Interfacial and emulsifying properties of amaranth (<i>Amaranthus hypochondriacus</i>) protein isolates under different conditions of pH

Amaranth proteins have adequate amino acid balance for substituting either partly or completely animal proteins in human nutrition. However, they present poor emulsifying properties in basic conditions corresponding to their extraction medium. Consequently their use in acidic conditions could be envisaged to better exploit their potentialities. To better understand their emulsifying properties we have studied their interfacial activities at pHs 2.0 and 8.0 and tried to make the link between 2D and 3D properties. Our results clearly demonstrate the better properties of AI at pH 2.0 than at pH 8 in terms of protein solubility, spreading, adsorption, viscoelastic properties of interfaces and emulsion stability. These results are in relation with the denaturated state of proteins at pH 2.0 where proteins form a harder interfacial film, as compared to pH 8.0. Thus the potential use of amaranth proteins in emulsifying applications should be oriented towards acidic applications.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1].Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7.For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations.No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids.These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1].Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7.For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations.No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids.These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

Phosvitin-calcium aggregation and organization at the air-water interface.

International audiencePhosvitin, an egg yolk protein constituted by 50% of phosphorylated serines, presents good emulsifying properties whereas its interfacial properties are not yet clearly elucidated and remain object of discussion. Phosvitin has a high charge density and naturally forms aggregates through phosphocalcic bridges in egg yolk. This high charge density, doubled by this capacity to aggregate, limits the adsorption of the protein at the air-water interface. In this work, we investigated the aggregation impact by calcium ions on the organization of the phosvitin interfacial film using the atomic force microscopy. Phosvitin interfacial films without calcium ions are compared to phosvitin interfacial films formed in the presence of calcium ions in the subphase. We demonstrated that phosvitin is able to anchor at air-water interfaces in spite of its numerous negative charges. In the compression isotherm a transition was observed just before 28 mN/m signifying a possible modification of the interfacial film structure or organization. Calcium ions induce a reorganization towards a greater compaction of the phosvitin interfacial film even at low surface pressure. In conclusion we suggest that, in diluted regime, phosvitin molecules could adsorb by their two hydrophobic extremities exhibiting loops in the aqueous phase, whereas in concentred regime (high interfacial concentration) it would be adsorbed at the interface by only one extremity (brush model)

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1]. Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7. For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations. No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids. These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

The phase and charge of milk polar lipid membrane bilayers govern their selective interactions with proteins as demonstrated with casein micelles

The biological membrane surrounding fat globules in milk (milk fat globule membrane; MFGM) is an interface involved in many biological functions and interactions with the surrounding proteins or lipolytic enzymes in the gastro-intestinal tract during digestion. The MFGM exhibits lateral heterogeneities resulting from the different phase states and/or head-group charge of the polar lipids, which were both hypothesized to drive interaction with the casein micelles that is the major milk protein assembly. Atomic force microscopy (AFM) imaging was used to track the interactions of casein micelles with hydrated supported lipid bilayers of different composition, phase state and charge. Zeta-potential and Langmuir isotherms of the different polar lipids offered additional information necessary to interpret AFM observations. We showed that the negatively-charged casein micelles did not interact with milk sphingomyelin in the gel or liquid-ordered phases but did interact with polar lipids in the liquid-disordered phase (unsaturated polar lipids and milk sphingomyelin above its melting point). A wide intermolecular distance between polar lipids allowed protein adsorption on the membranes. However, the presence of the anionic polar lipids phosphatidylserine and phosphatidylinositol prevented any interaction with the casein micelles, probably due to electrostatic repulsion. These results open perspectives for the preparation of tailored emulsions covered by polar lipids able to modulate the interfacial interactions with proteins

Deciphering the role of interfacial properties of milk fat globule membrane in its interaction with the surrounding proteins, the casein micelles

The interactions between the milk fat globule membrane (MFGM) and proteins (e.g. enzymes, caseins, whey proteins) are poorly known despite their importance in many interfacial mechanisms occurring at the surface of milk fat globules. The MFGM exhibits polar lipids lateral heterogeneities: the zwitterionic milk sphingomyelin (MSM) molecules form domains in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, that are surrounded by a fluid matrix of phospholipids in the liquid-disordered (Ld) phase composed of the unsaturated phosphatidylcholine (PC) and the anionic phosphatidylserine (PS) and phosphatidylinositol (PI) [1,2].AimThe aim of this study was to determine how the phase state and head-group charge of MFGM polar lipids drive interactions with the negatively charged casein micelles, as the major milk protein assembly.MethodsHydrated supported lipid bilayers with various compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. Real-time atomic force microscopy (AFM) imaging in liquid was performed before and after injection of casein micelles. Zeta-potential and Langmuir isotherms of the different polar lipids offered complementary information.ResultsThe interaction of casein micelles with milk polar lipids is hindered by the high lateral packing density of MSM domains in gel and Lo phases and by the negative charge of PI and PS, certainly due to electrostatic repulsions. But the casein micelles did adsorb onto the Ld phases of DOPC and of MSM above its phase transition temperature (T>Tm=34.3°C[3]), probably as a result of large inter-molecular distances.ConclusionThe absence of interaction between casein micelles and the complex MFGM polar lipids is due to the MSM-rich ordered domains and to the presence of anionic phospholipids in the Ld phase. These results open perspectives for the design of liposomes and emulsions with variable capacity for protein adsorption by the modulation of phase state and charge of the interfacial polar lipids.References: [1] Et-Thakafy et al., Food Chem., 2017, 220, 352–361 [2] Murthy et al., BBA-Biomembranes, 2016, 1858, 2181-2190[3] Murthy et al., BBA-Biomembranes, 2015, 1848, 2308-231

The interactions between milk polar lipid membrane bilayers and the casein micelles depend on the phase state and charge

The interactions between milk polar lipid membrane bilayers and the casein micelles depend on the phase state and charge. 16. Euro Fed Lipid Congress Fats, Oils and Lipids: Science, Technology and Nutrition in a Changing Worl