# In situ studies of plant-based meat analog texturization

Tong Guan, Corina Sägesser, Roman Villiger, Lisa Zychowski, Joachim

Kohlbrecher, Joseph Dumpler, Alexander Mathys, Patrick Rühs, Peter

Fischer, Olga Matsarskaia

► To cite this version:

Tong Guan, Corina Sägesser, Roman Villiger, Lisa Zychowski, Joachim Kohlbrecher, et al. In situ studies of plant-based meat analog texturization. Food Hydrocolloids, 2024, 155, pp.110215. 10.1016/j.foodhyd.2024.110215. hal-05520005

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# *In situ* studies of plant-based meat analog texturization

Tong Guan<sup>a, b, 1</sup>, Corina Sägesser<sup>c, 1</sup>, Roman Villiger<sup>d</sup>, Lisa Zychowski<sup>e</sup>, Joachim Kohlbrecher<sup>f</sup>, Joseph Dumpler<sup>c</sup>, Alexander Mathys<sup>c</sup>, Patrick Rühs<sup>e</sup>, Peter Fischer<sup>b, *</sup>, Olga Matsarskaia<sup>a, **</sup>

## ARTICLE INFO

### Keywords:

High-moisture extrusion cooking

Plant-based meat analogs

Custom-made cooling die

*In situ* small-angle neutron scattering

Fiber formation mechanism

## ABSTRACT

Plant-based meat analogs are the rising stars of the food industry as they promise to mimic the experience of eating meat without killing animals. They are popular due to their meat-like fibrous structures and typically obtained *via* high-moisture extrusion cooking. Texturization is believed to mainly take place during the solidification in a cooling die attached to the end of the extruder. However, the mechanisms behind this texturization are not fully elucidated yet. This work presents *in situ* studies of fiber evolution within a custom-designed extruder cooling die by small-angle neutron scattering on a length scale from 1.3 to 436 nm. The measurements demonstrated that macroscopic fibrous structures did not emerge from unfolding, elongation and orientation on molecular level. This contradicts existing hypothesis of the structuring mechanism underlying the meat-like fibers. Instead, the scattering patterns indicate the presence of densely packed protein nano-aggregates of around 40 nm consisting of globular proteins with a diameter of 9 nm. Based on that, chain-like arrangement of protein nano-aggregates, fractures of the viscoelastic mass in the flow field and sharp temperature-dependent solidification are proposed as underlying mechanisms for macroscopic fiber formation.

## 1. Introduction

Global meat consumption has significantly increased over the last several decades (Parlasca and Qaim, 2022). Meat production has a large share of environmental impacts, e.g., the production of animal-based foods has significantly higher greenhouse gas emission compared to plant-based foods, and the nitrogen emission from livestock is equal to one-third of the current human-induced nitrogen emission (Smetana et al., 2023). An elevated consumption of meat may also enhance the risk of chronic diseases, such as stroke, colorectal cancer, cardiovascular disease, and type 2 diabetes (Battaglia Richi et al., 2015; Godfray et al., 2018; Yang et al., 2016). Moreover, meat consumption is inevitably linked to ethical issues and animal welfare concerns (Parlasca and Qaim, 2022). For these reasons, people increasingly turn to vegetarian or flexitarian diets, gradually making meat substitutes more mainstream (Boukid, 2021). Among vegetarian food choices, plant-based meat

analogs (PBMAs) nowadays hold an important share of the global meat substitutes market due to their resemblance to animal meat, including the texture, appearance, flavor, and taste (Anusha Siddiqui et al., 2022; Chen, Feng, Blank, and Liu, 2022; Kyriakopoulou, Dekkers, and van der Goot, 2019). Although significant efforts have been made to improve the imitation of animal meat, an exact replicate of the sensory experience of animal meat is still a challenge for the food industry (Boukid, 2021; Hoek et al., 2011). Thus, pushing the boundaries to mimic meat experience as closely as possible will continue into the foreseeable future of PBMAs. The PBMA products that most closely resemble animal meat are characterized by their prominent fibrous structures similar to the muscle structures of animal meat (Dekkers, Boom, and van der Goot, 2018a). This fibrous structure can be achieved by several structuring processes. Here, we focus on the high-moisture extrusion cooking (HMEC) technique.

HMEC describes extrusion with a moisture content of 40 up to 70 wt

<footnote><sup>a</sup> Large Scale Structures Group, Institut Laue-Langevin, 71 Avenue des Martyrs, 38000, Grenoble, France</footnote>

<footnote><sup>b</sup> Food Process Engineering, Department of Health Science and Technology, ETH Zürich, Schmelzbergstrasse 7, 8092, Zürich, Switzerland</footnote>

<footnote><sup>c</sup> Sustainable Food Processing, Department of Health Science and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092, Zürich, Switzerland</footnote>

<footnote><sup>d</sup> Three-Tec GmbH, Birren 20, 5703, Seon, Switzerland</footnote>

<footnote><sup>e</sup> Planted Foods AG, Kemptpark 32-34, 8310, Kemptthal, Switzerland</footnote>

<footnote><sup>f</sup> Laboratory for Neutron Scattering, Paul Scherrer Institut, Forschungsstrasse 111, 5232, Villigen, Switzerland</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: peter.fischer@hest.ethz.ch (P. Fischer), matsarskaia@ill.fr (O. Matsarskaia).</footnote>

<footnote n="1">¹ Equal Contribution.</footnote>
9%. Plant protein powders and water, optionally mixed with other in-
gredients, are fed separately into an extruder barrel, mixed within the
barrel and conveyed towards the cooling die, which is attached to the
end of the extruder. Temperatures between 130 and 170 °C are applied
in the cooking zone to plastify the protein mass (Osen and
Schweiggert-Weisz, 2016). To avoid expansion, the hot mass is cooled
down to below 100 °C in a cooling die, where it solidifies. The flow
profile in the cooling die is similar to the Hagen-Poiseuille flow
(Akdogan, 1999; Sandoval Murillo, Osen, Hiermaier, and Ganzenmüller,
2019). Based on the properties of its shear stress and velocity profiles
(Vlachopoulos & Strutt, 2003), structural differences between the re-
gions near the wall and in the center of the cooling die are expected.
During solidification, a unique fibrous structure is formed, which gives
the final PBMA products a meat-like appearance and texture (Osen and
Schweiggert-Weisz, 2016).

The mechanism behind the formation of this fibrous structures is still a subject of discussion and ongoing research (van der Sman and van der Goot, 2023; Wittek, Zeiler, Karbstein, and Emin, 2021b). It has been hypothesized that the fibres are the result of spinodal phase separation during cooling followed by elongation in the flow field (Dekkers et al., 2018a; Sandoval Murillo et al., 2019). The morphology of freeze-dried extrudates obtained with X-ray tomography (XRT) showed the contrast of a multi-phase system, where a water-rich (i.e., protein-poor) and a water-poor (i.e., protein-rich) phase were oriented along the flow direction in the die on a millimeter scale, with a spatial resolution of 20 µm (Nieuwland, Heijnis, van der Goot, and Hamoen, 2023; Wittek, Zeiler, et al., 2021b). This pattern is also visible with cryo-imaging (Wittek, Ellwanger, et al., 2021a). However, these two phases could also be identified when working with relatively pure homogeneous materials such as soy protein isolate (SPI). It is therefore believed that these two phases are more likely the result of syneresis, whereby increased cross-linking between proteins displaces water which then forms a separate phase (van der Sman and van der Goot, 2023). Furthermore, the two phases demonstrate lamellar, not fibrous, structures and can therefore not fully explain the fibrous tearing pattern of extrudates. Besides XRT and cryo-imaging, labelled confocal laser scanning microscopy (CLSM) imaging studies indicate more or less oriented macro-compositional inhomogeneities on the microscale (Godschalk-Broers, Sala, and Scholten, 2022; Grabowska et al., 2016; Taghian Dinani, van der Harst, Boom, and van der Goot, 2023). Similar inhomogeneities of the microscale are also visible with scanning electron microscopy (SEM) (Dekkers, Hamoen, et al., 2018b). These inhomogeneities may play a role in the structure formulation, but cannot solely explain the presence of the macroscopic fibers as their anisotropy is less pronounced than what is macroscopically visible.

Other authors suggest that the fibrous structure is a result of mo-
lecular unfolding, alignment and network formation (Akdogan, 1999;
Schmid, Farahnaky, Adhikari, and Torley, 2022; Zhang et al., 2022).
Chen, Wei, and Zhang (2011) studied the changes of molecular
cross-linking during HMEC by means of chemical bond-breaking solu-
tions and found that hydrophobic interactions, hydrogen bonds as well
as disulfide bridges hold the extrudate structure together, while other
authors attribute only minor relevance to protein interactions (Wittek,
Zeiler, et al., 2021b). Moreover, the degree of protein unfolding, elon-
gation and orientation during the process is debated. Under extrusion
conditions, proteins most likely behave like random but compact,
globular polymers (Erni, Windhab, and Fischer, 2011; Mezzenga and
Fischer, 2013; van der Sman and van der Goot, 2023) instead of
open-chain random-coils as often illustrated. This is supported by Zink
et al. (2023) who showed, using scanning Wide Angle X-ray Scattering
(sWAXS), that the protein structure of soy protein concentrates does not
change during HMEC. Shifts in secondary structure from α-helices to-
wards β-sheets were identified in gluten by Fourier-transform infrared
spectroscopy (FTIR) (Wang et al., 2017). More recently, it was hypoth-
esized that the final structure might be a combined result of the mech-
anisms mentioned above (van der Sman and van der Goot, 2023; Zhang

et al., 2022). van der Sman and van der Goot (2023) reasoned that
proteins tend to aggregate into fibrillar networks due to shear, enforcing
phase separation as well as syneresis.

In summary, extrudate structures have been well studied on the
milli- and microscale as well as by analyzing their chemical bonds.
Nevertheless, the mechanisms behind the structure formation and the
bearing structure of the fibers are not clearly identified yet. The estab-
lished properties of raw materials and extrudate structure and the three
most common hypotheses explaining the structure development are
illustrated at different length scales in Fig. 1a. The challenge of studying
the mechanisms of the fibrous structure formation is due to the “black-
box” nature of extruder and cooling die, which impedes the application
of in-line detection techniques during extrusion, such as near infrared
(NIR) spectroscopy, Raman spectroscopy, nuclear magnetic resonance
(NMR) spectroscopy, rheological techniques, etc. (Saerens, Vervaet,
Remon, and De Beer, 2014). Dead-stop operation, a method which al-
lows to obtain samples from different zones inside the extruder barrel
and cooling die, has been widely used to investigate the change of the
physico-chemical properties and the morphological transformation of
the material (Chen et al., 2011; Yao, Liu, and Hsieh, 2004). The draw-
back of this method is that it requires at least a few minutes to cool down
the barrel and access the samples. During this time, the sample structure
may change. This limitation can be best addressed by in situ structure
analysis. To this end, Velichko et al. (2019) designed a shear cell with
X-ray-transparent and neutron-transparent windows for small-angle
X-ray scattering (SAXS) and small-angle neutron scattering (SANS) ex-
periments to investigate the structure evolution of plant proteins in situ.
However, such in situ studies have not yet been executed within an
extruder or under extrusion-like temperatures. Moreover, the fibrous
structure of PBMA has rarely been studied at the nanoscale. SANS pro-
vides access to study structures at that scale, and has been widely
applied to food systems (Gilbert, 2019; Lopez-Rubio and Gilbert, 2009),
such as food-protein gels (Bayrak et al., 2023; Napieraj et al., 2022; Tian
et al., 2020) and protein-polysaccharide systems (Schmidt, Cousin,
Huchon, Boué, and Axelos, 2009; Wang, Li, Wang, Lal, and Huang,
2007).

In this work, we present a custom-designed cooling die with three
neutron-transparent windows. Its design allows to perform *in situ* SANS
measurements along the cooling die, thereby studying the texturization
mechanism of the PBMA at the nanoscale. Additionally, the different
interaction of neutrons with hydrogen and its isotope deuterium allows
to selectively study the PBMA components (proteins, dietary fiber, and
lipids) *via* so-called contrast variation (Castellanos, Mcauley, and Curtis,
2017). With this, we can demonstrate that no molecular elongation and
orientation occurs in the cooling die but protein nano-aggregates of 40
nm size are present throughout the cooling die. Based on this, we pro-
pose three underlying mechanisms for structure formation within the
cooling die.

## 2. Materials and methods

2.1. *Materials*

Soy protein concentrate (SPC) Alpha® 8 IP was purchased from Solae (St. Louis, USA). According to the manufacturer, it is composed of 67.5% protein, 19.6% carbohydrates, 7% ashes, 4% water, and 1.6% fat. Deuterium oxide (D₂O) with 99.8 atom% D was sourced from Thermo Scientific™ (Loughborough, UK). Different ratios of D₂O/H₂O solutions were prepared with ultrapure water.

## 2.2. *High-moisture extrusion cooking process*

The *in situ* small-angle neutron scattering (SANS) measurements were performed at SANS-I (SINQ) at the Paul Scherrer Institut (PSI) in Villigen, Switzerland. The space available at SANS-I allowed for an extruder with a screw diameter of 12 mm or smaller to be placed. A co-
<figure><img src="image_4.png" /><figcaption>Fig. 1. From plant protein extract to meat analog. (a) Illustration of structural raw material properties, prevalent structure formation hypotheses: 1. Protein unfolding, elongation & cross-linking, 2. Phase separation and 3. Syneresis, and known properties of PBMA from nano-to macroscale. (b) and (c) Representative SEM image of the extrudate. (d) Photo of internal structure of the extrudate. (e) Photo of pulled extrudate.</figcaption></figure>

rotating twin-screw extruder of the type ZE12 HMI (Three-Tec GmbH, Seon, Switzerland) with a length-to-diameter (L/D) ratio of 40 was used (see Fig. 2a). The screw was mainly composed of conveying elements (pitch length of 1.5 L/D until 16 L/D followed by a pitch length of 1 L/D) and contained two kneading blocks (1.5 L/D each; first after 20 L/D, second after 30 L/D). The surface-to-volume ratio of such a small extruder is significantly higher compared to industrial ones. Processing parameters were therefore carefully defined in preliminary trials to optimize product fibrosity. While it is known that bigger extruders generally form more fibrous extrudates, the underlying mechanisms are the same and insights of our findings should therefore be applicable to all extruder sizes.

The custom-made cooling die mounted on the extruder has a channel cross-section of 15 × 4 mm² and a length of 250 mm as shown in Fig. 2b (schematically illustrated in Fig. S1). The cooling die consists of aluminium and has three neutron-transparent windows along its length to observe the structure development during the cooling process. Titanium was chosen as the window material due to its high neutron-transparency. The scattering from the titanium windows was found to be negligible compared to the scattering from the samples (see 2D and 1D SANS scattering patterns of the titanium windows in Fig. S2). Furthermore, its thermal conductivity of approximately 20 W/(m·K) is closer to the one of conventional cooling dies (normally made of steel) compared to other neutron-transparent materials such as glass and aluminium. The window dimensions were constructed based on three conditions: (i) The window needs to be as small as possible to not profoundly disturb the cooling, but large enough to record the scattering pattern in a timely manner. (ii) The structure development is influenced by the flow velocity and shear forces which substantially differ between channel edge and middle. Therefore, a field of view at the edge and in the middle needed to be explorable. (iii) SANS signals shall be collected for scattering angles of 0–15° and thus on the exit side the window frame

is angled by 15° as shown in Fig. 2c. Titanium is not only used for the windows but over the whole length of the cooling die to avoid flow disturbances at the die surface. Hence two titanium platelets cover the widths of the channel (see Fig. S1). Thus, the cooling water temperature was reduced compared to the temperature in the conventional steel die to compensate for the lower thermal conductivity. In contrast to conventional dies, it is tilted by 90° to orient the whole channel width to the beam. This rotation is obtained by a discontinuous channel profile switch (illustrated in Fig. S1).

The extrusion process parameters were kept constant during the whole beamtime. Throughput was set to 900 g/h of which 300 g/h was powder and 600 g/h was water. The screw speed was fixed to 400 rpm. The first barrel element was unheated, the following barrels heated to 80, 97, 130, 150, 150, and 150 °C, while the cooling die was set to 50 °C. Product temperature and pressure were monitored in the second-to-last and last barrel. Additionally, the torque was recorded. With these parameters the specific mechanical energy input (SME) and the specific thermal energy input (STE) were calculated according to Eq. (1) and Eq. (2), respectively.

$$SME = \frac{2\pi \times n \times M \times 60}{\dot{m}} \qquad (1)$$

where n is the screw speed (rpm), M is the torque (Nm), and $\dot{m}$ is the mass flow (kg/h).

$$STE = c_p(T) \times dT \qquad (2)$$

where $c_p(T)$ is the specific heat capacity (kJ/[kg·°C]), it was estimated based on the raw material components mass fraction as described by Singh and Heldmann (Singh and Heldman, 2015) and integrated from raw material temperature (room temperature) to highest product temperature (measured in the last barrel), where $dT$ is the incremental
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** *In situ* setup at the beamline and schematic illustration of sampling. (a) The twin-screw extruder ZE12 from Three-Tec mounted on SANS-I at PSI (1: D₂O bottle, 2: powder feeder, 3: extruder, 4: pump, 5: barrel, 6: cooling die, 7: incoming neutron beam, 8: detector vacuum tube). (b) Zoom of the custom-made cooling die mounted on the extruder placed at the beamline, the peripheries of windows were covered by cadmium pieces. (c) *In situ* sampling: at each window, two positions were measured. Static sampling: The extrudate was cut longitudinal to the flow direction with half of the thickness.</figcaption></figure>

temperature difference (°C). In addition to the monitoring, optical appearance of the extrudate was evaluated from time to time.

## 2.3. Small-angle neutron scattering (SANS)

The SANS measurements were performed *in situ* and statically to compare both types of samples. For the *in situ* SANS measurement, three configurations were used: (i) sample-to-detector distance (SDD) = 1.6 m with wavelength $\lambda$ = 5 Å, (ii) SDD = 8 m with $\lambda$ = 5 Å, (iii) selected measurements at SDD = 18 m with $\lambda$ = 10 Å. These three configurations cover a q-range from 0.0014 to 0.4761 Å⁻¹, which corresponds to 1.3–436 nm in real space. Here, $q = 4\pi\sin(\theta)/\lambda$ is the magnitude of the scattering vector, and $2\theta$ is the scattering angle (Pynn, 1990). The static measurements were performed on D22 at Institut Laue-Langevin (ILL) in

Grenoble, France. Two detectors with SDD = 1.4 m and SDD = 17.6 m, and a wavelength at 6 Å were used to cover a q-range similar to the one of the *in situ* measurement (from 0.0029 to 0.6409 Å⁻¹).

A schematic illustration of *in situ* and statically sampling methods is shown in Fig. 2c. In the *in situ* measurement, a circular beam with a diameter of 4 mm was used. Two circular fields of view for the middle and edge positions of the channel as illustrated in Fig. 2c. The center of the “middle” position is the geometric center of the flow channel, the center of the “edge” position is 4.5 mm away from the center of the “middle” position. Contrast variation was applied to study the roles of each component separately. Based on the respective scatteringlength densities, 45 wt% D₂O solutions were used to match out the proteins, 30 wt% D₂O solutions were used to match out the polysaccharides (Jacrot, 1976). 100 wt% and 45 wt% D₂O solutions were used to produce the
extrudate during the *in situ* measurement.

For the static measurements, the extrudates were produced under conditions identical to those of the *in situ* measurements, except that H₂O was used. The extrudates were cooled down and packed in vacuum bags to avoid moisture loss. For contrast variation, extrudates were soaked in 100, 45, and 30 wt% D₂O solutions for 24 h. The soaked-extrudates were cut longitudinally to the flow direction to get a thickness of about 2 mm as shown in Fig. 2c. The thickness difference between samples was corrected in the data reduction. The samples were measured in quartz cuvettes with 2 mm path length. The cuvettes were sealed with parafilm to avoid moisture loss during the measurement.

The data was corrected by the instrumental background, the detector efficiency, the transmission and scattering contributions from the empty titanium window for the *in situ* measurement, or from the quartz cuvette for the static measurement. The two-dimensional (2D) data was radially averaged into $I(q)$ (cm⁻¹) versus $q$ (Å⁻¹) for the one-dimensional (1D) curves by BerSANS (Keiderling, 2002). The 1D curves were brought to an absolute scale (cm⁻¹) using the scattering of pure water with a path thickness of 1 mm. The incoherent scattering was subtracted. Anisotropic 2D patterns were analyzed in sectors. Two perpendicular sectors were applied to each of the anisotropic 2D pattern. In order to capture the highest anisotropy, the annular intensity was calculated based on the 2D patterns as a function of the azimuthal angle φ over a narrow q-range (0.012–0.013 Å⁻¹) (Burger, Hsiao, and Chu, 2010; Tian et al., 2020; Weigandt, Porcar, and Pozzo, 2011). One of the sectors was averaged over ±10° along the direction of the highest annular intensity, another one was averaged over ±10° perpendicular to the direction of the highest annular intensity. The data fitting was done by SasView 5.0.4 (http://www.sasview.org).

## 2.4. Scanning electron microscopy (SEM)

A rectangular piece of sample was cut longitudinally to the flow direction from the extrudate. The sample preparation was done in a PELCO BioWave® Pro + microwave system (Ted Pella, USA). The sample was fixed on ice with 2.5/2% glutaraldehyde/paraformaldehyde in PBS followed with a wash, and then was post-fixed with 1% OsO₄ in double-distilled water followed by another wash. Then the dehydration was done with a graded series of ethanol on ice followed with critical point drying (CPD 931, Tousimis, USA). Then the cross-fractured sample was mounted on SEM aluminum stubs, and coated with 5 nm thick of platinum/palladium (CCU-10, Safematic, Switzerland). The scanning electron microscope (Merlin, Zeiss, Germany) was operated at an accelerating voltage of 1.5 kV with a working distance at around 4–5 mm, SE-inlens and Everhart-Thornley (ET) SE-images were obtained.

## 2.5. Statistical analysis

The uncertainties of the fitting parameters were calculated using SasView (sasview.org). The fit quality was assessed by the “goodness-of-fit” parameter, reduced chi-square $χ²$, as shown in Eq. (3):

$$χ^2_{\text{Reduced}} = \frac{\sum \left[ \frac{\left| I(q)_{\text{meas}} - I(q)_{\text{calc}} \right|^2}{E(q)^2} \right]}{N_{\text{pts}} - N_{\text{par}}} \quad (3)$$

where $I(q)_{\text{meas}}$ is the measured scattering intensity, $I(q)_{\text{calc}}$ is the scattering intensity calculated by fitting, $E(q)$ is the error on the intensity value, $N_{\text{pts}}$ is the number of data points being considered, $N_{\text{par}}$ is the number of free (i.e., fitted) parameters http://www.sasview.org (Pedersen, 1997). For a good fit, $χ²$ tends to 1.

# 3. Results

## 3.1. High-moisture extrusion cooking process

Small-angle neutron scattering (SANS) measurements were executed

once the extrusion process reached steady-state. The steady-state melt pressure was 22 ± 2 bar, the melt temperature evened out at 149.1 ± 0.2 °C. SME reached 422 ± 37 kJ/kg while STE reached 435 ± 1 kJ/kg. When pulled apart, the extrudate has a Hagen-Poiseuille-like flow profile showing in Fig. 1e. The internal meat-like fibrous structure was visible after tearing as shown in Fig. 1d. The representative scanning electron microscopy (SEM) images in Fig. 1b and c show a uniform matrix with fractures. The white dots are most probably proteins, while the bigger roundish structures are probably small starch granules. Inhomogeneities are randomly oriented at the detected length scale.

## 3.2. *In situ* small-angle neutron scattering

SANS measurements were performed at three windows of the neutron-transparent cooling die while producing extrudates with 100% D₂O. At each window, the middle and edge positions were measured as illustrated in Fig. 3. The radially averaged one-dimensional (1D) SANS scattering curves are also shown in Fig. 3. Curves from the 2nd and 3rd windows are offset by scale factors for the sake of visibility. The 1D curves were fitted with the empirical “broad peak” model (Tian et al., 2020). The model reflects two structural features at two length scales (low-q and high-q ranges) and a broad peak at around $q = 0.01–0.04$ Å⁻¹. The model is described by Eq. (4):

$$I(q) = \frac{A}{q^n} + \frac{C}{1 + (|q - q_0|\xi)^m} + B \quad (4)$$

where A is the Porod law scale factor, n the exponent of the Porod law, C the Lorentzian scale factor, m the exponent of the Lorentzian function, and B the scattering background. ξ is the Lorentzian screening length, which is interpreted as the gel mesh size or as the polymer diameter in the case of a polymer gel system or aggregated, non-interpenetrated polymers (Mendes, Oeser, Hayes, Boué, and Bastide, 1996; Saffer et al., 2014; Tian et al., 2020). $q_0$ is the peak position, which is related to the d-spacing as $d_0$ in $q_0 = 2\pi/d_0$. The d-spacing is again either a gel mesh size or the center-to-center distance between two neighboring aggregates which corresponds to the aggregate size in a dense system (Tian et al., 2020).

The parameters calculated from the fit are similar for all three windows at both edge and middle positions (all fitting parameters are shown in Table S1). In general, the Lorentzian exponents are at 1.6–1.8, the screening length is 8–10 nm and the d-spacing is 35–41 nm. The Porod exponents are 2.5–2.6, except at the 3rd window near the exit of the cooling die, where a slight increase of the Porod exponent to 3.1–3.2 was observed (see Table S1). Apart from fitting with the broad peak model, a polydisperse spherical form factor combined with a sticky hard sphere structure factor was implemented to further understand the shape of the polymers and their organization. The corresponding theory is described in the Supplementary information section C. The fits are shown in Fig. S3 and the fitting parameters are shown in Table S3. The good fit with the spherical form factor indicates that the Lorentzian screening length of the broad peak model corresponds to the diameter of globular, but polydisperse polymers. The inconsistencies at high q are due to the unknown polydispersity which is impossible to measure in such a dense system. The polydispersity calculated from fitting is shown in Fig. S4. The reduced second virial coefficient (Kern and Frenkel, 2003) calculated from the stickiness parameter is negative, indicating overall attractive interactions between the polymers in the system. In addition, Kratky plots ($I(q) \times q^2$ versus $q$) of the data are shown in Fig. S5. A peak at mid q is found, which indicates compact structures, such as aggregates. Since minor anisotropies were found in the 2D patterns, and the material was structured in a shear field, this compact structure has most probably a uniformly round shape. The combination of all modelling approaches (broad peak, spherical form and structure factor and Kratky plot) implies the presence of nano-aggregates of roughly 40 nm assembled from polymers with a diameter of around 9 nm as illustrated in
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** 1D SANS curves of measurements at all three windows with insets of 2D patterns measured at second and first windows and schematic illustration of nano-aggregate organization derived from curve fitting. The position of three windows in the cooling die and the middle and edge positions of each window are shown in the cooling die illustration. The 1D curves of the different windows are offset by scale factors for better readability. 2D patterns measured at SDD = 8 m ((i) and (ii): The edge and middle position of the second window, (iii) and (iv): The edge and middle position of the first window) and SDD = 1.6 m ((v) and (vi): the edge and middle position of the second window, (vii) and (viii) the edge and middle position of the first window).</figcaption></figure>

**Fig. 3.** The Porod exponent of 2.5–3.2 indicates that this system is densely packed at larger length scale (Hammouda, 2008). The similarity of the fitting parameters from each of the three windows at middle and edge positions demonstrates that, from the 1st window to the 3rd window in the cooling die, there is no pronounced structural development on the length scale observed.

At the 3rd window, minor anisotropies were found in the 2D patterns at both middle and edge positions as illustrated in Fig. 4. Thus, the 2D patterns of the 3rd window at the edge and middle positions were further analyzed in sectors. For each position, two perpendicular sectors were chosen based on their most pronounced anisotropy, and they were applied to all three 2D patterns obtained at three different SDDs. Since the scattering patterns are in reciprocal space, the orientation of structures in real space is perpendicular to the scattering patterns. The highest anisotropic scattering intensities were found at 61 and 141° (0° is defined as the flow direction in Fig. 4, it rotates clockwise) in the middle and at the edge of the cooling die, respectively. For each sector, the 1D curves from each SDD were stitched together in order to generate 1D curves covering the entire q-range as shown in Fig. 4. A scaling factor of 10 is applied to the data measured in the middle position for better visualization. The two 1D curves averaged from two sectors for measurements in the middle differ less than those of the measurements at the edge. This is further confirmed by the fitting parameters (see complete set of parameters in Table S2). At the edge position, different Lorentzian screening lengths of 9.5 and 8.2 nm are found for the two different sectors, while a with 8.1–8.2 nm similar screening lengths are found at the middle position. The orientation of polymers within the nano-aggregate and the nano-aggregate itself are schematically illustrated in the bottom of Fig. 4.

### 3.3. Contrast variation and the comparison between *in situ* and static data

The extrudate system studied here is a mixture of different components. In SANS, contrast variation can elucidate the role of each component separately (Jacrot, 1976). Extrudates produced with 100 and 45 wt% D₂O solutions during the *in situ* measurement are shown in Fig. 5a. With 100 wt% D₂O, a bump at around 0.01–0.04 Å was visible. When using 45 wt% D₂O to match out the contribution from proteins, the bump was not visible. This finding indicates that this feature can be attributed to proteins (as illustrated in Fig. 5c).

Static measurements on extrudates were intended to compare the structural differences of the fully extruded material and *in situ* measurements. As shown in Fig. 5b, the same contrast variations were applied to the static extrudates. Static samples show 1D SANS patterns similar to those of *in situ* measurements. With 100 wt% D₂O, the bump at around 0.01–0.04 Å is also found, but it is more pronounced in the static extrudate. When matching out the contribution from proteins with 45 wt % D₂O, both static and *in situ* show 1D curves with slopes similar to the curves of 100 wt% D₂O, but without a bump. In contrast, the scattering pattern of the samples in which carbohydrates are matched out at 30 wt % D₂O has identical features to non-matched curves, but with a lower scattering intensity which is due to the drop of scattering length density of SPC in 30 wt% D₂O. This indicates that the bump feature is due to scattering from proteins, indicating that the described polymers and their agglomerates are proteins. However, for all components the Porod exponent is around 3, indicating all components are densely packed at larger length scale.

The data from static samples was also fitted with the broad peak model in Eq. (4). The parameters obtained from the fits (see Table S1) are similar to those of the *in situ* measurements. The Porod exponent of 3.1 is equal to the one at the 3rd window in the *in situ* measurement. A similar Lorentzian exponent of 1.9 is found. However, a slight increase
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** 1D SANS curves and fittings of the third window at middle and edge positions averaged by sectors with insets of underlying 2D SANS pattern and schematic illustration of structure orientation. (i)-(iii) 2D SANS patterns with the corresponding sectors of measurements at the middle position at SDD = 18, 8, and 1.6 m; (iv)-(vi) 2D SANS patterns with the corresponding sectors of measurements at the edge position at SDD = 18, 8, and 1.6 m.</figcaption></figure>

of the Lorentzian screening length to 10.1 nm and an increase of the d-spacing to 55 nm is observed. Overall, the results of the static and *in situ* measurements are comparable. This does not only reveal the nano-structural similarity of the solidifying mass in the cooling die to the fully extruded material, but it also evidences the successful execution of

*in situ* experiments with the self-designed custom-made neutron-transparent cooling die.
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** 1D SANS curves of contrast variation *in situ* measurements and static samples and corresponding illustration of the features studied. (a) *In situ* measurements at the 3rd window middle and edge positions, when produced with 100 and 45 wt% D₂O. (b) Measurements of static samples soaked in 100, 45, and 30 wt% D₂O, Porod exponents are shown next to curves. (c) Schematic illustration of a protein nano-aggregate.</figcaption></figure>

## 4. Discussion

To elucidate the structural development of plant-based meat analogs (PBMAs) in the cooling die during high-moisture extrusion cooking (HMEC), we designed a cooling die with three neutron-transparent windows. This allowed for the *in situ* observation of PBMA structural development on the nanoscale along the cooling die by small-angle neutron scattering (SANS) without compromising thermal performance or interfering with the extrusion process. Minor anisotropy was observed towards the exit of the cooling die mainly at its edges, indicating a slight structural orientation of 141° to the flow direction. Apart from that, all *in situ* scattering patterns were similar and their 2D curves showed a broad peak between 0.01 and 0.04 Å⁻¹ which was attributed to proteins. Fits using a broad peak model with the aid of sphere form factor and sticky hard sphere structure factor implied that globular proteins with a diameter of 9 nm were organized in densely packed aggregates. This is well in line with previous observations of soy protein aggregates. Tang (2019) described that soy glycinin, which has a radius of gyration of 4.4 nm and remains globular even upon denaturation, tends to aggregate and form nano- up to microgels. While pure soy glycinin may self-assemble into stacks of discs, β-conglycinin acts as a chain terminator resulting in predominantly globular aggregates (Tang, 2019). These nano-aggregates form a viscoelastic mass with all other components of soy protein concentrate and do not display any significant growth, elongation and orientation in the shear field of the cooling die (Tang, 2019).

The *in situ* scattering patterns were similar to the one of the end product, which was measured statically after being refrigerated subsequent to exiting the cooling die. While Porod and Lorentzian exponents were in the same range, the curve features were more pronounced in the static measurement and the characteristic lengths were up to 30% larger

compared to the ones of *in situ* measurements. In contrast to refrigerated extrudates, fresh ones are still warm and more flexible. Thus, solidification seems to continue during refrigeration and could be the reason for the more pronounced features visible in the mid-q range of static samples. In addition, the static sample had to be soaked in D₂O and this might result in a slight swelling of the gel-like structures.

The described protein nano-aggregates are formed either during protein extraction or HMEC. Within the cooling die, no structuring on the nanoscale was observed. Thus, structure development must take place at larger length scales. However, no clear anisotropy was observed by scanning electron microscopy (SEM) in a fracture plane on a micrometer scale either. This is in accordance with literature as, to date, no scientific observation elucidated the anisotropic properties of plant-based meat analog structures. Taking these observations into account, three hypotheses are proposed regarding the formation of the fibrous macro-structure: protein nano-aggregate chains, viscoelastic mass fracture, and distinct solidification.

**Protein nano-aggregate chains:** Fibers form because of the self-assembly of the observed protein nano-aggregates into chain-like structures as illustrated in Fig. 6a. This assembly pattern is likely to occur in a laminar flow field during cooling. It has been observed before that sphere-like calcium caseinate aggregates with a diameter of 48 nm formed cylindrical micro-fibres in a shear field, resulting in meat-like macro-structures (Tian et al., 2020). The spherical aggregates assembled into chain-like fibers which did not lead to anisotropy in the size range of a single aggregate. For calcium caseinate aggregate fibres, anisotropy could only be detected on length scales of 100 nm and higher. In our system, no anisotropy was detected at this scale. Thus, nano-aggregate chains of our system must have bigger dimensions and be visible only above 436 nm, out of the measurement range. Since nano-aggregate chains were not visible by SEM after rupture (see
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Proposed hypotheses on formation of the fibrous macro-structure: (a) Protein nano-aggregate chains, (b) viscoelastic mass fracture and (c) distinct solidification along the isotherm.</figcaption></figure>

Fig. 1b), they must be well embedded in the matrix.

**Viscoelastic mass fracture:** Fibers are the result of fractures in the viscoelastic mass as visualised in Fig. 6b. Different distortions of flowing viscoelastic materials have been described. Most prominently, melt fracture is caused by non-laminar flow resulting in surface defects, such as a “shark skin”-like appearance, up to integral structural changes of extrudates such as the transition from the smooth strand to a chain of discrete fragments (Cogswell, 2003). Melt fractures occur at critical flow rates which are specific to different combinations of geometry, polymers and temperatures. They are presumably caused by dead-spaces, lacking wall adhesion or normal stresses resulting from exposure to tensile stress (Cogswell, 2003). Thus, in HMEC, melt fractures can be caused by normal forces, dead-spaces in the die-entry region, wall-slip, or by exceeding a temperature-dependent critical flow rate during cooling. Shark skin-like surface distortions at the die entry result in propagating cracks while bulk distortions such as discrete fragments are stretched into fibers in the flow field of the cooling die. Additionally, viscoelastic masses can crack due to shear stress between the flow and friction. For example, crevasses in glaciers occur at an angle of 135° to the valley wall as the ice in the middle tears away from the more stagnant ice at the edge (Jarrin, 2016). A similar fracture scenario may happen to the protein matrix in the cooling die. Prerequisites for fractures in a flowing mass are its viscoelastic properties. Further research is needed to fully understand the underlying mechanisms. However, the formation of a cohesive melt that contains components which exert normal stress upon tensile stress is clearly relevant. The protein nano-aggregates which we observe take this role in our system.

**Distinct solidification:** Fibers result from distinct local

solidification triggered by a sharp solidification temperature of the matrix. Due to cooling under flow, a temperature gradient between different mass fractions emerges along the flow field and the matrix solidifies successively into fibrous fragments as shown in Fig. 6c.

## 5. Conclusions

In summary, the custom-made cooling die described here allowed to observe the cooling process of HMEC *in situ*. Neither structural changes nor relevant orientations were observed at the nanoscale during solidification in the cooling die. This contradicts theories of protein unfolding and alignment as well as nucleation of bimodal phase separation (hypothesis 1 and 2 of Fig. 1a). Our observations are more in line with recently discussed multi-layer structuring theories (van der Sman and van der Goot, 2023). Comprehension of the mechanisms underlying fibrous structure formation enables targeted structure engineering and holds potential to design tailored machinery explicitly suited to produce meat-like structures and thereby transform industrial processes and prospects. We suggest that protein nano-aggregate chains, viscoelastic mass fracture and/or distinct solidification contribute to fibrous structure formation in meat analogs. Thus, it appears appropriate to further study viscoelastic behaviour of raw materials under HMEC-like conditions to better elaborate underlying dynamics as viscoelastic properties are relevant in all proposed mechanisms. Moreover, studies of the formation of nano-aggregates are imperative as they might well be the pivotal feature for the structuring. Understanding whether nano-aggregates are already present in the HMEC raw materials or if they develop during extrusion is critical. These insights could elucidate
how to appropriately pre-process raw materials that have not yet been successfully high-moisture-extruded.

## CRediT authorship contribution statement

**Tong Guan:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **Corina Sägesser:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Data curation, Conceptualization, Formal analysis. **Roman Villiger:** Resources, Conceptualization. **Lisa Zychowski:** Supervision, Project administration, Conceptualization. **Joachim Kohlbrecher:** Writing – review & editing, Investigation. **Joseph Dumpler:** Writing – review & editing, Investigation. **Alexander Mathys:** Writing – review & editing, Supervision. **Patrick Rühs:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Peter Fischer:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Olga Matsarskaia:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

## Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 847439. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union's Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000. The authors thank Paul Scherrer Institut for the provision of neutron beaming time at SANS-I beamline of SINQ under the proposal 20212450. We thank L. Porcar for the allocation of internal beaming time on D22 (ILL). Help with the in situ extrusion by T. Mair, E. Larsson and L. Sridharan is gratefully acknowledged. We also acknowledge P. Bigler and D. Kiechl for the help with extrusion trials. We thank S. F. Prévost, R. Schweins, S. Fuard and A. Robert for valuable discussions. We thank S. Handschin for taking the SEM images. We also thank V. Lütz-Bueno for valuable discussions and help with SANS measurements. The partnership of Soft Condensed Matter (PSCM) is thankfully acknowledged.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodhyd.2024.110215.

## References

* Akdogan, H. (1999). High moisture food extrusion. *International Journal of Food Science and Technology*, **34**, 195-207.

* Anusha Siddiqui, S., Bahmid, N. A., Mahmud, C. M., Boukid, F., Lamri, M., & Gagoua, M. (2022). Consumer acceptability of plant-, seaweed-, and insect-based foods as alternatives to meat: A critical compilation of a decade of research. *Critical Reviews in Food Science and Nutrition*, **63**, 6630-6651.

* Battaglia Richi, E., Baumer, B., Conrad, B., Darioli, R., Schmid, A., & Keller, U. (2015). Health risks associated with meat consumption: A review of epidemiological studies. *International Journal for Vitamin and Nutrition Research*, **85**, 70-78.

* Bayrak, M., Whitten, A. E., Mata, J. P., Conn, C. E., Floury, J., & Logan, A. (2023). Real-time monitoring of casein gel microstructure during simulated gastric digestion monitored by small-angle neutron scattering. *Food Hydrocolloids*, **144**, Article 108919.

* Boukid, F. (2021). Plant-based meat analogues: From niche to mainstream. *European Food Research and Technology*, **247**, 297-308.

* Burger, C., Hsiao, B. S., & Chu, B. (2010). Preferred orientation in polymer fiber scattering. *Journal of Macromolecular Science: Part C: Polymer Reviews*, **50**, 91-111.

* Castellanos, M. M., Mcauley, A., & Curtis, J. E. (2017). Investigating structure and dynamics of proteins in amorphous phases using neutron scattering. *Computational and Structural Biotechnology Journal*, **15**, 117-130.

* Chen, Y. P., Feng, X., Blank, I., & Liu, Y. (2022). Strategies to improve meat-like properties of meat analogs meeting consumers' expectations. *Biomaterials*, **287**, Article 121648.

* Chen, F. L., Wei, Y. M., & Zhang, B. (2011). Chemical cross-linking and molecular aggregation of soybean protein during extrusion cooking at low and high moisture content. *LWT - Food Science and Technology*, **44**, 957-962.

* Cogswell, F. (2003). Adventitious flow phenomena. In *Polymer melt rheology: A guide for industrial practice* (pp. 91-110). Woodhead Publishing.

* Dekkers, B. L., Boom, R. M., & van der Goot, A. J. (2018a). Structuring processes for meat analogues. *Trends in Food Science & Technology*, **81**, 25-36.

* Dekkers, B. L., Hamoen, R., Boom, R. M., & van der Goot, A. J. (2018b). Understanding fiber formation in a concentrated soy protein isolate - pectin blend. *Journal of Food Engineering*, **222**, 84-92.

* Erni, P., Windhab, E. J., & Fischer, P. (2011). Emulsion drops with complex interfaces: Globular versus flexible proteins. *Macromolecular Materials and Engineering*, **296**, 249-262.

* Gilbert, E. P. (2019). Small-angle X-ray and neutron scattering in food colloids. *Current Opinion in Colloid & Interface Science*, **42**, 55-72.

* Godfray, H. C. J., Aveyard, P., Garnett, T., Hall, J. W., Key, T. J., Lorimer, J., et al. (2018). Meat consumption, health, and the environment. *Science*, **361**, 243.

* Godshalk-Broers, L., Sala, G., & Scholten, E. (2022). Meat analogues: Relating structure to texture and sensory perception. *Foods*, **11**, 2227.

* Grabowska, K. J., Zhu, S., Dekkers, B. L., De Ruijter, N. C. A., Gieteling, J., & Van Der Goot, A. J. (2016). Shear-induced structuring as a tool to make anisotropic materials using soy protein concentrate. *Journal of Food Engineering*, **188**, 77-86.

* Hammouda, B. (2008). Probing nanoscale structures. *The SANS Toolbox*. Gaithersburg: National Institute of Standards and Technology.

* Hoek, A. C., Luning, P. A., Weijzen, P., Engels, W., Kok, F. J., & de Graaf, C. (2011). Replacement of meat by meat substitutes. A survey on person- and product-related factors in consumer acceptance. *Appetite*, **56**, 662-673.

* Jacrot, B. (1976). The study of biological structures by neutron scattering from solution. *Reports on Progress in Physics*, **39**, 911.

* Jarrin, D. (2016). Crevasse orientation with respect to flow velocity. https://juneauicefield.org/blog/2016/8/19/crevasse-orientation-with-respect-to-flow-velocity. (Accessed 19 September 2023).

* Keiderling, U. (2002). The new 'BerSANS-PC' software for reduction and treatment of small angle neutron scattering data. *Applied Physics A: Materials Science & Processing*, **74**, S1455-S1457.

* Kern, N., & Frenkel, D. (2003). Fluid-fluid coexistence in colloidal systems with short-ranged strongly directional attraction. *The Journal of Chemical Physics*, **118**, 9882-9889.

* Kyriakopoulou, K., Dekkers, B., & van der Goot, A. J. (2019). Plant-based meat analogues. In C. M. Galanakis (Ed.), *Sustainable meat production and processing* (1st ed., pp. 103-126). Academic Press.

* Lopez-Rubio, A., & Gilbert, E. P. (2009). Neutron scattering: A natural tool for food science and technology research. *Trends in Food Science and Technology*, **20**, 576-586.

* Mendes, E., Oeser, R., Hayes, C., Boué, F., & Bastide, J. (1996). Small-angle neutron scattering study of swollen elongated gels: Butterfly patterns. *Macromolecules*, **29**, 5574-5584.

* Mezzenga, R., & Fischer, P. (2013). The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions. *Reports on Progress in Physics*, **76**, Article 046601.

* Napieraj, M., Brület, A., Lutton, E., Randrianarisoa, U., Boire, A., & Boué, F. (2022). Monitoring food structure in plant protein gels during digestion: Rheometry and Small Angle Neutron Scattering studies. *Food Structure*, **32**, Article 100270.

* Nieuwland, M., Heijnis, W., van der Goot, A. J., & Hamoen, R. (2023). XRT for visualizing microstructure of extruded meat replacers. *Current Research in Food Science*, **6**, Article 100457.

* Osen, R., & Schweiggert-Weisz, U. (2016). High-moisture extrusion: Meat analogues. In *Reference module in food science*. Elsevier.

* Parlasca, M. C., & Qaim, M. (2022). Meat consumption and sustainability. *Annual Review of Resource Economics*, **14**, 17-41.

* Pedersen, J. S. (1997). Analysis of small-angle scattering data from colloids and polymer solutions: Modeling and least-squares fitting. *Advances in Colloid and Interface Science*, **70**, 171-210.

* Pynn, R. (1990). Neutron scattering: A primer, los alamos. *Sciences*, **19**, 1-31.

* Saerens, L., Vervaat, C., Remon, J. P., & De Beer, T. (2014). Process monitoring and visualization solutions for hot-melt extrusion: A review. *Journal of Pharmacy and Pharmacology*, **66**, 180-203.

* Saffer, E. M., Lackey, M. A., Griffin, D. M., Kishore, S., Tew, G. N., & Bhatia, S. R. (2014). SANS study of highly resilient poly(ethylene glycol) hydrogels. *Soft Matter*, **10**, 1905-1916.

* Sandoval Murillo, J. L., Osen, R., Hiermaier, S., & Ganzenmüller, G. (2019). Towards understanding the mechanism of fibrous texture formation during high-moisture extrusion of meat substitutes. *Journal of Food Engineering*, **242**, 8-20.
Schmid, E.-M., Farahnaky, A., Adhikari, B., & Torley, P. J. (2022). High moisture extrusion cooking of meat analogs: A review of mechanisms of protein texturization. *Comprehensive Reviews in Food Science and Food Safety, 21*, 4573-4609.

Schmidt, I., Cousin, F., Huchon, C., Boué, F., & Axelos, M. A. (2009). Spatial structure and composition of polysaccharide-protein complexes from small angle neutron scattering. *Biomacromolecules, 10*, 1346-1357.

Singh, R. P., & Heldman, D. R. (2015). Heat transfer in food processing. In *Introduction to food engineering* (5th ed., p. 277). Academic Press.

Smetana, S., Ristic, D., Pleissner, D., Tuomisto, H. L., Parniakov, O., & Heinz, V. (2023). Meat substitutes: Resource demands and environmental footprints. *Resources, Conservation and Recycling, 190*, Article 106831.

Taghian Dinani, S., van der Harst, J. P., Boom, R., & van der Goot, A. J. (2023). Effect of L-cysteine and L-ascorbic acid addition on properties of meat analogues. *Food Hydrocolloids, 134*, Article 108059.

Tang, C. (2019). In *Nanostructures of soy proteins for encapsulation of food bioactive ingredients* (1st ed., pp. 247-285). Academic Press.

Tian, B., Wang, Z., de Campo, L., Gilbert, E. P., Dalgliesh, R. M., Velichko, E., et al. (2020). Small angle neutron scattering quantifies the hierarchical structure in fibrous calcium caseinate. *Food Hydrocolloids, 106*, Article 105912.

van der Sman, R. G., & van der Goot, A. J. (2023). Hypotheses concerning structuring of extruded meat analogs. *Current Research in Food Science, 6*, Article 100510.

Velichko, E., Tian, B., Nikolaeva, T., Koning, J., van Duynhoven, J., & Bouwman, W. G. (2019). A versatile shear cell for investigation of structure of food materials under shear. *Colloids and Surfaces A, 566*, 21-28.

Wang, X., Li, Y., Wang, Y. W., Lal, J., & Huang, Q. (2007). Microstructure of β-lactoglobulin/pectin coacervates studied by small-angle neutron scattering. *Journal of Physical Chemistry B, 111*, 515-520.

Vlachopoulos, J., & Strutt, D. (2003). *The role of rheology in polymer extrusion. Preceedings of New Technology for Extrusion Conference* (pp. 20-21). Milan, Italy.

Wang, K., Li, C., Wang, B., Yang, W., Luo, S., Zhao, Y., et al. (2017). Formation of macromolecules in wheat gluten/starch mixtures during twin-screw extrusion: Effect of different additives. *Journal of the Science of Food and Agriculture, 97*, 5131-5138.

Weigandt, K. M., Porcar, L., & Pozzo, D. C. (2011). In situ neutron scattering study of structural transitions in fibrin networks under shear deformation. *Soft Matter, 7*, 9992-10000.

Wittek, P., Ellwanger, F., Karbstein, H. P., & Emin, M. A. (2021a). Morphology development and flow characteristics during high moisture extrusion of a plant-based meat analogue. *Foods, 10*, 1753.

Wittek, P., Zeiler, N., Karbstein, H. P., & Emin, M. A. (2021b). High moisture extrusion of soy protein: Investigations on the formation of anisotropic product structure. *Foods, 10*, 102.

Yang, C., Pan, L., Sun, C., Xi, Y., Wang, L., & Li, D. (2016). Red meat consumption and the risk of stroke: A dose-response meta-analysis of prospective cohort studies. *Journal of Stroke and Cerebrovascular Diseases, 25*, 1177-1186.

Yao, G., Liu, K. S., & Hsieh, F. (2004). A new method for characterizing fiber formation in meat analogs during high-moisture extrusion. *Journal of Food Science, 69*, E303-E307.

Zhang, Z., Zhang, L., He, S., Li, X., Jin, R., Liu, Q., et al. (2022). High-moisture extrusion technology application in the processing of textured plant protein meat analogues: A review. *Food Reviews International, 39*, 4873-4908.

Zink, J. I., Lutz-Bueno, V., Handschin, S., Dutsch, C., Diaz, A., Fischer, P., et al. (2023). Structural and mechanical anisotropy in plant-based meat analogues. *Food Research International, 179*, Article 113968.