Improvement of the functionality of vegetable proteins by controlled enzymatic hydrolysis

Vegetable proteins are available to the food industry in various forms, such as flours, concentrates, isolates, and TVP (textured vegetable protein). However, the functional properties of these products are not always optimal and need improvement for certain applications. In recent years it has been demonstrated that a limited enzymatic hydrolysis offers a convenient and specific means of improving certain functional properties of vegetable proteins. The hydrolysis reaction is controlled by the degree of hydrolysis (DH) which is defined as the percentage of peptide bonds cleaved. Under neutral or slightly alkaline conditions, DH can be monitored continuously by the pH-stat technique, since the amount of base necessary to maintain a constant pH is proportional to DH. The reaction is terminated at a preset DH-value to obtain the desired properties of the hydrolysate.Protein products from soya bean, faba bean, and potato were hydrolysed to DH 0%, 3% and 5%, and certain functional properties (iso-electric solubility, emulsification capacity, whipping expansion, and foam stability) were evaluated. The general picture observed is an increase, often pronounced, in these four properties with increasing DH — however, there are also distinct differences between the protein sources as well as between the different forms of a particular protein. This work demonstrates that protein sources indigenous to Europe are fully comparable to soya protein with respect to potential functional uses.     

The proteins from leaves

The nature and main characteristics of the leaf proteins are reviewed, stressing the even aminoacid composition of the different crops suitable for protein extraction. A satisfactory pattern of essential aminoacids is also shown to be a general characteristic. The principles of the wet fractionation of green vegetables are then reviewed; details are given on the squeezing stage and the criteria for the recovery of the proteins from the expressed juice are reported. The different physico-chemical status of such proteins requires different processes for their recovery as a whole or in fractionated bulk. Therefore, three different leaf protein concentrates (LPCs) can be obtained: cytoplasmatic (white), chloroplastic (green) and whole LPC, the last being a combination of the other two. Processes for LPC production as animal feed have already been established at the industrial level; procedures giving LPC for human consumption are at the laboratory or pilot scale. In this paper, attention has been paid to the criteria for the preparation of edible LPC, which are essentially based on both the decoloration of green whole LPC and the fractionated recovery of the cytoplasmatic and chloroplastic proteins present in the juice. In this second process, the white fraction is reserved for humans and the green LPC for animals. Technological aspects of the production of the above-mentioned LPCs are described and some nutritional and functional properties of the final products are briefly illustrated.     

Minor components and wheat quality: Perspectives on climate changes

Minor compounds of cereal grains such as lipids and cell wall polysaccharides play an important role in their milling properties, their transformation into baked cereal products and their nutritional properties. These effects are mainly due to their interactive properties, such as their ability to absorb large amounts of water for polysaccharides or to interact with starch polymers and proteins for lipids. Environmental stress induced by climate change and regulations for environmentally friendly agriculture can interfere with the biosynthesis of these minor grain compounds as well as the main compounds starch and protein. Indeed, the metabolic networks of all components of the endosperm of grains are closely associated. Therefore, in the context of climate change and agricultural sustainability, lipids and cell wall polysaccharides may be affected. Contrasting with a plethora of studies reporting the impact of environment and fertilizer on cereal proteins, there is a real lack of information on this particular topic for these minor compounds. However, our knowledge of the physico-chemical properties and biosynthesis of these minor compounds allows us to assess the likely strong impact of environmental and agronomic constraints on the relationships between cell wall polysaccharides, lipids and cereal quality.     

Seaweed Protein Hydrolysates and Bioactive Peptides: Extraction,Purification, and Applications

Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich composition in fiber, minerals, and proteins, has pointed them as a useful source of these components. Seaweed proteins are nutritionally valuable and include several specific enzymes, glycoproteins, cell wall-attached proteins, phycobiliproteins, lectins, or peptides. Extraction of seaweed proteins requires the application of disruptive methods due to the heterogeneous cell wall composition of each macroalgae group. Hence, non-protein molecules like phenolics or polysaccharides may also be co-extracted, affecting the extraction yield. Therefore, depending on the macroalgae and target protein characteristics, the sample pretreatment, extraction and purification techniques must be carefully chosen. Traditional methods like solid–liquid or enzyme-assisted extraction (SLE or EAE) have proven successful. However, alternative techniques as ultrasound- or microwave-assisted extraction (UAE or MAE) can be more efficient. To obtain protein hydrolysates, these proteins are subjected to hydrolyzation reactions, whether with proteases or physical or chemical treatments that disrupt the proteins native folding. These hydrolysates and derived peptides are accounted for bioactive properties, like antioxidant, anti-inflammatory, antimicrobial, or antihypertensive activities, which can be applied to different sectors. In this work, current methods and challenges for protein extraction and purification from seaweeds are addressed, focusing on their potential industrial applications in the food, cosmetic, and pharmaceutical industries.     

Properties of lupine proteins relevant to their nutritional performance

Composition, solubility, bound sugar content and quality, subunit composition and structure of the storage proteins of seeds ofLupinus albus are discussed. Aminoacid composition is also given for each protein, the various protein classes and the whole flour. These data allow for the characterization of the molecules of the various storage proteins and their contributions to the nutritional properties of the seed.In vitro digestibility (by mammalian endopeptidases) is reported and is less than for animal proteins. Possible causes, at the molecular level, for this behaviour and possible means to overcome these effects are examined. The relationships between the above data are considered in view of the nutritional performance of the proteins and of the genetical, agronomic and technological approaches most suited to improve the nutritional quality of lupine seeds as a protein source.     

Extraction processes and their effect on protein functionality

The refinement of vegetable protein sources involves processes which manipulate the mechanical and physicochemical properties of the source materials to obtain selective removal or destruction of undesirable components. However, the choice of conditions of temperature, water content, pH, solvent hydrophobicity etc., does not necessarily identify the eventual functional properties of the proteins in human foods as a primary constraint. This can produce less than optimal raw materials, and uncontrolled damage to the proteins during extraction, concentration or drying, making cross comparison of the intrinsic properties of different source materials quite meaningless. Alternatively, the proper choice of the process conditions used, with respect to the physical chemistry of the component macromolecules, can produce a wide variety of protein states from the same source material, and hence a spectrum of functional performance. While the details of process control for a given protein source may not be exactly defined, the principles are understood. The major problem now concerns the specification of the properties appropriate to a given functional use.     

Characterisation of gluten-free pasta through conventional and innovative methods: Evaluation of the uncooked products

Formation of a gluten protein network is fundamental for the texture and the overall quality of pasta. Replacement of the gluten network in gluten-free pasta is a major technological challenge, and the conventional technological processes have to be adapted to non-gluten formulations. The wide variety of raw materials and technologies used in the production of commercial gluten-free pasta stems from the – still on-going – search for solutions to these problems. The aim of this study was to evaluate the characteristics of different commercial gluten-free spaghetti currently available on the market, focusing on starch and protein organisation. Taking into account the chemical and biochemical properties of the samples, and their relationships to the physical characteristics of these products we looked at how some molecular properties relate to the final structure and quality of gluten-free pasta. Phenomena related to starch retrogradation were found to play a central role for the final texture of the products. At the same time, the origin of proteins included in the formulation was found to govern the protein–protein interactions, especially in those samples including proteins from different vegetable sources.     

Improving wheat to remove coeliac epitopes but retain functionality

Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a “coeliac-safe” wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research.     

Characterization of rice storage proteins by SE-HPLC and micro z-arm mixer

While the effect of protein content and composition on the functional properties of wheat flour is well studied, our knowledge on the same properties of rice flour is limited. This work was conducted to study the relationship between the dough mixing properties of flour from different rice cultivars and protein content and composition. An efficient sonication-based two-step extraction procedure was applied to isolate rice flour proteins. The size-exclusion HPLC (SE-HPLC) method, originally developed for separating wheat proteins, was applied with some minor modifications in order to study the size distribution of rice flour proteins. Four fractions were distinguished on the SE-HPLC profile and were further characterized by SDS-PAGE. Fractions I–III consisted of glutelins, while fraction IV contained albumin, globulin and prolamin proteins. When rice dough was characterized on the basis of mixing parameters in a micro z-arm mixer, significant differences were observed depending on the protein composition of the flour. Statistical analysis results indicated that the functional properties of the flour from different rice cultivars were associated with the amount of polymeric proteins and their size distribution.     

The S-poor prolamins of wheat,barley and rye: Revisited

The last review of the S-poor prolamins was published in 1995. Since then there has been a considerable increase in out knowledge of this interesting and unique group of proteins. The advances in the understanding of genetics and polymorphisms of the proteins are discussed including the available gene sequences and their alignments and consensus sequences. This group of prolamins are implicated as major allergens in WDEIA and wheat allergy and as immunodominant proteins in coeliac disease. The epitopes and their distribution throughout the protein sequences are reviewed. Their structure and physical chemistry is discussed in relation to their functional properties.     

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.     

Nutritional Yield: A Proposed Index for Fresh Food Improvement Illustrated with Leafy Vegetable Data

Consumer interest in food products, including fresh vegetables, with health promoting properties is rising. In fresh vegetables, these properties include vitamins, minerals, dietary fiber, and secondary compounds, which collectively impart a large portion of the dietary, nutritional or health value associated with vegetable intake. Many, including farmers, aim to increase the health-promoting properties of fresh vegetables on the whole but they face at least three obstacles. First, describing crop composition in terms of its nutrition-based impact on human health is complex and there are few, if any, accepted processes and associated metrics for assessing and managing vegetable composition on-farm, at the origin of supply. Second, data suggest that primary and secondary metabolism can be ??in conflict?? when establishing the abundance versus composition of a crop. Third, fresh vegetable farmers are rarely compensated for the phytochemical composition of their product. The development and implementation of a fresh vegetable ??nutritional yield?? index could be instrumental in overcoming these obstacles. Nutritional yield is a function of crop biomass and tissue levels of health-related metabolites, including bioavailable antioxidant potential. Data from a multi-factor study of leaf lettuce primary and secondary metabolism and the literature suggest that antioxidant yield is sensitive to genetic and environmental production factors, and that changes in crop production and valuation will be required for fresh vegetable production systems to become more focused and purposeful instruments of public health.     

Legume seed protein extraction,processing, and end product characteristics

Because of the difficulties in growing soybean in many parts of the world, other leguminosae crops (fababean, pea, lentil, lupine, bean chickpea, cow pea, etc.) are now being studied as new protein sources. They generally have a high protein content and a satisfactory amino acid composition. The studies which have led to the development of industrial flow sheets for protein extraction have mainly been carried out on pea (Pisum sativum), fababean (Vicia faba), and lupine (Lupinus albus). The processes generally used are (1) pin-milling plus air-classification which when applied to starch-rich legume seeds (pea, fababean), results in concentrates (defined as having protein contents of 60–75%), and (2) wet processes which produce isolates (defined as having protein contents of 90% to 95%). By air-classification, concentrates having 68% and 65% protein can be obtained, respectively, from fababean (31% protein) and pea (21% protein). Isolates, prepared by extraction of the flour proteins with alkaline solution followed by acid precipitation, have a protein content (N × 6.25) generally between 90% and 96% and a protein recovery yield varying between 60% and 65%. As a rule, isolates resulting from ultrafiltered extracts have a higher protein content. From the nutritional evaluation of these two types of products, concentrates and isolates, it appears that wet processes are more efficient for eliminating antinutritional factors. α-galactosides and glycosides are present in isolates only in traces. As for trypsin inhibitors and haemagglutinins, only one third of the activities in the flour remained in the isolates (in dry processes, the residual levels of these antinutritional factors were higher). From the studies of their functional properties, it appears that isolates and concentrates from sources such as fababean and peas, produced by the new processes described, are to some extent complementary or equivalent in their functional properties to those of the soybean, particularly for emulsifying and foaming purposes. These observations should encourage the development of these new processes.     

Biologische oder Physikalisch-Chemische Bestimmung der Nahrungsqualtität—eine Einführung

Some of the benefits as well as problems arising from biological methods in their application to the evaluation of the ‘internal’ or nutritional quality of food are discussed in the following papers. It, therefore, seems worth-while to put the question whether biological methods are the better ones compared with physico-chemical procedures or not. As food is destined for men it seems well reasonable to use human beings for the evaluation of food and food compounds. Really, this has been practised since the earliest time of mankind, and perhaps the first written report in this field is found in the bible. But human beings show a widely divergent genetic background and psychological influences too may give rise to misinterpretations. Beside of this human metabolic experiments are very expensive and longlasting. Feeding experiments with different animal species may partly overcome these difficulties, but the significance of the results for human being are uncertain. By means of physico-chemical methods food components may be determined fast and with great accuracy, but the problem of bioavailability rests unresolved as well as the question about the activity of these components within their natural matrix. Taking all this together the question: ‘biologicalor physico-chemical evaluation’ seems unrealistically formulated and should be exchanged against the statement: ‘biologicaland physico-chemical evaluation’. Both methods must be combinated with respect to the special problems which are to be solved and can thus complete one another in a favourable manner.     

Effects of species and breeding on wheat protein composition

Wheat proteins are characterized by their excellent contribution to technological and baking properties. However, wheat proteins, especially gluten and amylase-trypsin inhibitors (ATIs), are also known to be responsible for a broad range of intolerances and allergies. In order to evaluate the impact of genetic variability on the composition of these functional but immunogenic protein types, a set of different Triticum species, including common wheat, durum, spelt, emmer and einkorn, was examined regarding ATI and gluten concentrations by RP-HPLC. Additionally, inhibition towards trypsin was determined by an enzymatic assay. Based on the results, none of the investigated wheat species can be considered to be less ‘immunogenic’. Nevertheless, due to the large variability of ATI and gluten amounts among different genotypes, the selection of less-immunoreactive wheat varieties for individuals that suffer from wheat related diseases (WRDs) might be possible. The impact of breeding was assessed for different varieties of common wheat from different breeding periods. Results revealed significant improvement of technologically valuable parameters such as the amount of high molecular weight (HMW) glutenins and the gliadin to glutenin ratio, but no increase in immunogenic proteins.     

Increasing the utilisation of sorghum,millets and pseudocereals: Developments in the science of their phenolic phytochemicals,biofortification and protein functionality

There is considerable interest in sorghum, millets and pseudocereals for their phytochemical content, their nutritional potential and their use in gluten-free products. They are generally rich in a several phenolic phytochemicals. Research has indicated that the phenolics in these grains may have several important health-promoting properties: prevention and reduction of oxidative stress, anti-cancer, anti-diabetic, anti-inflammatory, anti-hypertensive and cardiovascular disease prevention. However, increased research on the actual health-promoting properties of foods made from these grains is required. Biofortified (macro and micronutrient enhanced) sorghum and millets are being developed through conventional breeding and recombinant DNA technology to combat malnutrition in developing countries. Enhanced nutritional traits include: high amylopectin, high lysine, improved protein digestibility, provitamin A rich, high iron and zinc, and improved mineral bioavailability through phytate reduction. Some of these biofortified cereals also have good agronomic characteristics and useful commercial end-use attributes, which will be important to their adoption by farmers. Knowledge of the structure of their storage proteins is increasing. Drawing on research concerning maize zein, which shows that it can produce a visco-elastic wheat-like dough, it appears that the storage proteins of these minor grains also have this potential. Manipulation of protein β-sheet structure seems critical in this regard.     

Damage to nutritional value of plant proteins by chemical reactions during storage and processing

The nutritive function of proteins for monogastric animals may be simply to supply the body with amino acids. However, the complexities of digestion, absorption and metabolism imply that amino acid composition, determined chemically after hydrolysis, can only be a very rough guide (usually setting upper limits) to the nutritional value of proteins. Chemical reactions which may affect nutritional values of proteins are discussed at length. Chemical tests, for determining the extent to which these reactions have occurred, are increasing, both in number and usefulness. However, to derive practical benefits from chemical tests, frequent parallel nutritional assessments must be conducted using animals.     

Wheat seed storage proteins: Advances in molecular genetics,diversity and breeding applications

Wheat seed storage proteins, especially glutenins and gliadins, have unique functional properties giving rise to a wide array of food products for human consumption. The wheat seed storage proteins, however, are also the most common cause of food-related allergies and intolerances, and it has become crucially important to understand their composition, variation and functional properties and interface this knowledge with the grain handling industry as well as the breeders. This review focuses on advances in understanding the genetics and function of storage proteins and their application in wheat breeding programs. These include: (1) The development and validation of high-throughput molecular marker systems for defining the composition and variation of low molecular weight glutenin subunits (LMW-GS) genes and a summary of the more than 30 gene-specific markers for rapid screening in wheat breeding programs; (2) The identification of more than 100 alleles of storage proteins in wild species provide candidate genes for future quality improvement; (3) The documentation of quality effects of individual LMW-GS and HMW-GS for improving end-use quality; and (4) The analysis of α-gliadin genes on chromosomes 6A and 6D with non-toxic epitopes as potential targets to develop less toxic cultivars for people with celiac disease. Genomic and proteomic technologies that will continue to provide new tools for understanding variation and function of seed storage proteins in wheat are discussed.     

Effects of cooking and screw-pressing on functional properties of protein in milkweed (Asclepias spp.) seed meals and press cakes

This study determined the effects of oil processing conditions on functional properties of milkweed seed proteins to evaluate their potential for value-added uses. Flaked milkweed seeds were cooked at 82 °C (180 °F) for 30, 60 or 90 min in the seed conditioner, and then screw-pressed to extract the oil. Proximate composition and protein functional properties of cooked flakes and press cakes were determined and compared with those of unprocessed ground, defatted milkweed seeds. Milkweed seed protein was most soluble at the pH range of 7–10, had excellent emulsifying properties, and produced substantial but highly unstable foams. Heat applied during seed cooking and screw-pressing did not reduce protein solubility and improved emulsifying, foaming, and water-holding capacities. Emulsifying capacity was much higher at pH 10 than at pH 7. These results showed that the protein in both the milkweed seed and its press cake from oil processing has useful functional properties that could be utilized in applications such as paint emulsifier and adhesive extender.     

Rye (Secale cerealeL.) Arabinoxylans: A Critical Review

The arabinoxylans of rye are of considerable importance for rye processing, breadmaking quality and nutritional properties. We review their occurrence in the rye kernel, the variability in their structure and their physico-chemical, nutritional and technological properties, including oxidative gelation. Where appropriate, they are compared with wheat arabinoxylans. Finally, we emphasise that progress in the understanding of their functionality is to be expected on the basis of an understanding of the variability in their structure.