High moisture extrusion of wheat gluten: Relationship between process parameters, protein polymerization, and final product characteristics

Journal of Food Engineering 259 (2019) 3–11

Contents lists available at ScienceDirect

Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng

High moisture extrusion of wheat gluten: Relationship between process parameters, protein polymerization, and final product characteristics

T

Valerie L. Pietscha, Romy Wernera, Heike P. Karbsteina, M. Azad Emina,∗ a

Institute of Process Engineering in Life Sciences, Chair of Food Process Engineering, Karlsruhe Institute of Technology, Karlsruhe, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Wheat gluten High moisture extrusion Texture properties Anisotropic structures SDS-Extractable protein

The polymerization of wheat gluten induced by the formation of disulfide bonds can be considered as one of the decisive mechanisms leading to the formation of meat analog products with anisotropic structures. Accordingly, the influence of barrel temperature (100, 125, 155 °C), screw speed (180, 400, 800 rpm) and feed rate (10, 20 kg/h) on wheat gluten polymerization and final product characteristics was investigated. Analysis of total SDS-extractable protein (SDS-EP) confirmed that wheat gluten polymerization increased with increasing thermomechanical treatment. The observed increase in polymerization could be correlated with the formation of anisotropic product structures and an increase in hardness and Young's modulus. Altogether, these results demonstrate that defining polymerization reactions of wheat gluten and their relation to extrusion process conditions can provide essential information towards tailoring final product characteristics.

1. Introduction In recent years, meat consumption has been increasingly associated with concerns towards environmental, ethical, and health issues (Boland et al., 2013; Post, 2012). As a consequence, eating behavior of consumers has changed towards a vegetarian or vegan diet (Asgar et al., 2010; Kumar et al., 2017). Nevertheless, a lot of consumers still do not want to dispense with the sensory properties of muscle meat in their daily diet (Elzerman et al., 2013; Hoek et al., 2011). Therefore, meat analog products have been developed that aim at resembling muscle meat. A prerequisite of such products is their fibrous, anisotropic structure that contributes to the meat-like texture and sensory perception of meat analogs (Elzerman et al., 2011; Kumar et al., 2011). A process that can be used to produce meat analog products is the extrusion process that is operated at high moisture contents and with a long cooling die attached to the end of the extruder. During this process, final product characteristics can be controlled by varying different process parameters such as barrel temperature, screw speed, die geometry, and die temperature (Noguchi, 1989). Varying those independent variables will result in different process conditions in the screw and die section of the extruder. Depending on these process conditions, plant proteins undergo several superimposing structuring mechanisms that cause the formation of anisotropic structures (Arêas, 1992; Tolstoguzov, 1993). First, the plant proteins are transported

along two co-rotating screws, where thermomechanical treatment generated by the screws and heated barrels alter the molecular structure of the protein. These changes in molecular structure usually occur in two successive steps involving the denaturation of protein subunits and the formation of new non-covalent and covalent interactions. Herein, the formation of protein-protein interactions results in the formation of protein aggregates that are, for example, soluble or insoluble in water. For soy and pea protein as well as wheat gluten, the formation of disulfide bonds has been reported to play a major role during high moisture extrusion processing (Chen et al., 2011; Fang et al., 2013; Li et al., 2018; Li and Lee, 1996; Liu and Hsieh, 2007; Osen et al., 2015; Pietsch et al., 2017; Prudêncio-Ferreira and Arêas, 1993; Wang et al., 2017). When the extruded material subsequently passes the cooling die, the proteins can form networks and phases that are oriented in flow direction (Arêas, 1992; Cheftel et al., 1992; Noguchi, 1989; Tolstoguzov, 1993). The orientation of the protein network accordingly depends on the flow characteristics in the die section, that are known to be a function of rheological properties, flow rate, material temperature, and die geometry (Akdogan, 1999; Noguchi, 1989). Moreover, Tolstoguzov (1993) emphasized that the formation of anisotropic structures during extrusion requires three fundamental steps: (1) the occurrence of a melt consisting of immiscible biopolymers that can be described as a water-in-water type emulsion; (2) the deformation of the emulsion-dispersed phase in flow direction; and (3) the

∗ Corresponding author. Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Chair of Food Process Engineering, Kaiserstr. 12, 76131, Karlsruhe, Germany. E-mail address: [email protected] (M.A. Emin).

https://doi.org/10.1016/j.jfoodeng.2019.04.006 Received 24 January 2018; Received in revised form 7 September 2018; Accepted 13 April 2019 Available online 17 April 2019 0260-8774/ © 2019 Elsevier Ltd. All rights reserved.

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

solidification of anisotropic fibrous or porous structures. Herein, the occurrence of a dispersed phase can result from different phenomena such as: free water that is enclosed in the biopolymer matrix or the presence of thermodynamic incompatible biopolymers. The incompatibility can lead to a polysaccharide- and/or protein-rich dispersed phase in a protein-rich continuous phase (Arêas, 1992; Tolstoguzov, 1993). In the presence of a dispersed phase, flow characteristics in the die section influence deformation, coalescence, and orientation of the dispersed phase in flow direction. In general, the occurrence of a dispersed phase in highly concentrated, plant protein based biopolymer mixtures has been confirmed by several experimental studies (Dekkers et al., 2016; Grabowska et al., 2014, 2016; Habeych et al., 2008; Noguchi, 1989). To the best of our knowledge, however, the process conditions and material compositions necessary for the formation of a desired dispersed phase are still unknown for the majority of plant proteins used for high moisture extrusion. To be able to design the product characteristics of meat analog products, it is necessary to gain a better understanding of the abovementioned structuring mechanisms. Above of all, the change in the molecular structure of plant proteins in the screw section of the extruder plays a decisive role on all subsequent mechanisms. It affects the thermodynamic compatibility and the rheological properties affecting the flow characteristics in the die section. It is, therefore, essential to understand the relationship between the change in molecular structure of plant proteins and final product characteristics. For wheat gluten, Wang et al. (2017) and Li et al. (2018) were able to relate the formation of fibrous structures with enhanced polymerization of wheat gluten subunits by the formation of disulfide bonds. Both studies focus on the use of additives to enhance the polymerization behavior of wheat gluten. To the best of our knowledge, only Pietsch et al. (2017) investigated the influence of process parameters on resulting process conditions and wheat gluten polymerization. The authors showed that different process conditions in the screw section of the extrusion process will have a pronounced effect on wheat gluten polymerization. No information, however, was gained on the relationship between process parameters, wheat gluten polymerization, and final product characteristics from this study. The objective of the present study was, therefore, to elucidate this relationship by characterizing the change in wheat gluten polymerization, visual anisotropy of the product, and texture properties. To relate the change in final product characteristics to the change in wheat gluten polymerization induced by process conditions in the screw section, only process parameters in this part of the process were varied. Process parameters in the die section of the process were kept constant.

the experimental setup. The extruder barrel is divided into 7 sections which can be heated separately, except the first one. A gravimetricallycontrolled feeder (DDW-DDSR40 from Brabender, Duisburg, Germany) was used to add material to the first section of the extruder. Water was fed to the second section of the extruder by a piston-membrane pump (model KM 251, Alldos, Pfinztal, Germany). The screw configuration used for all trials consisted of forward and reverse transport elements. A cooling die with the dimensions of 9 × 30 × 380 mm (H x W x L) was attached to the end of the extruder. The cooling temperature TC was set to 50 °C by a refrigerated circulator (type Presto Plus LH 47, Julabo, Seelbach, Germany). The interactions between various process parameters in the screw section of the extrusion process, wheat gluten polymerization, and resulting product texture were investigated at various barrel temperatures, screw speeds, and feed rates. These process parameters were selected to achieve different thermomechanical treatments in the screw section of the extrusion process. Barrel temperature settings are given in Table 1. Screw speeds of 180, 400, and 800 rpm, and feed rates of 10 and 20 kg/h were applied. Water content was adjusted to 54 ± 1% (w/ w) after measuring the water content of wheat gluten before each extrusion trial. Due to the limited availability of wheat gluten from one batch, extrusion trials were repeated twice except otherwise stated. Process conditions resulting from various process parameter settings were characterized by extruder temperature TE, extruder pressure pE, and specific mechanical energy input (SME). As depicted in Fig. 1, the extruder temperature TE specifies the material temperature of the melt before entering the die section and was measured using a thermocouple type J (Ahlborn, Holzkirchen, Germany). Likewise, a melt pressure sensor (type M3, max. pressure < 30 MPa, Gerfran, Provaglio d’Iseo, Italy) was mounted at extruder exit to measure extruder pressure pE. SME was calculated according to the following equation (Meuser and van Lengerich, 1984):

SME (Wh/ kg ) =

n nmax

×

Md − Md, unload 100

m˙

× Pmax

where n and nmax are the actual and maximum screw speed (1800 rpm). Md and Md,unload are the actual and idle torque (%). m˙ represents the total mass flow (kg/h) and Pmax the maximum engine power (40 kW). 2.3. Analysis of extractable wheat gluten under non-reducing and reducing conditions via SE-HPLC The change in extractability of wheat gluten under non-reducing and reducing conditions can be used to evaluate the polymerization between wheat gluten subunits caused by the formation of disulfide bonds (Delcour et al., 2012; Lagrain et al., 2008; Li and Lee, 1996). To analyze the change in the amount of extractable wheat gluten, sizeexclusion chromatography was performed using high performance liquid chromatography (Shimadzu, Kyoto, Japan). First, extraction of extruded samples under non-reducing and reducing conditions was carried out according to Lagrain et al. (2008), with some modifications. For the analysis, samples were collected from extrusion experiments and dried in a vacuum dryer (Heraeus, Hanau, Germany) at 40 °C and 8 mbar and ground to a particle size < 0.14 mm. The extractability of wheat gluten under non-reducing conditions was measured by extracting 0.02 g of ground sample with 10 ml of 0.2 M sodium phosphate buffer solution (pH 6.9) containing 3.5 mM SDS and 8 M urea. After using a vortex mixer to disperse the samples in buffer solution, samples were extracted for 24 h on a rotary shaker at 200 rpm. After the extraction step, samples were centrifuged at 4637×g for 50 min and the supernatants were taken for analysis of the extractable protein content. Wheat gluten extractability under reducing conditions was determined by adding 10 mM of the reducing agent dithiotreitol (DTT) to the buffer solution used for non-reducing conditions applying the same extraction procedure. The supernatants (100 μl) were loaded onto an analytical

2. Material and methods 2.1. Material Commercial vital wheat gluten was kindly supplied by Kröner Stärke (Ibbenbüren, Germany) with a water content of 8% (w/w). According to the manufacturer, protein content of wheat gluten was 83% on dry matter basis. Furthermore, wheat gluten contained 4.8 g/ 100 g polysaccharides, 3.8 g/100 g dietary fiber, and 6.0 g/100 g fat. Initial pH value of wheat gluten was 5.7. The molecular protein composition of the wheat gluten used in this study was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are attached as supplementary material. Chemicals for protein extractability analysis were obtained from Carl Roth (Karlsruhe, Germany). 2.2. Extrusion process All extrusion trials were carried out on a co-rotating twin screw extruder ZSK 26 Mc (Coperion, Stuttgart, Germany) with a screw diameter of 25.5 mm and length to diameter ratio of 29. Fig. 1 illustrates 4

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 1. Schematic illustration of the experimental setup depicting the constant parameters (water content, screw configuration, die geometry, and die temperature) as well as the measurement positions of extruder temperature TE, extruder pressure pE, and motor torque M. Variable process parameters were screw speed (180, 400, 800 rpm), feed rate (10, 20 kg/h), and barrel temperature in section 7 (100, 125, 155 °C).

Table 1 Settings for barrel temperature TB in section 1-7. Section 1

Section 2

Section 3

Section 4

Section 5

Section 6

Section 7

– – –

40 °C 40 °C 40 °C

60 °C 60 °C 60 °C

90 °C 90 °C 90 °C

100 °C 100 °C 100 °C

100 °C 100 °C 100 °C

100 °C 125 °C 155 °C

size exclusion column (TSKgel QC-PAK GFC 300, Tosoh Biosience, Tokyo Japan). A mixture of acetonitrile/water (1:1) containing 0.1% trifluoracetic acid (TFA) was used as elution solvent. The SE-HPLC measurements were conducted at a flow rate of 1.0 ml/min and an oven temperature of 27 °C. The eluted protein was detected by an UV/Vis detector at 214 nm. Extraction of extruded samples was repeated twice. For every supernatant, SE-HPLC analysis was repeated twice yielding four measurements per extruded sample. Fig. 2. Cutting direction for preparation of light microscopy samples.

2.4. Calculation of total SDS-extractable protein (SDS-EP)

samples were prepared by cutting 20 μm thin cuts from the middle of the sample parallel to flow direction using a cryomicrotome HM 500 OM (Microm international GmbH, Walldorf, Germany) at −20 °C. Afterwards, samples were fixated on microscope slides by drying the samples at 60 °C. Micrographs were taken using an Eclipse LV100ND (Nikon, Düsseldorf, Germany) in 40-fold magnification.

To evaluate the polymerization between wheat gluten subunits caused by disulfide bonds, the change in extractable protein under nonreducing and reducing conditions was calculated from the peak areas of the elution profiles. Under non-reducing conditions, all non-covalent interactions, i.e. electrostatic and hydrophobic interactions and hydrogen bonds, are cleaved. Thus, a decrease in extractability can be attributed to the formation of covalent bonds such as disulfide and isopeptide bonds. Under reducing conditions, all disulfide bonds will be reduced by adding a reducing agent. In the presence of disulfide bonds, extraction under reducing conditions will cause an increase in extractable wheat gluten as compared to extractable wheat gluten under non-reducing conditions. Accordingly, the ratio of the extractable protein content under non-reducing and reducing conditions will depict the change in protein extractability caused by the formation or rupture of disulfide bonds. Similar to Lagrain et al. (2008), we define this ratio as the total SDS-extractable protein (SDS-EP):

2.6. Analysis of texture properties Texture analysis methods such as compression or tensile tests are commonly used to characterize the product properties of meat-analog products (Chen et al., 2010; Dekkers et al., 2016, 2018; Fang et al., 2014; Grabowska et al., 2014, 2016; Noguchi, 1989; Osen et al., 2014). In this study, texture properties of extruded samples were evaluated using a texture analyzer (Z2.5 TS from Zwick Roell, Ulm, Germany). Samples were collected from extrusion trials and stored immediately in airtight plastic bags. Texture analysis was performed at room temperature within one day after extrusion. Tensile tests were used to characterize the change in Young's modulus E (elasticity) of the extruded samples. Therefore, a bone shaped specimen with the dimensions depicted in Fig. 3 was cut from the extruded sample in flow direction. Force-distance curves were recorded at a speed of 1.5 mm/s using a probe type 8201 (Zwick Roell, Ulm, Germany). Young's Modulus E was calculated from the slope of the linear region of the forcedistance curves (Bourne, 2002):

peak area (non reducing conditions ) per g sample ⎞ SDS − EP (%) = ⎜⎛ ⎟⋅100 ⎝ peak area (reducing conditions ) per g sample ⎠ 2.5. Visual analysis of anisotropy at macroscopic and microscopic length scales To visualize anisotropic product structures on a macroscopic scale, extruded samples were first cut on the edges, which resulted in a smooth cut surface. Then, the cuts were used to tear the samples apart in order to visualize their fracture pattern, which can be taken as an indicator for different anisotropic structures (Pietsch et al., 2017; van Vliet, 1996). Light microscopy was used to analyze the anisotropic product structures on a microscopic scale. As visualized in Fig. 2,

Young′s Modulus E (MPa) =

F ⋅L 0 F = 0.58⋅ A⋅ΔL ΔL

where A as the actual cross-sectional area and L0 as the original length of the specimen are geometrical parameters of the specimen (see 5

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 3. Dimensions of specimen for tensile tests adapted from Chen et al. (2010).

produced at 100 °C (see Fig. 4.2a), these void spaces exhibit predominantly round shapes and show no orientation in flow direction. When the sample was produced at a barrel temperature of 155 °C (see Fig. 4.2b), these spaces appear to be elongated in flow direction. The appearance of these void spaces shown in the micrographs in Fig. 4.2 corresponds to the theory of Tolstoguzov (1993) who pointed out that the presence of a dispersed phase contributes to the development of anisotropic structures during the extrusion process. According to these micrographs, the dispersed phase could comprise free water and/or air bubbles (Dekkers et al., 2018; Grabowska et al., 2014). The observed differences in deformation and orientation of the dispersed phases (see Fig. 4) are expected to depend mainly on die geometry, flow rate, cooling temperature, and rheological properties (Akdogan, 1999; Noguchi, 1989). Die geometry, flow rate, and cooling temperature were kept constant in this study. Accordingly, the difference in deformation and orientation is expected to result from different rheological properties after changing barrel temperature. This can simply result from a different molecular mobility of proteins at different temperatures. On the other hand, a change in temperature can also result in the polymerization of wheat gluten leading to a significantly different rheological behavior (Attenburrow et al., 1990; Emin et al., 2017; Kokini et al., 1994; Li and Lee, 1996; Pommet et al., 2004; Redl et al., 2003). To gain further insights, the influence of process parameters on resulting process conditions and wheat gluten polymerization is addressed in the following sections.

Fig. 3). The ratio between the force F exerted on the sample under tension and the change in original length of the sample ΔL is given by the slope of the force-distance curve. Besides tensile tests, compression tests were used to characterize the hardness of the extruded samples. Therefore, samples were pre-cut parallel and perpendicular to the flow direction of the extruded samples through the cooling die. Force-distance curves were recorded at constant deformation speed of 2 mm/s using a probe with a flat rectangular blade. From this, hardness σmax of the sample parallel and perpendicular to flow direction was calculated as following (Figura and Teixeira, 2007):

Hardness σmax (Pa) =

Fmax A

where Fmax is the maximum force necessary to break the sample and A is the contact area given by the thickness of the sample (1.5 mm) and the width of the rectangular cutting blade (1 mm). All texture analyses were repeated at least 5 times per sample. 2.7. Statistical analysis OriginPro Software, version 9.4G (OriginLab Corporation, Northampton, USA) was used for statistical analysis. The influence of process parameters on extruder temperature TE and differences between hardness measured perpendicular and parallel to flow direction were evaluated by 1-way-ANOVA using Scheffé’s test for comparison of means. A probability of p < 0.05 was used to identify significant differences. Linear and exponential regression analysis was used to identify a significant influence of extruder temperature on the degree of polymerization and a significant influence of the degree of polymerization on texture properties.

3.2. Correlation between total SDS-extractable protein (SDS-EP) and wheat gluten polymerization The results from extractability analysis of untreated and extruded wheat gluten (same samples as in Fig. 4) are given in Table 2. Under non-reducing conditions, untreated wheat gluten exhibits the highest extractability. In comparison, the extractability of the extruded samples under non-reducing conditions shows a 2-fold decrease at 100 °C and a 4-fold decrease at 155 °C barrel temperature. Since under non-reducing conditions all protein-protein interactions except from covalent bonds (e.g., disulfide bonds and isopeptide bonds) are cleaved, a decrease in extractable protein is recognized to correlate with an increase in covalent bonds (Cuq et al., 2000; Lagrain et al., 2008; Pommet et al., 2004). To further distinguish between the formation of disulfide and isopeptide bonds, samples were extracted under reducing conditions to cleave all disulfide bonds. In this case, the difference between the resulting extractability of untreated wheat gluten and samples treated at barrel temperatures of 100 °C and 155 °C was not significant (p > 0.05). From this result, it can be concluded that except from disulfide bonds, no other covalent bonds were formed by the extrusion conditions applied. This applies to all extruded samples as the extractability under reducing conditions was not significantly different from the untreated wheat gluten (p > 0.05). The resulting total SDS-extractable protein (SDS-EP) of samples as shown in Fig. 4 accounted for 50% of the sample showing no floworiented fracture pattern. For the sample exhibiting a flow-oriented

3. Results and discussion 3.1. Formation of anisotropic product structures To identify if anisotropic product structures were produced in the parameter range chosen, Fig. 4shows exemplary macro- and microstructures of samples produced at barrel temperatures of 100 °C (a) and 155 °C (b) in the last section of the extruder. Screw speed and feed rate were kept constant at 400 rpm and 10 kg/h, respectively. Pictures in Fig. 4.1 depict the fracture patterns of the extruded samples on a macroscopic scale. The fracture patterns visualize the inner structure of the extruded samples. Differences in fracture patterns can be explained by a change in the protein network orientation, e.g. towards flow direction in the cooling die (Noguchi, 1989; van Vliet, 1996). From these pictures, it can be observed that the sample produced at a barrel temperature of 100 °C (Fig. 4.1a) shows no orientation in flow direction (from left to right). At an increased barrel temperature of 155 °C (Fig. 4.1b), however, the fracture pattern appears to be more oriented in flow direction. In Fig. 4.2, micrographs of both samples show that void spaces are embedded in the extruded product matrix. In case of the sample 6

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 4. Macrostructure (1) and microstructure (2) of samples produced at barrel temperatures in the last section of the extruder of 100 °C (a) and 155 °C (b), screw speed of 400 rpm, and feed rate of 10 kg/h.

(Singh and Macritchie, 2004).

Table 2 Calculated peak areas per g sample; samples were taken at 400 rpm, 10 kg/h and barrel temperatures of 100 °C and 155 °C. An asterisk indicates significant differences. sample

untreated TB = 100 °C TB = 155 °C

peak area per g sample/a.u. non-reducing

reducing

976 ± 95∗ 527 ± 44∗ 238 ± 20∗

1036 ± 10 1060 ± 25 1040 ± 30

3.3. Influence of independent process parameters on extrusion process conditions

total SDS-extractable protein SDSEP/%

To characterize the processing conditions leading to a change in wheat gluten polymerization, extruder temperature, extruder pressure, and specific mechanical energy input (SME) were monitored. Depending on the barrel temperature, screw speed, and feed rate applied, extruder temperature varied between 100 and 143 °C, extruder pressure between 0.4 and 1.5 MPa, and SME between 2 and 82 kJ/kg. Fig. 5 shows the influence of screw speed on extruder temperature at different barrel temperature settings and feed rates. As expected, barrel temperature caused a significant increase (p < 0.05) in extruder temperature. At a feed rate of 10 kg/h, an increase in screw speed from 180 to 800 rpm at barrel temperatures of 100 °C and 125 °C resulted in a significant (p < 0.05) increase in extruder temperature. The results at the highest barrel temperature setting of 155 °C indicate the same tendencies. Due to an increase in standard deviation, which resulted from repeated extrusion trials, however, the influence was not significant (p < 0.05). The observed increase in extruder temperature TE can be related to an increase in viscous energy dissipation with increased screw speed. As consequence of viscous energy dissipation, an increase in thermal energy input results in higher material temperatures measured at the exit of the screw section. Increasing the feed rate to 20 kg/h resulted in an overall lower temperature range of 95–132 °C (see Fig. 5.2b). These results suggest that wheat gluten experienced a lower thermal and mechanical

90.6 ± 0.7∗ 49.7 ± 0.4∗ 22.9 ± 0.2∗

fracture pattern, SDS-EP decreased to 23%. These initial results suggest that wheat gluten polymerization can play a decisive role on the formation of anisotropic product structures. The findings are furthermore in good accordance with findings from Li et al. (2018) and Wang et al. (2017), who also showed that a change in wheat gluten polymerization coincides with the formation of anisotropic product structures. Furthermore, many studies reported a similar effect of temperature on wheat gluten polymerization reactions. In these studies, polymerization reactions between the major wheat gluten subunits, gliadin and glutenin, have been shown to be initiated at temperatures above 90 °C (Cuq et al., 2000; Lagrain et al., 2008; Langstraat et al., 2015; Singh and Macritchie, 2004). Depending on the time-temperature-profile, these polymerization reactions include the formation of intramolecular disulfide bonds between glutenin subunits. Moreover, glutenin and gliadin subunits have been shown to form intermolecular cross-links via thioldisulfide-interchange reactions at elevated temperatures > 120 °C 7

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 5. Effect screw speed on extruder temperature TE at different barrel temperatures in section 7 (100, 125, 155 °C) and feed rates of 10 kg/h (a) and 20 kg/h (b). Extrusion trials at 180 rpm, 10 kg/h, and various barrel temperatures settings were repeated three times. Maximum standard deviation of extruder temperature TE resulting from repeated extrusion trails was 4%. 1-way-ANOVA (p < 0.05) was used to identify significant differences. Results at different feed rates and otherwise constant process parameter settings were significantly different (p < 0.05).

treatment at a feed rate of 20 kg/h as compared to samples produced at a feed rate of 10 kg/h. This result can be explained by the fact that increasing feed rate will reduce the residence time of the material in the screw section of the extrusion process (Ganjyal and Hanna, 2002). Similar to a feed rate of 10 kg/h, increasing screw speed also caused an increase extruder temperature at 20 kg/h. Differences in extruder temperature, however, were not significant (p > 0.05) due to the increased standard deviations from repeated extrusion trials. Similar to extruder temperature, extruder pressure and SME increased with increasing barrel temperature and screw speed. Results are not shown since similar results on the interrelation between extruder temperature, extruder pressure, and SME were depicted in our previous study (Pietsch et al., 2017). It is discussed in this study that, SME and extruder pressure are expected to be influenced by a change in material properties and not vice versa. This interrelation makes it difficult to use SME and extruder pressure to describe the influence of process conditions on wheat gluten polymerization. For this reason, extruder temperature was used in the following to describe the effect of process conditions on wheat gluten polymerization.

Thus, the different temperature dependencies of wheat gluten polymerization are expected to result from different residence times and mechanical treatments induced by these process parameters in the screw section of extruder. It is known from literature that polymerization kinetics are influenced by the time-temperature-profile experienced by wheat gluten (Cuq et al., 2000; Lagrain et al., 2008; Langstraat et al., 2015; Singh and Macritchie, 2004). In addition, polymerization kinetics can also be a function of the mechanical stress applied (Emin et al., 2017; Pommet et al., 2004; Redl et al., 2003; Strecker et al., 1995). To gain a better understanding on the extent of different time-temperature-profiles and additional mechanical treatment on wheat gluten polymerization during the extrusion process, change in local time-temperature profiles will have to be considered in future studies (Emin et al., 2016; Emin and Schuchmann, 2016). Furthermore, investigations at defined thermal and mechanical treatment can give further insight on the influence of additional mechanical treatment on the polymerization kinetics of wheat gluten at high moisture extrusion conditions (Emin and Schuchmann, 2016; Pommet et al., 2004; Strecker et al., 1995).

3.4. Influence of extruder temperature on wheat gluten polymerization

3.5. Influence of wheat gluten polymerization on product hardness

Fig. 6 depicts the influence of extruder temperature on the total SDS-extractable protein (SDS-EP) for all extruded samples at flow rate of 10 kg/h (Fig. 6a) and 20 kg/h (Fig. 6b). All samples showed a decrease in SDS-EP of at least 20% as compared to the untreated wheat gluten having an initial SDS-EP of 91%. As already stated in section 3.2, wheat gluten polymerization reactions are clearly dependent on the extruder temperature. The results in Fig. 6, moreover, show that wheat gluten polymerization reactions are also influenced by screw speed and feed rate. For instance, at a constant extruder temperature of 140 °C, increasing screw speed from 180 to 800 rpm resulted in a decrease in SDS-EP from 30 to 17%, respectively. To compare the influence of screw speed and feed rate on the temperature dependency of wheat gluten polymerization, linear regression analysis was conducted. The fit parameters are given in Table 3, whereas the slope k1 of the linear fit reflects the dependence of SDS-EP on the extruder temperature. At a feed rate of 10 kg/h, the slope ranges between 0.54 and 0.71. Increasing the feed rate to 20 kg/h resulted in slopes ranging from 0.37 to 0.51 showing that the feed rate influences the temperature dependency of wheat gluten polymerization reactions. At the same time, the slope at a screw speed of 800 rpm was increased as compared to the other screw speeds for both feed rates.

Fig. 7 compares the hardness of all extruded samples measured parallel (a) and perpendicular (b) to flow direction with the change in total SDS-extractable protein (SDS-EP). Results show that hardness of the product is a function of SDS-EP as the hardness perpendicular and parallel to flow direction decreased with an increase in SDS-EP. Moreover, the hardness of products with more pronounced anisotropic structures (see Fig. 5) increased significantly (p < 0.05) when measured parallel and perpendicular to flow direction. To describe the relationship between SDS-EP and product hardness, a linear fit was used. The fit parameters are given in Table 4. From this, the relationship between hardness measured parallel to flow direction and change in SDS-EP can be described by a linear process function with a slope of 0.036. Whereas the relationship between hardness measured perpendicular to flow direction and SDS-EP results in a linear process function with a slope of 0.028. The measurement of hardness in two directions was chosen as a quantitative measure for the change in anisotropy. According to Dekkers et al. (2018), the change in texture properties will be determined by the mechanical properties of the continuous and dispersed phase as well as the volume fraction and orientation of the dispersed phase. Therefore, the resulting product texture will reflect the sum of 8

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 6. Effect of extruder temperature TE on total SDS-extractable protein (SDS-EP) at different screw speeds (180, 400, 800 rpm) and feed rates of 10 kg/h (a) and 20 kg/h (b). Data points depict results from single extrusion trials. Averages and standard deviations were calculated from four measurements per extruded sample. Extrusion trials at 180 rpm, 10 kg/h, and various barrel temperatures settings were repeated three times. All other parameter settings were done in duplicate. Maximum standard deviation resulting from repeated extrusion trails was 9%. According to linear regression analysis (see Table 3), differences between single data points are significant (p < 0.05).

3.6. Influence of wheat gluten polymerization on Young's modulus

these variables. Due to the dependency of product texture on the deformation, coalescence, and orientation of the protein network, it is expected that the product texture measured parallel and perpendicular to flow direction will differ when anisotropy occurs in the samples tested. Given the fact, however, that the measurements parallel and perpendicular to flow direction were described by linear process functions, this tendency is not reflected by the results in Fig. 7. No significant difference (p > 0.05) between hardness measured parallel and perpendicular can be found between all extruded samples. Nevertheless, anisotropic structures were confirmed by visual analysis (see Fig. 4). We therefore assume that the change in texture properties caused by the sum of the abovementioned variables was not sufficient to measure a significant difference by the method used in this study. Although the results in Fig. 7 cannot be used to quantify the extent of anisotropic structure formation, they clearly depict that the hardness is a strong function of polymerization and that the products with anisotropic structures show higher hardness. Similar results were reported by Li et al. (2018) who showed that the enhancement of polymerization reactions between wheat gluten by addition of alkali resulted in an increased hardness of the final products. The authors propose that an increase in wheat gluten polymerization will promote the formation of a denser protein network on a molecular level contributing to an increase in product hardness. At the same time, a subsequent increase in melt viscosity due to an increase in degree of polymerization will also cause an increase in product density and with this product hardness (Lin et al., 2000).

The relation between total SDS-extractable protein (SDS-EP) and Young's modulus is depicted in Fig. 8. In the range investigated, Young's modulus decreases with an increase in SDS-EP. Below a SDS-EP of 30%, however, no significant differences (p > 0.05) in Young's moduli were observed and the samples exhibited larger standard deviations. In this range, the extruded products exhibited more pronounced anisotropic structures (see Fig. 5). The presence of inhomogeneities caused by the increased anisotropy of the samples, possibly resulted in a higher deviation in Young's modulus. In all, the results show that a decrease in SDS-EP, and therefore an increase in wheat gluten polymerization, relates to an increase in Young's modulus and the formation of an anisotropic structure. The interrelation between Young's modulus and SDSEP can be described by an exponential fit given in Table 5. The Young's modulus reflects the ratio between tensile stress and extensional strain. An increase in Young's modulus implies an increase in rigid components on different length scales in the material tested (Cuq et al., 2000). In this context, the observed increase in Young's modulus can be correlated with a change in material properties from elastic to stiff or rigid (Figura and Teixeira, 2007). For wheat gluten, the progress of polymerization reactions can lead to an increased rigidity of the protein chains by the formation of intramolecular disulfide bonds. Similar to the findings in Fig. 8, Cuq et al. (2000) reported for bioplastic films made of wheat gluten that an increase in tensile strength and decrease in extensional strain corresponded with increased polymerization reactions by the formation of disulfide bonds. At the same time, an increase in Young's modulus is promoted by the formation of rigid structures on a micro- and/or macroscopic level.

Table 3 Linear regression analysis describing the relation between extruder temperature TE and total SDS-extractable protein (SDS-EP) at different screw speeds and feed rates. An asterisk indicates significant differences. Equation

Screw speed

Feed rate

k0 ± SE

k1 ± SE

R2corr

SDS − EP=k 0 + k1⋅TE

180 rpm 400 rpm 800 rpm 180 rpm 400 rpm 800 rpm

10 kg/h

114.7 ± 5.2∗ 98.5 ± 12.4∗ 110.8 ± 16.7∗ 101.8 ± 8.8∗ 95.2 ± 15.1∗ 106.7 ± 8.9∗

- 0.61 ± 0.04∗ - 0.54 ± 0.09∗ 0.71 ± 0.14∗ 0.40 ± 0.08∗ 0.37 ± 0.13∗ 0.51 ± 0.07∗

0.94 0.84 0.83 0.75 0.53 0.92

20 kg/h

9

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Fig. 7. Relation between total SDS-extractable protein (SDS-EP) and hardness σmax measured parallel (a) and perpendicular (b) to flow direction. Samples were produced at different barrel temperatures, screw speeds (180, 400, 800 rpm), and feed rates of 10 kg/h and 20 kg/h. Data points depict results from single extrusion trials. Averages and standard deviations were calculated from a minimum of five measurements per extruded sample. Extrusion trials at 180 rpm, 10 kg/h, and various barrel temperatures settings were repeated three times. All other parameter settings were done in duplicate. Maximum standard deviation resulting from repeated extrusion trails was 13%. According to 1-way ANOVA, differences between hardness measured parallel and perpendicular to flow direction were not significantly different for all samples (p > 0.05). The dashed line depicts linear regression analysis given in Table 4.

According to the theory of Tolstoguzov (1993), the formation of anisotropic structures is propagated by the presence and deformation of the dispersed phase in flow direction. As anisotropy was shown to occur with increased wheat gluten polymerization (see Figs. 4 and 6), it is very likely that the deformation and orientation of these anisotropic structures in flow direction caused an increased rigidity.

4. Conclusion High moisture extrusion processing was used to attain products from wheat gluten that exhibited different anisotropic structures. These structures were achieved by varying process parameters in the screw section of the extrusion process. Microscopic analysis revealed that the presence of a dispersed phase contributed to the formation of anisotropic product structures. Analysis of the relationship between process parameters, process conditions, molecular structure, and resulting product characteristics highlighted that, in the range of extrusion conditions investigated, an increase in wheat gluten polymerization could be correlated with the formation of anisotropic product structures as well as an increase in hardness and Young's modulus. Linear and exponential process functions were used to describe these correlations. To be able to control the formation of anisotropic structure formation, it is necessary to control the change in wheat gluten polymerization during the extrusion process. Thermal treatment during extrusion plays a dominant role on wheat gluten polymerization as expected, but the results also show that the extruder temperature alone does not suffice to explain the relationship between extruder temperature and total SDSextractable protein content. Consequently, further studies are needed to explain how local process conditions during extrusion processing influence the polymerization behavior of wheat gluten.

Fig. 8. Relation between total SDS-extractable protein (SDS-EP) and Young modulus E. Samples were produced at different barrel temperatures, screw speeds (180, 400, 800 rpm), and feed rates of 10 kg/h and 20 kg/h. Data points depict results from single extrusion trials. Averages and standard deviations were calculated from a minimum of five measurements per extruded sample. Extrusion trials at 180 rpm, 10 kg/h, and various barrel temperatures settings were repeated three times. All other parameter settings were repeated in duplicate. Maximum standard deviation of Young's modulus resulting from repeated extrusion trails was 13%. The dashed line depicts the exponential regression analysis given in Table 5.

Table 4 Linear regression analysis describing the relation between total SDS-extractable protein (SDS-EP) and hardness measured parallel and perpendicular to flow direction. An asterisk indicates significant differences. Equation

Parameter y

k0 ± SE

k1 ± SE

R2corr

y = k 0 + k1⋅SDS − EP

hardness parallel to flow direction hardness perpendicular to flow direction

3.02 ± 0.17∗ 2.51 ± 0.12

0.037 ± 0.004∗ 0.028 ± 0.003∗

0.70 0.72

10

Journal of Food Engineering 259 (2019) 3–11

V.L. Pietsch, et al.

Table 5 Exponential regression analysis describing the relation between total SDS-extractable protein (SDS-EP) and Young modulus E. An asterisk indicates significant differences. Equation

k0 ± SE

k1 ± SE

R2corr

E = k 0⋅exp(k1⋅SDS − EP )

219 ± 40∗

−0.049 ± 0.006∗

0.65

protein aggregation during extrusion process studied by size exclusion chromatography coupled with multi-angle laser light scattering. J. Food Eng. 115 (2), 220–225. Fang, Y., Zhang, B., Wei, Y., 2014. Effects of the specific mechanical energy on the physicochemical properties of texturized soy protein during high-moisture extrusion cooking. J. Food Eng. 121, 32–38. Figura, L.O., Teixeira, A.A., 2007. Food Physics: Physical Properties - Measurement and Applications. Springer-Verlag Berlin Heidelberg, Berlin, Heidelberg. Ganjyal, G., Hanna, M., 2002. A review on residence time distribution (RTD) in food extruders and study on the potential of neural networks in RTD modeling. J. Food Sci. 67 (6), 1996–2002. Grabowska, K.J., Tekidou, S., Boom, R.M., van der Goot, A.-J., 2014. Shear structuring as a new method to make anisotropic structures from soy–gluten blends. Food Res. Int. 64, 743–751. Grabowska, K.J., Zhu, S., Dekkers, B.L., Ruijter, N.C.A.de, Gieteling, J., van der Goot, A.J., 2016. Shear-induced structuring as a tool to make anisotropic materials using soy protein concentrate. J. Food Eng. 188, 77–86. Habeych, E., Dekkers, B., van der Goot, A.J., Boom, R., 2008. Starch–zein blends formed by shear flow. Chem. Eng. Sci. 63 (21), 5229–5238. 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 (3), 662–673. Kokini, J.L., Cocero, A.M., Madeka, H., de Graaf, E., 1994. The development of state diagrams for cereal proteins. Trends Food Sci. Technol. 5 (9), 281–288. Kumar, P., Sharma, B.D., Kumar, R.R., 2011. Product profile comparison of analogue meat nuggets versus chicken nuggets. Fleischwirtschaft Int. 1, 72–75. Kumar, P., Chatli, M.K., Mehta, N., Singh, P., Malav, O.P., Verma, A.K., 2017. Meat analogues: health promising sustainable meat substitutes. Crit. Rev. Food Sci. Nutr. 57 (5), 923–932. Lagrain, B., Thewissen, B.G., Brijs, K., Delcour, J.A., 2008. Mechanism of gliadin–glutenin cross-linking during hydrothermal treatment. Food Chem. 107 (2), 753–760. Langstraat, T.D., Jansens, K.J.A., Delcour, J.A., van Puyvelde, P., Goderis, B., 2015. Controlling wheat gluten cross-linking for high temperature processing. Ind. Crops Prod. 72, 119–124. Li, M., Lee, T.-C., 1996. Effect of extrusion temperature on solubility and molecular weight distribution of wheat flour proteins. J. Agric. Food Chem. 44 (3), 763–768. Li, T., Guo, X.-N., Zhu, K.-X., Zhou, H.-M., 2018. Effects of alkali on protein polymerization and textural characteristics of textured wheat protein. Food Chem. 239 (Suppl. C), 579–587. Lin, S., Huff, H.E., Hsieh, F., 2000. Texture and chemical characteristics of soy protein meat analog extruded at high moisture. J. Food Sci. 65 (2), 264–269. Liu, K.S., Hsieh, F.-H., 2007. Protein–protein interactions in high moisture-extruded meat analogs and heat-induced soy protein gels. J. Am. Oil Chem. Soc. 84 (8), 741–748. Meuser, F., van Lengerich, B., 1984. Systems analytical model for the extrusion of starches. In: Zeuthen, P., Cheftel, J.C., Eriksson, C., Jul, M., Leniger, H., Linko, P., Farela, G., Vos, G. (Eds.), Thermal Processing and Quality of Foods. Elsevier Applied Science Publishers, London, pp. 175–179. Noguchi, A., 1989. Extrusion cooking of high-moisture protein food. In: Mercier, C., Linko, P., Harper, J.M. (Eds.), Extrusion Cooking. American Association of Cereal Chemists, St. Paul, Minnesota, USA, pp. 343–370. Osen, R., Toelstede, S., Wild, F., Eisner, P., Schweiggert-Weisz, U., 2014. High moisture extrusion cooking of pea protein isolates: raw material characteristics, extruder responses, and texture properties. J. Food Eng. 127, 67–74. Osen, R., Toelstede, S., Eisner, P., Schweiggert‐Weisz, U., 2015. Effect of high moisture extrusion cooking on protein–protein interactions of pea (Pisum sativum L.) protein isolates. Int. J. Food Sci. Technol. 50 (6), 1390–1396. Pietsch, V.L., Emin, M.A., Schuchmann, H.P., 2017. Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat analog products. J. Food Eng. 198 (Suppl. C), 28–35. Pommet, M., Morel, M.-H., Redl, A., Guilbert, S., 2004. Aggregation and degradation of plasticized wheat gluten during thermo-mechanical treatments, as monitored by rheological and biochemical changes. Polymer 45 (20), 6853–6860. Post, M.J., 2012. Cultured meat from stem cells: challenges and prospects. Meat Sci. 92 (3), 297–301. Prudêncio-Ferreira, S.H., Arêas, J.A.G., 1993. Protein-protein interactions in the extrusion of soya at various temperatures and moisture contents. J. Food Sci. 58 (2), 378–381. Redl, A., Guilbert, S., Morel, M.-H., 2003. Heat and shear mediated polymerisation of plasticized wheat gluten protein upon mixing. J. Cereal Sci. 38 (1), 105–114. Singh, H., Macritchie, F., 2004. Changes in proteins induced by heating gluten dispersions at high temperature. J. Cereal Sci. 39 (2), 297–301. Strecker, T.D., Cavalieri, R.P., Zollars, R.L., Pomeranz, Y., 1995. Polymerization and mechanical degradation kinetics of gluten and glutenin at extruder melt‐section temperatures and shear rates. J. Food Sci. 60 (3), 532–537. Tolstoguzov, V.B., 1993. Thermoplastic extrusion—the mechanism of the formation of extrudate structure and properties. J. Am. Oil Chem. Soc. 70 (4), 417–424. van Vliet, T., 1996. Large deformation and fracture behaviour of gels. Curr. Opin. Colloid Interface Sci. 1 (6), 740–745. Wang, K., Li, C., Wang, B., Yang, W., Luo, S., Zhao, Y., Jiang, S., Mu, D., Zheng, Z., 2017. Formation of macromolecules in wheat gluten/starch mixtures during twin-screw extrusion: effect of different additives. J. Sci. Food Agric. 97 (15), 5131–5138.

Acknowledgements This research project was supported by the German Ministry of Economics and Energy (via AiF) and FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn) in the scope of project AiF 18727 N. Further, the authors would like to express their thanks to Kerstin Sauther, Andrea Butterbrodt, and Rosa Förster for supporting the extrusion experiments and sample analyses. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jfoodeng.2019.04.006. References Akdogan, H., 1999. High moisture food extrusion. Int. J. Food Sci. Technol. (34), 195–207. Arêas, J.A.G., 1992. Extrusion of food proteins. Crit. Rev. Food Sci. Nutr. 32 (4), 365–392. Asgar, M.A., Fazilah, A., Huda, N., Bhat, R., Karim, A.A., 2010. Nonmeat protein alternatives as meat extenders and meat analogs. Compr. Rev. Food Sci. Food Saf. 9 (5), 513–529. Attenburrow, G., Barnes, D.J., Davies, A.P., Ingman, S.J., 1990. Rheological properties of wheat gluten. J. Cereal Sci. 12 (1), 1–14. Boland, M.J., Rae, A.N., Vereijken, J.M., Meuwissen, M.P.M., Fischer, A.R.H., van Boekel, M.A.J.S., Rutherfurd, S.M., Gruppen, H., Moughan, P.J., Hendriks, W.H., 2013. The future supply of animal-derived protein for human consumption. Trends Food Sci. Technol. 29 (1), 62–73. Bourne, M.C., 2002. Food Texture and Viscosity: Concept and Measurement. Elsevier Academic Press Inc., San Diego. Cheftel, J.C., Kitagawa, M., Quéguiner, C., 1992. New protein texturization processes by extrusion cooking at high moisture levels. Food Rev. Int. 8 (2), 235–275. Chen, F.L., Wei, Y.M., Zhang, B., Ojokoh, A.O., 2010. System parameters and product properties response of soybean protein extruded at wide moisture range. J. Food Eng. 96 (2), 208–213. 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 Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 44 (4), 957–962. Cuq, B., Boutrot, F., Redl, A., Lullien-Pellerin, V., 2000. Study of the temperature effect on the formation of wheat gluten network: Influence on mechanical properties and protein solubility. J. Agric. Food Chem. 48 (7), 2954–2959. Dekkers, B.L., Nikiforidis, C.V., van der Goot, A.J., 2016. Shear-induced fibrous structure formation from a pectin/SPI blend. Innov. Food Sci. Emerg. Technol. 36, 193–200. Dekkers, B.L., Hamoen, R., Boom, R.M., van der Goot, A.J., 2018. Understanding fiber formation in a concentrated soy protein isolate - pectin blend. J. Food Eng. 222 (Suppl. C), 84–92. Delcour, J.A., Joye, I.J., Pareyt, B., Wilderjans, E., Brijs, K., Lagrain, B., 2012. Wheat gluten functionality as a quality determinant in cereal-based food products. Annu. Rev. Food Sci. Technol. 3 (1), 469–492. Elzerman, J.E., Hoek, A.C., van Boekel, M.A.J.S., Luning, P.A., 2011. Consumer acceptance and appropriateness of meat substitutes in a meal context. Food Qual. Prefer. 22 (3), 233–240. Elzerman, J.E., van Boekel, M.A.J.S., Luning, P.A., 2013. Exploring meat substitutes: consumer experiences and contextual factors. Br. Food J. 115 (5), 700–710. Emin, M.A., Schuchmann, H.P., 2016. A mechanistic approach to analyze extrusion processing of biopolymers by numerical, rheological, and optical methods. Trends Food Sci. Technol. 1–8. Emin, M.A., Teumer, T., Schmitt, W., Rädle, M., Schuchmann, H.P., 2016. Measurement of the true melt temperature in a twin-screw extrusion processing of starch based matrices via infrared sensor. J. Food Eng. 170 (Suppl. C), 119–124. Emin, M.A., Quevedo, M., Wilhelm, M., Karbstein, H.P., 2017. Analysis of the reaction behavior of highly concentrated plant proteins in extrusion-like conditions. Innov. Food Sci. Emerg. Technol. (44), 15–20. Fang, Y., Zhang, B., Wei, Y., Li, S., 2013. Effects of specific mechanical energy on soy

11