Opportunities and challenges of plant proteins as functional ingredients for food production

Significance

Growing demand for sustainable food has intensified interest in plant-based proteins as functional ingredients. By comprehensively analyzing the chemical composition, amino acid profiles, and functional properties of commercial plant protein ingredients, we provide insights into their suitability for food applications, influenced by both protein sources and ingredient processing. The variability in functional properties, even among batches, complicates standardized end-product manufacturing in the food industry. Our research emphasizes the importance of understanding the relationship between structure, processing, functionality, and applicability, which requires interdisciplinary innovation. This research contributes to the emerging field of plant-based food technology, paving the way for the sustainable production of high-quality, protein-rich food that aligns with evolving dietary preferences and global sustainability goals.

Abstract

Consumer interest in meat and dairy alternatives drives demand for plant-based protein ingredients. While soy and gluten dominate the market, there is a trend to explore alternative crops for functional ingredient production. The multitude of ingredients poses challenges for food manufacturers in selecting the right protein. We investigated 61 commercially available protein ingredients from various sources, categorizing them based on their protein content into protein-rich flours (protein content less than 50%), protein concentrates (protein content between 50% and 80%), and protein isolates (protein content higher than 80%). Methionine, cysteine, and lysine were decisive for the amino acid score, which even varied between ingredients produced from the same raw material. Such differences were also observed in the protein solubility profiles, characterized by their raw material–specific protein pattern. By focusing on soy, pea, and fava bean ingredients, the broad spectra of emulsifying and foaming properties were illustrated. These ranged from non–emulsifying and non–foaming to high emulsifying capacities of 737 mL/g ingredient and foaming activities of 2,278%, accompanied by a foam stability of 100%. Additionally, we demonstrated that the functionality of ingredients obtained from different batches could vary by up to 24% relative SD. Protein solubility, powder wettability, color, and particle size were determined as key properties for the differentiation of soy, pea, and fava bean protein ingredients by principal component analysis. In our study conclusion, we propose essential measures for overcoming challenges in protein ingredient production and utilization to realize their full potential in fostering sustainable and innovative plant-based food production.

Plant-based meat and dairy alternatives have become increasingly important due to growing consumer demand for sustainable food and a shift toward plant-based diets (1, 2). In these alternatives, plant proteins substitute traditional animal-sourced proteins (2, 3). While soy and gluten ingredients have long dominated the plant protein market (4), there has been a rising trend to explore other plants as sources of proteins (5). Crops such as peas, fava beans, canola, rice, sunflower, and others are gaining traction as viable protein sources due to their availability, nutritional and sensory profiles, and functional properties, encompassing physicochemical and techno-functional characteristics (6). This diversification not only provides options for individuals with specific dietary restrictions or preferences but also offers a wider range of functional properties that can be used in food product development (7).

Commercial protein ingredients are classified as flours, concentrates, and isolates based on their protein content. Soy protein isolates contain over 90% proteins, concentrates have 65 to 90%, and protein-rich flours range from 50 to 65%, with a strict definition existing only for soy ingredients (8). Protein concentrates and isolates are produced using dry and wet fractionation processes. Dry fractionation involves grinding followed by sieving or sifting to separate larger starch particles from smaller protein bodies (9) and is mainly employed for protein- and starch-rich materials. Although it can also be used for oil-rich crops such as soy and lupine, while providing similar protein enrichment, much lower product quantities are obtained than with starch-rich materials (10). The increasing market presence of dry fractionated ingredients is attributed to their resource and cost efficiency, sustainability, and the elimination of a final drying step (11, 12). The gentle production process also enhances their functionality, primarily attributed to the degree of denaturation of the protein fraction (12). It is noteworthy that the use of thermal treatment for debittering before fractionation is sometimes used in industry and can impact functionality (4, 13).

Wet processes are more commonly applied to produce protein concentrates from oil-rich grain legumes and oilseeds. Soluble components such as carbohydrates or hydrophilic secondary plant metabolites are removed from defatted meals or press cakes using methods like acidic extraction, aqueous ethanolic extraction, or moist heat denaturation. The latter method immobilizes the proteins through denaturation (14, 15). For protein isolate production, proteins are usually extracted under alkaline conditions during wet fractionation, with additional steps such as acidic pre-extraction to enhance protein purity. Solubilized proteins are concentrated by ultrafiltration or isoelectric precipitation and finally dried (16). However, wet fractionation requires substantial amounts of water and energy, which conflicts with the growing importance of sustainability in consumer decision-making.

Protein ingredients exhibit differences not only in composition, encompassing total protein content, accompanying substances such as carbohydrates, and secondary plant metabolites, but also in their protein pattern (17). The protein fractions, categorized by their solubility according to Osborne (18), include albumins (soluble in demineralized water), globulins (soluble in dilute salt solutions), prolamins (soluble in (aqueous) alcohol), and glutelins (soluble in dilute acids or alkaline solutions) (19). Albumin and globulins are predominantly present in grain legumes, oilseeds, and tubers, while prolamins and glutelins make up the majority of the total protein in cereals (20). Globulins are further classified as legumin-like or vicilin-like based on the main pea protein fractions' nomenclature, or known as the 11S and 7S fractions, respectively. Both fractions are present in grain legumes independently of the source (21, 22). The structural organization of globulin proteins is intricate and dependent on environmental conditions like ionic strength or pH. Under certain conditions, their association can lead to the formation of larger aggregated states, affecting their solubility and interfacial properties (23, 24). Therefore, variations in functional performance can be anticipated in diverse environmental conditions.

Various raw materials and processing techniques, yielding diverse product types, contribute to ingredients with a wide range of nutritional profiles and functional properties. Even products from the same raw material, but different batches, can exhibit distinct functional properties, influenced by factors such as the botanical origin/plant variety and cultivation conditions, as well as variations in process parameters during ingredient production (25–27). The underlying causes of these differences remain insufficiently investigated. Current research often focuses on individual processing methods (28, 29), evaluating their impact on functionality, or analyzing specific attributes using different methods (27). This complexity complicates the food industry's task in identifying the most suitable protein options for their specific needs.

Understanding the composition as well as functional properties of plant protein ingredients, is crucial for their successful incorporation into foods. This study analyzed 61 commercially available protein ingredients using standardized methods to allow direct comparison of their protein, fat, and ash content and solubility behavior. Focusing on pea, soy, and fava bean, we explored further functional properties and uncovered correlations among them. This analysis aims to illuminate the complexity and challenges associated with plant proteins. The article also presents future perspectives including supply chain considerations, ingredient availability, sustainability, and cost implications, providing a holistic understanding of the challenges and opportunities in the plant protein ingredient market.

Results and Discussion

The 61 protein ingredients were derived from oilseeds (16%), cereals (12%), tubers (5%), and grain legumes (67%) with soy, pea, and fava bean being the major raw material sources (Fig. 1A). Grain legume ingredients, especially soy protein, have long been easily accessible due to their established commercial availability. In recent years, pea and fava bean proteins, marketed intensively, have gained popularity as non-genetically modified organisms (non-GMO), allergen-free, and gluten-free alternatives. However, sourcing products from less common raw materials, like chickpeas, lentils, and lupines, proved challenging, likely due to limited cultivation driven by relatively low market demand. Although protein-rich by-products from oilseeds are abundant in the form of press cakes, the challenge is to enrich protein while reducing undesirable phytochemicals such as polyphenols. However, the commercialization of oilseed protein ingredients such as sunflower, hemp, and linseed is underway. Rice protein ingredients extracted from rice bran or endosperm and potato protein ingredients, obtained from potato fruit juice following starch extraction, are also available on the market, but only a few companies are currently processing these raw materials for food protein production.

Fig. 1.

Overview (A, n = 61) and protein contents (B) of plant protein flours, concentrates (conc.), and isolates (iso.). Mean values of individual ingredients are displayed as data points to the Left of the box plots (different batches of the same ingredient are marked in green). For sample sizes for Fig. 1B, see SI Appendix, Table S1.

Protein Content of Plant Protein Ingredients.

Protein content was evaluated using a standard nitrogen-to-protein conversion factor of 6.25, which is consistent with the factor used in the specifications and labeling of commercial protein ingredients and was, therefore, chosen to ensure comparability. However, it should be noted that this approach is likely to overestimate protein contents of plant protein ingredients (30). According to the protein content, we categorized the ingredients into high protein flours (less than 50% protein), protein concentrates (protein content between 50% and 80%), and protein isolates (protein content higher than 80%) (Fig. 1B) differing from the classification for soy ingredients, but in line with commercially available product specifications. Of the total ingredients studied, protein flours, concentrates, and isolates accounted for 10%, 41%, and 49%, respectively. Additional information on the chemical composition of the ingredients (dry matter, ash, and fat content) is provided in supporting information (SI Appendix, Table S1).

Oilseeds.

Protein ingredients derived from defatted press cakes carry protein contents of 35 to 65%, depending on the type of raw material (14). Variations in protein contents observed in our study, such as 49 to 71% for hemp, 36 to 47% for linseed, and 50 to 62% for sunflower ingredients, could primarily be explained by differences in the fat content of the ingredients (SI Appendix, Table S1). Different pressing methods or the addition of further deoiling steps, such as solvent extraction, to remove residual oil from the press cake may account for the differences in fat content.

Cereals.

Commercially available cereal protein ingredients from rice and wheat exhibited much higher protein contents compared to oilseed ingredients, but similar to that of grain legume isolates. This is attributed to the protein fractions in both cereal and grain legume isolates being extracted through wet fractionation, enabling higher protein purity. Rice protein ingredients, with protein contents ranging from 83 to 92%, presumably employ conventional extraction methods such as wet milling, alkaline extraction, or enzymatic hydrolysis. Enzymatic hydrolysis addresses the low solubility of rice proteins in water, primarily due to the high glutelin content (31). The examined wheat ingredients had comparable protein contents ranging between 84% and 87%. Wheat gluten (gliadin and glutenin), a by-product of wheat processing yielding starch as the main product, is produced through washing processes that separate gluten and starch. Gluten is unique due to its viscoelastic and adhesive properties, which differentiate its usage from other plant-based protein ingredients (32).

Potato.

Potato proteins exhibited protein contents ranging from 93 to 100%. Notably, variations in ash content, ranging from 0 to 14% (SI Appendix, Table S1), suggest the presence of nonprotein constituents, despite the given protein contents being close to 100%. Potato proteins consist of two main fractions: patatin, constituting up to 40% and classified as a globulin, and protease inhibitors, making up about 50% (33, 34). Both fractions are commercially available in isolated forms, providing options for patatin-rich and protease inhibitor-rich ingredients (35). The native potato proteins of the patatin fraction, in particular, are described as exhibiting favorable functional properties, making them attractive for use in food applications (34).

Grain legumes.

Grain legume ingredients were derived from oil-rich grain legumes like lupine and soy, and starch-rich grain legumes such as chickpea, fava bean, lentil, and pea. For soy ingredients, three levels of protein purity were found: one concentrate with 50% protein, three concentrates with 71 to 74% protein, and four isolates with 92 to 94% protein. Variations in protein content among concentrates suggest differences in processing methods. The lowest purity concentrate likely originated from defatted soybean meal, while further concentration (71 to 74%) involves targeted removal of soluble compounds, particularly carbohydrates, most likely through wet fractionation (acidic or aqueous alcohol extraction). Soy protein isolates like isolates from other grain legumes are typically produced by aqueous or mildly alkaline extraction followed by isoelectric precipitation, resulting in an enrichment of globulin content (15, 36).

Variations in purity were observed among lupine (94%), fava bean (91 to 93%), and pea protein isolates (82 to 88%). Since similar nitrogen-to-protein factors are described for these grain legumes, the use of the general factor of 6.25 cannot account for these differences. Thus, the differences can be attributed to variances in raw material composition and processing. Starch-rich grain legumes are usually not defatted prior to protein extraction, resulting in fat accumulation in the isolate and a decreased total protein content (25). Our study quantified fat contents of 6 to 8% for fava bean protein isolates and 8 to 10% for pea protein isolates (SI Appendix, Table S1) comparable to previous work (25, 37). Disparities in ash content (4 to 16%) suggest variation in extraction parameters, with high pH extractions increasing salt content after neutralization (38). Protein concentrates from fava beans, peas, and chickpeas showed protein contents of 55 to 67%, 51 to 55%, and 44 to 70%, respectively. The protein enrichment in the fava bean and pea concentrates aligned with values reported for dry fractionated concentrates, where the higher protein content in fava bean concentrates can be attributed to more favorable seed characteristics, in particular to larger starch granules (10, 39). Conversely, the wide range of protein contents (44 to 70%) and the differences in fat contents (9 to 23%) of the chickpea concentrates indicate the use of both dry and wet fractionation processes (39).

Amino Acid Score of Plant Protein Ingredients.

Optimizing plant-based diets to meet human nutritional requirements necessitates a thorough understanding of the nutritional composition of plant protein ingredients. Unlike animal proteins, plant-based alternatives often lack some indispensable amino acids (40), which are present in lower ratios compared to reference profiles. Scoring patterns, established by the Food and Agriculture Organization (FAO) to align with amino acid requirements across different age groups and the recommended daily protein intake, guide this assessment. Fig. 2 depicts the amino acid scores (AAS) and identified limiting amino acids in the investigated protein ingredients based on current FAO recommendations for all population groups except infants [preschool child, 2013, SI Appendix, Tables S2 and S3, (41)]. Additional AAS based on further reference patterns and the amino acid composition of the plant protein ingredients are provided in SI Appendix, Tables S2–S4.

Fig. 2.

Amino acid score and limiting amino acids of plant protein flours, concentrates (conc.), and isolates (iso.). Mean values of individual ingredients are displayed as data points to the Left of the box plots (different batches of the same ingredient are marked in green). (Lys: lysine; Val: valine; Met: methionine). For sample sizes, see SI Appendix, Table S1.

Grain legume–derived proteins displayed expected limitations in the indispensable amino acids cysteine and methionine, while lysine limitations were notable in cereal and oilseed ingredients (42). Potato protein demonstrated the most complete coverage of indispensable amino acids, with the AAS ranging from 0.88 to 1.01, showing lower ratios for valine (n = 2) or cysteine and methionine (n = 1), in line with a previous report (43). The lowest AAS was observed for wheat (gluten) ingredients, consistent with reported compositional characteristics (44). Among grain legumes, soy, chickpea, and pea concentrates exhibited the highest AAS reaching up to 0.84, up to 0.79, and up to 0.77, respectively. Similar findings were reported for chickpea and soy (45, 46). Clear differences in limiting amino acid content within ingredient categories led to AAS fluctuations of up to 27% (e.g., pea concentrates ranging from 0.50 to 0.77). These variations could be due to different manufacturing processes enriching distinct protein fractions or reducing amino acid content, potentially due to elevated thermal treatments (42). Raw material differences, influenced by factors like climate, location, soil diversity, agricultural practices, and varietal distinctions, may also contribute to these observations (42), as evidenced by distinct variations between different production batches of the same pea (0.43 to 0.51) and potato (0.93 to 1.01) isolates.

To address amino acid limitations, grain legumes, low in methionine but high in lysine, can be combined with cereals rich in methionine but deficient in lysine. Lysine contents above the reference level were observed for all grain legume ingredients except for the lupine protein isolate (SI Appendix, Fig. S1). Furthermore, sunflower, rice, and potato protein ingredients showed cysteine and methionine contents higher than the reference. Through ingredient blending, it could thus be possible to fully compensate for the limitations or to require only a slight increase in total protein intake to provide sufficient amounts of all indispensable amino acids (47). However, to ensure the nutritional value of protein, consideration should be given not only to amino acid composition but also to the digestibility and bioavailability of protein and amino acids. Recent research indicates that postprandial plasma concentration of certain amino acids may decrease after consuming plant protein blends, even with optimized compositions (48). Additionally, antinutritive compounds such as phytic acid and trypsin inhibitors may interfere with the absorption of divalent cations during digestion or affect the protein´s bioavailability (49). Processing methods, in particular heat and/or alkaline treatments, can induce chemical changes like Maillard reactions or oxidation, thereby significantly reducing bioavailability (50–53). In our study, we did not differentiate between active and inactive forms of amino acids, indicating a need for further investigation in future studies to fully understand these aspects.

From Chemical Composition to the Ingredients’ Functional Properties.

To elucidate the relationships between different functional properties, we first considered the protein solubility of all examined protein ingredients. Subsequently, the emulsifying and foaming properties of soy, pea, and fava bean are employed to highlight raw material and ingredient-dependent variations. Additional parameters including powder wettability, color, oil binding capacity, and particle size were also analyzed and included in the Spearman rank correlation analysis to reveal correlations between the individual properties of the grain legume ingredients.

Protein Solubility of Plant Protein Ingredients.

Protein ingredient solubility, notably considered as resolubility, is a crucial property that contributes to diverse functional characteristics, particularly interfacial properties at oil–water or air–water interfaces (54). It is important to note that, strictly speaking, a distinction should be made between the solubility and dispersibility of protein components (55). The analysis employed here, which assesses the nitrogen content in the supernatant, does not allow for differentiation between dispersed and soluble proteins. Therefore, the term ‘protein solubility’ will continue to be used hereafter. Fig. 3 depicts protein solubility profiles across the pH range of 3.0 to 8.0 for oilseeds, cereals, potato, and grain legumes, revealing significant variation among individual ingredients.

Fig. 3.

Protein resolubility curves across the pH range of 3.0 to 8.0 of protein ingredients derived from oilseeds (A), cereals (B), potato (C), and grain legumes (D–H). Filled and empty dots represent protein isolates and flours/concentrates, respectively. Dots are connected for better visibility. Values are means ± SD (n = 3).

Oilseeds.

Protein solubility in oilseed ingredients is notably low in the acidic and slightly acidic range (5 to 22% at pH 3.0 to 6.0), with all sunflower and one linseed ingredient exhibiting increased solubility in the more alkaline region (50 to 70% at pH 8.0 for sunflower and up to 40% for linseed). Differences in the solubility of the two linseed ingredients could be attributed to variations in the oil extraction processes. The nearly insoluble ingredient had an average fat content of 14%, whereas the more soluble preparation had a residual fat content of 0.6%. The extremely low residual fat content suggests the use of solvent extraction or a combination of cold pressing followed by solvent extraction for oil removal, while the residual fat content of 14% indicates mechanical pressing, likely at higher temperatures, resulting in partial protein denaturation and hence poorer resolubility. Hemp ingredients consistently exhibited low solubility (4 to 16%) across the entire pH range.

Cereals.

The solubility curves of three rice proteins showed very low solubility across the investigated pH range. Two ingredients consistently had extremely low solubility, remaining below 5% regardless of pH. The third ingredient exhibited improved solubility, ranging from 15 to 21%, probably indicating partial enzymatic hydrolysis during processing (31). In contrast, wheat (gluten) ingredients displayed a distinct solubility profile, with high solubility in the acidic range, reaching up to 80% at pH 3.0, and lower solubility at pH levels of 6.0 and higher. This distinction is attributed to the composition of protein fractions in gluten products (44).

Potato.

Two distinct solubility profiles were observed for potato ingredients. Two batches of the same ingredient exhibited a similar U-shaped curve, with a minimum in the pH 4.0 to 5.0 range, typical for globulin-rich proteins. This indicates a patatin-rich ingredient (33, 35). In contrast, the third ingredient showed a strongly deviating solubility profile with the highest solubility in the acidic range. This aligns with reported results for the protease inhibitor-rich fraction of potato protein, known to have an isoelectric point of 8.0 and a strong positive charge under acidic conditions (35).

Grain legumes.

All grain legume ingredients displayed the characteristic U-shaped curves. These curves demonstrated elevated solubility at strongly acidic and alkaline pH levels, corresponding to the negative and positive net charge of globulins, respectively. Low protein solubility was observed in the isoelectric range between pH 4.0 and pH 6.0, corresponding to the isoelectric points of 7S and 11S globulins (56).

A notable variability in solubility is evident among commercial grain legume ingredients, even within the same raw material category, with more pronounced differences in the alkaline compared to the isoelectric region. For pea ingredients, solubility ranged from 6 to 65% at pH 7.0 and 9 to 79% at pH 8.0. Even three batches of the same pea protein ingredient exhibited a wide variation in protein solubility at pH 8.0 (Fig. 3G), with values of 14%, 21%, and 26%, representing a relative SD (%RSD) of 24%. Soy preparations exhibited solubility between 12% and 61% at pH 8.0, while fava beans displayed an even wider range from 23 to 100% at pH 8.0. Although the exact production processes for each protein remain unknown, variations in solubility profiles are likely attributed to differences in the processing of raw materials, even for the same protein ingredients. For instance, proteins undergo partial denaturation during alkaline extraction and isoelectric precipitation, intensifying with a greater shift in pH (57), implying that a higher degree of denaturation corresponds to a higher irreversible aggregation and, therefore, a lower solubility. Additionally, in the case of wet fractionation, the drying process can significantly influence protein solubility, as drying conditions, particularly spray drying, affect both surface hydrophobicity and particle size of the protein ingredients (58). Both factors influence the functional properties, as corroborated by our correlation analyses for particle size.

The smallest variations in protein solubility occurred in the range of the lowest solubilities, especially notable for soy protein isolates and concentrates with 27% RSD. More pronounced fluctuations in this area, indicated by a 43% RSD, were observed for fava bean ingredients. The soy protein ingredients likely underwent aqueous treatment given their lack of starch, removing all soluble compounds, including albumins. Fava bean protein ingredients, primarily concentrates, were probably obtained through dry fractionation, indicating reduced protein denaturation and simultaneous enrichment of various protein fractions (albumins and globulins) and, thus, might explain the greater variation in the isoelectric region.

A significant correlation was found between protein solubility and powder properties of protein ingredients. Spearman rank correlation analysis of grain legume ingredients revealed a significant positive correlation between protein solubility and powder wettability (SI Appendix, Fig. S2). This correlation was particularly pronounced for protein concentrates and isolates at pH 7.0, with correlation factors of ρ = 0.83 and ρ = 0.34, respectively. Additionally, larger powder particles resulted in lower protein solubility at pH 7.0 for concentrates (correlation factors of ρ = −0.51 and ρ = −0.41 for Dx (90 µm) and span, respectively).

Emulsifying Properties of Soy, Pea, and Fava Bean Ingredients.

Proteins stabilize oil–water interfaces due to their amphiphilic nature and cohesive layer formation through protein–protein interactions upon adsorption (59). Fig. 4A illustrates the emulsifying capacities of soy, pea, and fava bean ingredients in oil-in-water emulsions. Emulsifying capacity varied with pH, being lower at pH 4.0 (near the isoelectric point) than at pH 7.0, consistent with previous findings (60, 61). During initial emulsification stages, protein particle diffusion and adsorption are controlled by protein solubility, charge, and flexibility (60). Differences between ingredients were more pronounced at pH 7.0, attributed to increased solubility enhancing the flexibility of soluble proteins in stabilizing oil–water interfaces (62).

Fig. 4.

Emulsifying capacity (A) and foaming activity (B) of soy, pea, and fava bean protein concentrates (conc.) and isolates (iso.). Mean values of individual ingredients are displayed as data points to the Left of the box plots (different batches of the same ingredient are marked in green). Different letters indicate significant differences on a P ≤ 0.05 level basis.

To analyze emulsification factors, distinctions were made between protein isolates and concentrates, recognizing that concentrates may form Pickering emulsions with insoluble particles, leading to distinct stabilization mechanisms (63). At pH 4.0, minimal differences were observed, with some ingredients (n = 11) falling below the method´s detection limit of 125 mL/g ingredient. However, at neutral pH (7.0), differences between isolates and concentrates became more pronounced, resulting in a wider range of emulsifying capacities. Concentrates showed a broader range at pH 7.0 (203 to 737 mL/g) compared to isolates (328 to 673 mL/g) at pH 7.0. This suggests that protein isolates' emulsifying capacities are primarily influenced by the protein itself, while nonprotein compounds in concentrates, along with processing and raw material variations, play a significant role. When comparing isolates at pH 7.0, soy, pea, and fava bean showed similar emulsifying capacities (500 to 650 mL/g) attributed to globulin enrichment and removal of accompanying substances during wet fractionation.

At both pH values, isolates exhibit higher emulsifying capacity than concentrates within the same grain legume species. Emulsification is strongly influenced by protein surface hydrophobicity and charge (22). The exposure of hydrophobic amino acid residues at the protein surface, facilitated by the partial unfolding during isolate production, is a prerequisite for protein adsorption to oil droplets through hydrophobic interactions (64).

Spearman’s rank correlation revealed a moderate correlation between emulsifying capacity and protein solubility at pH 7.0 for isolates (ρ = 0.45), with an improved correlation for concentrates at the same pH (ρ = 0.62) (SI Appendix, Fig. S2). In concentrate processing, where proteins maintain their native state, the impact of protein solubility on emulsification is more pronounced. The intensive processing involved in isolate production magnifies the influence of other protein characteristics like surface hydrophobicity and molecular flexibility, overlapping the effects of solubility (64). This suggests a need to re-evaluate protein solubility as a general prerequisite for emulsification. As the protein solubility of grain legume proteins is generally lower at pH 4.0, particle size [(Dx (90 µm)] (ρ = −0.60), oil binding capacity (ρ = 0.51), and powder wettability (ρ = 0.70) became more decisive in emulsification at pH 4.0. A larger particle size [Dx (90 µm)] is negatively correlated with emulsifying capacity, indicating that powders with larger particles exhibit poorer emulsification performance. However, when considering the correlations between protein solubility and emulsifying, and later also foaming properties, it must be emphasized that no distinction is made between colloidally dispersed protein and soluble, fully dissolved protein (55), potentially resulting in lower correlations. This is because soluble proteins play a superior role in these functionalities.

At pH 7.0, a moderate correlation (ρ = 0.46) was observed between emulsifying capacity and powder wettability. Positive correlations between powder wettability and protein solubility were found at both pH values. These correlations emphasize the essential role of hydration properties, particularly protein ingredient wettability, in achieving high emulsifying capacities, especially at low pH. The results suggest a complex interplay between proteins, nonprotein constituents (e.g., starch and fibers), and the flexibility of proteins at the oil–water interface.

Foaming Properties of Soy, Pea, and Fava Bean Ingredients.

Foaming activity.

Foam formation, structure, and stability are influenced by various factors. Fig. 4B illustrates the foaming activity of individual grain legume protein ingredients. Values range from 0% (no foam formation) to 1,132% and 2,278% at pH 4.0 and pH 7.0, respectively. One fava bean product exhibited exceptional foaming activity – 1,132% at pH 4.0 and 2,278% at pH 7.0 - while protein solubility was not exceptionally higher compared to the other fava bean protein ingredients. Notably, pH variations did not result in significant differences in foam activity. However, mean values showed a trend toward higher foaming activity at pH 4.0, particularly for protein isolates.

Protein concentrates exhibited slightly higher foam activities at pH 7.0 compared to pH 4.0, indicating the presence of additional components, besides proteins, influencing foaming activity. This underscores that protein content does not correlate with foaming activities at either pH value, as observed in the Spearman’s rank correlation analysis. It is evident that the protein content alone does not exclusively determine foam formation; rather the presence of soluble or hydratable components is crucial. Protein solubility, a fundamental criterion for selecting foaming agents, determines the diffusion rate and available protein (65). However, a significant correlation between foaming activity and protein solubility could not be found at pH 4.0 and only a moderate correlation was noted at pH 7.0 for all grain legume ingredients (ρ = 0.31), consistent with literature (60) (SI Appendix, Fig. S2). The correlation improved to ρ = 0.61 when focusing on protein concentrates at pH 7.0. Most protein isolates exhibited better foaming properties at pH 4.0, close to the isoelectric point, where reduced surface net charge and accelerated adsorption kinetics enhance foam formation (65, 66). However, in concentrate processing, where proteins likely remain in their native state, the role of protein solubility in foam formation becomes more prominent. Fava bean concentrates, commonly produced by dry fractionation, showed a high correlation between foaming activity and protein solubility (ρ = 0.69) at pH 7.0. Thus, factors beyond solubility, such as molecular flexibility and hydrophobicity, are crucial for foaming (60).

Furthermore, a robust correlation was observed between foaming activities at pH 4.0 and pH 7.0, with ρ = 0.85 for all grain legume protein ingredients and ρ = 0.94 for all concentrates. However, the multitude of factors influencing foam properties poses a challenge in distinctly identifying raw material-specific effects on these properties.

Foaming stability.

Proteins maintain foam by stabilizing interfaces, forming cohesive films, and retaining liquid in foam lamellae (SI Appendix, Fig. S3A). Despite varying stability (from rapid collapse to nearly 100%), isolates and concentrates showed challenges in detecting significant differences. Soy preparations consistently exhibited superior stability compared to pea and fava bean ingredients. On examining foams with at least 100% foaming activity, soy ingredients showed minimal stability variation (70 to 98%) at both pH values, in contrast to pea (3 to 97%) and fava bean protein ingredients (4 to 98%). This difference may arise from soy's lower fat content compared to pea and fava bean (SI Appendix, Table S1), as lipids could interfere with the adsorbed protein layer at the air/water interface, reducing foaming stability (65).

The foaming stability of the examined protein ingredients, particularly soy-derived ones, showed minimal dependence on pH values. Pea and fava bean concentrates exhibited a slight improvement in foaming stability at pH 4.0. Near the isoelectric point, reduced electrostatic repulsion allowed for a higher packing density through protein–protein interactions, forming closely packed, thick layers (66). This denser adsorbed protein layer likely provides steric stabilization, preventing bubble rupture and yielding a more stable foam. Conversely, electrostatic repulsion can hinder contact between adjacent bubbles, preventing foam destabilization through coalescence (65, 66).

Spearman’s rank correlation revealed moderate correlations between emulsifying capacity and foaming stability at pH 4.0 (ρ = 0.50) and pH 7.0 (ρ = 0.40) (SI Appendix, Fig. S2). This implies the influence of similar factors, including surface hydrophobicity, protein folding, molecular flexibility, surface charge, and hydrophilic/lipophilic balance, on both foaming and emulsion properties. A previous study has also demonstrated correlations between foaming and emulsifying properties (67).

Foaming stabilities at pH 4.0 and pH 7.0 exhibited strong correlations: ρ = 0.80 for all grain legume protein ingredients, ρ = 0.84 for all protein isolates, and ρ = 0.69 for all protein concentrates. This suggests that effective foaming agents at pH 4.0 also perform well at pH 7.0 and vice versa. However, high foaming activity does not guarantee high foaming stability. Among grain legume raw materials, soy protein ingredients showed a strong correlation between foaming activity and foaming stability at pH 4.0 (ρ = 0.91) and at pH 7.0 (ρ = 0.97). In the case of pea and fava bean protein ingredients, no significant correlations or only moderate correlations, ρ = 0.30 at pH 7.0 for pea and ρ = 0.66 at pH 4.0 for fava bean, were demonstrated.

Foam density.

Foam density, a crucial parameter for characterizing foam structure and quantifying protein and water content in the foam lamellae, is an indicator for gas entrapment. SI Appendix, Fig. S3B illustrates immediate postfoaming foam density measurements. In all three grain legume species, certain protein ingredients showed undeterminable foam densities due to low foaming activity and stability. At pH 4.0, foam densities ranged between 90 and 177 g/L for pea, 123 and 245 g/L for soy, and 84 and 287 g/L for fava bean products, with most falling within 120 to 200 g/L, except for one fava (287 g/L) and one soy (245 g/L) concentrate. Elevating pH to 7.0 increased densities, aligning with previous findings (68). At pH 7.0, increased protein solubility may lead to more proteins in the lamellae, fostering the formation of smaller bubbles with larger interfacial surfaces, thus, creating denser foams (69). Additionally, lamella thickness influences foam density, with greater protein repulsion at pH 7.0 causing thicker lamellae due to increased distance between adsorbed proteins (66, 69).

The foaming stability results indicate that proteins with high foam densities may not consistently form stable films. Spearman’s rank correlation revealed no significant association between foam density and foaming stability for all grain legume protein ingredients. Moreover, a significant negative correlation between foam density and foaming activity was observed for all grain legume protein ingredients: ρ = −0.42 at pH 4.0 and ρ = −0.25 at pH 7.0, with a more pronounced effect for the concentrates: ρ = −0.51 at pH 4.0 and ρ = −0.68 at pH 7.0 (SI Appendix, Fig. S2). This implies that higher foaming activity is linked to lower foam density, consistent with existing literature (70). Larger gas entrapment enables higher foaming activities with the same initial volume of protein suspension.

Pea isolates from different batches showed less variation in foam properties compared to the entire set of analyzed pea isolates. The %RSD within batches ranged from 7 to 19% for foaming activity, 6 to 9% for foaming stability, and 3 to 9% for foam density. In contrast, across all pea isolates, %RSD was higher: 49 to 51% for foaming activity, 63 to 92% for foaming stability, and 13 to 37% for foam density.

The grain legume ingredients investigated displayed similar foaming behavior due to the presence of comparable protein fractions. Furthermore, it was demonstrated that accompanying substances, process parameters, and factors like pH value influence foaming properties, pose challenges in predicting a reliable foaming or nonfoaming agent.

Exploring Functional Differentiation of Soy, Pea, and Fava Bean Ingredients.

Differences in the composition and properties of protein ingredients can significantly impact their suitability in alternative products. Principal component analysis (PCA) identified key properties and relationships between soy, pea, and fava bean protein ingredients. Examined properties (P ≤ 0.05) were visualized using PCA score and loading plots (Fig. 5 and SI Appendix, Fig. S3), where the first three PC explained 62.5% of the total variation. PC1 (35.9%) was influenced by protein solubilities, powder wettability, color (L*-, a*-, b*-values), and particle size [Dx (90 µm)]. PC2 (17.8%) contribution was provided by protein content, AAS, particle size (span), and foaming properties such as foaming stability and foam density at pH 7.0 (Fig. 5), while protein content, protein solubility at pH 3.0, and foaming activity and foam density at pH 4.0 and pH 7.0 were involved in PC3 (8.8%) (SI Appendix, Fig. S4). Despite differences in the functional properties, there was a raw material-specific grouping of the protein ingredients. However, some protein ingredients fall into the group of other raw materials, as processing-specific factors also play a role in functionality in addition to raw material-specific effects. For example, protein solubility was higher in pea concentrate (Pea 13) than in pea isolates, resulting in the pea concentrate being classified in the group of fava bean ingredients, which are generally characterized by high protein solubility.

Fig. 5.

Principal component analysis score and loading plot (PC1 and PC2), describing relationships and variations in functional properties of soy, pea, and fava bean protein ingredients. Filled and empty dots represent protein isolates and concentrates, respectively (SOL: protein solubility; WET: powder wettability; Protein: protein content; FA: foaming activity; FS: foaming stability; FD: foam density; OBC: oil binding capacity; EC: emulsion capacity; AAS: amino acid score).

Soy.

The centered position of soy protein ingredients in the score plot complicated the correlation of individual properties, except for Soy 2 (Fig. 5). Soy 2's distinct position arose from its notable low protein solubility (14% at pH 7.0) and low powder wettability [17 s/(0.02 g/cm²)] in contrast to the other soy protein ingredients with mean values of 39% and 865 s/(0.02 g/cm²), respectively. Considering the previously described contribution of solubility and hydration properties to foam formation, Soy 2 exhibited a significant negative correlation with the foam properties. Additionally, the high proportion of accompanying substances of almost 50% strongly affected the functional properties. The other soy protein ingredients, besides Soy 2, were characterized by moderate protein solubilities in the alkaline range (mean value 44%) compared to the protein ingredients derived from pea and fava bean, with mean values for protein solubility at pH 8.0 of 29% and 62%, respectively. As a result of the significant involvement of protein solubility in PC1, the soy protein ingredients were, therefore, placed in a central position.

Pea.

Pea protein ingredients, as characterized by PC1, exhibited higher color values for a* and b* and a lower lightness (L* value) compared to soy and fava bean protein ingredients. Differences in color characteristics were attributed to variations in pigments, particularly in cotyledons after dehulling. Raw material and process-specific effects, such as pigment removal during defatting of oil-rich legumes or pigment concentration during isolate production contributed to color variations together with particle size (71). The lower L* values were consistent with the larger particle size [Dx (90 µm)] of the pea protein ingredients, being in accordance with another study (72). The pea protein ingredients were spread along PC2, influenced by both the protein content and the AAS. Consequently, the pea protein concentrates exhibited a higher AAS compared to the pea protein isolates. The positioning on PC1 was due to the comparatively lower protein solubility and powder wettability of the pea protein ingredients compared to soy and fava bean protein ingredients across all pH values. Interestingly, no direct effects of the reduced protein solubility on the foaming and emulsifying properties were observed.

Fava bean.

Protein ingredients from fava bean exhibited the highest protein solubilities (pH 4.0 to 8.0) and superior powder wettability, indicative of excellent hydration properties along PC1. A negative correlation with particle size [Dx (90 µm)] further highlighted enhanced hydration, attributed to smaller particles. Notably, a subset of fava bean protein ingredients, including isolates 12 and 13, were excluded due to lower protein solubility, presumably resulting from partial denaturation during alkaline extraction and isoelectric precipitation (73). The positions of the fava bean protein ingredients along PC2 were mainly driven by the protein content, separating the protein isolates from the protein concentrates.

Concluding Remarks and Future Directions.

The landscape of plant proteins for the production of meat, milk, and egg product alternatives is undergoing a considerable transformation, driven by the consumers’ demand for sustainable and nutritious food. Traditionally, soy and gluten have dominated the market, but concerns about these sources, combined with growing consumer demand for alternatives, have paved the way for research into a wider range of plant-based materials. Further plant-derived protein ingredients are currently commercially available with the majority coming from grain legumes such as pea and fava beans due to their longstanding role in feed production. When sourcing ingredients for our study, it was easy to obtain a variety of soy, fava bean, and pea protein ingredients. In contrast, the availability of different lupine, chickpea, lentil, and oilseed protein ingredients, in particular with elevated protein content, was significantly more limited. To foster a more diverse ingredient market and promote the cultivation of underestimated crops, essential research support is needed for their breeding and optimization of growing conditions, but also for their processing into protein ingredients with standardized composition and functional properties. A comprehensive understanding of the specific properties and functional profiles is also crucial. This knowledge has the potential to accelerate the transformation of the food system toward a more plant-based diet, to increase demand from the food industry, and to establish a viable market for farmers.

Our study revealed that individual protein ingredients diverge in their protein content, facilitating their classification into flours, concentrates, and isolates. However, analysis of the amino acid content also showed that the amino acid composition and, therefore, the amino acid score can vary even within a raw material group with similar protein contents. The variation is much greater for the functional properties—in our study the emulsifying and foaming characteristics. The concurrent high variability between batches presents an additional challenge for food manufacturers and for the production of standardized end-products. This underscores the importance of future research focusing on the impact of raw material variations and individual process steps on the functional properties of plant proteins as maintaining low variations in ingredients’ functionality is crucial for their industrial application. In addition, functional properties are often assessed using different analytical methods, making direct comparisons of research results nearly impossible, and apparently identical products can vary in their functionality.

While the processing of animal proteins for food production is very well understood, exploration of the functionality of plant-based proteins, with the exception of gluten and soy, is still in its early stages. With an increasing choice of protein ingredients, the food industry faces the challenge of selecting the most suitable plant-based protein ingredients for specific applications. Further studies are needed to elucidate the relationships between the functional properties presented and application-specific requirements, to assess the impact of processing and also storage on ingredient functionality, and finally to ensure the maintenance of protein quality throughout ingredient and food processing. Systematically cataloging commercially available ingredients could help in the selection process, while leveraging AI to predict protein behavior within the food matrix is a promising approach (11). This opens the door to innovation and has the potential to accelerate the development of innovative, high-quality, and nutritionally optimized plant-based products in the future.

The properties of protein ingredients analyzed in our study are important for their food application. However, additional factors like sensory properties, availability, and sustainability parameters also play a significant role. Plant proteins, for instance, may exhibit beany and bitter aromas and flavors, potentially deterring consumers or necessitating the use of suitable flavorings. The original raw materials flavors remain perceptible, particularly in less processed concentrates. Higher processing levels typically remove off-flavor components, yielding ingredients with plainer flavors suitable for a broader range of food applications.

However, extensive processing can negatively impact sustainability factors, as demonstrated by Lie-Piang et al. (74). To enhance the sustainability of protein ingredient production, utilizing by-products is crucial. This is evident in practices like producing pea or potato protein as by-products of starch production, or sunflower protein as a by-product of oil production. While these processes are often optimized for the primary product (starch or oil), future adaptations may be necessary to prioritize the product with the highest added value, ensuring complete raw material utilization and improved production efficiency. Moreover, the success or failure of an ingredient hinges on its price competitiveness with large-scale alternatives like soybean ingredients or wheat gluten, produced at significantly lower prices. Particularly during periods of high inflation, this factor prompts many small enterprises and start-ups to withdraw from the highly competitive protein ingredients and alternative products market.

In many Life Cycle Assessment calculations, sustainability factors are often related to either kilograms of raw material or protein. Incorporating protein quality would enable a more holistic evaluation of ingredients. Our study reveals that, even from the same crop, protein ingredients exhibit varying AAS, generally falling below the FAO's recommended value for single protein sources. Consequently, more protein would need to be consumed to achieve an equivalent protein value if single plant protein sources are used. Utilizing protein blends from different sources in plant-based products can help overcome amino acid limitations. However, it is important to consider that protein quality is not only determined by the amino acid profile but also by digestibility and bioavailability. Currently, there is a lack of clarity on how to integrate all these complex aspects of protein quality into LCA analyses, and interdisciplinary approaches are needed to address this in an evidence-based manner.

The future of plant-based protein ingredients holds exciting opportunities and complex challenges. Overcoming these challenges requires ongoing innovation and also a sophisticated understanding of consumer preferences. Strategic positioning is crucial in the competitive landscape, emphasizing the need for a harmonious blend of scientific innovation, sustainable processes, and strategic market management for the future success of plant protein ingredients. Anticipating further developments and advances, interdisciplinary collaboration will play a critical role in shaping the future of plant-based nutrition.

Materials and Methods

A total of 61 commercial protein ingredients from different raw material sources and suppliers were collected (SI Appendix, Table S1). Chemicals and analytical standards were purchased from Merck KGaA (Germany) and Agilent Technologies Inc., ensuring the highest available purity.

Chemical Composition.

Dry matter and ash contents were determined using a thermogravimetric method at 105 °C and 550 °C following Association of Official Agricultural Chemists Official Method 923.03 (75). Protein content was measured based on the Dumas combustion method in accordance with AOAC Official Method 968.06 using the average nitrogen-to-protein conversion factor of N × 6.25. Fat content was assessed using Soxtherm^® fat extraction following the procedure described in Weibull–Stoldt as outlined in AOAC 963.15 (75). Amino acid composition was determined after protein hydrolysis according to the Commission Regulation (EC) No 152/2009 (2009) (76) by ortho-phthalaldehyde/9-fluorenylmethoxycarbonyl chloride (OPA/FMOC) precolumn derivatization and subsequent quantification by high-performance liquid chromatography using a C18 reversed-phase column.

Functional Properties.

Emulsifying capacity and foaming properties were analyzed as described in detail by Schlegel et al. (77) at pH 4.0 and 7.0 in distilled water. Emulsification was determined by continuously adding corn oil during homogenization with an Ultra-Turrax until phase inversion, as indicated by an abrupt drop in electrical conductivity. The amount of oil required to induce phase inversion was used to calculate the emulsifying capacity. Foaming activity and stability of 5% (w/w) protein dispersions were assessed after 8 min of mechanical whipping at the highest speed using a planetary mixer (Hobart System N50). Foaming activity was determined by measuring foam height, while foam density was calculated by weighing a specific volume of foam in a measuring cylinder. Foam stability was observed over 60 min. Protein solubility was measured in distilled water in the range from pH 3.0 to 8.0 according to the literature (78). The content of solubilized protein in the supernatant was determined by the Dumas combustion method and related to the protein content of the ingredient. Powder wettability, adapted from International Dairy Federation 87:1979 (79) was assessed using a 2.89 cm³ sample in an automatic feeder (Ø 3.5 cm). The weight sample was gently deposited on 100 g distilled water in a crystallizing dish (Ø 8.0 cm). Wetting time, normalized to 1 g of powder with a surface area of 0.02 g/cm², was recorded. Color (CIE L*a*b* values) was measured using a DigiEye Imaging System V2.60 (VeriVide Ltd., Leicester, United Kingdom) with prior calibration (illuminant D 65). Oil binding capacity was analyzed after dispersion with corn oil and centrifugation at 700 g for 15 min (80). Particle size Dx (90 µm) and span were determined via Malvern laser diffraction particle size analyzer as described before (58, 81).

Amino Acid Score.

AAS were computed by comparing the concentrations (mg/g protein) of indispensable amino acids in the test protein to those in a reference (scoring) pattern. The minimum ratio, known as AAS, identifies the first-limiting amino acid. Three scoring patterns were used: Preschool child patterns were selected to align with the recommendations for the Protein Digestibility-Corrected Amino Acid Score (82) and Digestible Indispensable Amino Acid Score (41) for all age groups, except infants. Furthermore, the scoring pattern considering the amino acid requirements of adults was applied (SI Appendix, Table S2).

Statistical Evaluation.

All analyses of chemical composition and functional properties were performed in triplicate for each protein ingredient tested. Box plots are used to visualize the variability in the datasets, where the boxes represent the interquartile range (IQR, 25 to 75%), and the horizontal line represents the median. The whiskers of the boxplots indicate data within 1.5 IQR, and outliers are shown as black dots. In Fig. 3, values are means ± SD (n = 3). Statistical analysis and PCA were conducted using XLSTAT software version 2022.3.1 (Addinsoft, Paris, France). An ANOVA with Tukey post hoc test was performed to determine significant differences (P ≤ 0.05). Prior to PCA, ANOVA with Tukey post hoc test was also performed to select properties for PCA with significant differences between the groups of grain legume protein ingredients (P ≤ 0.05), with n = 15 for pea, n = 6 for soy, and n = 13 for fava bean ingredients. A Spearman correlation (ρ) analysis was done to identify significant correlations by the utilization of a two-tailed significance test (P ≤ 0.05). Individual tests were done to determine significant correlations between the physicochemical and functional properties of all grain legume protein ingredients (n = 36), of all grain legume protein concentrates (n = 17), of all grain legume protein isolates (n = 19), and of all protein ingredients derived from pea (n = 15), soy (n = 8), and fava bean (n = 13), respectively.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

The names of the products cannot be disclosed because we want to treat the manufacturers of the proteins confidentially and do not want to pit one product against another. If anyone is interested, they can contact the authors and the individual data can be passed on.