CHAPTER 1: Food Protein Structures, Functionality and Product Development

The unique combinations of biological, nutritional and functional properties in proteins make them important food ingredients with the capacity to provide a range of diverse and highly versatile products following processing.¹  Proteins contain amino acids, which provide essential nutritional support, and certain fractions such as whey and casein are good sources of functional proteins.^1,2  The ability of proteins in whey such as α-lactalbumin, β-lactoglobulin and bovine serum albumin to interact and form aggregates contributes to the techno-functional properties of protein products such as whey protein isolate and whey protein concentrate and therefore to their use in emulsions, gels, coatings, films and protein-based encapsulation materials.^1,3 

To obtain pure proteins or to design food products that contain (added) proteins, it is necessary to separate the desired protein from unwanted proteins and non-protein components present in the starting material. With variations that depend on the protein raw material, the process of producing biofunctional protein hydrolysates and peptides from food proteins typically involves extracting crude proteins from the protein source using aqueous or organic solvents and centrifuging the extract to further purify and separate the isolated proteins from unwanted and often insoluble non-protein materials.⁴  Further purification steps may include dialyzing the supernatant of the protein extract against distilled water to remove residual salt and precipitate salt-soluble contaminants or treating the extract with dilute acid to initiate precipitation of the protein of interest (or that of the impure sediment) while leaving the impurities (or the desired protein) in solution.⁴  The proteins obtained from this kind of extraction and precipitation process are referred to as protein isolates and concentrates and could undergo additional purification based on their size, affinities for certain ligands, hydrophobicity and ionic properties in order to obtain a purer, more homogeneous protein product.⁴ 

The techno-functional properties of proteins such as their water- and oil-binding capacities, solubility and emulsification and foaming properties influence food processing, preparation and storage, in addition to contributing to the quality and organoleptic properties of foods.⁵  Since food processing can affect the techno-functional properties of proteins, a knowledge of the structural arrangement of proteins and the capacity of chosen processing methods to modify protein functionality is important not only to researchers but also to the chemical, pharmaceutical and food industries.⁵  In this chapter, we review various sources of food proteins, the capacity of structure to influence protein function, the use of enzymes to modify structure and thus function, emerging food protein processing methods, the influence of the consumer and ethical and sustainability considerations in shaping food protein product development.

Food proteins and their component bioactive peptides have been isolated from a variety of plant foods, including cereals such as rice, maize, wheat, barley, oat and sorghum,^6–10  pseudocereals such as buckwheat, amaranth and quinoa,^9,11,12  pulses such as peas, chickpeas, lentils and kidney beans,^13–16  oilseed crops such as flaxseed, soybean, peanuts, sunflower, cotton, hempseed and sesame,^17–25  tuber crops such as potato,²⁶  edible seeds such as chia and pumpkin,^27,28  tree nuts such as almonds, chestnuts, pecans and walnuts^29–32  and by-products of fruits and vegetables such as peach, cherry, date and tomato seeds.³³  The wide distribution and heterogeneity of these plant protein sources not only demonstrate the structural diversity, abundance and diverse origins of plant food proteins but also the enormous potential for isolating novel peptides with various important bioactive properties from these plant protein sources. The isolation of proteins from plants distinctly differs from that of non-plant sources such as animals, fish and algae in not involving the additional steps required to remove vegetal non-protein materials such as fiber in addition to antinutritional factors such as enzyme inhibitors, saponins, uricogenic nucleobases, vicine, gossypol, metal chelators, convicine, cyanogenic glycosides and polyphenolics, all of which have the potential to limit protein availability and utilization.^34–37  Given their status as the staple foods in many regions of the world, certain cereals such as wheat, rice and maize are among the most important sources of plant proteins.³⁸  Although animal proteins are generally thought to be of higher quality than vegetal proteins, soybean protein is notable for being of extremely high quality [with a perfect protein digestibility-corrected amino acid score (PDCAAS) of 1.0], containing all the essential amino acids and being low in saturated fats.³⁸  In terms of food product development, various soy-derived foods such as miso, tofu, soy yogurt, yuba, toasted soy protein powders, natto, soy sauces, soy burger, tempeh, soy milk and soy-based sausages have not only achieved great commercial success but also are widely consumed by consumers who see them as nutritious and healthy alternatives to proteins from animal sources.^39–41 

Animal proteins are an important and often essential component of various food products where their physicochemical and biological characteristics serve to enhance the nutritional, organoleptic and even health-promoting properties of those foods.⁴²  The high nutritional quality and excellent physicochemical properties of the dairy proteins casein and whey are central to their wide use in many food products. From infant formulas, sour cream, yogurt, marmalade and coffee whiteners containing whey protein concentrates to mayonnaise, ice cream, desserts, fabricated meats, pizza cheese, whipping cream and salad dressings containing casein and/or egg-white proteins,^2,42  examples of widely consumed food products composed of integral animal protein ingredients abound. Similarly, the physicochemical and structural properties of muscle meat proteins are critical to their use in preparing surimi, a crude myofibrillar protein concentrate, derived from under-utilized marine fishes such as mackerel, croaker, Pacific whiting and Alaska pollock and used for making seafood analogs such as lobster and imitation crab meat.⁴³  The biological properties of protein hydrolysates and peptides of animal origin have also contributed to their use in the food industry for the formulation of medical foods designed to manage food allergies and control conditions such as cystic fibrosis, liver disease, Crohn's disease and phenylketonuria.⁴⁴  Bioactive hydrolysates and peptides have been derived from a myriad of diverse animal and marine protein sources and protein by-products including salmon, oyster, milk, eggs, snow crab, seahorse, giant squid, sea urchin, shrimp, half-fin anchovy, blood clam muscle, jellyfish collagen, rockfish gelatin, cheese, chicken breast muscle, dry-cured ham, tuna cooking juice, abalone viscera, beef, Kacang goat and tunicate, as previously reviewed.^45–49  Such food protein hydrolysates and bioactive peptides of animal origin have been found to possess a range of health-promoting properties, including hypolipidemic, antioxidant, antiproliferative, immunomodulatory, antimicrobial and antihypertensive activities.⁵⁰ 

Although the heightened demand for high-quality food proteins and growing food security concerns in recent years have contributed to the increased use of proteins from insects for both food and feed,⁵¹  the consumption of insects, or entomophagy, is hardly a novel idea given that insects were a part of the diet of the evolutionary precursors of humans.⁵²  It is estimated that up to 2000 different insect species are edible and could be consumed at different stages of development, such as egg, larva or pupa, with some of the most popular including locusts, crickets, caterpillars, bees, wasps and ants.^51,53,54  Insects are relatively rich in high-quality proteins with an essential amino acid content of 46–96%.⁵⁵  Various studies have identified a number of enzymatically derived peptides from insect proteins with different bioactive properties, including anticancer, antihypertensive, antimicrobial, immunomodulatory, antidiabetic and antioxidant activities.^53,56–58  In a recent in silico study, cuticular structural proteins of the edible yellow mealworm (Tenebrio molitor) were found to be more effective precursors of dipeptidyl peptidase-IV (DPP-IV)-inhibiting peptides than other protein types following hydrolysis with pepsin and papain.⁵⁹  The enzymatic hydrolysis of insect proteins could have positive effects on functional attributes of the proteins, as shown by the improvement of solubility in the protein concentrates of Gryllodes sigillatus and Locusta migratoria following hydrolysis.^60,61  Although many studies have reported the production of bioactive peptides (BAPs) and protein hydrolysates from various edible insects, most of them were focused on Bombyx mori (silkworm). Furthermore, as a result of the greater biomass arising from a higher feed conversion ratio, the use of insects for animal feed is thought to be more cost-effective than using traditional livestock feed from soy and fish protein.⁵³  In addition, insect farming is reported to be a more sustainable practice, which leaves a less damaging ecological footprint, than the breeding of animals for food since insects breed faster, do not require as much water and land area and produce comparatively less greenhouse gases.^53,55 

Although records indicate that algae were used for human nutrition more than 2000 years ago in ancient China,⁶²  there has been a resurgent interest in them in recent years, making algae such as seaweeds and microalgae increasingly important sources of food proteins.⁶³  Factors contributing to the growing use of algae include their relative ease of cultivation even on non-arable lands, high sunlight utilization efficiency and capacity to be grown using seawater and on residual nutrients.⁶⁴  Apart from their protein content, algae are known to contain substantial amounts of other nutrients such as B vitamins and polyunsaturated fatty acids,⁶²  as was shown by a study which reported that in addition to its protein content of 26–32%, the unicellular red microalga Galdieria sulphuraria also contained certain B vitamins and β-carotene.⁶⁵  Proteins from algal sources have also been used for the production of health-promoting bioactive peptides, as demonstrated by the isolation of an antioxidant and antiproliferative peptide fraction from the protein waste of the microalga Chlorella vulgaris.⁶⁶  In that study, in which the peptide fraction showed dose-dependent antiproliferative activity, better antioxidant activity than the control Trolox and induced cell cycle arrest in AGS gastric cancer cells, VECYGPNRPQF, an antioxidant and anticancer decapeptide with the capacity to inhibit NO production, was also produced from the microalgal protein.⁶⁶  In other studies, angiotensin-converting enzyme (ACE)-inhibitory peptides were produced from the microalgae Gracilariopsis lemaneiformis⁶⁷  and Mazzaella japonica.⁶⁸  The novel peptide from G. lemaneiformis, Gln–Val–Glu–Tyr, which was obtained following tryptic digestion of the seaweed protein, was found to have an IC₅₀ value of 474.36 µM or 0.255 mg mL⁻¹.⁶⁷  In the latter study, up to 11 distinct potent ACE-inhibitory peptides were obtained following the hydrolysis of M. japonica protein and identification by means of reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS).⁶⁸  Finally, apart from human consumption, seaweed proteins are also used for animal feed and for their sensory value.⁶³ 

Protein isolates and concentrates are regularly used for the formulation of food products and whereas protein isolates have a minimum protein content of 90% on a dry weight basis (dwb), the minimum protein content of concentrates on a dwb is 65%.⁴  The polypeptide composition and structure of the proteins in concentrates and isolates greatly influence their physicochemical properties and functions and thus their possible applications, as demonstrated in various studies.^69–71  For instance, in a study investigating the effect of heat treatment on the physicochemical and emulsifying properties of whey protein isolate and concentrate, the fluorescence intensity of heated whey protein samples was higher than that of the unheated proteins with heating leading to the unfolding of the polypeptide chains and exposing buried hydrophobic groups at the core of the globular protein, thereby resulting in a higher fluorescence intensity.⁷²  In addition, heat treatment also reduced the content of free sulfhydryl groups and decreased the intensity of native β-lactoglobulin and α-lactalbumin [as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)] in the whey protein concentrate thermal polymers compared with the unheated concentrate.⁷²  The difference between the more homogeneous, dense and stable network of the whey protein isolate thermal polymers and that of the heat-polymerized whey protein concentrate was further demonstrated by the differing zeta potential values. Commonly used as a measure of protein suspension stability, the zeta potentials of the heated isolate and concentrate were significantly lower than that of the untreated sample at protein concentrations of 8 and 10%. More interestingly, the zeta potential of the heated concentrate at 12% protein concentration increased as the temperature rose from 80 to 90 °C, whereas the higher protein concentration of 12% had no effect on the zeta potential of the heat-polymerized isolate, underlining the effect of differences in protein composition and the impact of heating on the protein's hydrophobic or hydrophilic character.⁷² 

Apart from heating, succinylation, which is a posttranslational chemical modification involving the derivatization of the ε-amino group of lysine residues in proteins and used to improve solubility, has been shown to alter the structure and thus functional properties of food proteins.⁷³  The succinylation of poorly soluble milk protein concentrate by Shilpashree et al.⁷³  enhanced its solubility by altering the charge on the protein and reducing the particle size of the native milk protein concentrate. Furthermore, succinylation significantly increased the water-binding capacity, foam capacity, foam stability, emulsifying activity and emulsifying stability of milk protein concentrate compared with the untreated sample, in addition to substantially increasing its viscosity.⁷³  It is thought that the transfer of succinyl groups to the lysine residues of proteins leads to unfolding of polypeptide chains as a result of the electrostatic repulsion between the added carboxyl groups of the succinyl groups and the neighboring native carboxyl groups. Therefore, buried amino acid residues within the protein core become exposed, making them accessible for enhanced interactions with the aqueous medium and thus enhancing their water-binding capacity.⁷³  The increase in foam capacity was also partly attributed to this enhanced water-binding capacity. Furthermore, protein isolates from conophor nut (Tetracarpidium conophorum) were reported to have a higher emulsion stability and foaming capacity but lower emulsifying, gelling and oil- and water-absorbing capacities than the oilseed's protein concentrates.⁷⁴  In another study, Ajibola et al.⁶⁹  attributed the relatively low intrinsic fluorescence value of African yam bean protein concentrates to the presence of highly denatured proteins, the denaturation of which possibly resulted in the considerable exposure of the tryptophan and tyrosine residues to the polar aqueous environment, which then led to greater fluorescence quenching. Other studies investigating the structure–function relationships of food protein isolates and concentrates from pea, amaranth, quinoa, chickpea, chia and lentils have also been reported.^70,75 

The solubility of proteins under various extraction conditions has been exploited to obtain different protein fractions with diverse properties and applications, viz. albumins, globulins, prolamins (or gliadins) and glutelins.⁴  The desired protein fraction is usually obtained using a solution with carefully determined properties, such as specific ionic strength and pH, and thus precisely designed to remove the protein of interest while keeping other proteins in solution.⁴  Using a modification of the classical Osborne procedure, successive extraction with distilled water, 5% NaCl, 60–90% ethanol and 0.4% NaOH yields albumins, globulins, prolamins and glutelins, respectively.⁴  Each extract is then centrifuged, filtered and dialyzed against distilled water, followed by centrifugation of the dialysate in order to obtain a supernatant or precipitate, which is freeze-dried as the desired protein fraction.⁴  In a more recent study by the authors, the significantly higher surface hydrophobicity values of flaxseed globulins, compared with the albumin proteins, were attributed to their greater content of hydrophobic amino acids.⁷⁶  Moreover, the higher solubility of the albumins was linked to their content of low molecular weight polypeptides, which enhanced structural flexibility more than the larger polypeptides present in the globulins. The superior albumin solubility was also related to the significantly (p < 0.05) higher levels of negatively charged amino acids and lower contents of hydrophobic and aromatic amino acids.⁷⁶ 

In a recent study examining the structure–function relationship of proso millet storage protein fractions, the significantly higher solubility of the albumin proteins relative to globulins at neutral pH was credited to the presence of low molecular weight protein subunits and the attachment of large carbohydrate moieties to the former and also the greater content of high molecular weight protein subunits in the latter.⁷⁷  Conversely, the presence of large hydrophobic amino acid residues in the prolamins and glutelins of proso millet was linked to their poor solubility. A similar correlation between higher hydrophobic and aromatic amino acid contents and comparatively lower solubility this time in cumin seed (Cuminum cyminum) albumin relative to its glutelin has also been reported.⁷⁸  The cumin seed albumin fraction also had smaller emulsion particle sizes but higher emulsifying activity and stability compared with the glutelin.

In a study of the physicochemical and structural properties of albumin and globulin proteins from buckwheat (Fagopyrum esculentum Moench), Tang and Wang⁷⁹  reported a higher content of uncharged polar amino acids and lower acidic amino acid levels in the albumin relative to the globulin. This may have contributed to the higher solubility of the albumin compared with globulin proteins, a result that highlights the potential for adding buckwheat albumin proteins to acidic protein-fortified beverages.

In a very important study highlighting the structure–function effect of protein fractions and the influence of food protein fractions on other macromolecules, Baxter et al.⁸⁰  reported that glutelins and globulins obtained from rice endosperm reduced the water absorption of rice starch during cooking. Critically, whereas globulin initially accelerated the rate of water uptake by starch and glutelin initially slowed it down, both protein fractions had the overall effect of significantly reducing the total amount of absorbed water compared with the pure starch control.⁸⁰  Furthermore, the addition of glutelin to rice starch increased the pasting temperature whereas globulin lowered the pasting and textural properties other than gel hardness in a concentration-dependent manner.⁸⁰  A possible explanation for this effect of the non-water-soluble glutelin is a direct interaction with starch granules given that it can bind reversibly to amylose and amylopectin, unlike the water-soluble albumin, which in an earlier study⁸¹  had a similar effect on starch during cooking. The effect of albumin was likely mediated by limiting the proportion of free water available through the formation of hydrogen bonds with the water molecules.⁸⁰  Given the striking differences in the effects of the protein fractions on the textural properties of foods, this study highlights the potential application of food protein fractions in product formulations.⁸⁰ 

Enzymes have the capacity to alter or modify the structure of proteins and hence their function. Enzyme crosslinking, the use of enzymes as catalysts to promote the formation of covalent bonds between protein molecules, and enzymatic hydrolysis of proteins to yield hydrolysates and peptides are two strategies for using enzymes to modify protein structure and function.^82,83  Of the many enzyme types that have found wide applications in crosslinking reactions, transglutaminase (a transferase), which catalyzes transamidation reactions between the glutamyl and lysyl side chains of target proteins, has been used most successfully.⁸³  In the food industry, transglutaminase has been successfully used as a catalyst for the formation of protein networks in various foods and for producing protein hydrogels.⁸³  Other enzymes that are commonly used to introduce crosslinks into protein matrices include oxidoreductases such as tyrosinase, laccase and peroxidases.⁸³ 

In a study examining the effect of enzymatic crosslinking on the digestibility of β-casein peptic digests, fungal tyrosinase and bacterial transglutaminase were used as crosslinking enzymes and the proteolytic digestion by pepsin was performed under conditions mimicking the gastric environment.⁸⁴  SDS-PAGE results showed that crosslinked β-casein was stable at acidic pH and more resistant to the proteolytic action of pepsin than the native milk protein, thus highlighting the application of this technique for modifying food structures and developing novel food products.⁸⁴  In addition, the degree of peptic hydrolysis of the native β-casein was found to be 50% greater than that of the fully crosslinked milk protein, a result that further draws attention to the impact of crosslinking on digestibility and the prospects for exploiting this technique in designing food products for satiety, appetite control and controlled energy intake.⁸⁴  Using transglutaminase, the yield of Maillard reaction peptides was considerably increased and the amount of bitter amino acids was significantly decreased, contributing to the improved mouthfulness of Maillard reaction products from soybean protein hydrolysates.⁸⁵  In another study, microbial transglutaminase-mediated crosslinking was used to enhance the functional properties of soy protein isolate, including properties such as surface hydrophobicity, apparent viscosity and emulsion and foaming stability.⁸⁶  Crosslinking reactions catalyzed by laccase have also been used to improve the protein emulsifying properties of sodium caseinate,⁸⁷  enhance the gelation, water-holding capacity and antioxidant properties of α-lactalbumin⁸⁸  and reduce potential allergenicity in fish by modifying amino acid residues in fish protein allergenic epitopes.⁸⁹ 

Although bioactive peptides can be released from food proteins in a number of ways, including microbial fermentation, during gastric digestion and by the action of enzymes during endogenous proteolysis,⁴⁸  the in vitro exogenous enzymatic hydrolysis of food proteins represents by far the most important, common and prolific means of producing bioactive peptides for research, given the potential of this technique to produce many novel active peptide sequences.⁴⁶  Since peptide sequences are essentially inactive while encrypted within primary parent protein structures, they must be released and activated by enzymatic action in order to exert their bioactive properties.⁹⁰  Some of these bioactive functions include antihypertensive, antioxidant, antidiabetic, antimicrobial, anticancer, hypocholesterolemic, immunomodulatory and anti-inflammatory activities.⁴⁵  The biological function of food protein-derived peptides is known to depend on their structural and chemical properties, including amino acid composition, sequence, peptide chain length and charge.⁹¹  For instance, peptides with immunomodulatory effects tend to be short (2–10 residues) and to contain negatively charged, hydrophobic and aromatic amino acids.⁹¹  It was reported that the peptide PTGADY released from Alaska pollock proteins following tryptic digestion significantly improved humoral, cellular and non-specific immunity in immunosuppressed mice.⁹²  The most frequently reported immunomodulatory peptides are composed of proline, phenylalanine, glycine, valine, leucine, glutamic acid and tyrosine residues.⁹¹  Conversely, low molecular weight peptides containing branched-chain amino acids, proline and/or aromatic amino acids have been shown to be effective antihypertensive agents in general.⁸² 

Since mammalian blood pressure is mainly regulated by ACE and renin, two principal enzymes in the renin–angiotensin pathway, differences exist in the specific manner in which antihypertensive peptides inhibit the activity of each enzyme, as previously discussed in detail.^82,93  In brief, whereas the presence of peptides containing proline, aromatic, hydrophobic and branched-chain amino acids seems to enhance ACE inhibition, renin inhibition by bioactive peptides does not seem to depend on the presence of proline residues. Moreover, renin inhibition appears to be enhanced by the presence of a bulky amino acid residue at the C-terminus and a hydrophobic amino acid residue at the N-terminus.^82,93  In addition, a high acidic amino acid content, which confers a net negative charge on a peptide, seems to promote ACE inhibition since the negatively charged peptide could diminish enzyme activity by interacting with the ACE active site, causing a chelation of the zinc atom essential for the activity of the metalloprotease.⁸² 

For antioxidant peptides, a study showing the importance of amino acid sequence to antioxidant activity found that the composition of the side-chain terminal amino acid residues affects the capacity of antioxidative peptides to chelate metal ions.⁹⁴  Despite having the same chain length, QEKLE (S35–N2) demonstrated a stronger metal ion chelating ability than DAQEK (S35–C2) due to the Gln residue present at its N-terminus. Since Gln contains a carbamoyl group (–CONH₂) in its structure, the carbonyl group (–CO–) has the ability to function as a ligand molecule, thus enhancing the formation of a stable complex with the metal ion and enabling S35–N2 to trap metal ions more effectively.⁹⁴ 

Other examples of structure–function mechanisms of antioxidant peptides are given in a comprehensive review including the report that the –COO– group of glutamine next to tyrosine in the peptide Ala–Glu–Glu–Glu–Tyr–Pro–Asp–Leu derived from dry-cured ham enhanced its antioxidant activity by inducing the donation of the hydrogen atom from the –OH group in the tyrosine residue, whereas Glu, Asp, His and Lys residues, which are known to interact with metal ions, enhanced metal chelation by rice bran protein hydrolysates.⁴⁶  On their part, food protein-derived anticancer peptides mostly range from 3 to 25 amino acid residues in length and contain predominantly hydrophobic amino acids such as leucine, proline, glycine and alanine, in addition to one or more of tyrosine, serine, lysine, threonine, arginine and glutamic acid.⁹¹ 

Techno-functional properties of proteins such as their emulsion, foaming, gelation, solubility, rheological, viscosity and water-binding properties could have an impact on their use in the development of new food products. Proteins play a critical role as surfactants in edible foams and emulsions, with proteins derived from milk and egg occupying a central position as foam and emulsion stabilizers.⁹⁵  During the process of microparticulation, which treats whey protein concentrate with dynamic high-pressure shearing in the presence and absence of heat, the whey protein concentrate particles could become aggregated and attain enhanced emulsification, gelation and foaming functional properties.⁹⁶  Simplesse, the most popular protein-based fat replacer, which is used in a variety of food products such as ice cream, salad dressing, yogurt, butter and margarine spreads, cheese spreads, vegetable dips, mayonnaise and sour cream, is produced following such a microparticulation process. With the increase in the number of consumers choosing a vegetarian or vegan lifestyle, there has been a boost in the push to explore the use of plant proteins such as pulses as foaming and emulsification materials.⁹⁵  Aquafaba, the viscous slurry-like water in which legume seeds such as chickpeas have been cooked, has also been studied for its capacity for use as an egg-white substitute in food foams and emulsions.⁹⁷  The study reported that centrifuged aquafaba produced more stable emulsions compared with egg white and that the functional properties of the aquafaba were not affected by increased levels of NaCl in the foam and emulsifying capacity tests conducted.⁹⁷ 

Supercritical fluid extrusion (SCFX), a hybrid food processing method that combines the distinct advantages of supercritical fluid and extrusion technologies, has emerged as a very important technique for food processing.⁹⁸  The obvious advantages of CO₂, including its ability to reach its supercritical conditions easily and being chemically inert, non-toxic and non-flammable, make it the fluid of choice for SCFX.⁹⁹  Unlike conventional steam-based extrusion, which is carried out under extreme processing conditions, such as temperatures >130 °C and high shear values (>150 rpm screw speed), which can adversely affect the structure and conformation of thermolabile biomolecules such as proteins, the comparatively milder SCFX conditions ensure minimal processing losses.⁹⁸  The benefit of mild processing conditions and other advantages, such as the possibility of using a variable number and size of cells for texture control and the ability to encapsulate flavor compounds, colorants and bioactives on the interior surface of cellular structure, have contributed to the assortment of (protein-containing) products made using SCFX, such as ready-to-eat breakfast cereals, pasta, snack foods, whey protein crisps and confectionery.^98,100–103  Gopirajah et al.¹⁰¹  reported that the SCFX of milk protein using a co-rotating twin-screw extruder (Figure 1.1) resulted in a new product with higher emulsion activity index and lower creaming index compared with unextruded commercial milk protein concentrate and commercial sodium caseinate, and that this new ingredient was able to form stable emulsions at room temperature. In another study, whey protein was found to exhibit higher viscosity and shear thinning behavior and to provide a better oil-in-water emulsion that was stable against creaming and sedimentation compared with emulsions formed by unextruded whey protein concentrate and commercial sodium caseinate, which were used as controls.¹⁰⁴  In addition, the report that SCFX reduced the trypsin inhibitor content and increased the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of lentils¹⁰²  is an example of the capacity of structural changes introduced following processing to influence not only a food protein's techno-functionality but also its bioactivity.

Ohmic (or Joule) heating ensures rapid and uniform heating of foods while maintaining food quality and extending shelf life by preventing microbiological deterioration.¹⁰⁵  Although conventional thermal processing helps to provide microbiologically safe food of high quality, the intensity of thermal processing has been known to diminish the nutrient content and organoleptic properties of food such as color, aroma, flavor, appearance and texture.¹⁰⁰  Whereas fortification could help limit nutrient losses via traditional thermal treatment, there is hardly a similar remedy for the loss of sensory attributes.¹⁰⁵  However, it has been reported that OH processing does not significantly impair the sensory properties of foods.¹⁰⁵  There was no effect on either amino acid content or protein quality following OH treatment of vegetable baby food compared with conventional retort sterilization, which decreased the amounts of both essential and non-essential amino acids in the baby vegetable purées.¹⁰⁶  It is thought that the decrease in amino acid content following conventional sterilization is due to the heightened susceptibility of vegetable proteins to the more intense conditions of conventional heat treatment.¹⁰⁶ 

Demonstrating the promising prospects and innovative potential of this emerging processing technology, Pereira et al.¹  used a combination of OH and iron cold gelation to produce whey protein hydrogels, which made possible the incorporation of considerable amounts of iron into the protein network. The study took advantage of the distinct physicochemical properties of whey proteins, the relatively higher strength and higher water-holding capacity of gels produced by cold gelation, the ability of iron to induce aggregation and gel formation in preheated proteins and the opportunity to use a variation of OH moderate electric fields (MEFs) to modify protein structure and thus influence techno-functionality.¹  In an earlier study by the same group, images produced using transmission electron microscopy showed that the aggregation of whey proteins during OH thermal treatment can be minimized with a simultaneous reduction of the heating charge.¹⁰⁷  It has also been reported that the application of MEFs of different intensities during the OH treatment of sodium caseinate caused subtle conformational changes in protein structure and yielded a protein with enhanced solubility, especially at the isoelectric point.¹⁰⁸  Given the use of sodium caseinate as an emulsifier, thickener and/or foaming agent in the production of baked foods, whipped toppings, meat products, cheese analogs and desserts, this result suggests a potential application of sodium caseinate in liquid foods.¹⁰⁸  Lastly, ohmic heating was shown to preserve nutritional quality in a study in which certain flavor-enhancing compounds were detected in OH-treated infant formula samples but not in samples undergoing conventional thermal processing.¹⁰⁹  In addition, the OH-treated samples also formed a smaller amount of 5-hydroxymethylfurfural, an antinutritional agent and intermediate product from the Maillard reaction, than the control samples.

Although the non-uniform heat distribution of microwave heating is an obvious drawback in comparison with an alternative emerging processing technology such as ohmic heating,¹⁰⁵  microwave heating has a number of advantages, including high heating rates, ease of control, convenience, thermal efficiency and short heating times.¹¹⁰  Microwave technology is based on the application of electromagnetic irradiation in the 200 MHz–300 GHz frequency range.¹⁰⁰  An investigation of the effect of processing on the micromorphology, microstructure and techno-functional attributes of Alaska pollock surimi polysaccharide–protein mixed gels showed that microwave treatment enhanced the textural properties and water-holding capacity of gels.¹¹¹  In addition, microwave-heated gels had relatively higher gel strength and much stronger interactions between the molecular chains of their polysaccharides and proteins compared with water bath-heated surimi mixed gels.¹¹¹  Compared with a control, microwave-assisted enzymatic hydrolysis was shown to improve significantly the protein solubility, degree of hydrolysis and antioxidant activity of fish frame protein hydrolysates while reducing their antigenicity.¹¹²  Although both microwave treatment and water bath heating altered the α-helix conformation of beef myofibrillar protein, it was the former that resulted in a significantly higher binding ability of ketone flavor compounds and thus a more classic meaty taste.¹¹³  Also, conformational changes attributed to microwave pretreatment are thought to have contributed to the higher ACE and DPP-IV inhibition and lower allergenicity of Alcalase-hydrolyzed cricket (Gryllodes sigillatus) proteins compared with samples obtained by conventional enzymatic hydrolysis.⁶⁰  The often reported issues of texture deterioration and higher cooking loss encountered during microwave processing of surimi products were found to be minimized in a study in which a combination of microwave and traditional conduction heating with a water bath was used.¹¹⁰  Not only did microwave heating inhibit protein denaturation and expand protein aggregates, but also the temperature preservation mode of microwave heating yielded proteins with desirable morphology and improved texture.¹¹⁰ 

Food processing by means of ultrasound technology is based on acoustic waves with frequency >20 kHz¹¹⁴  and is divided into low-intensity or high-frequency (100 kHz–1 MHz) ultrasound and high-intensity or low-frequency (20–100 kHz) ultrasound.¹¹⁵  Ultrasound treatment has the capacity to alter protein structure and, therefore, influence techno-functionality and bioactivity.¹¹⁴  The intensity of the ultrasound treatment applied influences the effects on structure and techno-functionality as seen with whey proteins, where intense ultrasound treatment resulted in protein aggregation and reduced solubility, whereas mild ultrasonication exposed hydrophilic domains and thus increased water–protein interaction, resulting in an increase in solubility.^116,117  When applied to aggregated soybean β-conglycinin (7S) and glycinin (11S) fractions, high-intensity ultrasound treatment modified the tertiary and quaternary structures of 7S and 11S and decreased their sulfhydryl group content, but did not significantly alter their secondary structures.¹¹⁸  In addition, high-intensity ultrasonication reduced the turbidity and particle size of soybean β-conglycinin and increased its solubility, surface hydrophobicity, emulsifying activity and emulsion stability, while also reducing the turbidity and increasing the emulsifying activity of the glycinin fraction.¹¹⁸ 

Ultrasound-treated black soldier fly (Hermetia illucens) protein isolates and hydrolysates showed the highest antioxidant activity and exhibited excellent solubility and foam expansion capacity over a wide pH range compared with the untreated samples.¹¹⁹  The high-intensity ultrasound treatment of sunflower protein isolates led to a significant increase in surface hydrophobicity, solubility, emulsifying capacity, emulsion stability, foaming capacity, foam stability, oil-binding capacity and sulfhydryl content, but a decrease in water-binding capacity and lysine content of the isolates.¹¹⁴  The sonication of sunflower protein samples apparently resulted in molecular unfolding, thereby exposing previously buried hydrophobic groups and regions.¹¹⁴  The change in conformation has an implication for solubility and other functional attributes since increased surface hydrophobicity has been known to result in higher solubility.¹²⁰  Other studies reporting the use of ultrasound treatment to influence protein microstructure and functional properties include those of canola protein isolate¹²¹  and beef myofibrillar proteins.¹²² 

Food processing using PEF technology (Figure 1.2) typically involves the application of short pulses of high electric field and intensity in the order of 15–50 kV cm⁻¹ every few micro-to milliseconds to a product contained in a treatment chamber placed between electrodes.^123–125  As a novel, non-thermal, green technology, PEF treatment does not result in the type of loss of nutritional and sensory quality observed with traditional thermal treatment.^100,126  In the early days, when it was almost wholly focused on providing microbiologically safe foods by inactivating microbes, the ability of PEF to limit the loss of food quality made it ideal for the processing of high-protein foods and liquid food products including yogurt drinks, apple sauce, salad dressing, milk, milk products, egg products, juices and other liquid foods.¹²⁵ 

It has been reported that PEF treatment (0–28 kV cm⁻¹) significantly improved the solubility, sulfhydryl group content and surface hydrophobicity of myofibrillar proteins isolated from pale, soft, exudative (PSE)-like chicken breast and that this improvement was directly proportional to increasing PEF intensity up to >18 kV cm⁻¹, when the physicochemical properties started to deteriorate.¹²⁷  In addition, the application of PEF to the chicken breast myofibrillar proteins modified their rheological properties but not their primary structure. PSE-like chicken is a serious concern in poultry processing and marketing because of its subpar color, poor water-holding capacity and soft texture.¹²⁸  That study, which reported the contribution of PEF to the improvement of meat protein functionality, is important to the food industry given that PSE-like chicken is a critical meat quality issue that could severely affect sensory attributes and undermine consumer willingness to purchase chicken products.¹²⁷  A study comparing the effect of PEF and conventional thermal processing on ovomucin-depleted egg-white proteins over a range of pH found that PEF treatment (0.7–1.8 kV cm⁻¹) minimized protein aggregation during the processing of heat-sensitive egg-white proteins compared with traditional heating for 10 min at 60 °C.¹²⁹  This result is of great relevance to the food industry, where turbidity is a serious consideration in the protein fortification of drinks.^129,130 

In another study, PEF treatment resulted in unfolding of protein structure and an increase in the superficial hydrophobicity of rehydrated whey protein isolate.¹²⁶  PEF processing of proteins could also have an impact on other macromolecules, as shown in a study by Zhang et al.,¹³¹  where PEF pretreatment of canola proteins prior to oil extraction resulted in an increase in oil yield, in addition to improvements in the physicochemical properties of the protein such as solubility, foaming capacity, foam stability, emulsifying capacity, emulsion stability, oil-holding capacity and water-holding capacity. Other studies have reported the use of PEF treatment to rupture microbial cell membranes and enhance safety,¹³²  inactivate endogenous enzymes such as alkaline phosphatase in milk and lipoxygenase in beans during food processing,¹³²  improve meat and fish tenderization and aging¹³³  and improve drying temperature and freezing time¹³⁴  during food protein processing.

HHP processing involves the exposure of food products, often in custom-made vessels containing a pressure-transmitting fluid, to high pressures in the 100–1000 MPa range for 5–30 min,¹⁰⁰  and was initially developed to eliminate microorganisms and extend food shelf life.¹³⁵  HHP treatment has also been used to enhance the process of enzymatically producing bioactive peptides from food proteins and to increase the yield and bioactivity of the peptides.¹³⁵  HHP treatment significantly enhanced the antioxidant activity and the degree of hydrolysis of Alcalase-derived sweet potato protein hydrolysates.²⁶  In separate studies, HHP altered the structure and reduced the allergenicity of α-casein¹³⁶  and fresh squid (Todarodes pacificus) proteins.¹³⁷  HHP treatments at various pressures resulted in changes in the secondary structure components of α-casein, including an increase in α-helices and β-turns, and also a statistically significant decrease in β-sheets, and thus contributed to changes in allergenicity, with the lowest concentration of allergenic peptides being recorded at 200 MPa.¹³⁶  Using a range of high-pressure treatments (200–600 MPa), Jin et al.¹³⁷  established that the allergenicity of the tropomyosin Tod p1 (TMTp1) protein in fresh squid could be reduced following HHP-induced modifications of structure. In addition, HHP processing disrupted secondary structure conformation, improved simulated gastrointestinal digestibility and increased surface hydrophobicity (H₀) at 200 and 400 MPa, but not at 600 MPa, where H₀ was reduced.¹³⁷  It was also recently reported that HHP processing of cod (Gadus morhua) enhanced protein digestibility and attenuated the binding capacities of IgE and IgG with no protein oxidation, unlike two traditional thermal processing methods, which actually increased allergenicity and induced protein oxidation in the sample.¹³⁸  Given the frequent and substantial consumption of milk and other dairy products and the widespread popularity of squid and cod on seafood menus, these results could prove consequential in the processing of protein foods. In buckwheat grains exposed to HHP treatment, phytic acid, trypsin inhibitor, tannin and saponin levels were reduced by 45.5, 13, 19.9 and 14.6%, respectively, compared with untreated controls in a study demonstrating a potential application of this emerging processing technology in reducing antinutritional factors.¹³⁹  Lastly, HHP treatment of red abalone (Haliotis rufescens) muscle protein led to β-sheet and β-turn conformational changes, which contributed to an increase in protein digestibility,¹⁴⁰  and HHP-induced modifications of bovine serum albumin structure resulted in changes in the protein's physicochemical properties such as solubility and emulsifying and foaming capacities.⁵ 

In general, microparticles (which range in size from 0.1 to 1000 µm) are designed to encapsulate bioactives, shield encapsulated bioactives from degradation and control their release.¹⁴¹  Microencapsulation carriers can be designed in a variety of ways, including as mononuclear microcapsules, in which the core, i.e. the particles, are coated with or surrounded by the shell material, polynuclear microcapsules, which have many cores enclosed within the shell material, and matrix microcapsules, in which there is a uniform or homogeneous distribution of the core material within the shell (Figure 1.3).^5,142  Certain physicochemical properties of proteins, such as their aggregation, solubility and gelation capacities, have been exploited for the design of novel encapsulation systems that help shield unstable, sensitive or unpleasant-tasting bioactive compounds and food ingredients, including probiotics, enzymes, flavors, vitamins, antioxidants, colors, polyunsaturated fatty acids and acidulants, from harsh storage, processing and gastrointestinal tract transport conditions to ensure their delivery while enhancing bioavailability.^141,143  Proteins are excellent candidates for the design of such bio-based macro-, micro- and nanoscale delivery systems given their high nutritional value, biocompatibility, biodegradability and their GRAS (generally recognized as safe) status, making them the most widely used biomaterials in food technology.¹⁴¹ 

The use of pea protein as a natural, hypoallergenic, sustainable and low-cost protein-based encapsulation material was demonstrated in a study in which a spray drying technique was adopted for the microencapsulation of conjugated linoleic acid.¹⁴⁴  It was found that pea protein concentrate yielded a more effective encapsulation wall system than pea protein isolate and a blend of the protein concentrate and maltodextrin provided the most promising microparticles with respect to solubility, dispersibility and surface topography.¹⁴⁴  In another study, Selmer et al.¹⁴⁵  exploited the foaming, water-binding, emulsification and heat-induced coagulation properties of egg-white proteins and the novel technology of supercritical drying to design protein-based aerogels as potential carrier materials. On testing pasteurized and spray-dried egg-white proteins at different pH, ionic concentrations and protein contents, it was found that the most mechanically stable aerogels were obtained at alkaline pH.¹⁴⁵  Another study showed that the solubility and bioavailability of curcumin were enhanced following its encapsulation in whey protein microparticles, suggesting that the design of a soluble curcumin–whey protein complex containing water-insoluble ingredients such as curcumin could potentially be beneficial in the formulation of curcumin-based pharmaceuticals and functional foods.¹⁴⁶  It has also been reported that the susceptibility of tuna oil to oxidative degradation could be curtailed by combining it with peppermint oil in microcapsules formed with whey protein isolate and carboxymethylcellulose.¹⁴⁷  Thus, taking advantage of the emulsification capacity of whey protein, the beneficial effects of the docosahexaenoic acid (DHA)-rich tuna oil could be preserved and optimally utilized. Similar studies include the use of novel matrices provided by chia seed protein isolate–chia seed gum and flaxseed protein isolate–flaxseed gum complex coacervates to microencapsulate chia seed and flaxseed oil, respectively, thus enhancing the oxidative stability of the essential fatty acids found in the oils.^148,149 

Compared with microencapsulation, nanotechnology including the development of nanoscale (ranging in diameter from 10 to 1000 nm) carrier materials for food applications is considered recent.¹⁴¹  Such nanoparticles are designed to respond to changes in environmental stimulus such as pH and temperature and are therefore ideal for delivering compounds and bioactives at a precise instant and location in the gastrointestinal tract.¹⁵⁰  Using a multiple emulsions and spray-drying technique, saffron extracts (<100 nm) were nanoencapsulated with a whey protein concentrate–pectin complex, which yielded spray-dried powders of up to 96% encapsulation efficiency.¹⁵¹  Yellow mealworm (Tenebrio molitor L.) proteins were recently used to design biopolymer nanocapsules for the delivery and controlled release of curcumin.¹⁵²  Uncoated nanoparticles and chitosan-coated nanocomplexes were used and the results showed that the stabilization of curcumin was more efficient in the coated nanoparticles and that over 90% of encapsulated curcumin was released following exposure to simulated model gastrointestinal conditions.¹⁵²  Using nanoscale carrier materials formed by a whey protein–pectin complex, Ghasemi et al.¹⁵³  encapsulated d-limonene, a volatile food flavoring substance known to be chemically unstable upon exposure to air, light, moisture or high temperatures. They reported an encapsulation efficiency of 88% and found that nanocomplexes made with a blend of 4% whey protein concentrate and 1% pectin had the lowest stability and highest viscosity at pH 3.0 ¹⁵³. The physicochemical properties of whey protein and its mixture of globular proteins are thought to have contributed to the formation of optimal whey protein-based nanocapsules and are thus potentially important for the production of d-limonene-containing food products such as muffins, cakes, juices and biscuits.¹⁵³  Additional information can be obtained from a recently published excellent review on the use of protein-based biomaterials for designing nanoscale carrier materials.¹⁵⁴ 

In choosing a food product, today's consumer goes beyond palatability to consider other issues such as sustainability, inherent healthfulness and ability of a food product to provide health benefits supplemental to its nutritive function. In a study that captures the importance of these considerations, rice–soy crisps were supplemented with an alternative algal protein source, spirulina, and fortified with the micronutrients zinc, iron, vitamin A and vitamin C.¹⁰³  As has been highlighted in this chapter, unicellular organisms such as the red microalga Galdieria sulphuraria have continued to receive significant research attention as sustainable sources of food proteins because of their high protein density since simple production systems can yield considerable amounts.^65,90  The high protein content, which is comparable to that of vegetal proteins, and also high eicosapentaenoic (EPA) and DHA levels have helped promote their marketability as health foods.⁶³  The popularity of clean meat (also referred to as cultured meat or in vitro meat), which is produced from animals after cell isolation, culture and tissue engineering manipulations,¹⁵⁵  has continued to rise. This is not surprising considering that the prospect of producing meat without slaughtering animals is appealing for obvious environmental benefits and also to certain consumers for ethical reasons.⁶³  Given that this form of meat production uses potentially 45% less energy and emits 96% less greenhouse gases, in addition to the argument that an in vitro meat bioreactor the size of a swimming pool could feed 40 000 people annually while taking up 99% less land area than the average for farmed beef, such sources of food protein are bound to attract growing interest in the years ahead.^63,156  Reports of the better emulsifying capacity and stability of emulsions stabilized by SCFX-processed protein extrudates compared with unextruded controls^101,104  highlight the benefits and importance of the more sustainable extrusion process, given the wide applications of emulsions in food products such as ketchup, sauces, salad dressings, dips, mayonnaise and creams.¹⁰⁴  A combination of the green processing technology of ultrasound treatment and the sustainable choice of using an underutilized edible insect protein source for enzymatic peptide production resulted in the release of peptides with increased antidiabetic activity.¹⁵⁷  In the study, subtilisin was used to cleave the native mealworm (Tenebrio molitor) protein prior to the tryptic treatment of the hydrolysate for the release of peptides with enhanced α-glucosidase inhibitory activity, which have the potential for use as ingredients in formulating foods for glycemic index control.¹⁵⁷ 

Although meat is generally considered a good source of high-quality protein, it is often regarded as unhealthy and unsustainable, and the last 5 years have seen a dramatic increase in plant-based meats.^158,159  Red meat and processed meats such as bacon, ham and sausages have been associated with cardiovascular diseases, cancer, type 2 diabetes and obesity, and livestock cultivation takes up a considerable amount of arable land and contributes immensely to the emission of copious amounts of greenhouse gases.^159,160  These considerations and the reported 350% increase in the number of consumers choosing to adopt a vegan lifestyle in recent years for ethical and health reasons have contributed to the interest in and development of so-called ‘meatless meats’.¹⁶¹  Plant-based meats currently on the market, such as Beyond Meat^® and Impossible Foods^® burger patties, are designed to mimic the texture, flavor, color, taste, nutritional characteristics and experience of eating specific types of meat and thus seek to appeal to a wider and more diverse consumer base than the relatively smaller vegan and vegetarian market that was the target of earlier meat alternatives.^158,159  A number of issues have been raised with regard to recently introduced meatless meats, examples being their high sodium content compared with a traditional beef burger patty and the addition of high levels of heme to the Impossible Foods burger patty to enhance its meaty flavor and appearance.¹⁵⁹  A high intake of heme has been linked to an increase in body iron stores and elevated type 2 diabetes risk.^159,162  Also, there is the argument that simply replacing meats with plant-based meat alternatives does not in and of itself translate to improved diet quality and health, since meat analogs such as Beyond Meat and Impossible Foods burger patties are often consumed in fast food settings along with French fries, white bread buns and sugary drinks.¹⁵⁹ 

The growing demand for protein foods presents both opportunities and challenges for researchers and food product developers. For instance, in seeking to produce sustainable, more affordable but also nutritious protein-rich foods from insects, scientists must also confront the microbiological, chemical, physical and allergenic risks inherent in using members of the class Insecta for food.^55,58  Given the significant microbial load of Enterobacteriaceae in insects such as Tenebrio molitor and Acheta domesticus, the capacity of insects to accumulate chemical contaminants, the risk of choking from insufficiently ground insect particles and the presence of certain allergens in insects such as mealworms, it is imperative to analyze critically the risks involved in developing food products containing insect proteins and to also devise innovative ways for preventing or minimizing such risks.⁵⁵ 

Although the use of emerging green technology for food processing continues to enjoy growing popularity, there is a need to increase efforts towards scaling up reported beneficial results. It is also important to understand the mechanisms by which the alteration of protein structure results in functional changes, an example being the need to elucidate the precise means through which the modification of α-casein structure could regulate allergenicty.¹³⁶  In addition, gaps in research on the impact of SCFX on protein conformation and on the viability of using a combination of different emerging technologies in place of one aggressive processing method need to be explored.¹⁰⁰  Similarly, concerns have been raised regarding the production process for cultured meats that requires the use of synthetic additives such as hormones and certain nutrients, which, although they are of food grade, could be unappealing to certain consumers who place a premium on naturally produced foods.⁶³  Finally, further research is required on enzyme crosslinking of proteins, given the possible risk of creating foods with enhanced potential for allergenicity.¹⁶³