A comprehensive review on pulse protein fractionation and extraction: processes, functionality, and food applications.

The increasing world population requires the production of nutrient-rich foods. Protein is an essential macronutrient for healthy individuals. Interest in using plant proteins in foods has increased in recent years due to their sustainability and nutritional benefits. Dry and wet protein fractionation methods have been developed to increase protein yield, purity, and functional and nutritional qualities. This review explores the recent developments in pretreatments and fractionation processes used for producing pulse protein concentrates and isolates. Functionality differences between pulse proteins obtained from different fractionation methods and the use of fractionated pulse proteins in different food applications are also critically reviewed. Pretreatment methods improve the de-hulling efficiency of seeds prior to fractionation. Research on wet fractionation methods focuses on improving sustainability and functionality of proteins while studies on dry methods focus on increasing protein yield and purity. Hybrid methods produced fractionated proteins with higher yield and purity while also improving protein functionality and process sustainability. Dry and hybrid fractionated proteins have comparable or superior functionalities relative to wet fractionated proteins. Pulse protein ingredients are successfully incorporated into various food formulations with notable changes in their sensory properties. Future studies could focus on optimizing the fractionation process, improving protein concentrate palatability, and optimizing formulations using pulse proteins.

Bioinformatics tools for the study of bioactive peptides from vegetal sources: evolution and future perspectives.

Bioactive peptides from vegetal sources have been shown to have functional properties as anti-inflammatory, antioxidant, antihypertensive or antidiabetic capacity. For this reason, they have been proposed as an interesting and promising alternative to improve human health. In recent years, the numerous advances in the bioinformatics field for in silico prediction have speeded up the discovery of bioactive peptides, also reducing the associated costs when using an integrated approach between the classical and bioinformatics discovery. This review aims to provide an overview of the evolution, limitations and latest advances in the field of bioinformatics and computational tools, and specifically make a critical and comprehensive insight into computational techniques used to study the mechanism of interaction that allows the explanation of plant bioactive peptide functionality. In particular, molecular docking is considered key to explain the different functionalities that have been previously identified. The assumptions to simplify such a high complex environment implies a degree of uncertainty that can only be guaranteed and validated by in vitro or in vivo studies, however, the combination of databases, software and bioinformatics applications with the classical approach has become a promising procedure for the study of bioactive peptides.

Analysis of ingredient list and nutrient composition of plant-based burgers available in the global market.

The nutrient composition of plant-based burgers is a key factor when making their purchase/consumption decision to maintain a balanced diet. For this reason, ingredient list and nutritional information of burgers launched in the global market were retrieved from their labels. Products were classified based on the technology development, market position and region of the manufacturer. From the ingredient analysis, we observed a high heterogeneity in the ingredients used, a predominance of soy and wheat as main sources of proteins, and the increasing use of new protein sources (e.g. peas, other types of beans and pseudo-cereals). Oil was the most cited ingredient followed by salt. Nutritional composition varied mainly depending on the region with no clear pattern among countries. To less extent, technology development resulted in traditional products with lower amounts of protein and higher amounts of carbohydrates. Vegan and vegetarian products showed limited differences due to the high intra-heterogenicity.

Quality changes in high hydrostatic pressure treated enriched tomato sauce.

BACKGROUND: Use of high hydrostatic pressure (HHP) with reduced processing times is gaining traction in the food industry as an alternative to conventional thermal treatment. In order to enhance functional benefits while minimizing processing losses, functionalized products are being developed with such novel techniques. In this study, changes in quality parameters for HHP treated enriched tomato sauce were evaluated, with the aim to assess its viability as an alternative to conventional thermal treatment methods. RESULTS: HHP treatments at 500 MPa, 30 °C/50 °C significantly increased the total phenolic and lycopene content of the sauce samples, achieving 6.7% and 7.5% improvements over conventionally treated samples. The antioxidant capacity of the HHP-treated samples was also found to match or be better than conventionally treated samples. Furthermore, a T2 relaxation time study revealed that pressure-temperature processing treatments were effective in maintaining the structural integrity of water molecules. Microbiological analyses revealed that 500 MPa/50 °C 5 min treatment can offer 8 logs reduction colony formation, matching the results of conventional thermal treatment. CONCLUSION: Combined pressure-temperature treatments improve results, reduce time consumption. 500 MPa/50 °C treatments provided retention of quality parameters and significant reduction in microbial activity. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pea protein isolate-soluble soybean polysaccharides electrostatic assembly: effect of pH, biopolymer mass ratio and heat treatment.

BACKGROUND: In past years, thousands of protein-polysaccharide complexes have been investigated to modify protein characteristics and functionality in food systems. However, the interaction between pea protein isolate (PPI) and soluble soybean polysaccharide (SSPS) has not been thoroughly characterized yet. RESULTS: In the present study, the phase behavior of PPI and SSPS mixtures was analyzed as a function of PPI:SSPS mixing ratio (1:1 to 1:0.10) and pH (7.0 to 2.0), showing that these biopolymers could be electrostatically assembled at 1:1 to 1:0.25 mixing ratios and 4.0 to 3.0 pH values. Then, the characteristics of the PPI-SSPS complexes were studied before and after heating (90 °C and 30 min) by ζ-potential, surface hydrophobicity, protein solubility, particle size distribution and physical stability for 56 days. By lowering the pH and PPI:SSPS mixing ratio, the complexes showed increased solubility, changed 𝜁-potential and higher physical stability. By heating, the complexes presented increased hydrophobicity and physical stability. CONCLUSION: Overall, PPI-SSPS complexes increased the protein solubility, reduced the particle size, and changed both the ζ-potential and the surface hydrophobicity with respect to PPI control, allowing stabilization of the colloidal system and broadening the possible applications of these high-quality proteins in acidic food systems. © 2024 Society of Chemical Industry.

Comparative evaluation of milk proteins and oil-seed-cake-derived proteins extracted by chemical and biological methods for obesity management.

BACKGROUND: In light of the exponential rise in global population, there is a critical requirement to reduce food waste on a global scale. According to studies, agricultural wastes such as oil-seed cakes offer great nutritional value. Acid precipitation (A) and alkaline extraction methods (traditional methods) were used to extract protein from oil-seed cakes; however, both procedures are linked to decreased protein quality and quantity, which prompted the development of a novel strategy known as the biological/microbial/probiotic (B) method. Therefore, the present study aimed to highlight the optimal way of protein extraction from oil-seed cakes and the effect of extraction methods on protein efficacy against obesity. The outcomes were also compared with milk proteins. RESULTS: In vitro study provided evidence that proteins from both sources (plant and milk) suppressed adipogenesis and stimulated adipolysis in 3T3L-1 cells. For the in vivo study, mice were fed with different protein extracts: soya protein preparation (SPP), ground protein preparation (GPP), whey protein (WP) and casein protein (CP) containing 40% of their calories as fat. Body weight decreased significantly in all the rats except CP-fed rats. Body mass index, atherogenic index, plasma triglyceride and very-low-density lipoprotein cholesterol level decreased significantly in all the groups in comparison to the model group (high-fat-diet group), but the decrease was more pronounced in plant proteins than milk proteins. In hepatocytes, the expression of fasting-induced adipose factor, carnitine palmitoyltransferase I and peroxisome proliferator-activated receptor α genes was increased significantly in SPP-fed groups. Adiponectin gene expression was upregulated significantly in visceral fat tissue in groups fed SPP-B, GPP-A and CP, whereas leptin gene was downregulated significantly in all groups except SPP-A. CONCLUSION: This study demonstrates that SPP-B showed the most effective anti-obesity property, followed by WP. Additionally, we found that the biological precipitation approach produced better outcomes for plant proteins isolated from oil-seed cakes than the acid precipitation method. © 2023 Society of Chemical Industry.

Novel animal product substitutes: A new category of plant-based alternatives to meat, seafood, egg, and dairy products.

Many consumers are adopting plant-centric diets to address the adverse effects of livestock production on the environment, health, and animal welfare. Processed plant-based foods, including animal product analogs (such as meat, seafood, egg, or dairy analogs) and traditional animal product substitutes (such as tofu, seitan, or tempeh), may not be desirable to a broad spectrum of consumers. This article introduces a new category of plant-based foods specifically designed to overcome the limitations of current animal product analogs and substitutes: novel animal product substitutes (NAPS). NAPS are designed to contain high levels of nutrients to be encouraged (such as proteins, omega-3 fatty acids, dietary fibers, vitamins, and minerals) and low levels of nutrients to be discouraged (such as salt, sugar, and saturated fat). Moreover, they may be designed to have a wide range of appearances, textures, mouthfeels, and flavors. For instance, they could be red, orange, green, yellow, blue, or beige; they could be spheres, ovals, cubes, or pyramids; they could be hard/soft or brittle/pliable; and they could be lemon, thyme, curry, or chili flavored. Consequently, there is great flexibility in creating NAPS that could be eaten in situations where animal products are normally consumed, for example, with pasta, rice, potatoes, bread, soups, or salads. This article reviews the science behind the formulation of NAPS, highlights factors impacting their appearance, texture, flavor, and nutritional profile, and discusses methods that can be used to formulate, produce, and characterize them. Finally, it stresses the need for further studies on this new category of foods, especially on their sensory and consumer aspects.

In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Scalable Manufacturing of Environmentally Stable All-Solid-State Plant Protein-Based Supercapacitors with Optimal Balance of Capacitive Performance and Mechanically Robust.

The development of advanced biomaterial with mechanically robust and high energy density is critical for flexible electronics, such as batteries and supercapacitors. Plant proteins are ideal candidates for making flexible electronics due to their renewable and eco-friendly natures. However, due to the weak intermolecular interactions and abundant hydrophilic groups of protein chains, the mechanical properties of protein-based materials, especially in bulk materials, are largely constrained, which hinders their performance in practical applications. Here, a green and scalable method is shown for the fabrication of advanced film biomaterials with high mechanical strength (36.3 MPa), toughness (21.25 MJ m-3 ), and extraordinary fatigue-resistance (213 000 times) by incorporating tailor-made core-double-shell structured nanoparticles. Subsequently, the film biomaterials combine to construct an ordered, dense bulk material by stacking-up and hot-pressing techniques. Surprisingly, the solid-state supercapacitor based on compacted bulk material shows an ultrahigh energy density of 25.8 Wh kg-1 , which is much higher than those previously reported advanced materials. Notably, the bulk material also demonstrates long-term cycling stability, which can be maintained under ambient condition or immersed in H2 SO4 electrolyte for more than 120 days. Thus, this research improves the competitiveness of protein-based materials for real-world applications such as flexible electronics and solid-state supercapacitors.

Recent advances in bioengineered scaffold for in vitro meat production.

In vitro meat production via stem cell technology and tissue engineering provides hypothetically elevated resource efficiency which involves the differentiation of muscle cells from pluripotent stem cells. By applying the tissue engineering technique, muscle cells are cultivated and grown onto a scaffold, resulting in the development of muscle tissue. The studies related to in vitro meat production are advancing with a seamless pace, and scientists are trying to develop various approaches to mimic the natural meat. The formulation and fabrication of biodegradable and cost-effective edible scaffold is the key to the successful development of downstream culture and meat production. Non-mammalian biopolymers such as gelatin and alginate or plant-derived proteins namely soy protein and decellularized leaves have been suggested as potential scaffold materials for in vitro meat production. Thus, this article is aimed to furnish recent updates on bioengineered scaffolds, covering their formulation, fabrication, features, and the mode of utilization.

Plant Protein Can Be as Efficient as Milk Protein to Maintain Fat Free Mass in Old Rats, Even When Fat and Sugar Intakes Are High.

BACKGROUND: Alternative, sustainable, and adequate sources of protein must be found to meet global demand. OBJECTIVES: Our aim was to assess the effect of a plant protein blend with a good balance of indispensable amino acids and high contents of leucine, arginine, and cysteine on the maintenance of muscle protein mass and function during aging in comparison to milk proteins and to determine if this effect varied according to the quality of the background diet. METHODS: Old male Wistar rats (n = 96, 18 mo old) were randomly allocated for 4 mo to 1 of 4 diets, differing according to protein source (milk or plant protein blend) and energy content (standard, 3.6 kcal/g, with starch, or high, 4.9 kcal/g, with saturated fat and sucrose). We measured: every 2 mo, body composition and plasma biochemistry; before and after 4 mo, muscle functionality; after 4 mo, in vivo muscle protein synthesis (flooding dose of L-[1-13C]-valine) and muscle, liver, and heart weights. Two-factor ANOVA and repeated measures 2-factor ANOVA were conducted. RESULTS: There was no difference between protein type on the maintenance during aging of lean body mass, muscle mass, and muscle functionality. The high-energy diet significantly increased body fat (+47%) and heart weight (+8%) compared to the standard energy diet but had no effect on fasting plasma glucose and insulin. Muscle protein synthesis was significantly stimulated by feeding to the same extent in all groups (+13%). CONCLUSIONS: Since high-energy diets had little impact on insulin sensitivity and related metabolism, we could not test the hypothesis that in situations of higher insulin resistance, our plant protein blend may be better than milk protein. However, this rat study offers significant proof of concept from the nutritional standpoint that appropriately blended plant proteins can have high nutritional value even in demanding situations such as aging protein metabolism.

Modifying the plant proteins techno-functionalities by novel physical processing technologies: a review.

Plant proteins have recently gained market demand and momentum due to their environmentally friendly origins and health advantages over their animal-derived counterparts. However, their lower techno-functionalities, digestibility, bioactivities, and anti-nutritional compounds have limited their application in foods. Increased demand for physically modified proteins with better techno-functionalities resulted in the application of different thermal and non-thermal treatments to modify plant proteins. Novel physical processing technologies (NPPT) considered 'emerging high-potential treatments for tomorrow' are required to alter protein functionality, enhance bioactive peptide formations, reduce anti-nutritional, reduce loss of nutrients, prevention of damage to heat liable proteins and clean label. NPPT can be promising substitutes for the lower energy-efficient and aggressive thermal treatments in plant protein modification. These facts captivated the interest of the scientific community in designing novel functional food systems. However, these improvements are not verifiable for all the plant proteins and depend immensely on the protein type and concentration, other environmental parameters (pH, ionic strength, temperature, and co-solutes), and NPPT conditions. This review addresses the most promising approaches of NPPT for the modification of techno-functionalities of plant proteins. New insights elaborating the effect of NPPTs on proteins' structural and functional behavior in relation to other food components are discussed. The combined application of NPPTs in the field of plant-based bioactive functionalities is also explored.

Plant protein-based nanocomposite films: A review on the used nanomaterials, characteristics, and food packaging applications.

Consumer demands to utilize environmentally friendly packaging have led researchers to develop packaging materials from naturally derived resources. In recent years, plant protein-based films as a replacement for synthetic plastics have attracted the attention of the global food packaging industry due to their biodegradability and unique properties. Biopolymer-based films need a filler to show improved packaging properties. One of the latest strategies introduced to food packaging technology is the production of nanocomposite films which are multiphase materials containing a filler with at least one dimension less than 100 nm. This review provides the recent findings on plant-based protein films as biodegradable materials that can be combined with nanoparticles that are applicable to food packaging. Moreover, it investigates the characterization of nanocomposite plant-based protein films/edible coatings. It also briefly describes the application of plant-based protein nanocomposite films/coating on fruits/vegetables, meat and seafood products, and some other foods. The results indicate that the functional performance, barrier, mechanical, optical, thermal and antimicrobial properties of plant protein-based materials can be extended by incorporating nanomaterials. Recent reports provide a better understanding of how incorporating nanomaterials into plant protein-based biopolymers leads to an increase in the shelf life of food products during storage time.

Plant-derived proteins as a sustainable source of bioactive peptides: recent research updates on emerging production methods, bioactivities, and potential application.

The development of novel protein sources to compensate for the expected future shortage of traditional animal proteins due to their high carbon footprint is a major contemporary challenge in the agri-food industry currently. Therefore, both industry and consumers are placing a greater emphasis on plant proteins as a sustainable source of protein to meet the growing nutritional demand of ever increasing population. In addition to being key alternatives, many plant-based foods have biological properties that make them potentially functional or health-promoting foods, particularly physiologically active peptides and proteins accounting for most of these properties. This review discusses the importance of plant-based protein as a viable and sustainable alternative to animal proteins. The current advances in plant protein isolation and production and characterization of bioactive hydrolysates and peptides from plant proteins are described comprehensively. Furthermore, the recent research on bioactivities and bioavailability of plant protein-derived bioactive peptides is reviewed briefly. The limitations of using bioactive peptides, regulatory criteria, and the possible future applications of plant protein-derived bioactive peptides are highlighted. This review may help understand plant proteins and their bioactive peptides and provide valuable suggestions for future research and applications in the food industry.

Integration of machine learning with computational structural biology of plants.

Computational structural biology of proteins has developed rapidly in recent decades with the development of new computational tools and the advancement of computing hardware. However, while these techniques have widely been used to make advancements in human medicine, these methods have seen less utilization in the plant sciences. In the last several years, machine learning methods have gained popularity in computational structural biology. These methods have enabled the development of new tools which are able to address the major challenges that have hampered the wide adoption of the computational structural biology of plants. This perspective examines the remaining challenges in computational structural biology and how the development of machine learning techniques enables more in-depth computational structural biology of plants.

Currently Available Handouts for Low Phosphorus Diets in Chronic Kidney Disease Continue to Restrict Plant Proteins and Minimally Processed Dairy Products.

OBJECTIVES: The 2020 Kidney Disease Outcome Quality Initiative guidelines recommend adjusting phosphorus intake to achieve and maintain normal serum phosphorus levels for adults living with chronic kidney disease. These guidelines also recommend considering the dietary source of phosphorus as different sources have different bioavailability; however, phosphorus food lists are not provided. Therefore, the aim of this study is to investigate the current teaching materials in Canada regarding low phosphorus diet. DESIGN AND METHODS: Using a geographical approach, websites from each province and territories' government, health, and renal programs (where applicable) were reviewed for resources on dietary phosphorus restriction in chronic kidney disease. All publicly available handouts/booklets/printable webpages were obtained and reviewed for recommendations on how to implement a low phosphorus diet. RESULTS: Sixty-one resources in total met inclusion criteria (52 handouts from health agencies in 6 provinces and 9 handouts from the Kidney Foundation of Canada). Items with minimal nutrition value, such as cola, beer and cocoa, chocolate, and baking powder, were the most commonly restricted with 84% (51/61) resources making this recommendation. Plant proteins and minimally processed dairy were restricted in 80% (49/61) of resources. Processed animal meat was recommended to be restricted in 70% (43/61) of resources and whole grains in 65% (40/61). Sixty-three percent of the handouts (39/61) discuss avoiding phosphorus additives. CONCLUSIONS: Many resources restrict items with minimal nutrition value to lower phosphorus intake; however, plant foods, including plant proteins and whole grains, continue to be restricted in the majority of resources, despite having lower bioavailability. The 2020 Kidney Disease Outcome Quality Initiative guidelines recommend considering bioavailability of phosphorus source when implementing low phosphorus diets; current handouts in Canada would likely benefit from review.

Effects of cadmium stress on the morphology, physiology, cellular ultrastructure, and BvHIPP24 gene expression of sugar beet (Beta vulgaris L.).

To clarify the mechanism of the response of sugar beet (Beta vulgaris L.) to cadmium (Cd) stress, this study investigated changes in the phenotype, physiological indexes, and subcellular structure of B. vulgaris under Cd treatment and the transcriptional pattern of the BvHIPP24 gene (a heavy metal-associated isoprenylated plant protein involved in heavy metal detoxification). The plant height and shoot and root growth of B. vulgaris seedlings were inhibited to some extent under 0.5 and 1 mM Cd, with gradually wilting and yellowing of leaves and dark brown roots. When the Cd concentration was increased, malondialdehyde content and the activities of peroxidase, superoxide dismutase, and glutathione S-transferase increased differentially. qPCR indicated that the expression of BvHIPP24 was induced by different concentrations of Cd. Although transmission electron microscopy revealed damage to nuclei, mitochondria, and chloroplasts, B. vulgaris exhibited strong adaptability to 0.5 mM Cd according to a comprehensive analysis using the membership function. The results showed that B. vulgaris may reduce cell damage and improve its Cd tolerance by regulating functional gene expression and antioxidant enzymes. This study increases our understanding of the Cd-tolerance mechanism of B. vulgaris and provides insights into the use of B. vulgaris in Cd bioremediation.

Sugar beet is a novel energy crop with superior characteristics for both heavy metal phytoremediation and biomass energy development. This work is the first to investigate both the morphological, physiological, and ultrastructural response of sugar beet to cadmium stress and the induction of a functional metallochaperone gene by cadmium. This study explains the cadmium tolerance mechanism of sugar beet based on a comprehensive evaluation and provides an important theoretical basis for further application of beet in heavy metal bioremediation.

Bioengineered Enzymes and Precision Fermentation in the Food Industry.

Enzymes have been used in the food processing industry for many years. However, the use of native enzymes is not conducive to high activity, efficiency, range of substrates, and adaptability to harsh food processing conditions. The advent of enzyme engineering approaches such as rational design, directed evolution, and semi-rational design provided much-needed impetus for tailor-made enzymes with improved or novel catalytic properties. Production of designer enzymes became further refined with the emergence of synthetic biology and gene editing techniques and a plethora of other tools such as artificial intelligence, and computational and bioinformatics analyses which have paved the way for what is referred to as precision fermentation for the production of these designer enzymes more efficiently. With all the technologies available, the bottleneck is now in the scale-up production of these enzymes. There is generally a lack of accessibility thereof of large-scale capabilities and know-how. This review is aimed at highlighting these various enzyme-engineering strategies and the associated scale-up challenges, including safety concerns surrounding genetically modified microorganisms and the use of cell-free systems to circumvent this issue. The use of solid-state fermentation (SSF) is also addressed as a potentially low-cost production system, amenable to customization and employing inexpensive feedstocks as substrate.

Relationship between Protein Digestibility and the Proteolysis of Legume Proteins during Seed Germination.

Legume seed protein is an important source of nutrition, but generally it is less digestible than animal protein. Poor protein digestibility in legume seeds and seedlings may partly reflect defenses against herbivores. Protein changes during germination typically increase proteolysis and digestibility, by lowering the levels of anti-nutrient protease inhibitors, activating proteases, and breaking down storage proteins (including allergens). Germinating legume sprouts also show striking increases in free amino acids (especially asparagine), but their roles in host defense or other processes are not known. While the net effect of germination is generally to increase the digestibility of legume seed proteins, the extent of improvement in digestibility is species- and strain-dependent. Further research is needed to highlight which changes contribute most to improved digestibility of sprouted seeds. Such knowledge could guide the selection of varieties that are more digestible and also guide the development of food preparations that are more digestible, potentially combining germination with other factors altering digestibility, such as heating and fermentation. Techniques to characterize the shifts in protein make-up, activity and degradation during germination need to draw on traditional analytical approaches, complemented by proteomic and peptidomic analysis of mass spectrometry-identified peptide breakdown products.