Influence of Lowering the pH Value on the Generation of Fibrous Structures of Protein Gels with Different Network Types.

High-moisture extrusion of plant proteins to create meat-like structures is a process that has met with increasing attention in the recent past. In the process, the proteins are thermomechanically stressed in the screw section of the extruder, and the resulting protein gel is structured in the attached cooling die. Various protein sources, notably soy protein isolate (SPI) and wheat gluten, are used to form gels with different networks: SPI creates a physical, non-covalent network, while gluten forms a chemical, covalent one. The food industry frequently adds weak acids to modify taste and shelf life. However, it is known that a change in pH affects the gelation behavior of proteins because the repulsive forces within and between the proteins change. The research reported here was carried out to investigate for the two proteins mentioned the influence of pH modification by the addition of citric acid and acetic acid on gel formation and the meat-like structures produced. For this purpose, materials and parameters were screened using a closed cavity rheometer, followed by extrusion trials at pH 7.36-4.14 for SPI and pH 5.83-3.37 for gluten. The resulting extrudates were analyzed optically and mechanically, and protein solubility was tested in a reducing buffer. For both protein systems, the addition of acid results in less pronounced meat-like structures. At decreasing pH, the complex viscosity of SPI increases (from 11,970 Pa·s to 40,480 Pa·s at 100 °C), the generated gel becomes stronger (strain decreased from 0.62 to 0.48 at 4.5 × 105 Pa), and the cross-linking density grows. For gluten, a decreasing pH results in altered reaction kinetics, a more deformable resulting gel (strain increased from 0.7 to 0.95 at 4.5 × 105 Pa), and a decreased cross-linking density. Solubility tests show that no additional covalent bonds are formed with SPI. With gluten, however, the polymerization reaction is inhibited, and fewer disulfide bonds are formed.

Rheological properties of dry-fractionated mung bean protein and structural, textural, and rheological evaluation of meat analogues produced by high-moisture extrusion cooking.

A closed cavity rheometer was used to study the rheology of dry-fractionated mung bean protein -DFMB- (55% protein d.m.). Then, the high-moisture extrusion cooking at 40% and 50% moisture contents and different temperatures (115, 125, 135 and 145 °C) was performed, investigating the impact on structural, textural, and rheological properties of extrudates. When subjected to a temperature ramp (40-170 °C), DFMB showed an increase of G* from 70 °C, as a consequence of starch gelatinization and protein gelation. The peak, indicating the end of aggregation reactions, was at 105 °C and 110 °C for DFMB at 50% and 40% moisture content, respectively. The time sweep analysis described the protein behavior in no-shear/shear conditions, highlighting a more pronounced effect of the temperatures compared to moisture content. During the extrusion cooking, the temperature increase led to a decrease of pressure, indicating a reduction of the melt viscosity. The microstructure of the extrudates showed a more pronounced anisotropic profile when higher temperatures were applied. Hardness, chewiness, and cohesion were directly correlated with the temperature, which also affected the rheological properties of extrudates. A combination of textural and rheological analyses can offer a clear overview of the structural characteristics of meat analogues.

Highly protein-loaded melt extrudates produced by small-scale ram and twin-screw extrusion - evaluation of extrusion process design on protein stability by experimental and numerical approaches.

Understanding of generation, extent and location of thermomechanical stress in small-scale (< 3 g) ram and twin-screw melt-extrusion is crucial for mechanistic correlations to the stability of protein particles (lysozyme and BSA) in PEG-matrices. The aim of the study was to apply and correlate experimental and numerical approaches (1D and 3D) for the evaluation of extrusion process design on protein stability. The simulation of thermomechanical stress during extrusion raised the expectation of protein degradation and protein particle grinding during extrusion, especially when TSE was used. This was confirmed by experimental data on protein stability. Ram extrusion had the lowest impact on protein unfolding temperatures, whereas TSE showed significantly reduced unfolding temperatures, especially in combination with kneading elements containing screws. In TSE, the mechanical stress in the screws always exceeded the shear stress in the die, while mechanical stress within ram extrusion was generated in the die, only. As both extruder designs revealed homogeneously distributed protein particles over the cross section of the extrudates for all protein-loads (20-60%), the dispersive power of TSE revealed not to be decisive. Consequently, the ram extruder would be favored for the production of stable protein-loaded extrudates in small scale.

Novel glass capillary microfluidic devices for the flexible and simple production of multi-cored double emulsions.

HYPOTHESIS: Double emulsions with many monodispersed internal droplets are required for the fabrication of multicompartment microcapsules and tissue-like synthetic materials. These double emulsions can also help to optically resolve different coalescence mechanisms contributing to double emulsion destabilization. Up to date microfluidic double emulsions are limited to either core-shell droplets or droplets with eight or less inner droplets. By applying a two-step jet break-up within one setup, double emulsion droplets filled with up to several hundred monodispersed inner droplets can be achieved. EXPERIMENTS: Modular interconnected CNC-milled Lego®-inspired blocks were used to create two separated droplet break-up points within coaxial glass capillaries. Inner droplets were formed by countercurrent flow focusing within a small inner capillary, while outer droplets were formed by co-flow in an outer capillary. The size of inner and outer droplets was independently controlled since the two droplet break-up processes were decoupled. FINDINGS: With the developed setup W/O/W and O/W/O double emulsions were produced with different surfactants, oils, and viscosity modifiers to encapsulate 25-400 inner droplets in each outer drop with a volume percentage of inner phase between 7% and 50%. From these emulsions monodispersed multicompartment microcapsules were obtained. The report offers insights on the relationship between the coalescence of internal droplets and their release.

Blending Proteins in High Moisture Extrusion to Design Meat Analogues: Rheological Properties, Morphology Development and Product Properties.

High moisture extrusion (HME) of meat analogues is often performed with raw materials containing multiple components, e.g., blends of different protein-rich raw materials. For instance, blends of soy protein isolate (SPI) and another component, such as wheat gluten, are used particularly frequently. The positive effect of blending on product texture is well known but not yet well understood. Therefore, this work targets investigating the influence of blending in HME at a mechanistic level. For this, SPI and a model protein, whey protein concentrate (WPC), were blended at three different ratios (100:0, 85:15, 70:30) and extruded at typical HME conditions (55% water content, 115/125/133 °C material temperature). Process conditions, rheological properties, morphology development, product structure and product texture were analysed. With increasing WPC percentage, the anisotropic structures became more pronounced and the anisotropy index (AI) higher. The achieved AI from the extrudates with a ratio of 70:30 (SPI:WPC) were considerably higher than comparable extrudates reported in other studies. In all extrudates, a multiphase system was visible whose morphology had changed due to the WPC addition. The WPC led to the formation of a much smaller dispersed phase compared to the overlying multiphase structure, the size of which depends on the thermomechanical stresses. These findings demonstrate that targeted mixing of protein-rich raw materials could be a promising method to tailor the texture of extruded meat analogues.

Effect of Oil Content and Oil Addition Point on the Extrusion Processing of Wheat Gluten-Based Meat Analogues.

High-moisture extrusion is a common process to impart an anisotropic, meat-like structure to plant proteins, such as wheat gluten. The addition of oil during the process promises to enhance the sensory properties of the meat analogs. In this study, the influence of oil on extrusion-relevant parameters as well as the structure-related characteristics of extruded wheat gluten was investigated. Oil was added directly to the extruder at different contents (0, 2, 4, 6%) and addition points (front/end of the extruder barrel). Process conditions, complex viscosity, Young's modulus and oil phase morphology were determined as a function of oil content and oil addition point. With increasing oil content, material temperature, die pressure, and complex viscosity decreased. The addition of oil at the end of the extruder barrel reduced this effect compared to the addition of oil in the front part of the extruder. It was observed that the extrudate's tensile strength is a function of material temperature, resulting in an increase in tensile strength with increasing material temperature. The oil was dispersed in the gluten matrix as small droplets with irregular shape. As the oil content increased, the size of the oil droplets increased, while the addition of oil at the end of the extruder resulted in a decrease in droplet size.

Comparison of the Rheological Properties of Plant Proteins from Various Sources for Extrusion Applications.

Plant proteins in foods are becoming increasingly popular with consumers. However, their application in extruded products remains a major challenge, as the various protein-rich raw materials (e.g., from different plant origins) exhibit very different material properties. In particular, the rheological properties of these raw materials have a distinct influence on the extrusion process and must be known in order to be able to control the process and adjust the product properties. In this study, process-relevant rheological properties of 11 plant-based protein-rich raw materials (differing in plant origin, protein content, and manufacturer) are determined and compared. The results demonstrate distinct differences in the rheological properties, even when plant origin and protein content are identical. Time sweeps reveal not only large differences in development of viscosity over time, but also in magnitude of viscosity (up to 15-fold difference). All materials exhibit gel behaviour and strain thinning behaviour in the strain sweeps, whereas their behaviour in the non-linear viscoelastic range differs greatly. Typical relaxation behaviour of viscoelastic materials could be observed in the stress relaxation tests for all materials. Comparison of the maximum achieved shear stress, which correlates with the elastic properties, reveals an up to 53-fold difference. The results of this study could serve as a starting point for adapting raw material selection and composition to process and product design requirements and help to meet the challenge of applying plant-based proteins in food extrusion.

Morphology Development and Flow Characteristics during High Moisture Extrusion of a Plant-Based Meat Analogue.

Plant-based meat analogues that mimic the characteristic structure and texture of meat are becoming increasingly popular. They can be produced by means of high moisture extrusion (HME), in which protein-rich raw materials are subjected to thermomechanical stresses in the extruder at high water content (>40%) and then forced through a cooling die. The cooling die, or generally the die section, is known to have a large influence on the products' anisotropic structures, which are determined by the morphology of the underlying multi-phase system. However, the morphology development in the process and its relationship with the flow characteristics are not yet well understood and, therefore, investigated in this work. The results show that the underlying multi-phase system is already present in the screw section of the extruder. The morphology development mainly takes place in the tapered transition zone and the non-cooled zone, while the cooled zone only has a minor influence. The cross-sectional contraction and the cooling generate elongational flows and tensile stresses in the die section, whereas the highest tensile stresses are generated in the transition zone and are assumed to be the main factor for structure formation. Cooling also has an influence on the velocity gradients and, therefore, the shear stresses; the highest shear stresses are generated towards the die exit. The results further show that morphology development in the die section is mainly governed by deformation and orientation, while the breakup of phases appears to play a minor role. The size of the dispersed phase, i.e., size of individual particles, is presumably determined in the screw section and then stays the same over the die length. Overall, this study reveals that morphology development and flow characteristics need to be understood and controlled for a successful product design in HME, which, in turn, could be achieved by a targeted design of the extruders die section.

High Moisture Extrusion of Soy Protein: Investigations on the Formation of Anisotropic Product Structure.

The high moisture extrusion of plant proteins is well suited for the production of protein-rich products that imitate meat in their structure and texture. The desired anisotropic product structure of these meat analogues is achieved by extrusion at high moisture content (>40%) and elevated temperatures (>100 °C); a cooling die prevents expansion of the matrix and facilitates the formation of the anisotropic structure. Although there are many studies focusing on this process, the mechanisms behind the structure formation still remain largely unknown. Ongoing discussions are based on two very different hypotheses: structure formation due to alignment and stabilization of proteins at the molecular level vs. structure formation due to morphology development in multiphase systems. The aim of this paper is, therefore, to investigate the mechanism responsible for the formation of anisotropic structures during the high moisture extrusion of plant proteins. A model protein, soy protein isolate, is extruded at high moisture content and the changes in protein-protein interactions and microstructure are investigated. Anisotropic structures are achieved under the given conditions and are influenced by the material temperature (between 124 and 135 °C). Extrusion processing has a negligible effect on protein-protein interactions, suggesting that an alignment of protein molecules is not required for the structure formation. Instead, the extrudates show a distinct multiphase system. This system consists of a water-rich, dispersed phase surrounded by a water-poor, i.e., protein-rich, continuous phase. These findings could be helpful in the future process and product design of novel plant-based meat analogues.

Functionalization of Enzymatically Treated Apple Pomace from Juice Production by Extrusion Processing.

Food by-products can be used as natural and sustainable food ingredients. However, a modification is needed to improve the technofunctional properties according to the specific needs of designated applications. A lab-scale twin-screw extruder was used to process enzymatically treated apple pomace from commercial fruit juice production. To vary the range of the thermomechanical treatment, various screw speeds (200, 600, 1000 min-1), and screw configurations were applied to the raw material. Detailed chemical and functional analyses were performed to develop a comprehensive understanding of the impact of the extrusion processing on apple pomace composition and technofunctional properties as well as structures of individual polymers. Extrusion at moderate thermomechanical conditions increased the water absorption, swelling, and viscosity of the material. An increase in thermomechanical stress resulted in a higher water solubility index, but negatively affected the water absorption index, viscosity, and swelling. Scanning electron microscopy showed an extrusion-processing-related disruption of the cell wall. Dietary fiber analysis revealed an increase of soluble dietary fiber from 12.6 to 17.2 g/100 g dry matter at maximum thermo-mechanical treatment. Dietary fiber polysaccharide analysis demonstrated compositional changes, mainly in the insoluble dietary fiber fraction. In short, pectin polysaccharides seem to be susceptible to thermo-mechanical stress, especially arabinans as neutral side chains of rhamnogalacturonan I.

Extrusion Processing of Pure Chokeberry (Aronia melanocarpa) Pomace: Impact on Dietary Fiber Profile and Bioactive Compounds.

The partial substitution of starch with dietary fiber (DF) in extruded ready-to-eat texturized (RTE) cereals has been suggested as a strategy to reduce the high glycemic index of these food products. Here, we study the impact of extrusion processing on pure chokeberry (Aronia melanocarpa) pomace powder (CPP) rich in DF and polyphenols (PP) focusing on the content and profile of the DF fractions, stability of PP, and techno-functional properties of the extrudates. Using a co-rotating twin-screw extruder, different screw speeds were applied to CPP with different water contents (cw), which resulted in specific mechanical energies (SME) in the range of 145-222 Whkg-1 and material temperatures (TM) in the range of 123-155 °C. High molecular weight soluble DF contents slightly increase with increasing thermomechanical stress up to 16.1 ± 0.8 g/100 g dm as compared to CPP (11.5 ± 1.2 g/100 g dm), but total DF (TDF) contents (58.6 ± 0.8 g/100 g dm) did not change. DF structural analysis revealed extrusion-based changes in the portions of pectic polysaccharides (type I rhamnogalacturonan) in the soluble and insoluble DF fractions. Contents of thermolabile anthocyanins decrease linearly with SME and temperature from 1.80 ± 0.09 g/100 g dm in CPP to 0.24 ± 0.06 g/100 g dm (222 Whkg-1, 155 °C), but phenolic acids and flavonoids appear to be largely unaffected. Resulting techno-functional (water absorption and water solubility) and physical properties related to the sensory characteristics (expansion, hardness, and color) of pure CPP extrudates support the expectation that granulated CPP extrudates may be a suitable food ingredient rich in DF and PP.

The Influence of Extrusion Processing on the Gelation Properties of Apple Pomace Dispersions: Involved Cell Wall Components and Their Gelation Kinetics.

By-products of fruits and vegetables like apple pomace can serve as techno-functional ingredients in foods. Due to their physicochemical properties, e.g., viscosity, water absorption, or oil-binding, food by-products can modify the texture and sensory perception of products like yogurts and baked goods. It is known that, by extrusion processing, the properties of by-products can be altered. For example, by thermo-mechanical treatment, the capacity of food by-products to increase viscosity is improved. However, the mechanism and involved components leading to the viscosity increase are unknown. Therefore, the complex viscosity of apple pomace dispersions and the involved fractions as pectin (a major part of the water-soluble fraction), water-soluble and water-insoluble fraction, were measured. In the investigated range, an increase in the pectin yield and water solubility was observed with increasing thermo-mechanical treatment by extrusion processing. However, pectin and water-soluble cell wall components had only a limited effect on the complex viscosity of apple pomace dispersions. The insoluble fraction (particles) were investigated regarding their swelling behavior and influence on the complex viscosity. An intensification of thermo-mechanical treatment resulted in increasing swelling behavior.

Denaturation Behavior and Kinetics of Single- and Multi-Component Protein Systems at Extrusion-Like Conditions.

In this study, the influence of defined extrusion-like treatment conditions on the denaturation behavior and kinetics of single- and multi-component protein model systems at a protein concentration of 70% (w/w) was investigated. α-Lactalbumin (αLA) and ß-Lactoglobulin (ßLG), and whey protein isolate (WPI) were selected as single- and multi-component protein model systems, respectively. To apply defined extrusion-like conditions, treatment temperatures in the range of 60 and 100 °C, shear rates from 0.06 to 50 s⁻1, and treatment times up to 90 s were chosen. While an aggregation onset temperature was determined at approximately 73 °C for WPI systems at a shear rate of 0.06 s⁻1, two significantly different onset temperatures were determined when the shear rate was increased to 25 and 50 s⁻1. These two different onset temperatures could be related to the main fractions present in whey protein (ßLG and αLA), suggesting shear-induced phase separation. Application of additional mechanical treatment resulted in an increase in reaction rates for all the investigated systems. Denaturation was found to follow 2.262 and 1.865 order kinetics for αLA and WPI, respectively. The reaction order of WPI might have resulted from a combination of a lower reaction order in the unsheared system (i.e., fractional first order) and higher reaction order for sheared systems, probably due to phase separation, leading to isolated behavior of each fraction at the local level (i.e., fractional second order).

Influence of Thermomechanical Treatment and Ratio of ß-Lactoglobulin and α-Lactalbumin on the Denaturation and Aggregation of Highly Concentrated Whey Protein Systems.

The influence of thermomechanical treatment (temperature 60 °C-100 °C and shear rate 0.06 s-1-50 s-1) and mixing ratio of ß-lactoglobulin (ßLG) and α-lactalbumin (αLA) (5:2 and 1:1) on the denaturation and aggregation of whey protein model systems with a protein concentration of 60% and 70% (w/w) was investigated. An aggregation onset temperature was determined at approx. 80 °C for both systems (5:2 and 1:1 mixing ratio) with a protein concentration of 70% at a shear rate of 0.06 s-1. Increasing the shear rate up to 50 s-1 led to a decrease in the aggregation onset temperature independent of the mixing ratio. By decreasing the protein concentration to 60% in unsheared systems, the aggregation onset temperature decreased compared to that at a protein concentration of 70%. Furthermore, two significantly different onset temperatures were determined when the shear rate was increased to 25 s-1 and 50 s-1, which might result from a shear-induced phase separation. Application of combined thermal and mechanical treatment resulted in overall higher degrees of denaturation independent of the mixing ratio and protein concentration. At the conditions applied, the aggregation of the ßLG and αLA mixtures was mainly due to the formation of non-covalent bonds. Although the proportion of disulfide bond aggregation increased with treatment temperature and shear rate, it was higher at a mixing ratio of 5:2 compared to that at 1:1.

Modification of Apple Pomace by Extrusion Processing: Studies on the Composition, Polymer Structures, and Functional Properties.

By-products of fruit and vegetable processing are an inexpensive and sustainable source of dietary fiber, potentially offering valuable functional properties such as water binding and thickening. Due to these favorable properties, they can be utilized to reformulate widely-consumed foods, e.g., bakery products or beverages. In this study, apple pomace was used as a model system to study whether extrusion technology affects food by-product functionality and thus has the potential to broaden the application of by-products in foods. The effect of the process parameters and the extent of thermo-mechanical treatment on the structural and functional properties of apple pomace were analyzed after extrusion trials using various screw speeds, water contents, and barrel temperatures. Compared to the raw material, apple pomace extruded at Tbarrel = 100 °C, n = 700 min-1 and mH2O = 17% showed an increased water solubility up to 33%. The water absorption increased from 5 to 19 Pa·s and the paste viscosity from 5 to 339 Pa·s by extrusion processing. Analyses of dietary fiber contents and fiber polysaccharide structures revealed that thermo-mechanical stress (n = 700 min-1, mH2O = 22%) increased the content of soluble dietary fiber from 12.5 to 16.7 g/100 g dry matter, and that the harshest conditions even enabled the formation of low-molecular-weight dietary fiber. Arabinans (as neutral rhamnogalacturonan I side chains) appeared to be most sensitive to thermo-mechanical stress, whereas xylans (i.e., a group of minor polysaccharides) were an example of a more stable fiber polysaccharide. Also, the degree of methylation of the pectic polysaccharides was strongly reduced from 50% to 15% when thermo-mechanical stress was applied. Imaging and pore size analysis showed that extrusion processing could disrupt the rigid cell wall macromolecular structure.

Impact of defined thermomechanical treatment on the structure and content of dietary fiber and the stability and bioaccessibility of polyphenols of chokeberry (Aronia melanocarpa) pomace.

Dietary fiber is a potential replacement for other ingredients such as starch in reformulated extruded breakfast cereals. Analysis of chokeberry pomace powder revealed a total dietary fiber content of 57.8 ± 2 g/100 g with 76% being insoluble, 20% high molecular soluble and 4% low molecular soluble dietary fiber. The fiber polysaccharide composition was analyzed in detail by using a variety of analytical approaches. Extrusion-like processing conditions were studies in a Closed Cavity Rheometer enabling the application of defined thermal (temperature range 100-160 °C) and mechanical treatments (shear rates between 0.1 s-1 and 50 s-1) to chokeberry pomace powder. Application of temperatures up to 140 °C irrespective of the mechanical treatment does not remarkably alter dietary fiber structure or content, but reduces the initial content of total polyphenols by about 40% to a final content of 3.3 ± 0.5 g/100 g including 0.63 ± 0.1 g/100 g of anthocyanins, 0.18 ± 0.02 g/100 g of phenolic acids and 0.090 ± 0.007 g/100 g of flavonols, respectively. The retained polyphenols are fully bioaccessible after in vitro digestion, and antioxidant capacity remains unchanged as compared to the untreated pomace powder. Glucose bioaccessibility remains unaffected, whereas glucose content is reduced. It is concluded that chokeberry pomace powder is a good source of dietary fiber with the potential to partially substitute starch in extruded breakfast cereals.

Emulsifier Composition of Solid Lipid Nanoparticles (SLN) Affects Mechanical and Barrier Properties of SLN-Protein Composite Films.

Protein films can be applied to improve food quality and to reduce packaging waste. To overcome their poor water barrier properties, lipids are often incorporated. The function of incorporated lipid depends on the interface between filler and matrix. This study aimed to tailor the properties of a protein-lipid film by designing the oil/water interface to see if the concept of inactive/active filler is valid. Therefore, we varied the emulsifier stabilizing solid lipid nanoparticles (SLN) to promote (via ß-lactoglobulin) or to minimize (via Tween 20) interactions between particle surface and protein. SLN were incorporated into protein films and film properties were determined. Addition of SLN led to significantly decreased water vapor permeability (WVP) of protein films. However, WVP was mainly affected by the emulsifiers and not by the lipid. Protein-stabilized SLN (BS) replaced a lacking protein in the protein network and therefore did not influence the mechanical properties of the films at ambient temperature. BS-composite films were temperature sensitive, as lipid and sucrose palmitate melted at temperatures above 40 °C. Tween 20-stabilized SLN (TS) led to reduced tensile strengths, probably due to perturbative effects of TS and plasticizing effects of Tween 20. Dynamic mechanical analysis showed that TS and Tween 20 increased film mobility. Melting of lipid and emulsifiers, and temperature-dependent behavior of Tween 20 led to a strong temperature dependence of the film stiffness. By designing the interface, particles can be used to tailor mechanical properties of protein films. Tuned edible films could be used to control mass transfers between foods.