Soy protein isolates (SPIs) and concentrates (SPCs), which primarily consist of glycinin (11S globulin) and β[jls-end-space/]-conglycinin (7S globulin), are the dominant ingredients in the production of plant-based meat products using high-moisture extrusion (HME). Yet the links between their gelation and structure formation under HME are not fully understood. In this work, we employ small-angle scattering (SAS) techniques, complemented by rheology, on heat-set gels to elucidate the multiscale aggregation pathways of fractionated 11S and 7S globulins, and relate these to the extrudate structure. Heat-set gels revealed a three-level structural hierarchy: compact protein units that persist after heating; aggregates whose mid-scale size and morphology vary with pH; and networks characterised by clustering strength. Near the isoelectric point (pI), both globulin-enriched fractions formed particulate gels. Under these conditions, 7S globulin-enriched gels were stiffer because their smaller aggregates created a higher density of contacts per unit volume, resulting in a higher clustering strength. Away from the pI, fibrillar aggregation dominated, and 11S globulin-enriched gels became stiffer as a result of forming thicker and more tightly connected strands. At pH 7, blending experiments revealed that even small additions of 7S globulin weaken the gels; however, under HME, the same additions enhance anisotropy. Specifically, 7S globulin promotes deformability and alignment, while 11S globulin contributes to network strengthening through covalent crosslinks. Together, these results establish parallels between heat-induced gelation and extrusion, highlighting the complementary roles of 11S and 7S globulins in structuring high-moisture extrudates of soy proteins.

The development of novel plant-based meat alternatives that closely mimic the anisotropic structure of animal meat offers a solution to mitigate the adverse effects of animal meat consumption. The currently most widely adopted production route is shear processing through high-moisture extrusion (HME). The complex structure formation mechanisms that determine the final fibrous texture of extrudates have yet to be fully understood. The main obstacle is the lack of multiscale studies investigating the principles governing structure formation from the nano- to the macro-structural level. This work aims to address this knowledge gap by studying materials, collected after a dead-stop operation of an industrial pilot-plant scale extruder, with multiple characterisation techniques, such as Magnetic Resonance Imaging (MRI) and Small-Angle Scattering (SAS). We demonstrate that the nm- to μm-scale structure is formed already within the extruder barrels, and that sub-mm-scale anisotropy develops within the cooling die. Furthermore, we show that diffuse light reflectance (DR) probes the size and coarseness of the lamellar phase-separated regions.

High-moisture extrusion (HME) is a proven industrial food processing technique used to create textured plant-protein materials that can serve as alternatives for animal meat. The required multiscale anisotropic structure of the extrudate can be achieved by selecting suitable HME process conditions, as well as by pH-shifting. In this work, we explored pH-shifting via the water feed, which is an attractive industrially-scalable approach. Soy protein concentrate (SPC) was extruded on lab-scale and extrudates were characterized ex situ, from molecular to mm scale, using Diffuse Reflectance (DR), Magnetic Resonance Imaging (MRI), Small-Angle-Scattering of Neutrons (SANS) or X-rays (SAXS). pH-shifting had a non-monotonic effect on extrudate hardness and anisotropic structure at both sub-mm (MRI) and μm (DR) scale. At the sub-μm scale, SANS and SAXS data indicated that, at pH > pI, the radius of protein nano-aggregates monotonically increases, accompanied by a transition from particulate to fibrillar protein aggregation. When pH was further shifted to alkaline conditions, the decrease in clustering strength and nematic order parameter pointed to an increase in intra- and inter-fibrillar repulsion, respectively. Protein extractability experiments indicated that the effects of pH-shifting on anisotropic structure formation could not be attributed to covalent intermolecular crosslinking. Thus, repulsive non-covalent electrostatic protein-protein interactions play a dominant role in the formation of multiscale anisotropic structure during SPC extrusion. The formation of an optimal anisotropic SPC extrudate structure is determined by the pH-dependent balance between fibrillar nano-aggregate clustering and electrostatic repulsion. Alkalization or acidification via the water feed implies that protein charge and structure may not be in equilibrium yet with the imposed pH conditions. The transient nature of pH-shifting via the water feed results in an intricate interplay with extrusion conditions. Therefore, control of anisotropic structure formation, via the water feed, in SPC extrudates, is extruder specific.