In addition to the knowledge of thermophysical properties of proteinaceous plant-based materials (Högg and Rauh, 2023a, Högg and Rauh, 2023b), it is imperative to separate the intertwined effects of high pressure, elevated temperature and shear rate (also known as thermomechanical treatments) that occur in the extruder barrel during the high-moisture extrusion (HME). This separation is necessary to gain knowledge on the separate influence of these parameters to understand the texturization mechanisms and their influences during HME in detail. Based on the current literature, high pressure, shear rate and elevated temperature can significantly influence both the microstructure and macrostructure of high-moisture meat analogues (HMMA): i) high pressure applied during HME can affect its density and compactness, thus, pressures can lead to denser structures with reduced porosity (Choton, Gupta, Bandral, Anjum, & Choudary, 2020; Guyony, Fayolle, & Jury, 2022b). Furthermore, pressures between 30 bar to 100 bar may promote protein-protein interactions, resulting in enhanced protein aggregation and network formation (Akdogan, 1996; Cornet, Snel, Lesschen, van der Goot, & van der Sman, 2021; Noguchi, 1989; Wild, 2016); ii) shear rate refers to the velocity gradient and deformation (kneading, compression, mixing etc.) experienced by the material during HME. The shear rate plays a crucial role in protein orientation and alignment. Higher shear rates result in more significant protein alignment, which can lead to a more anisotropic and fibrous structure in the extrudate (Chen, Ker, & Wu, 1990; Cornet et al., 2021; Grabowska et al., 2016; Pietsch, Emin, & Schuchmann, 2017; Strecker, Cavalieri, Zollars, & Pomeranz, 1995); iii) elevated temperature can cause protein denaturation, leading to unfolding and restructuring of protein structures. This unfolding contributes to the formation of new protein networks, which can lead to the development of fibrous structures in the cooling die. (Chen et al., 1990; Cornet et al., 2021; Guyony et al., 2022b; Högg & Rauh, 2023a; Kiiru et al., 2020; Wild, 2016).

The approach to understand the extrusion process with experimental studies as well as using the ‘black box’ model to predict changes in the process for starch-, biopolymer- (Emin & Schuchmann, 2016) and protein-based products are described in the literature to a certain extent (Ferawati et al., 2021; Liu & Hsieh, 2008; Noguchi, 1989; Osen, Toelstede, Wild, Eisner, & Schweiggert-Weisz, 2014; Palanisamy, Franke, Berger, Heinz, & Töpfl, 2019; Pietsch et al., 2017; Pietsch, Bühler, Karbstein, & Emin, 2019; Ryu, 2020; Sandoval Murillo, Osen, Hiermaier, & Ganzenmüller, 2019; Vatansever, Tulbek, & Riaz, 2020; Wittek, Zeiler, Karbstein, & Emin, 2021; Yao, Liu, & Hsieh, 2004). These studies have contributed significantly to the development of extrusion food processing technology, but there are still fundamental knowledge gaps to understand the process, even though its importance is widely recognized (Guyony et al., 2022b; Leonard, Zhang, Ying, Xiong, & Fang, 2021; Zhang et al., 2022).

It is well accepted that process-related changes in proteins occur during HME, like protein melting and protein denaturation, which are considered to be the initial state for texturization (Guyony et al., 2022b; Harper, 1989; Högg & Rauh, 2023a; Riaz, 2000; Wild, 2016). Due to elevated temperature, high shear rate, and high pressure in the extruder barrel (= screw section), the hydrated proteins undergo denaturation and therefore lose their native state (Guyony et al., 2022b; Harper, 1989; Högg & Rauh, 2023a; Riaz, 2000; Wild, 2016). The denaturation of proteins leads to the formation of new bonds (cross-linking phase) since reactive groups of proteins become available (Wild, 2016; Zhang et al., 2019). In the literature, there is an ongoing discussion on which of the protein-protein interactions has the highest influence on the structure of HME samples. The current literature states that disulfide bonds, and to a certain extent also non-covalent bonds (e.g., hydrogen and hydrophobic bonds) play a primary role in creating the backbone structure of the extrudate rather than peptide bonds (Cheftel, Kitagawa, & Quéguiner, 1992; Guyony et al., 2022b; Liu & Hsieh, 2008; Noguchi, 1989; Osen & Schweiggert-Weisz, 2016; Pietsch et al., 2019; Zhang et al., 2019). The induced changes in protein-chemical properties, including protein melting and protein denaturation and subsequent rearrangement of partially or fully unfolded proteins, result in the formation of a new protein structure, leading to texturization. The extent of protein denaturation, aggregation, and depolymerization relies on the intensity of thermomechanical treatment, especially the resulting final material temperature, applied in the screw section (Verbeek & van den Berg, 2010; Zhang et al., 2019). The final material temperature, as highlighted by Högg and Rauh (2023a) serves as the criterion for surpassing the extrusion melting hurdle (EMH) to achieve the initial state for the formation of a fibrous structure in the cooling die.

However, during the extruder process, thermomechanical treatments occur simultaneously, and process, system, and product parameters are closely interlinked and interacting, wherefore the acquisition of product and process knowledge is complex (Cornet et al., 2021; Guyony et al., 2022b; Pietsch et al., 2019; Wild, 2016; Zhang et al., 2019).

In order to address the complexity of protein-protein interaction during HME, model process systems, such as closed cavity rheometer, shear cell or pressure cell can be employed. These systems allow the independent and controlled variation of thermal and mechanical stresses, similar to the stresses that occur during HME, thereby allowing monitoring changes in the protein-protein interactions by offline chemical analysis (Cornet et al., 2021; Emin & Schuchmann, 2016; Noguchi, 1989; Pietsch et al., 2019).

In one of the aforementioned studies, Pietsch et al. (2019) imitated thermomechanical treatments using a closed cavity rheometer, applying shear rates ranging from 0.1 to 50 1/s and temperatures ranging from 60 °C to 160 °C. They studied the influence of defined thermal and mechanical stresses on the rheological properties and protein-protein interactions of soy protein concentrate. However, pressure-induced effects were not investigated in their study. Their results suggested that thermomechanical treatment at maximum shear rates of 0.1 and 50 1/s in combination with temperatures in the range of 60 °C to 160 °C had no significant influence on changes in protein-protein interactions. It needs to be stated that the applied shear rates did not resemble the conditions present in an HME process. Usually the shear rate in an HME process is >50 s−1and < 410 s−1 (Chen, Zhang, Zhang, & Wang, 2022; Suparno, Dolan, Ng, & Steffe, 2011).

To elucidate the texturization mechanism during HME and the influence of elevated temperature, shear rate and high pressure on texturization, it is important to consider their influences separately. Since the shear rates investigated by Pietsch et al. (2019), were not representative HME conditions and did not exhibit any significant impact on protein-protein interactions, our study proceeded to focus on investigating the influence of high pressure and elevated temperature. This decision was driven by the lack of available data regarding the effects of high pressure and elevated temperature on plant-based proteins within the domain of HME conditions. Therefore, knowledge about the thermomechanical treatment in combination or alone and their effect (synergistic, additive, or antagonistic) on protein-chemical properties and on structure formation is necessary to produce high-moisture meat analogs (HMMA) with defined final structures.

The changes in protein-protein interactions induced by the application of model process systems in the domain of HME conditions have not been yet investigated by other studies. Therefore, due to limited research in that domain the influence of elevated temperature and high pressure on protein-protein interactions was investigated in this study using a specially designed temperature-controlled pressure chamber (Ulrich et al., 2016), among others.

As the aforementioned studies (Noguchi, 1989; Pietsch et al., 2019; Wittek et al., 2021) did not find any changes in protein-protein interactions in extruded samples, it was hypothesized in our study that the process parameters occurring during high moisture extrusion may have antagonistic effects. Subsequently, effects may disperse when they occur simultaneously. Therefore, it was deemed necessary to assess the parameters separately or in pairs, due to their mutual influence and parallel occurrence within the extruder barrel. This allows the effect of individual parameters on protein-reactions, and thus, subsequently on protein texturization to be deduced. Furthermore, it was hypothesized that the high pressure present inside the extruder barrel might not be sufficient enough to cause significant changes to protein. To substantiate this hypothesis, protein alterations under more extreme pressure conditions were studied.

To investigate the impact of extrusion parameters, namely temperature (95–140 °C) and pressure (30–60 bar), on SPC Alpha® 8 IP separately and in combination, and to evaluate any synergistic, additive or antagonistic effects on protein changes, a temperature-controlled pressure chamber was utilized. The cooling rate of the thermostat affixed to the pressure chamber was slower than the typical cooling rates observed in the cooling die during HME. To reduce the impact of the extended cooling phase within the pressure chamber, we conducted a different series of experiments involving an oil bath at the same temperature range but with rapid cooling using an ice bath. Additionally, our research aimed to explore the extreme point of protein texturization by investigating the combined influence of extreme pressure and elevated temperature using a high-pressure thermal sterilization system. Previous research has demonstrated that applying pressures of 6000 bar can induce protein texturization (Sim et al., 2019).

The purpose of this investigation was to analyze the effects of increased temperature and pressure, individually and in conjunction, to determine the predominant factor impacting protein reactions. The assessment employed similar temperature and pressure ranges to those found during HME to provide a thorough understanding of their overall impact on the HME process. SPC Alpha® 8 IP was selected as a raw material because it is one of the most commonly used ingredients for HME production. The aim of our study was not to generate extrudates without shear in the model process systems mentioned; instead, our goal was to draw potential conclusions on the specific impact of shear on the HME process based on our findings. In order to gain a comprehensive understanding of protein reactions, various protein chemical analyses were carried out. Changes in sample structures at high pressure and elevated temperatures on a macroscopic scale were visually captured, as well.