Cell wall as a barrier for protein extraction from tomato leaves: A biochemical study.

Solanum lycopersicum (Tomato) leaves and stems are considered waste. Valorization of this waste can be achieved by for example the extraction of proteins. This prospect is promising but currently not feasible, since protein extraction yields from tomato leaves are low, amongst other due to the (physical) barrier formed by the plant cell walls. However, the molecular aspects of the relationship between cell wall properties and protein extractability from tomato leaves are currently not clear and thus objective of this study. To fill this knowledge gap the biochemical composition of plant cell walls was measured and related to protein extraction yields at different plant ages, leaf positions, and across different tomato accessions, including two Solanum lycopersicum cultivars and the wildtype species S. pimpinellifolium and S. pennellii. For all genotypes, protein extraction yields from tomato leaves were the highest in young tissues, with a decreasing trend towards older plant material. This decrease of protein extraction yield was accompanied by a significant increase of arabinose and galacturonic acid content and a decrease of galactose content in the cell walls of old-vs-young tissues. This resulted in strong negative correlations between protein extraction yield and the content of arabinose and galacturonic acid in the cell wall, and a positive correlation between the content of galactose and protein extraction yield. Overall, these results point to the importance of the pectin network on protein extractability, making pectin a potential breeding target for enhancing protein extractability from tomato leaves.

Quantitative characterizations of visual fibrousness in meat analogues using automated image analysis.

A desirable quality of plant-based meat analogues is to resemble the fibrous structure of cooked muscle meat. While texture analysis can characterize fibrous structures mechanically, assessment of visual fibrous structures remains subjective. Quantitative assessment of visual fibrous structures of meat analogues relies on expert knowledge, is resource-intensive, and time-consuming. In this study, a novel image-based method (Fiberlyzer) is developed to provide automated, quantitative, and standardized assessment of visual fibrousness of meat analogues. The Fiberlyzer method segments fibrous regions from 2D images and extracts fiber shape features to characterize the fibrous structure of meat analogues made from mung bean, soy, and pea protein. The computed fiber scores (the ratio between fiber length and width) demonstrate a strong correlation with expert panel evaluations, particularly on a per-formulation basis (r2 = 0.93). Additionally, the Fiberlyzer method generates fiber shape features including fiber score, fiber area, and the number of fiber branches, facilitating comparisons of structural similarity between meat analogue samples and cooked chicken meat as a benchmark. With a simple measurement setup and user-friendly interface, the Fiberlyzer method can become a standard tool integrated into formulation development, quality control, and production routines of plant-based meat analogue. This method offers rapid, cheap, and standardized quantification of visual fibrousness, minimizing the need for expert knowledge in the process of quality control.

Advanced tensile testing as a new tool to quantify properties of food.

Mechanical properties of food products are regularly analysed by tensile tests. The aim of this study was to demonstrate the potential of using advanced tensile testing techniques to better understand the mechanical properties of anisotropic food products, such as meat analogues and certain dairy products. The effects of various tensile testing parameters, including tensile gauge length and deformation rate, on the interpretation of mechanical properties of meat analogues was studied. Additionally, digital image correlation, an image analysis technique, was used for true distance recording and analysis of fracturing behaviour of the products. An isotropic product was prepared from solely soy protein isolate, and an anisotropic product was prepared from soy protein isolate and pectin using the shear cell technology. The tensile properties of the products were studied with four different moulds with varying gauge lengths of 17.5, 15, 11.5, and 8.5 mm, and at three deformation rates of 46.2, 23.1, and 11.6 mm/min. A smaller gauge length and slower deformation rate improved visualization and interpretation of the multi-stage descending branch in force - distance curves of anisotropic products. Additionally, tensile parameters, specifically toughness, proved to be more accurate at small gauge length and slow deformation rate, because overestimation due to rapid crack propagation was prevented. True distance data obtained with digital image correlation further improved the interpretation of the fracturing behaviour of the products. Inhomogeneous strain distribution in anisotropic products was shown with digital image correlation, in contrast to the homogeneous strain distribution observed in isotropic products. Furthermore, the Poisson's ratio, obtained through digital image correlation, explained inherent differences in structure and plasticity between isotropic and anisotropic meat analogues. This study shows the importance of careful selection of testing parameters and techniques. Moreover, it advises the use of digital image correlation for better measurement of fracture mechanics and strain distribution.

Rework Potential of Soy and Pea Protein Isolates in High-Moisture Extrusion.

High-moisture extrusion (HME) is an effective process to make fibrous products that can be used as meat analogues. In this study, the effect of extrusion of already extruded products (i.e., re-extrusion) was tested with the aim to explore the potential of rework in HME. The rework of material is important because it is a route to reduce waste, which is always produced, for example during the start or at the end of a production run. Pea and soy protein isolates (PPI and SPI) were first extruded, then freeze-dried and ground, and extruded again. The visual and textural properties of the fibrous products were evaluated. Also, the rheological properties, solubility, and water-holding capacity (WHC) of the ingredients and the products after the first and second extrusion were quantified. The obtained freeze-dried powders after the first HME cycle had a reduction in solubility of 15% for PPI and 74% for SPI. Furthermore, WHC was reduced by 65% and 17% for PPI and SPI, respectively. After the second HME cycle, the reduction in solubility and WHC was augmented to 22% and 90% for PPI, and 79% and 63% for SPI. No effect on stock and loss moduli after heating and cooling were found, even after two HME cycles. SPI fibrous products did not differ in cutting strength, anisotropy index, or visual appearance after re-extrusion. Only, a decrease in hardness was detected, from 62.0 N to 51.1 N. For PPI, re-extrusion did reduce the cutting force and hardness but not the anisotropy index. It was concluded that even though HME induces a loss of solubility and WHC, this did not affect the fibrous texture formation of the protein. This means that the texture formed during HME does not depend on the process history and that rework is thus possible for fibrous products.

Flavor-protein interactions for four plant proteins with ketones and esters.

The interaction between flavors and proteins results in a reduced headspace concentration of the flavor, affecting flavor perception. We analyzed the retention of a series of esters and ketones with different chain lengths (C4, C6, C8, and C10) by protein isolates of yellow pea, soy, fava bean, and chickpea, with whey as a reference. An increase in protein concentration led to a decrease in flavor compound in the headspace as measured with atmospheric pressure chemical ionization time-of-flight mass spectroscopy (APCI-TOF-MS). Flavor retention was described with a flavor-partitioning model. It was found that flavor retention could be well predicted with the octanol-water partitioning coefficient and by fitting the hydrophobic interaction parameter. Hydrophobic interactions were highest for chickpea, followed by pea, fava bean, whey, and soy. However, the obtained predictive model was less appropriate for methyl decanoate, possibly due to its solubility. The obtained models and fitted parameters are relevant when designing flavored products with high protein concentrations.

Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein ß-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native ß-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with ß-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

XRT for visualizing microstructure of extruded meat replacers.

X-Ray Tomography (XRT) was used to visualize the microstructure of extruded meat replacers. The high moisture extrudates contained lamella, that became visible upon pulling the extrudate apart. In frozen state, these lamella could be visualized with XRT. The freezing increased the density difference between the water-rich and protein-rich layers, thus increasing the contrast obtained in the XRT. Differences in the physical structure were reflected in the measured structure. In non-frozen samples, no lamella were visible, indicating insufficient contrast. Because of the contrast obtained in frozen samples, we conclude that the XRT technique is a valuable addition to investigate extrudate structure, that can be used to quantify differences in extrudates obtained by for example variation in composition. Here we showed a higher lamella thickness for soy protein isolate (SPI) compared to more fibre-rich soy protein samples.

Challenges and solutions of extracting value-added ingredients from fruit and vegetable by-products: a review.

Every year, huge amounts of fruit and vegetable by-products in the food processing factories are produced. These by-products have great potential to be used for different targets especially the extraction of value-added ingredients. The target of this study is to review the challenges of extraction of value-added ingredients from fruit and vegetable by-products on the industrial scale and to describe current trends in solving these problems. In addition, some strategies such as multi-component extraction as well as application of fermentation before or after the extraction process, and production of biofuel, organic fertilizers, animal feeds, etc. on final residues after extraction of value-added ingredients are discussed in this review paper. In fact, simultaneous extraction of different value-added ingredients from fruit and vegetable by-products can increase the extraction efficiency and reduce the cost of value-added ingredients as well as the final volume of these by-products. After extraction of value-added ingredients, the residues can be used to produce biofuels, or they can be used to produce organic fertilizers, animal feeds, etc. Therefore, the application of several appropriate strategies to treat the fruit and vegetable by-products can increase their application, protect the environment, and improve the food economy.

The role of plant age and leaf position on protein extraction and phenolic compounds removal from tomato (Solanum lycopersicum) leaves using food-grade solvents.

The large availability and considerable amount of proteins (approx. 30 % on dry matter) make tomato leaves attractive as a potential new protein source. In this study, the feasibility of extracting proteins and removing phenolic compounds from tomato leaves using food-grade solvents as function of plant age and leaf position was investigated. Water and 50-50 % ethanol-water were used. We found that most proteins (>70 mg/g leaf protein) remained in the pellet after extraction. The protein purity of the dry matter present in the supernatant did not exceed the original leaf protein content. Additionally, leaf position had stronger effect than plant age on the leaf protein content and extraction yield. Ethanol-water was more efficient in removing phenolic compounds than water. The most phenolic compounds was removed from the top leaves. For future processing, the diversity of leaves has to be considered when striving for full utilization of tomato plants (fruits and leaves).

Fibrous Structures from Starch and Gluten.

Starch is added to meat analogues for binding and water holding. In this study, we investigate whether starch can have an additional role as a structuring agent. Therefore, different types of starch were combined with wheat gluten at various amounts and sheared in a High Temperature Shear Cell to determine how starch influences the structuring behavior of gluten-starch blends. The starches were chosen based on their diverse amylose contents, leading to different technological properties. Remarkable differences were found between the starches investigated. The addition of Amioca starch (containing 1% amylose) had a strong negative influence on the ability of gluten to form fibers. Maize starch (25% amylose) and Hylon VII (68% amylose) formed fibrous materials up to high starch additions. The pre-gelatinizing of maize starch further increased the ability of gluten-starch mixtures to form fibrous structures. The influence of the different types of starch on the hardness, deformability, and stiffness of the sheared samples was also assessed, revealing a spectrum of achievable properties through the addition of starch. Most remarkable was the formation of a material with anisotropy in Young's modules. This anisotropy is also found in chicken meat, but not in protein-based fibrous materials. Furthermore, it was observed that the pre-gelatinization of starch facilitated fiber formation. A similar effect of pre-gelatinizing the starch was found when using faba bean meal with added wheat gluten, where fibrous structures could even be formed in a recipe that previously failed to produce such structures without pre-treatment.

Quantifying water distribution between starch and protein in doughs and gels from mildly refined faba bean fractions.

The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We investigated the distribution of water between the protein and starch phase of doughs and heat-induced gels made from air-classified faba bean fractions by developing a method suited for investigation of such multi-component ingredients. The moisture contents of the protein and starch phases in the dough were determined using a method based on partial sorption isotherms of mixed doughs of protein- and starch-rich fractions at high water activity. Water content of the protein phase is higher than that of the starch phase in dough, showing that protein takes up more water than starch at room temperature. Also, the moisture content of the protein phase in the gels was calculated using a model based on the denaturation temperature of legumin. From the experiments and the modelling, it became evident that the moisture content of the protein phase in the gel is lower than the moisture content of the protein phase in the dough, showing the importance of considering moisture migration from the protein to the starch during heating.

Non-linear rheology reveals the importance of elasticity in meat and meat analogues.

The interest in plant-based meat analogues as an alternative to meat is currently growing. Rheological benchmarking is used to reveal how closely meat analogues resemble the original meat products. Texture maps and dissipation colour schemes were used to reveal similarities in and differences between rheological responses of meat and meat analogues (especially chicken analogues). Under heating, meat analogues differ in terms of their lower elasticity compared with heated meat. The changes caused by heating meat and meat analogues were different as well. Heating of meat resulted in a tougher and more elastic material, while heating has a minor effect on meat analogues. Future developments should therefore focus on routes to create more elasticity and possibly allow heating effects on texture to mimic meat characteristics even better.

Thermo-mechanical processing of plant proteins using shear cell and high-moisture extrusion cooking.

Consumption of plant-based meat analogues offers a way to reduce the environmental footprint of the human diet. High-moisture extrusion cooking (HMEC) and shear cell processing both rely on thermo-mechanical treatment of proteins to product fibrous meat-like products. However, the mechanisms underlying these processes are not well understood. In this review we discuss the effect of thermo-mechanical processing on the physicochemical properties and phase behavior of proteins and protein mixtures. The HMEC and shear cell processes are comparable in their basic unit operations, which are (1) mixing and hydration, (2) thermo-mechanical treatment, and (3) cooling. An often overlooked part of the extruder that could be crucial to fibrillation is the so-called breaker plate, which is situated between the barrel and die sections. We found a lack of consensus on the effect of heat on protein-protein interactions, and that the experimental tools to study protein-protein interactions are limited. The different mechanisms for structure formation proposed in literature all consider the deformation and alignment of the melt. However, the mechanisms differ in their underlying assumptions. Further investigation using novel and dedicated tools is required to fully understand these thermo-mechanical processes.

Characteristics of Soy Protein Prepared Using an Aqueous Ethanol Washing Process.

Currently, the predominant process for soy protein concentrate (SPC) production is aqueous ethanol washing of hexane-extracted soy meal. However, the use of hexane is less desired, which explains the increased interest in cold pressing for oil removal. In this study, cold-pressed soy meal was used as the starting material, and a range of water/ethanol ratios was applied for the washing process to produce SPCs. Washing enriched the protein content for the SPCs, regardless of the solvent used. However, we conclude that washing with water (0% ethanol) or solvents with a high water/ethanol ratio (60% and above) can be more advantageous. Washing with a high water/ethanol ratio resulted in the highest yield, and SPCs with the highest protein solubility and water holding capacity. The water-only washed SPC showed the highest viscosity, and formed gels with the highest gel strength and hardness among all the SPCs at a similar protein concentration. The variations in the functionality among the SPCs were attributed to protein changes, although the effects of non-protein constituents such as sugar and oil might also be important. Overall, the aqueous ethanol washing process combined with cold-pressed soy meal created SPCs comparable to commercial SPC in terms of composition, but with varied functionalities that are relevant for novel soy-food developments.

Removal of phenolic compounds from de-oiled sunflower kernels by aqueous ethanol washing.

Selective removal of phenolic compounds (PCs) from de-oiled sunflower kernel is generally considered a key step for food applications, but this often leads to protein loss. PC removal yield and protein loss were assessed during an aqueous or aqueous ethanol washing process with different temperatures, pH-values and ethanol contents. PC yield and protein loss increased when the ethanol content was < 60% or when a higher temperature was applied. Our main finding is that preventing protein loss should be the key objective when selecting process conditions. This can be achieved using solvents with high ethanol content. Simulation of the multi-step exhaustive process showed that process optimization is possible with additional washing steps. PC yield of 95% can be achieved with only 1% protein loss using 9 steps and 80% ethanol content at 25℃. The functional properties of the resulting concentrates were hardly altered with the use of high ethanol solvents.

Functionality of Ingredients and Additives in Plant-Based Meat Analogues.

Meat analogue research and development focuses on the production of sustainable products that recreate conventional meat in its physical sensations (texture, appearance, taste, etc.) and nutritional aspects. Minced products, like burger patties and nuggets, muscle-type products, like chicken or steak-like cuts, and emulsion products, like Frankfurter and Mortadella type sausages, are the major categories of meat analogues. In this review, we discuss key ingredients for the production of these novel products, with special focus on protein sources, and underline the importance of ingredient functionality. Our observation is that structuring processes are optimized based on ingredients that were not originally designed for meat analogues applications. Therefore, mixing and blending different plant materials to obtain superior functionality is for now the common practice. We observed though that an alternative approach towards the use of ingredients such as flours, is gaining more interest. The emphasis, in this case, is on functionality towards use in meat analogues, rather than classical functionality such as purity and solubility. Another trend is the exploration of novel protein sources such as seaweed, algae and proteins produced via fermentation (cellular agriculture).

Alternatives to Meat and Dairy.

The increasing size and affluence of the global population have led to a rising demand for high-protein foods such as dairy and meat. Because it will be impossible to supply sufficient protein to everyone solely with dairy and meat, we need to transition at least part of our diets toward protein foods that are more sustainable to produce. The best way to convince consumers to make this transition is to offer products that easily fit into their current habits and diets by mimicking the original foods. This review focuses on methods of creating an internal microstructure close to that of the animal-based originals. One can directly employ plant products, use intermediates such as cell factories, or grow cultured meat by using nutrients of plant origin. We discuss methods of creating high-quality alternatives to meat and dairy foods, describe their relative merits, and provide an outlook toward the future.

Effect of mechanical interaction on the hydration of mixed soy protein and gluten gels.

Mixed gels of plant proteins are being investigated for use as meat analogues. Juiciness is an important characteristic for the acceptability of meat analogues. The juiciness is assumed to be governed by the hydration properties, or water holding capacity, of the gel (WHC). We analysed the WHC of single-phase gels of respectively soy protein and gluten by applying Flory-Rehner theory. This enabled us to describe the WHC of more the complex mixed gels. The WHC of mixed soy protein - gluten gels is shown not to be a linear combination of their constituents. At high volume fractions, soy forms a continuous network and swells similarly to pure soy without being hindered by gluten. However, increasing gluten content leads to a gradual decrease in soy swelling. This is due to the mechanical interaction between soy and gluten. We propose that gluten-rich gels have a continuous gluten network that entraps soy and hinders its swelling. The elastic moduli of the gluten network were extracted from WHC data, and are in reasonable agreement with experimentally determined moduli. A better understanding of the effect of mixed gel composition on WHC is valuable for the development of the next generation meat analogues.

Modifying Faba Bean Protein Concentrate Using Dry Heat to Increase Water Holding Capacity.

We investigated the effect of dry-heat treatment on the properties of faba bean protein concentrate using soy protein concentrate as a benchmark. While soy protein-widely used as an ingredient in meat replacers-is recovered through a wet fractionation, protein recovery from starch bearing pulses like faba bean can be done via dry fractionation. This process does not require drying or heating steps and therefore, keeps the original protein functionality intact. This results in differences in properties such as water binding capacity of the protein fraction. Faba bean protein concentrate was dry-heated at temperatures from 75-175 °C, which resulted in higher water-holding capacity and less soluble protein, approaching values of soy protein concentrate. These changes were due to partial denaturation of protein, changing the structure of the protein, and exposing hydrophobic sites. This led to protein aggregation, as observed by light microscopy. Only noncovalent bonds caused the decrease of solubility of dry-heated faba bean protein concentrate. We conclude that dry-heating of dry fractionated faba bean protein can change the functional properties of the protein fraction to desired properties for certain applications. The effect is similar to that on soy, but the underlying mechanisms differ.

Oxidative stability of soy proteins: From ground soybeans to structured products.

The production of soy protein-based foods requires multiple-step, intensive processing and storage of soy ingredients, which can increase the product's susceptibility to oxidation. Therefore, we investigated the oxidative stability of soy protein-based products subjected to different relevant conditions or treatments: over storage of soy flours, over fractionation to yield soy protein isolate (SPI), and over subsequent thermomechanical processing to yield a model structured product. Soy flours were stable to lipid and protein oxidation over 250 days storage in chilled or ambient conditions. The fractionation process applied to make SPI did not increase substantially protein carbonylation, but increased surface-exposed hydrophobicity and decreased free thiols, compared to the starting defatted flour. Subsequent processing of hydrated SPI powder at 140 °C further increased protein carbonylation to a high extent. Therefore, we conclude that soy flours can be stable over long storage times, but processing to yield structured foods products promote protein oxidation.