Proteins for the future: A soft matter approach to link basic knowledge and innovative applications

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Introduction
In a context of global demographic and food transitions, a shift from the use of animal to alternative (out of which plant) proteins is expected in a near future. This increasing alternative protein content in human diet will bring positive effects on both climate and public health. But before these important changes on dietary practices, a significant research investment is necessary to lay the foundation of these future foods. At the same time and with this objective of sustainability, numerous wastes of animal processing have to be valorized for new non-food applications and this challenge also requires basic scientific knowledge. In this goal, teams of Science for Food and Bioproduct's INRA Division develop for a long time innovative research on food proteins and their interactions in view to imagine these new architectures and/or applications.
From proteins to complex architectures found either in natural (milk, meat, eggs, seeds, …) or in processed (emulsions, foams, gels, …) foods, one of the most important scientific challenge is to link the protein molecular structure, self-assemblies mechanisms, supramolecular architectures and their resulting properties. The challenge is to understand the properties of these natural and processed architectures at macroscopic scale, in relation with the organization of the basic components and their interactions at a mesoscopic scale. This involves multiscale approaches requiring methodological and theoretical developments. Furthermore in these disordered materials, energy scale of particle interactions is of the order of thermal energy and such weak interactions allow these colloidal self-assemblies to adopt different phase behavior at thermodynamic equilibrium that can Simplified diagram of the interfacial film organization according to the order of protein injection (Le Floch-Fouéré, C., Beaufils, S., Lechevalier, V., Nau, F., Pézolet, M., Renault, A. & Pezennec, S., 2010) ovalbumin

A C C E P T E D M A N U S C R I P T
6 Besides in systems where several proteins with different properties (size, hydrophobicity, charge, conformation flexibility, …) coexist, the interactions between proteins enhance interfacial behavior that cannot be considered as the sum of the single behaviors. Le Floch-Fouéré et al. (Le Floch-Fouéré, C., Beaufils, S., Lechevalier, V., Nau, F., Pézolet, M., Renault, A. & Pezennec, S., 2010;Le Floch-Fouéré, C., Pezennec, S., Lechevalier, V., Beaufils, S., Desbat, B., Pézolet, M. & Renault, A., 2009) using an original approach of sequential adsorption between ovalbumin and lysozyme, thus highlighted a specific and stratified organization of the two proteins inside the interfacial film (Fig. 2). These results incited researchers to understand protein-protein interactions in condensed systems, which will be detailed in the part 2 of this chapter, since protein crowding close to the interface could generate intermolecular interactions of prominent importance in the understanding of interfacial properties.
Work is in progress to relate these interfacial and foaming properties to bulk behaviors the issue over time being to control technological parameters to improve interfacial properties in relation to foaming properties.  Based on these results, Loiseleux et al. (2017) investigated the texture of low fat dairy emulsions stabilized by a mixture of Whey Protein Aggregates (WPA) (2 or 3% w/w) and 0.1% (w/w) of native whey protein (Loiseleux, T., Garnier C., Beaumal V., Croguennec T., Guilois S., Jonchère C., Roland-Sabaté A., Anton M. & Riaublanc, A., 2017). Surprisingly, these low oil content emulsions texturize over time without additives through two mechanisms: the first one occurs during homogenization and concerns the bridging flocculation between droplets by WPA and the second one appears during storage and corresponds to the formation of a protein-lipid network leading to emulsion gelling.
Molecular interactions and density of the protein-lipid network depends on storage temperature and

A C C E P T E D M A N U S C R I P T
11 pressure with protein fraction. Using this method, it is usually possible to cover about 3 decades of osmotic pressures (from 1 kPa to 1 MPa), achieving protein concentration up to 500-700 g/L at the highest pressures.
The variation of the osmotic pressure with protein concentration is given in Fig. 4 (B-D) for the three protein systems studied by the INRA division, Science for Food and Bioproduct's researchers until now. We give below a brief description of these results, with additional information about the textural (rheological) and structural changes experienced by the solutions as concentration increases.
It demonstrates how the diversity found in food proteins (including their assemblies) results in a rich range of behaviors in concentrated systems.

A. Lysozyme
Lysozyme is a globular protein that has been well characterized since its discovery in 1922 (Fleming, A., 1922). Numerous studies have been conducted on lysozyme, in particular its self-association characteristics when concentrated or submitted to physicochemical changes (Cardinaux, F., Gibaud,

A C C E P T E D M A N U S C R I P T
22 extraction remains to be assessed since those components can have great influence on collagen selfassembly, functionality and bioactivity.
Concerning elastin, this protein is a highly insoluble and stable polymer with an uncommon amino acids composition, about 75 % hydrophobic residues (Gly, Val, Ala); it is the longest lasting protein in vertebrates (140 years). A distinguishing feature of elastin is also its resistance to high temperature and pH that usually denature many proteins ( , 2010). The association of these two crops into a single food increases its protein content and be benefit from their complementary essential amino acid profiles (Laleg, K., Barron, C., Sante-Lhoutellier, V., Walrand, S. & Micard, V., 2016). Considered as a staple and popular cereal food, pasta appears as an interesting model for such an association.
The partial or total replacement of gluten by legume protein in pasta changed its structure which affected the delivery of its nutrients, mainly starch and proteins (Laleg, K  . We have studied the ability of -LG/LF to entrap efficiently a hydrophilic molecule, e.g. vitamin B9 (B9). The first step was to determine the efficient conditions for complex coacervation in the presence of B9 (Fig. 7). We showed that the formation of vitamin B9heteroprotein coacervates occurs at specific protein concentration range (Chapeau et al. 2016). The recovered dense phase (vitamin B9-whey protein coacervates) is highly concentrated in proteins ( ˷ 300 g/kg) and shows an interesting biocarrier efficiency of 4 to 10 g B9/kg coacervates. The vitamin B9-whey protein coacervates is stable and shows interesting potentiality to protect the vitamin B9 toward photodegradation (unpublished). Now, a challenging part is to study the scaling-up of HPCC loaded bioactives from laboratory and bench scales to pilot scale. Also, the conditions for a controlled release of bioactives from heteroprotein coacervates during their digestion have to be further investigated. These studies are currently undertaken.

A C C E P T E D M A N U S C R I P T
29 Until today, HPCC is a largely unexplored phenomenon. We are convinced that it can bring mechanistic information about protein assemblies useful for all areas of biology. They are also promising objects for various applications including, encapsulation, change of food texture and formation of biocompatible protein films. This fascinating research topic becomes of high interest for food scientists as several research groups start to publish on HPCC.

Conclusion
Animal or plant-based proteins are present in a wide variety of processed and non-processed food products. The nature and concentrations of the proteins involved vary massively from one product to another but in many of these products, the protein interactions play a central role in determining the final structure and properties of the product. In the past 15 years Science for Food and Bioproduct's researchers have led soft matter approaches to establish the link between food protein interactions, phase transitions and structural properties especially at the interfaces, in condensed state, or in complex coacervation conditions, in view to build functional structures.

A C C E P T E D M A N U S C R I P T
30 Now, in a context of demographic and food transitions, environmental and energy constraints, and links between these factors and public health, the challenge of Science for Food and Bioproduct's teams is to integrate food systems in all their dimensions from production to end use taking into account sourcing, production method, composition / structure, processing methods, digestive deconstruction, by-products, ... to reach to sustainability criteria. In this way alternative sources of food proteins (plant, algae, insect) and by-product valorization will require the development of extraction strategies, the screening and design of functional mixed protein assemblies, and the understanding of aggregation and self-assembly of proteins to generate functional assemblies taking into account the physiological conditions and the physics of condensed media. All the previous works of Science for Food and Bioproduct's INRA Division described in this chapter constitute solid foundations to address these future challenges.