Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat analog products

Accepted Manuscript Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat analog products

Valerie L. Pietsch, M.Azad Emin, Heike P. Schuchmann PII:

S0260-8774(16)30388-0

DOI:

10.1016/j.jfoodeng.2016.10.027

Reference:

JFOE 8704

To appear in:

Journal of Food Engineering

Received Date:

20 July 2016

Revised Date:

08 September 2016

Accepted Date:

29 October 2016

Please cite this article as: Valerie L. Pietsch, M.Azad Emin, Heike P. Schuchmann, Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat analog products, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.10.027

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Highlights: Differences in product appearance were related to wheat gluten polymerization.

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Wheat gluten polymerization only took place in the screw section of the extruder.

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Thermal treatment was the major parameter influencing wheat gluten polymerization.

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In the range investigated, SME and pressure had no influence on wheat gluten polymerization.

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Determining polymerization behavior at high moisture extrusion conditions will enable a more

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targeted design of the extrusion process for the production of meat analog products.

ACCEPTED MANUSCRIPT Process conditions influencing wheat gluten polymerization during high moisture extrusion of meat

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analog products

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Valerie L. Pietsch, M. Azad Emin, Heike P. Schuchmann

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Institute of Process Engineering in Life Sciences, Section of Food Process Engineering, Karlsruhe

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Institute of Technology, Karlsruhe, Germany

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Corresponding author: M.A. Emin. Tel: +49 (0)721 608-48311, Fax: +49 (0)721 608-45967, Email:

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[email protected], Mailing address: Karlsruhe Institute of Technology, Institute of Process

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Engineering in Life Sciences, Section I: Food Process Engineering, Kaiserstr. 12, 76131 Karlsruhe

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ACCEPTED MANUSCRIPT Abstract

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The aim of this study was to investigate the influence of process conditions on changes in

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polymerization behavior of wheat gluten during high moisture extrusion processing. By considering

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the process as two hierarchical sections (i.e. screw and die section), wheat gluten extractability

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analyses under non-reducing conditions showed that polymerization reactions were determined by

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process conditions in screw section. Process conditions were characterized by material temperature

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at extruder exit as measure for thermal treatment, extruder pressure at extruder exit and specific

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mechanical energy input (SME) as measure for mechanical treatment. The effect of extruder pressure

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from 1.5 to 3.5 MPa and SME from 32 to 206 kJ∙kg-1 was studied by varying die length and screw

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configuration. In the range investigated, extruder pressure and SME had no significant influence on

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polymerization reactions. Only extruder temperature from 90 to 160 °C induced significant changes in

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polymerization behavior which was moreover reflected by the visual appearance of the samples

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reflecting different anisotropic product structures.

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Keywords: plant protein, wheat gluten, protein polymerization, high moisture extrusion, meat analog

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ACCEPTED MANUSCRIPT 1. Introduction

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Health, ecological and ethical aspects are leading to a change in consumers’ dietary habits towards a

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reduction of meat consumption (Elzerman et al., 2013). Nevertheless, a vast majority of consumers

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does not want to dispense with the sensory attributes of meat products from animal origin (Hoek et

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al., 2011). To solve this conflict, products based on plant proteins such as wheat gluten have been

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developed to resemble the product properties of muscle meat. A prerequisite of such meat analog

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products is their anisotropic structure which can be achieved by altering the proteins native structure

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via high moisture extrusion processing (Arêas, 1992; Cheftel et al., 1992).

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During high moisture extrusion processing, proteins undergo thermal and mechanical stresses by

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heating of the barrel and shearing of the screws. As a result of this thermomechanical treatment,

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proteins native structures are altered by denaturation and a change in molecular structure leading to

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the formation of soluble and/or insoluble aggregates (Chen et al., 2011; Fang et al., 2013; Liu and Hsieh,

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2007; Osen et al., 2015). By attaching a long cooling die to the end of the extruder, proteins are

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assumed to realign in flow direction forming an anisotropic protein network (Cheftel et al., 1992;

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Nogouchi, 1989; Osen et al., 2014). Based on this general understanding of the process, it can be

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expected that different final product characteristics can be achieved by altering process conditions

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during high moisture extrusion processing. Process conditions in the screw section can be varied

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through independent process parameters, such as barrel temperature, screw speed and configuration,

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whereas process conditions in the die section can be varied through cooling rate and die geometry.

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This improves the flexibility of the process to a significant extent. Therefore, to be able to tailor the

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final product characteristics of meat analogs to match consumers’ expectations, a thorough

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understanding of underlying protein structuring mechanisms and their relation to process parameters

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in each process section is necessary.

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However, extrusion is a multivariate complex process, and the sections are directly linked to each

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other. Any change in one section (e.g. cooling rate in die section) results in a change in process

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conditions in the other section (e.g. pressure and filling degree in screw section). Therefore, studies

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ACCEPTED MANUSCRIPT performed on high moisture extrusion focus on this process as a whole.

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This way, several studies have been able to provide a better understanding on the effect of

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temperature and/or moisture content on molecular structure, physicochemical and final product

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properties for soy and pea proteins, respectively (Chen et al., 2010; Chen et al., 2011; Fang et al., 2013;

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Fang et al., 2014; Lin et al., 2002; Liu and Hsieh, 2007; Osen et al., 2014; Prudêncio-Ferreira and Arêas,

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1993). Furthermore, findings from Fang et al. (2013; 2014) indicate that thermal as well as mechanical

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treatment during extrusion processing affect soy proteins molecular structure and product properties

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during extrusion processing.

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Changes in molecular structure of wheat gluten subunits resulting from polymerization and

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depolymerization reactions have been described to influence dough mixing, film-forming and bread-

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making properties (Jansens et al., 2011; Jansens et al., 2013; Khatkar et al., 1995; Singh, 2005;

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Veraverbeke and Delcour, 2002). Further studies on the relevance of wheat gluten as quality

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determinant of food products have been summarized by Delcour et al. (2012). Despite the abundant

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literature in different fields of research, wheat gluten has not been used as major matrix material, but

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as minor ingredient in the production of meat analog products by high moisture extrusion processing

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(Liu and Hsieh, 2007). To the best of our knowledge, Grabowska et al. (2014) were the first to show

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that shear structuring of wheat gluten as single component results in the formation of anisotropic,

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meat-like structures. However, the mechanisms behind formation of such anisotropic wheat gluten

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networks and how network structures are influenced by process conditions during high moisture

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extrusion are still unknown.

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As reviewed by Delcour et al. (2012), several studies have been able to relate changes in functional

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properties of food products such as bread and pasta to wheat gluten polymerization by successively

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influencing rheological properties and protein network formation. Transferring this knowledge to the

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field of high moisture extrusion, we hypothesized that structuring mechanisms of meat analog

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products are also subjected to polymerization behavior of wheat gluten polymerization during high

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moisture extrusion processing. Therefore, the objective of this study was to investigate whether and,

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ACCEPTED MANUSCRIPT if so, how process conditions during high moisture extrusion influence wheat gluten polymerization

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and depolymerization reactions. Regarding the complexity of the process, we considered the process

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as two hierarchical sections: Screw section and die section. When investigating decisive process

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parameters influencing polymerization reactions taking place in both process sections, however, the

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interdependence between process conditions in screw and die section has to be taken into account.

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The objective of this study is to investigate the influence of temperature, pressure and mechanical

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treatment on changes in polymerization behavior of wheat gluten in screw and die section. Therefore,

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we present an experimental concept that is feasible to identify decisive process parameters influencing

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protein structuring mechanisms for each process section.

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2. Material and methods

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2.1. Materials

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Commercial vital wheat gluten was kindly supplied by Kröner Stärke (Ibbenbüren, Germany) with a

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moisture content of 8%. According to the manufacturer, protein content on dry matter basis was 83%.

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Chemicals for protein extractability analysis were obtained from Carl Roth (Karlsruhe, Germany).

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2.2. Extrusion process

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To investigate the influence of process conditions in screw and die section on changes in wheat gluten

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polymerization, different extruder setups were designed which are depicted in fig. 1. For all trials, a

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co-rotating twin screw extruder ZSK 26 Mc (Coperion, Stuttgart, Germany) with screw diameter of

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25.5 mm and length to diameter ratio of 29 was used. The extruder barrel is divided into 7 sections

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which can be heated separately, except the first one. Material and water were fed to the first section

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of the extruder by a gravimetrically controlled feeder (DDW-DDSR40 from Brabender, Duisburg,

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Germany) and a piston-membrane pump (model KM 251, Alldos, Pfinztal, Germany), respectively.

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Since the aim of this study was to investigate whether process conditions in screw and die section

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affect wheat gluten polymerization, constant process parameters were chosen in a range where the

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most pronounced effects of mechanical and thermal treatment on wheat gluten polymerization can

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ACCEPTED MANUSCRIPT be expected. Therefore, all trials were run at constant screw speed of 300 rpm, feed rate of 10 kg/h

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and moisture content of 40% (wet basis).

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2.2.1. Screw section

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The influence of extruder temperature, extruder pressure and mechanical treatment on wheat gluten

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polymerization was studied by attaching a short circular die instead of a rectangular cooling die to the

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end of the extruder (see fig. 1a). With this setup, samples could be taken directly after

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thermomechanical treatment in the screw section and before further processing in the cooling die

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section. Extruder temperature was varied by setting barrel temperatures in each section, except for

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the first one, as shown in table 1. Extruder temperature was measured at extruder exit (directly before

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the die) using a thermocouple type J (Ahlborn, Holzkirchen, Germany).

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To simulate the influence of backpressure from cooling die on protein polymerization, dies with various

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lengths of 5, 15, and 25 mm were used. All dies had a diameter of 3 mm. Resulting extruder pressure

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was measured at extruder exit (directly before the die) using a melt pressure sensor (type M3, max.

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pressure < 30 MPa, Gefran, Provaglio d’Iseo, Italy).

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To achieve different mechanical treatments, two different screw configurations were applied as shown

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in fig. 2. Mild process conditions are induced by using screw configuration A, whereas reverse transport

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elements included in screw configuration B lead to more intensive process conditions (Emin and

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Schuchmann, 2013). Change in mechanical treatment was monitored by calculating specific

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mechanical energy input (SME) according to the following equation:

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𝑀𝑑 ‒ 𝑀𝑑, 𝑢𝑛𝑙𝑜𝑎𝑑

AC 𝑛 𝑛𝑚𝑎𝑥

×

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𝑆𝑀𝐸 =

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where n and nmax are the actual and maximum screw speed (1800 min-1) Md and Md,unload are the actual

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and idle torque (%). 𝑚 represents the total mass flow (kg/h) and Pmax the maximum engine power (40

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kW).

𝑚

× 𝑃𝑚𝑎𝑥

(kJ∙kg-1)

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ACCEPTED MANUSCRIPT 2.2.2.Die section

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Processing of thermomechanically treated wheat gluten in the die section was conducted by attaching

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a long cooling die with dimensions of 15 x 30 x 380 mm (H x W x L) to the end of the extruder (see fig.

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1b). For this experimental setup, screw configuration B was used. The influence of cooling temperature

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on protein polymerization was investigated for different extruder temperatures. Cooling temperature

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was set to 0 °C, 20 °C, 50 °C and 80 °C by a refrigerated circulator (type Presto Plus LH 47, Julabo,

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Seelbach, Germany).

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2.3. Wheat gluten extractability

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Protein extractability in solvents containing denaturing agents can be used to monitor polymerization

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between wheat gluten subunits (Delcour et al., 2012; Lagrain et al., 2008; Li and Lee, 1996). For the

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analysis, samples were collected from extrusion experiments and dried in a vacuum dryer (Heraeus,

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Hanau, Germany) at 40 °C and 8 mbar and ground to a particle size < 0.28 mm. Wheat gluten

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extractability was measured by extracting 0.02 g of ground sample with 10 mL buffer containing 0.086

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M Tris, 0.09 glycine, 4 mM ethylenediaminetetraacetic acid sodium salt (Na2EDTA), 8 M urea and 0.5%

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(w/v) sodium dodecyl sulfate (SDS) at pH 9.1. Samples were dispersed in buffer solution using a vortex

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mixer. After 1 h of total extraction time, protein suspensions were centrifuged at 17,700 g for 20 min.

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The supernatant was taken to detect changes in extractable protein content by fluorescence analysis

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using an Infinite 200 Pro microplate reader (Tecan, Crailsheim, Germany). Intrinsic tryptophan

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fluorescence was excited at an excitation wavelength of 295 nm and resulting emission spectra were

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recorded from 300 to 450 nm (data not shown). For all samples, maximum fluorescence intensities

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were detected at an emission wavelength of 342 nm. Within preliminary studies, SDS-PAGE

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densitometry was used to ensure that fluorescence intensity at this wavelength is directly proportional

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to a decrease in amount of extractable wheat gluten. Since results from SDS-PAGE densitometry were

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consistent with results from fluorescence intensity measurements, data concerning SDS-PAGE analysis

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is not shown within this article.

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ACCEPTED MANUSCRIPT 2.4. Statistical analysis

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Extrusion trials were repeated at least twice and wheat gluten extractability analyses were done in

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triplicate. To estimate whether extruder pressure, temperature and SME, respectively, influenced

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changes in extractable wheat gluten after screw section, multiple linear and 1st order exponential

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regression analyses were compared using OriginPro Software, version 9.1G (OriginLab Corporation,

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Northampton, USA). The effect of extruder temperature and cooling temperature on extractable

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wheat gluten was evaluated by 1-way-ANOVA. Scheffé’s test was used for comparison of means. For

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all statistical analyses, a probability of p < 0.05 was used to identify significant differences.

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3. Results and Discussion

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3.1. Influence of cooling temperature on wheat gluten extractability and product structure after die

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The influence of cooling temperature on change in extruder pressure and extractable wheat gluten is

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depicted in fig. 3. As expected, results in fig. 3a show that decreasing cooling temperature resulted in

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a significant (p < 0.05) increase in backpressure from 1.0 to 3.5 MPa and 1.2 to 5.1 MPa at an extruder

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temperature of 110 °C and 145 °C, respectively. Specific mechanical energy input (SME) which is taken

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as measure of mechanical treatment was increased from 72 to 268 kJ∙kg-1. From this, it can be

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concluded that different cooling temperatures induced a change in process conditions in screw and

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die section. Nevertheless, results in fig. 3b show that extractable wheat gluten monitored by change

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in fluorescence intensity was not influenced by cooling temperature (p > 0.05). Instead, wheat gluten

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extractability was significantly (p < 0.05) decreased by an increase in extruder temperature from 110

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to 145 °C.

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The influence of extruder temperature on wheat gluten extractability was reflected by change in final

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product structure. Fig. 4 shows digital images of two different products resulting from 110 °C and

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145 °C extruder temperature. The samples were cut on the edges which resulted in a smooth cut

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surface. The cuts were then used to tear the samples carefully apart in order to visualize the inner

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structure, whereas differences in fracture behavior can be explained by change in the protein network

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ACCEPTED MANUSCRIPT orientation, e.g. towards flow direction in the cooling die (Nogouchi, 1989; van Vliet, 1996). At an

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extruder temperature of 145 °C, the inner structure of the sample appears to be anisotropic which is

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indicated by a flow-oriented fracture behavior (flow direction was from right to left). This has been

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described as characteristic attribute for structured plant proteins from high moisture extrusion

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processing (Osen et al., 2015). At an extruder temperature of 110 °C, however, fracture behavior of

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the sample appeared to be rather isotropic lacking a characteristic flow pattern.

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For high moisture extrusion processing, it is recognized that the orientation of the protein network in

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direction of the laminar flow in the cooling die depends on die geometry, cooling temperature and

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rheological properties of the melt (Akdogan, 1999; Nogouchi, 1989). Die geometry and cooling

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temperature were kept constant for the products depicted in fig. 4. As a consequence, difference in

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visual product appearance is most likely to derive from a change in rheological properties. Whereby,

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rheological properties are known to be directly connected to a change in the molecular structure of

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wheat gluten, e.g. via polymerization reactions (Attenburrow et al., 1990; Kokini et al., 1994; Redl et

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al., 2003).

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In this study, wheat gluten polymerization reactions were monitored by protein extractability analyses.

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The extractability in solvents containing denaturing agents is often used to monitor polymerization

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reactions between wheat gluten subunits (Lagrain et al., 2008; Schofield et al., 1983; Wieser et al.,

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1998). These subunits are comprised of two major fractions, gliadins and glutenins. Since gliadins are

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only soluble in aqueous ethanol, only glutenin subunits can be extracted by solutions containing

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denaturing agents such as SDS and urea (Schofield et al., 1983; Wieser, 2007). These substances were

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used in the present study and are known to cleave all non-covalent interactions (Shimada and Cheftel,

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1988). As a consequence, it is recognized that a decrease in extractable protein under non-reducing

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conditions can be subjected to an increase in polymerization of glutenin subunits via disulfide bonds

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(Strecker et al., 1995; Wrigley, 1996).

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According to findings from fig. 3 and 4, it can be concluded that different extruder temperatures

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affected wheat gluten polymerization and with this final product appearance. Polymerization reactions

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ACCEPTED MANUSCRIPT of wheat gluten have been described to be induced by temperatures above 90 °C and mainly involve

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the formation of disulfide bonds between glutenins, as well as glutenins and gliadins (Delcour et al.,

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2012; Lagrain et al., 2008; Li and Lee, 1996). Further studies on wheat gluten polymerization under

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controlled conditions show that this polymerization behavior is influenced by temperature, pressure

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and mechanical treatment (Kieffer et al., 2007; Pommet et al., 2004; Strecker et al., 1995). Thus,

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regarding results from fig. 3 and 4, it can be assumed that different thermomechanical treatment in

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screw section resulted in different degrees of wheat gluten polymerization and with this different

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rheological properties influencing final product properties, such as product appearance. These results

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underline our initial hypothesis suggesting a relation between wheat gluten polymerization and final

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product properties. Furthermore, they point out that wheat gluten polymerization during high

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moisture extrusion processing should to be controlled in order to obtain certain product properties.

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Therefore, it is necessary to understand how process parameters in screw and die section affect certain

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process conditions which will lead to different degrees of wheat gluten polymerization.

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Nevertheless, it is difficult to conclude how temperature, pressure and mechanical treatment affected

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wheat gluten polymerization during high moisture extrusion processing from results in fig. 3. Although

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these results depict the influence of extruder temperature and cooling temperature on wheat gluten

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polymerization, it needs to be taken into account that both parameters simultaneously interrelate with

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SME and extruder pressure. Furthermore, SME and extruder pressure depict major changes in process

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conditions. As they are integral values, they cannot give full information on local process conditions.

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Thus, changing cooling temperatures could have resulted in different local process conditions causing,

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however, an overall similar decrease in wheat gluten extractability. To investigate the influence of

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temperature, pressure and mechanical treatment on wheat protein polymerization in a more targeted

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way, extrusion trials without reciprocal effects from process conditions in cooling die were conducted.

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ACCEPTED MANUSCRIPT 3.2. Change in wheat gluten extractability after screw section

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3.2.1.Influence of die pressure and extruder temperature

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Fig. 5 describes the effect of die length on extruder pressure and wheat protein polymerization at

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different extruder temperatures. By varying die lengths from 5 to 25 mm, pressure was significantly (p

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< 0.05) increased from 1.5 to 3.5 MPa (see fig. 5a). In addition, an increase in extruder pressure with

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increasing extruder temperature from 90 to 160 °C can be observed at constant die length. With

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increasing extruder temperature from 90 to 160 °C, fluorescence intensity showed a 4-fold decrease

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and with this a decrease in extractable wheat gluten.

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To evaluate the relation between change in wheat gluten extractability and extruder temperature by

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varying barrel temperature and pressure by varying die length, different regression analyses were

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conducted. Besides single and multiple linear regression analyses, 1st order exponential regression

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analysis was considered since polymerization reactions of gluten has previously been described to

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follow 1st order kinetics (Domenek et al., 2003). Results from regression analyses are compared in table

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2. For linear and 1st order exponential regression analyses, extruder temperature is assumed as single

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influencing parameter. Here, coefficients of determination R2corr amount 0.89 and 0.90, respectively,

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indicating that there is a significant correlation between extruder temperature and change in

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extractable wheat gluten.

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The interdependence between extruder temperature, pressure and wheat gluten extractability was

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considered by multiple linear regression analysis. An R2corr of 0.91 indicates that a change in extractable

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wheat gluten can be related to both, extruder temperature and pressure. However, similar correlation

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was observed for single linear and 1st order exponential regression analyses, in which the effect of

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extruder pressure is not included. Since results in fig. 5a show that extruder pressure was also

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influenced by extruder temperature, the increase in R2corr for multiple regression analysis should be

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considered to originate from this interrelation and not the direct effect of extruder pressure on

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extractable wheat gluten.

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Pressure at the end of the extruder is dependent on volumetric flow rate, melt viscosity and die

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ACCEPTED MANUSCRIPT geometry. Since volumetric flow rate was kept constant during all extrusion trials, an increase in

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pressure at constant die length can only be related to an increase in melt viscosity with increasing

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extruder temperature. An increase in melt viscosity with temperature is expected to be a result of an

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increase in polymerization degree. Thus, our findings from fig. 5 indicate that an increase in extruder

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pressure and melt viscosity was due to an increase in wheat gluten polymerization.

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Regarding the relation between extruder temperature, melt viscosity and wheat gluten

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polymerization, comparable results have been obtained from Attenburrow et al. (1990), Kokini et al.

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(1994) and Redl et al. (2003) who focused on rheological behavior of wheat gluten during

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thermomechanical treatment. Results from these studies indicated that an increase in viscosity upon

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heating originates from an increase in molecular weight due to polymerization reactions. Although

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different plasticizers and moisture contents were used in the studies mentioned, findings in fig. 5

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suggest similar reactions. More detailed extraction analysis, however, would be necessary in order to

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gain further information on polymerization and depolymerization pathways of wheat gluten subunits

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during high moisture extrusion processing (Wieser et al., 1998).

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According to Kieffer et al. (2007), pressure treatment of wheat gluten can affect the proteins molecular

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structure. Compared to atmospheric conditions (0.1 MPa), the effect of pressure > 200 MPa was shown

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to alter molecular structure including polymerization reactions of wheat gluten subunits by cleavage

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and rearrangement of disulfide bonds. Moreover, the effect of pressure treatment can be increased

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by additional heating. Up to now, the effect of pressure in a range relevant for extrusion conditions in

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screw section (< 200 MPa) has not been studied.

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In conclusion, findings from fig. 5 indicate that, in the range investigated, wheat gluten polymerization

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was only influenced by extruder temperature. However, increasing pressure at extruder exit also leads

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to a change in degree of fill and residence time. The extent of this interrelation can lead to different

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thermomechanical treatment in screw section. Since SME varied between 84 and 206 kJ∙kg-1 with

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ACCEPTED MANUSCRIPT changing die length and extruder temperature (see fig. 5), not only thermal, but also mechanical

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energy input needs to be considered as further parameter influencing wheat gluten polymerization.

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3.2.2.Influence of mechanical energy input and barrel temperature

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Screw configurations with (screw configuration B) and without reverse elements (screw configuration

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A) were used to study the influence mechanical energy input on wheat gluten polymerization. The

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results are depicted in fig. 6. For screw configuration A, SME varied from 32 to 66 kJ∙kg-1 at extruder

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temperatures from 90 to 130 °C (fig. 6a). As expected, SME was significantly increased (p < 0.05) and

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varied from 80 to 148 kJ∙kg-1 at extruder temperatures from 90 to 160 °C by using reverse elements in

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screw configuration B. At same barrel temperature settings, measured extruder temperatures were

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reduced between 3 and 10 K when using screw configuration A instead of B. This trend can be

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explained by the effect of reverse elements in screw configuration B where material experiences higher

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mechanical input when passing the tips of reverse screw elements (Emin and Schuchmann, 2013). The

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observed increase in SME for screw configuration B in comparison to screw configuration A gives

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further evidence supporting this explanation. As consequence of viscous energy dissipation, thermal

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energy input increases resulting in higher material temperatures measured at the exit of the screw

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section.

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Although change in screw configuration from A to B lead to a significant increase in SME, results in fig.

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6b depict that change in extractable wheat gluten was only a function of extruder temperature. This

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result is supported by regression analyses in table 3 that show high coefficients of determinations R2corr

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of 0.94 for both, 1st order exponential and single linear regression analysis. The interrelation between

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temperature, SME and change in extractable protein content was estimated by multiple linear

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regression analysis with an R2corr of 0.97. SME is indirectly influenced by extruder temperature due to

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change in material properties. Since change in rheological properties due to temperature induced

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polymerization was shown in fig. 5, the increase in R2corr for multiple regression analysis results are

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explained rather from this interrelation than the direct effect of mechanical energy input on

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extractable wheat gluten.

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ACCEPTED MANUSCRIPT Although results from fig. 5 and 6 indicate that, for the range investigated, extruder temperature was

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the major parameter influencing wheat gluten polymerization, Pommet et al. (2004) and Strecker et

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al. (1995) found that the combination of heating and shearing reduces activation energies for wheat

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gluten polymerization as well as depolymerization reactions. These studies were conducted in specific

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rheometers applying defined thermal and mechanical stresses. Such devices can be used to gain

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accurate models of polymerization behavior under extrusion-like conditions allowing to vary shear rate

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and temperature independently. However, the authors investigated wheat gluten polymerization

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either at low moisture contents or with glycerol as plasticizer. Thus, their findings cannot be

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transferred directly to explain the influence of process conditions during high moisture extrusion on

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wheat gluten polymerization. Nevertheless, they indicate that, depending on the range investigated,

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wheat gluten polymerization and depolymerization reactions can be influenced by the combination of

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thermal and mechanical treatment.

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Evidence on the effect of mechanical treatment during high moisture extrusion processing of other

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plant proteins than wheat gluten is given by Fang et al. (2013; 2014) who found molecular weight of

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soy proteins decreased with increasing SME. Here, SME was varied between 839 and 1277 kJ∙kg-1 which

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is significantly higher compared to the range investigated in the present study. Besides this difference

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in SME, the results from Fang et al. (2013) cannot be compared directly to the findings of this study

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because soy proteins and wheat gluten are composed of different amino acids. Thus, reaction behavior

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during thermal and mechanical treatment will be different due to the composition of the particular

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plant protein and the reactive functional groups available. To be able to explain the different responses

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of plant proteins to certain process conditions, first of all, the reaction behavior of a specific plant

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protein needs to be understood thoroughly. To gain this basic knowledge on wheat gluten, it is

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necessary to obtain information on the effect of combined mechanical and thermal treatment on

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wheat gluten polymerization in a wider range of process conditions. This can be achieved by including

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the effect of screw speed, flow rate and moisture content on thermal and mechanical treatment of

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wheat gluten in following studies.

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ACCEPTED MANUSCRIPT Furthermore, determining polymerization behavior of wheat gluten at high moisture extrusion

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conditions will enable a more targeted design of the extrusion process for the production of meat

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analog products. To accomplish this goal, detailed investigations on resulting product structures will

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be necessary to define an optimal degree of wheat gluten polymerization in relation to product

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properties of meat analog products.

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4. Conclusion

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By considering the process as two hierarchical sections, it was shown that wheat gluten polymerization

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reactions were mainly taking place in the screw section of the extruder. Change in the polymerization

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reactions in the screw section affected visual appearance of final products. To exclude the influence of

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reciprocal effects from process conditions in cooling die, the influence of temperature, pressure, and

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mechanical treatment on wheat gluten polymerization in screw section was studied by varying die

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length and screw configuration. These trials showed that, in the range investigated, only thermal

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treatment in the screw section influenced wheat gluten polymerization, whereas the die temperature,

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pressure, and specific mechanical energy input (SME) had no significant influence.

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Acknowledgements

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This research project was supported by the German Ministry of Economics and Energy (via AiF) and FEI

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(Forschungskreis der Ernährungsindustrie e.V., Bonn) in the scope of project AiF 18727 N. Further, the

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authors would like to express their thanks to Kerstin Sauther and Andrea Butterbrodt for supporting

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the extrusion experiments and protein extractability analyses.

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ACCEPTED MANUSCRIPT Figure 1. Schematic illustration of the experimental setup to investigate the influence of process conditions in screw and die section on changes in wheat gluten polymerization after screw section (a) and die section (b)

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Figure 2. Schematic illustration of screw configurations A and B used to study the effect of SME on wheat gluten polymerization

Figure 3. Influence of cooling temperature TC on extruder pressure (a) and fluorescence intensity Imax at 342 nm (b) at different extruder temperatures TE. Constant parameters: screw speed: 300 rpm, feed rate: 10 kg/h, moisture: 40%. Significant differences were identified via 1-way-ANOVA. In fig. 3a, all pressure values at TE = 107 ± 2 °C () are significantly different (p < 0.05) from pressure values at TE = 145 ± 4 °C (). At same extruder temperatures TE, significant differences (p < 0.05) between extruder pressure at different cooling temperatures TC were compared and are indicated by different letters. In

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fig. 3b, fluorescence intensities at TE = 107 ± 2 °C () are significantly different (p < 0.05) from fluorescence intensities at TE = 145 ± 4 °C (). At same extruder temperature TE, fluorescence

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intensities Imax are not significantly different (p > 0.05).

Figure 4. Digital images of products resulting from 110 °C extruder temperature (left) and 145 °C

80 °C.

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extruder temperature (right) after processing in screw and die section at a cooling die temperature of

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Figure 5. Influence of 5, 15 and 25 mm die length on extruder pressure pE (a) and fluorescence intensity Imax at 342 nm (b) at different extruder temperatures TE. Constant process parameters: screw speed:

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300 rpm, feed rate: 10 kg/h, moisture: 40%, screw configuration: B. All trails were repeated at least twice. Values from repeated trials were not averaged because, at same process parameter settings, different values were measured for extruder temperature TE due to minor changes in process conditions. Accordingly, data points depict results from single extrusion trials. Figure 6. Influence of screw configuration A and B on SME (a) and fluorescence intensity Imax at 342 nm (b) at different extruder temperatures TE. Constant parameters: screw speed: 300 rpm, feed rate: 10

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depict results from single extrusion trials.

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ACCEPTED MANUSCRIPT Table 1 Barrel temperature settings for section 1-7 used to study effect of extruder temperature on wheat gluten polymerization. Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 40 °C

60 °C

80 °C

90 °C

90 °C

90 °C

-

40 °C

60 °C

100 °C

100 °C

100 °C

100 °C

-

40 °C

60 °C

100 °C

100 °C

100 °C

120 °C

-

40 °C

60 °C

100 °C

100 °C

100 °C

155 °C

-

40 °C

60 °C

100 °C

100 °C

120 °C

170 °C

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Table 2 Linear, multiple and 1st order exponential regression analyses describing the relation between temperature, die length and fluorescence intensity. Significant differences are indicated by an asterisk. parameter xi = 1;2 ki = 1;2 ± SE temperature TE -8.5 ± 0.6*

𝑦 = 𝑘 0 + 𝑘 1 ∙ 𝑥1 + 𝑘 2 ∙ 𝑥2 ln 𝑦 = ‒ 𝑘1 ∙ 𝑥1 ∙ ln 𝑘0

temperature TE

-8.6 ± 0.6* 1.6*

k0 ± SE 1354 ± 85*

R2corr 0.89

1276 ± 81*

0.91

pressure pE

4.3 ±

temperature TE

-1041 ± 72* 5309 ± 353* 0.90

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Equation 𝑦 = 𝑘 0 + 𝑘 1 ∙ 𝑥1

equation 𝑦 = 𝑘 0 + 𝑘 1 ∙ 𝑥1

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Table 3 Linear, multiple and 1st order exponential regression analyses describing the relation between temperature, SME and fluorescence intensity. Significant differences are indicated by an asterisk. parameter xi = 1,2 ki = 1;2 ± SE k0 ± SE * temperature TE -6.66 ± 0.40 1095 ± 47* temperature TE

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𝑦 = 𝑘 0 + 𝑘 1 ∙ 𝑥1 + 𝑘 2 ∙ 𝑥2

SME

-3.87 ±

temperature TE

-762 ± 44*

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ln 𝑦 = ‒ 𝑘1 ∙ 𝑥1 ∙ ln 𝑘0

-5.60 ± 0.40* 1.05*

1062 ± 37*

R2corr 0.94 0.97

3929 ± 215* 0.94