CPC unfolding and reversibility from two oligomeric states of the protein as a function of pH. Acknowledgements LLB, SOLEIL, and I2BC facilities are acknowledged for beamtime and proteomic expertise. This work was also supported by ANSO Project No. ANSOCR-PP-2021- 01. References 1. Annighöfer B, et al. High pressure cell to investigate protein unfolding up to 600 MPa by small-angle neutron scattering. Rev Sci Instr 2019;90:025106. 2. Minic S, et al. Effect of ligands on HP-induced unfolding and oligomerization of β-lactoglobulin. Biophysical J 2020;119:2262-74. 3. Minic S, et al. Structure of proteins under pressure: covalent binding effects of biliverdin on beta-lactoglobulin. Biophysical J 2022;121:2514-25

Combined hydrogels of starch and β-lactoglobulin as matrices for the preservation of C-phycocyanin

The color of food products is an important aspect in food industry, and its preservation remains a big challenge. We aim to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae. For this purpose, we incorporated C-PC in combined starch and β-lactoglobulin (BLG) hydrogels by using a high-pressure (HP) process. Indeed, in thermal treatment, the color derived from C-PC is entirely lost. We characterized the obtained HP gels by both rheology and small-angle X-ray scattering (SAXS). Various formulations of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were tested under HP up to 4,500 bar. A good preservation of the C-PC pigment was established by mixing BLG and starch with C-PC at pH 7, with concentrations of 180, 5, and 10 mg/mL, respectively. Identical component concentrations were maintained in the binary systems. Structure of gels was characterized by SAXS providing insight of C-PC interactions with BLG and starch after HP process which leads to the formation of solid gels with larger mesh compared to two-component systems. This results in enhanced mechanical properties, which were determined by amplitude and frequency sweep measurements using a rheometer with applied plane/plane geometry. Therefore, adding starch, even at small concentration, significantly improves gel visual appearance and mechanical properties. Our study reveals that preservation through HP treatment is more effective than high temperature treatment, as visually observed through the sustained color integrity of C-PC blue dye

The use of starch and β-lactoglobulin composite hydrogels as frameworks for preserving c-phycocyanin

Our study aimed to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae due to its importance in the food industry. We incorporated C-PC into hydrogels formed by combining starch and β-lactoglobulin (BLG) using high-pressure (HP) processing to achieve this objective. Notably, thermal treatment resulted in the complete loss of colour derived from C-PC. We performed a comprehensive characterization of the resulting HP gels by rheology measurements, texture profile analysis (TPA), small-angle X-ray scattering (SAXS), and scanning electron microscopy (SEM). Different compositions of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were processed under high-pressure (HP) conditions reaching up to 4,500 bar. The C-PC pigment was effectively preserved by mixing BLG and starch with C-PC at pH 7, maintaining concentrations of 180, 5, and 10 mg/mL, respectively. The same concentrations of components were retained in the binary systems. Rheological properties of the gels were determined using a rheometer with plane/plane geometry, and texture analysis was conducted through TPA. These findings enabled the assessment of food gel's properties, such as hardness, springiness, chewiness, and cohesiveness. The structural characteristics of the gels were determined by SAXS, offering insights into the interactions between C-PC, BLG, and starch after HP processing. Adding CPC and starch formed solid gels with a larger mesh than the pure BLG gels. SEM scans of the gel surface revealed that all components influenced the overall morphology of gels. Even at low concentrations, the addition of starch notably influenced the gels' visual appearance and mechanical properties. Our investigation highlights the superior effectiveness of HP treatment in the preservation of C-PC compared to high-temperature treatment, evident in the sustained colour integrity of the C-PC blue dye

Influence of pressure on the low-frequency vibrational modes of lysozyme and water: A complementary inelastic neutron scattering and molecular dynamics simulation study

International audienceWe performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low‐frequency vibrational modes of lysozyme in aqueous solution in the 1 atm–6 kbar range. Increasing pressure induces a high‐frequency shift of the low‐frequency part (<10 meV = 80 cm−1) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water

The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn2+ ions

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn 2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn 2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in-situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn 2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In-situ visible absorption study of BV chromophore confirmed the ability of Zn 2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ~100 MPa in the presence of Zn 2+. By combining HPfluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket

Neutron scattering study of lysozyme solution under high pressures

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Structure of proteins under pressure: Covalent binding effects of biliverdin on β-lactoglobulin

International audienceHigh pressure (HP) is a particularly powerful tool to study protein folding/unfolding, revealing subtle structural rearrangements. Bovine β-lactoglobulin (BLG), a protein of interest in food science, exhibits a strong propensity to bind various bioactive molecules. We probed the effects of the binding of biliverdin (BV), a tetrapyrrole linear chromophore, on the stability of BLG under pressure, by combining in situ HP-small-angle neutron scattering (SANS) and HP-UV absorption spectroscopy. Although BV induces a slight destabilization of BLG during HP-induced unfolding, a ligand excess strongly prevents BLG oligomerization. Moreover, at SANS resolution, an excess of BV induces the complete recovery of the protein “native” 3D structure after HP removal, despite the presence of the BV covalently bound adduct. Mass spectrometry highlights the crucial role of cysteine residues in the competitive and protective effects of BV during pressure denaturation of BLG through SH/S-S exchange

First steps toward the development of SONATE, a Compact Accelerator driven Neutron Source

Facilities providing bright thermal neutron beams are of primary importance for various research topics such as condensed matter experiments, neutron-imaging or medical applications. Currently these are mainly spallation sources and nuclear reactors. However, these later facilities are ageing and the political context does not favor the building of new ones. This is the case in CEA-Saclay (France), where the Orphee reactor is planned to shutdown in 2019. Therefore, another local facility, affordable by one country, able to provide high brilliance neutron beams has to be built. At CEA-Saclay, a compact accelerator driven neutron source, SONATE, is investigated in taking advantage of the IPHI accelerator able to deliver a 3 MeV proton beam with an intensity up to 100 mA. In the future, SONATE is foreseen to operate with 20 MeV protons to increase the neutron brightness. In addition to the difficulties to operate such high intensity accelerators, the other challenges regard the target-moderator-reflector (TMR) design which is crucial to maximize the neutron flux at the detector location. At CEA-Saclay, several experiments were performed between 2016 and 2019 with the IPHI accelerator. Geant4 simulations were also developed. They demonstrate the feasibility of such concept and enable to find the best TMR configuration for the future SONATE facility. These developments are reported in this article

First steps toward the development of SONATE, a Compact Accelerator driven Neutron Source

Facilities providing bright thermal neutron beams are of primary importance for various research topics such as condensed matter experiments, neutron-imaging or medical applications. Currently these are mainly spallation sources and nuclear reactors. However, these later facilities are ageing and the political context does not favor the building of new ones. This is the case in CEA-Saclay (France), where the Orphee reactor is planned to shutdown in 2019. Therefore, another local facility, affordable by one country, able to provide high brilliance neutron beams has to be built. At CEA-Saclay, a compact accelerator driven neutron source, SONATE, is investigated in taking advantage of the IPHI accelerator able to deliver a 3 MeV proton beam with an intensity up to 100 mA. In the future, SONATE is foreseen to operate with 20 MeV protons to increase the neutron brightness. In addition to the difficulties to operate such high intensity accelerators, the other challenges regard the target-moderator-reflector (TMR) design which is crucial to maximize the neutron flux at the detector location. At CEA-Saclay, several experiments were performed between 2016 and 2019 with the IPHI accelerator. Geant4 simulations were also developed. They demonstrate the feasibility of such concept and enable to find the best TMR configuration for the future SONATE facility. These developments are reported in this article

Effect of Ligands on HP-Induced Unfolding and Oligomerization of β-Lactoglobulin

International audienceTo probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine b-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HPsmall-angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to ab. 300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by ab. 100 MPa. Increasing temperature from 30 to 37°C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to ab. 300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization