A review on the extraction and processing of natural source-derived proteins through eco-innovative approaches

processes Review A Review on the Extraction and Processing of Natural Source-Derived Proteins through Eco-Innovative Approaches Giselle Franca-Oliveira, Tiziana Fornari and Blanca Hernández-Ledesma *   Citation: Franca-Oliveira, G.; Fornari, T.; Hernández-Ledesma, B. A Review on the Extraction and Processing of Natural Source-Derived Proteins through Eco-Innovative Approaches. Processes 2021,9, 1626. https://doi.org/10.3390/pr9091626 Received: 29 July 2021 Accepted: 3 September 2021 Published: 9 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Institute of Food Science Research (CIAL, CSIC-UAM, CEI UAM+CSIC), Nicolás Cabrera, 28049 Madrid, Spain; [email protected] (G.F.-O.); [email protected] (T.F.) *Correspondence: [email protected]; Tel.: +34910017970 Abstract: In addition to their nutritional and physiological role, proteins are recognized as the major compounds responsible for the rheological properties of food products and their stability during manufacture and storage. Furthermore, proteins have been shown to be source of bioactive peptides able to exert beneficial effects on human health. In recent years, scholarly interest has focused on the incorporation of high-quality proteins into the diet. This fact, together with the new trends of consumers directed to avoid the intake of animal proteins, has boosted the search for novel and sustainable protein sources and the development of suitable, cost-affordable, and environmentally friendly technologies to extract high concentrations of valuable proteins incorporated into food products and supplements. In this review, current data on emergent and promising methodologies applied for the extraction of proteins from natural sources are summarized. Moreover, the advantages and disadvantages of these novel methods, compared with conventional methods, are detailed. Additionally, this work describes the combination of these technologies with the enzymatic hydrolysis of extracted proteins as a powerful strategy for releasing bioactive peptides. Keywords: food proteins; novel extraction methodologies; enzymatic hydrolysis; bioactive peptides 1. Introduction Proteins are essential macronutrients involved in the growth and development of the body. In addition to their nutritional and physiological properties, the techno-functional characteristics of proteins are responsible for the appearance, texture, and stability of food products. Moreover, proteins have been demonstrated to be a source of peptides capable of exerting multiple biological activities after their release by hydrolysis, gastrointestinal digestion, and/or food processing [1]. The incorporation of high-quality proteins into the everyday diet is a prevalent theme in current research. To meet consumer trends in limiting the intake of animal proteins, nutritionists and food industries are exploring the use of novel and sustainable protein sources from plants, insects, and algae. However, satisfying this demand requires the simultaneous development of suitable, cost-effective, and eco-friendly technologies to extract higher concentrations of valuable proteins to be incorporated into food and supplements [ 2 ]. The application of conventional methods generally results in lower extraction yields due to protein degradation from extreme pH, temperature, solvent conditions, and long extraction times. Therefore, researchers are currently focused on non-thermal green technologies for improving extraction efficiency and reducing protein degradation. These methods generally have no damaging effect on the environment. Moreover, the minimal use of toxic chemicals and reactants makes extracted proteins safe for animal and human consumption. Other than improving the protein yield, these innovative techniques can enhance their nutritional and techno-functional properties as well as their potential as a source of bioactive peptides. Understanding the principles of each of these new methodologies, and their advantages and disadvantages in comparison with conventional methods, is essential to advance in the knowledge of their applications in the food industry, and to Processes 2021,9, 1626. https://doi.org/10.3390/pr9091626 https://www.mdpi.com/journal/processes Processes 2021,9, 1626 2 of 24 achieve extractions, from new sources, of high-purity proteins with interesting properties at high concentrations. Thus, this review aims to respond to this need by providing all the existing evidence on the emergent extraction technologies applied on natural protein sources. Moreover, the application of some novel technologies, in combination with the enzymatic hydrolysis of extracted proteins with the aim to release bioactive peptides, is also described. 2. Extraction of Proteins from Natural Sources 2.1. Chemical Extraction Techniques The classification of chemical methods is based on the solvent used, such as water, alkali, organic solvents, and acids. Although the efficiency of the methodology primarily depends on the nature of the protein sample, the processing conditions have also been demonstrated to show their influence on protein recovery [ 3 ]. Aqueous extraction is a frequently used method due to the high protein solubility and stability of isolated proteins in water. Moreover, this methodology shows other advantages such as its easy operation conditions and low cost [ 4 ]. Generally, extraction with water is performed under basic conditions, as it has been described for proteins from different plant sources such as mung bean (Vigna radiata) [ 5 ], grass pea (Lathyrus sativus) [ 6 ], rice and rice bran [ 7 , 8 ], and tomato [ 9 , 10 ], among others. However, extreme extraction conditions, such as high temperature or high alkaline conditions, could influence the thermal, conformational, and functional properties of protein fractions, reducing their nutritional value and degrading their bioactive compounds [ 11 ]. Mild acidic conditions have also been reported to efficiently extract proteins from sunflower with a 23–26% rate recovery [12]. To extract proteins containing non-polar, hydrophobic and/or aromatic amino acid residues, organic solvents like ethanol, butanol, and acetone are required [ 13 ]. Thus, a recent study has described the application of an ethanol-petroleum ether combination in water to extract proteins from Moringa olifera seeds, reaching 33% of recovery after the purification of the protein [4]. 2.1.1. Aqueous Two-Phase System (ATPS) Currently, aqueous two-phase system (ATPS) is being used for efficient protein extraction due to the associated properties, such as the hydrophobicity of the phase system, the electrical potential between phases, molecular size, and the bioaffinity of the protein [ 2 ]. ATPS is a multifunctional technique that allows separating, concentrating, and purifying proteins. It is based on the mixture of two components of different natures. The appropriate choice of these two components guarantees the completion of two defined and equilibrated layers. The benefits of the ATPS technique are of great interest to the scientific community. Its attributes of rapidity, flexibility, economical convenience, and biocompatibility ensure a higher selectivity, purity, and extraction yield than with conventional systems. On top of that, phase constituents do not denature proteins; they can stabilize protein structures within their biological activity [ 14 ]. A recent review summarizing the existing evidence on the application of ATPS for the recovery of valuables, as well as the elimination of contaminants from industrial waste discharges, has been published [ 15 ]. Among these high-value components, proteins have been demonstrated to be efficiently extracted by ATPS. Therefore, two-phase systems constituted by sodium citrate and polyethylene glycol, or sodium citrate and ethanol, have been recently reported as useful in extracting proteins from shrimp (Litopenaeus vannamei) waste and microalgae (Arthrospira platensis) [16,17]. 2.1.2. Subcritical Water Extraction (SWE) Subcritical water (SW) extraction (SWE) is a technique based on the use of hot water in the range from water’s normal boiling point (100 ◦ C) to water’s critical temperature (374 ◦ C), while using a high pressure to maintain water in its liquid state within those temperatures (usually 220–230 bar). The increase in temperature significantly modifies water’s properties. For example, its viscosity and density decrease, but its compressibility remains low, and Processes 2021,9, 1626 3 of 24 indeed the most important change is the temperature-dependent decrease of water’s dielectric constant. Thus, the hydrogen bonding structure of water is weakened, which enables the solubilization of moderately polar and nonpolar compounds. Furthermore, SW produces a high-ion product, a property suitable for hydrolysis reactions and thus, when compared with water at lower temperatures and ambient pressures, SW conditions enhance the depolymerization of polysaccharides and the generation of smaller soluble protein fractions [18]. Mlyuka et al. [ 19 ] presented SWE as a strategic alternative for the food industry with promising potential in the selective extraction of bioactive compounds and in green hydrolysis reactions, while stressing scaling as the major challenge facing its commercial use. SWE is an efficient, cheap, fast, and environmentally friendly technology. Several reviews [ 19 – 21 ] analyzed the tuning of operation conditions in different types of raw materials, to maximize extraction yield and/or to avoid the degradation and decomposition of desired products. In general, in comparison with conventional alkali or enzymatic hydrolysis, SW provides comparable or higher yields in shorter times. In a very recent contribution, Álvarez-Viñas et al. (2021) reviewed the most important features in the SWE/hydrolysis of proteins, the different modes of operation, and analyzed the effect of process conditions on the product properties [ 22 ]. The authors highlighted the necessity of establishing optimal conditions as a compromised solution for processing proteins derived from agro-food wastes and algal biomass, suggesting that could be desirable in the stage wise operation to sequentially obtain high-valued fractions. Nevertheless, the formation of allergenic and/or toxic peptides from wastes and biomasses during protein extraction and hydrolysis should be prudently tested. 2.2. Enzyme-Assisted Extraction Enzyme-assisted extraction (EAE) is a green technology based, firstly, on the action of degrading enzymes within the major components of the cell wall, such as cellulose, hemicellulose, and/or pectins, resulting in the disruption of the wall and the release of cellular proteins [ 23 ]. Secondly, proteases break down the high molecular weight cell proteins into smaller and more soluble portions, thus providing valuable extraction conditions [ 2 ]. Although EAE has been characterized by its long processing time, high costs, elevated energy consumption, and irreversible carbohydrate-protein matrix disruption, it has become an emergent strategy showing advantages in comparison with conventional solvent-based extraction methods [ 23 ]. Moreover, products obtained using this technology evidence a higher purity and suitability for human consumption [ 24 – 26 ]. Thus, Sari et al. demonstrated the higher protein yield resulting from the extraction of proteins from soybean (Glycine max) and rapeseed (Brassica napus subsp.napus) meals when serine, endo, and exoproteases were used in comparison to the protein yield obtained without enzyme addition [ 24 ]. Similarly, Rommi et al. [ 27 ] demonstrated the beneficial effects of pectinolytic enzymes acting on pectic polysaccharides and glucans on the extraction yield of proteins from rapeseed press cakes made from cold oil processing. The use of enzymes increased the protein yield by 1.7 times in comparison with that obtained without enzymes. In addition, these authors found that the enzymatic hydrolysis of carbohydrates at a pH of 6 allowed for the extraction of rapeseed press cake proteins with a higher solubility and dispersion stability than those obtained using an alkaline extraction that provoked their partial denaturation [ 28 ]. Chirinos et al. also found that the EAE (alcalase) of proteins from sacha inchi (Plukenetia volubilis L.) kernel meal resulted in a higher ( ≈ 1.5-fold) protein recovery than that obtained through alkaline extraction [ 29 ]. More recently, bi-( α - amylase and amyloglucosidase) and tri-enzyme ( α -amylase, amyloglucosidase, and β -1,3,4glucanase) treatments were applied to extract proteins from defatted barley flour, obtaining a protein yield of 49% and 78.3%, respectively [ 30 ]. Similarly, defatted soybean flour was treated with xylanase, pectinase, cellulase, and a cocktail of commercial carbohydrases within alkaline extraction, resulting in the increase of the protein yield by 21% compared to the 2 h alkaline extraction without enzymatic treatment [ 31 ]. In a recent study, four Processes 2021,9, 1626 4 of 24 enzyme preparations, followed by assisted alkaline extraction, were tested for protein extraction from the seaweed Palmaria palmata [ 32 ]. These authors found that the most efficient treatments were the combination of Celluclast ® 0.2% w/wplus Alcalase ® 0.2% w/w(90.0% extraction efficiency), or Shearzyme ® 0.2% w/wplus Alcalase ® 0.2% w/w (85.5% extraction efficiency). Moreover, these methods allowed for improving the amino acid profile, the essential amino acid score, and the ratio of the extracted proteins, in which their potential as source of bioactive peptides was also improved, making this methodology suitable for extracting proteins with interesting nutritional and functional properties. Recent studies have reported on the suitability of enzyme-assisted extraction to recover proteins from other sources, such as sugar beet (Beta vulgaris L.) leaves [ 33 ] and almond cake [34]. 2.3. Novel Assisting Cell Disruption Techniques 2.3.1. Microwave-Assisted Extraction (MAE) Microwave-assisted extraction (MAE) is a novel cell disruption technique that uses electromagnetic waves of frequency in the range from 300 MHz to 300 GHz [ 23 ]. These waves are absorbed by the matrix and converted into thermal energy, which heats the moisture inside the cells. This generates a high pressure on the cells’ walls, increasing their porosity and thus facilitating the extraction of their compounds [ 35 ]. The main MAE parameters required for optimization and scaling up the extraction process are the sample solubility, the solid-liquid ratio, the extraction process time and temperature, the microwave power, the system agitation, the dielectric constant, and the dissipation factor [ 36 – 38 ]. In comparison with conventional technologies, MAE presents some advantages, such as a higher reproducibility in a shorter period of time, and a lower solvent and energy consumption. These advantages make MAE a suitable technology for extracting different compounds, such as proteins, carbohydrates, and antioxidant polyphenols [ 39 ]. Studies on the MAE of proteins from various biological sources are presented in Table 1. This Table shows a comparison of the yield values, protein content, and other characteristics of the resulting products with those obtained from traditional extraction technologies, such as solvent extraction or steam infusion, among others. According to these studies, the use of MAE results in higher plant protein yields compared to the standard alkaline procedure [ 26 , 40 ]. Electromagnetic microwaves also provide other potential advantages over conventional hydro-thermal treatments, such as uniform heating, an enhanced extraction rate, lower solvent consumption, and a higher extraction speed [ 3 , 41 ]. Moreover, the improvements of functional properties and the digestibility of extracted proteins when using MAE have been reported [ 26 , 42 ]. Thus, MAE has been recommended as an approach to extracting protein from structurally rigid biological samples, which are difficult to digest using enzymes and/or ultrasound waves, such as bran, or other by-products of the milling industry (i.e., sesame, rice, and wheat) [43–45]. 2.3.2. Ultrasound-Assisted Extraction (UAE) Although ultrasound-assisted extraction (UAE) has been studied since the 1950s as a suitable approach to obtain proteins from natural sources, its application in food science is very recent, being recognized as a clean and novel technology. UAE is based on the propagation of pressure oscillations in a liquid medium at the speed of sound, which results in the formation, growth, and collapse of microbubbles, allowing cell disruption and a mass transfer to the medium [ 46 ]. The bubbles generated are relatively large and their collapse provokes cell wall breakup, a reduction of in particle size, and a mass transfer across cell membranes, allowing the extraction of substances from the medium. UAE’s performance is affected by different parameters, such as the food matrix, extraction solvent, exposure time and temperature, ultrasound frequency, power, amplitude, and the type of equipment used [ 47 ]. As a green technology, UAE is energy efficient, easy to install, with minimal environmental impact, and its maintenance costs are low. Moreover, it requires Processes 2021,9, 1626 5 of 24 a low investment and shorter extraction times, thus reducing the process time and the associated costs [48]. Thus, as a fast, cost-effective, and environmentally friendly technology, UAE has been used to extract and modify vegetable proteins, improving the efficiency of the extraction process. Recently, the existing evidence for the UAE of plant-based protein has been summarized by Rahman et al. [ 47 ]. Moreover, comparison between UAE and conventional technologies used to extract proteins from vegetal and animal food sources is shown in Table 1. Ochoa-Rivas et al. compared alkali extraction, MAE, and UAE to extract protein from peanut flour. The highest protein yield resulted from UAE, which also improved the techno-functional properties (the water absorption, foaming and emulsifying activities) and the in vitro protein digestibility of the extracted proteins [ 26 ]. Furthermore, UAE was recently applied to extract individual arachin and conarachin from defatted peanut protein, resulting in an increase in the extraction yield, a shortening of the extraction time and temperature, and an improvement of the emulsifying properties of arachin [ 49 ]. Similarly, increases in the protein yield and the enhancement of the functional and biological properties were reported after the UAE of proteins from pea and brewer’s spent grain proteins [ 50 , 51 ]. Although the number of studies applying UAE to extract proteins from animal sources is more limited, this technology has been also recognized as a means to increase the extraction yield and improve the functional properties of extracted proteins from chicken liver and common carp (Cyprinus carpio) byproducts [52,53]. Despite being considered a novel and suitable approach to extracting proteins from different food materials, UAE has been associated with some weaknesses, such as the formation of radicals responsible for the release of degradation products affecting protein quality, resulting in protein oxidation, loss of aroma, changes in protein color, structure changes, texture alterations, free radical formation, and a metallic flavor [54]. Table 1. Comparison between conventional and emergent assisting cell disruption technologies (microwave and ultrasoundassisted) for the extraction of food proteins. Food Protein Source Results of the Extraction Process Reference Conventional Extraction Microwave-Assisted Ultrasound-Assisted Rice bran Extraction yield: 12.85% Protein content: 75.32% Extraction time: 60 min Extraction yield: 15.68% Protein content: 79.98% Extraction time: 2 min [41] Protein yield: 2.92% Protein yield: 4.37% Protein content: 71.27% [40] Rice Extraction yield: 38.0% Protein purity: 64.12% Extraction yield: 65.0–86.0% (combined with α-amylase degradation) Protein purity: 77.47–92.99% Higher solubility, emulsifying activity and foaming capacity [55] Sesame bran Protein content: 24.5% TPC: 3.45 mg GAE/g Protein content: 43.8 to 61.6% (91.7% by MAEE) TPC: 4.20 mg GAE/g (8.04 mg GAE/g by MAEE) Highest recovery of antioxidant compounds [44] Protein yield: 24.5% (alkaline extraction)-79.3% (enzymatic-assisted extraction) Protein yield: 59.8 (ultrasound-assisted)-87.9% (combined with enzymatic treatment) [45] Processes 2021,9, 1626 6 of 24 Table 1. Cont. Food Protein Source Results of the Extraction Process Reference Conventional Extraction Microwave-Assisted Ultrasound-Assisted Protein yield: 58.0% (vacuum-ultrasound assisted)-65.9% (vacuum-ultrasound assisted enzymatic extraction) Higher total phenolic capacity and antioxidant capacity [56] Peanut flour Protein yield: 42.4% Protein yield: 55.0% Improvement of water absorption, foam activity, emulsifying activity, and in vitro digestibility Protein yield: 57.6% Improvement of water absorption, foam activity, emulsifying activity, and in vitro digestibility [26] Defatted peanut protein Arachin extraction yield: 37.53% Conarachin extraction yield: 7.57% Shortening of the extraction time and temperature Improvement of emulsifying properties of arachin [49] Defatted wheat germ protein Extraction yield: 24.0–37.0% Extraction yield: 45.6% (combined with reverse micelles) [43] Pea protein Extraction yield: 71.6% Extraction yield: 82.6% Shortening of extraction times and reduction of water consumption Improvement of functional properties and biological activities [50] Alfalfa protein Extraction yield: 14.5% (Ultrasound-ultrafiltration-assisted alkaline ioelectric precipitation) Potein content: 91.1 g/100 g Increase of solubility, water-holding and oil-binding capacities Reduction of emulsifying and foaming properties [51] Brewer’s spent grain protein Extraction yield: 45.71% Extraction yield: 86.16% Protein purity: 57.84% Enhancement of the fat absorption capacity, emulsifying and foaming properties [57] Soy milk Extraction yield: 3.86% (steam infusion) Protein content: 7.38% Extraction yield: 4.83% Protein content 13.12% Improvement of characteristics of soy milk Increase of protein solubility and digestibility [58] Soy okra Extraction yield: 0.35% (steam infusion) Protein content: 25.0% Extraction yield: 0.23% Protein content 18.5% [58] Jackfruit leaves Protein content: 8.41% Protein content: 9.56% Protein content: 9.63% [59] Processes 2021,9, 1626 7 of 24 Table 1. Cont. Food Protein Source Results of the Extraction Process Reference Conventional Extraction Microwave-Assisted Ultrasound-Assisted Eurycoma longifolia roots Extraction yield: 9.76% in 38 min (heat assisted) Extraction yield: 9.54% in 5 min [60] Coffee silverskin Protein yield: 24.35% (alkali extraction)-32.52% (sequential alkaline-acid extraction) Protein yield: 43.53% Protein yield: 14.04% [61] Dolichos lablab L. protein Extraction yield: 40.95% Extraction yield: 69.98% Enhancement of functional characteristics and antioxidant capacity of the protein [62] Common carp by-products Extraction yield: 0.82–1.27% Protein content: 87.63 to 88.19% Reverse correlation between the extraction time and the gel strength and viscosity of gelatin Extraction yield: 19.80–27.0% Protein content: 86.15 to 90.21% Decrease of the gel strength and viscosity of gelatin [53] Duck feet gelatin Extraction yield: 51.83% (water bath) and 22.06% (electric pressure cooker) Extraction yield: 17.58% Improvement of gel strength, melting point, and viscosity [42] Bighead carp Protein yield: 19.15–36.39% (water bath) Protein content: 84.15–88.67% Protein yield: 30.94–46.67% Protein content: 89.17–91.85% [63] Chicken liver protein Extraction yield: 43.5% Protein content: 63.9% Extraction yield: 67.6% Protein content: 61.8% Improvement of the water/oil holding capacity and emulsifying properties [52] TPC: total phenolic content; GAE: gallic acid equivalent; MAEE: Microwave-assisted enzymatic extraction. In addition, when UAE is applied at high intensities, it generates heat which provokes protein denaturation and unfolding. Thus, the intensity and synergy of ultrasound need to be optimized before their application. Recently, UAE has been combined with other technologies to reduce its limitations and improve the extraction of food proteins. Among these combinations, those including UAE and EAE have been reported to be effective to extract target compounds with the advantages of enhanced extraction yield, reduced extraction time, and physiological activity. Görgüç et al. compared alkali-EAE with ultrasound-EAE, reporting an increase in the sesame bran protein yield from 79.3% to 87.9% [ 45 ]. These authors also found an increase in the protein yield, phenolic content, and antioxidant capacity of the protein extracts when vacuum-ultrasound-EAE was applied to the sesame bran [56]. Processes 2021,9, 1626 8 of 24 2.3.3. Pulsed Electric Field and High Voltage Electrical Discharge Extraction Pulsed electric field (PEF) and high voltage electrical discharge (HVED) are emerging non-thermal technologies that use high voltage to generate an electric field to perform an extraction. Both technologies are based on the pores formed on the cell membrane (electroporation) resulting from the application of an electric field, thus allowing the extraction of intracellular components through a diffusion process [ 64 ]. In PEF technology, a material is placed between two electrodes through which high voltage pulses (from 100 to 300 V/cm to 20–80 kV/cm) are applied in short time periods. Under the effect of PEF, the cell membrane is electrically pierced, losing its permeability in a reversible or irreversible manner depending on the electrical parameters, the cell characteristics (size, age, and shape), and the pulsing media composition [ 65 ]. These features have made PEF into an extensive technology for microorganisms’ inactivation, recovering high-value compounds, like proteins and polyphenols, and improving freezing and drying processes [ 66 ]. In HVED technology, a current of high-voltage electrical discharge is applied between two electrodes forming a plasma channel where energy is then presented directly in a liquid. Because PEF and HVED are non-thermal techniques, they present some advantages over conventional heat-assisted extraction techniques, such as their ability to preserve thermolabile food constituents as proteins and increasing the quality of the extracts during processing and throughout the storage period [ 13 ]. Moreover, the low energy consumption of these extraction processes agrees with the principles of green extraction [67]. Recent studies have applied PEF and HVED to recover proteins from different natural sources. For example, Roselló-Soto et al. compared the effects of PEF, HVED, and ultrasound pre-treatments before the extraction of intracellular compounds from olive kernels [ 68 ]. In this study, HVED technology was found to be more effective than others in terms of the energy and treatment time required to extract the phenolic compounds and proteins. Sarkis et al. also reported on the efficiency of the PEF and HVED approaches in increasing the yield of polyphenol, lignin, and protein extracts from sesame cake, and in improving the kinetics of diffusion [ 69 ]. More recently, PEF technology was applied to release and extract proteins from the microalgae Chlorella vulgaris and Neochloris oleoabundans. The protein yield was much lower than that obtained using mechanical approaches with a higher energy input [ 70 ]. Although PEF technology has been associated with changes in the solubility and functional properties of extracted proteins, these modifications can be beneficial. Thus, Zhang et al. reported that the PEF treatment of canola (Brassica napus) seeds increases the solubility, emulsifying, and foaming properties of the extracted proteins [ 71 ]. Changes in the secondary structure and in the antioxidant activity of peptides released from pine nut proteins have also been reported [72]. 2.3.4. High Hydrostatic Pressure-Assisted Extraction High hydrostatic pressure (HHP) technology is based on the application of an isostatic pressure ranging from 100 to 1000 MPa transmitted instantaneously and uniformly through a fluid, generally water. This pressure provokes the deformation of cells and the damage of their membranes and protein structures, thus allowing for the penetration of the solvent within cells and increasing the transfer rate of its intracellular components [ 73 ]. HHP processing, also known as “cold pasteurization”, has been traditionally used in the food industry to reduce the microbial charge and improve the shelf-life of different food systems [ 74 ]. However, in recent years, HHP processing has been extended to other innovative uses, such as the selective recovery of phenolic compounds, polysaccharides, fats, and proteins, among others [ 75 – 77 ]. Moreover, HHP processing has become one of the most efficient methods for modifying the properties of proteins, such as thermal and rheological properties, gelation solubility, water holding and foaming capacity, stability, surface hydrophobicity, emulsifying activity, and stability [ 78 ]. It also induces the reversible denaturation of native proteins and the modulation of protein-protein and protein-solvent interactions, stimulating the formation of oligomeric and aggregated species that can negatively affect the digestibility of the protein [ 79 , 80 ]. However, in some cases, the alteration Processes 2021,9, 1626 9 of 24 of the food proteins profile caused by HHP results in the higher exposure of susceptible digestion sites and, consequently, in a higher hydrolysis and yield of hydrolytic products, thereby reducing both time and costs [ 81 ]. In addition to its ability to modify proteins, HHP also shows an ability to modulate the conformation and activity of different enzymes as well as their interaction with their target proteins [ 82 ]. Additionally, in the recent years HHP has been employed in combination with enzymatic treatments to hydrolyze proteins from different food sources, such as kidney and pinto bean (Phaseolus vulgaris), soy and flaxseed (Linum usitatissimum) protein, and whey protein isolate, and to release bioactive peptides (see the review of Ulug et al. [ 83 ]). Since the efficiency of hydrolysis depends on the protein system, pressurization conditions, and the enzyme used, optimizing these parameters to produce the maximum amount of bioactive peptides from a complex food matrix is essential to accelerate the development of bioactive peptides-based food products. 3. Release of Bioactive Peptides Bioactive peptides are sequences of amino acids inactive within the source protein. Once released, they can exert antioxidant, anti-inflammatory, antihypertensive, anti-obesity, antimicrobial, and immunomodulatory biological activities. These activities suggest their potential as novel, safe, and effective ingredients for functional foods and/or nutraceuticals to prevent and manage chronic disorders. Enzymatic hydrolysis and fermentation are the most known conventional methods to release bioactive peptides from their source protein. However, their use in the food industry is limited due to some disadvantages. Acid and alkaline hydrolysis are low-cost methodologies, but they affect some amino acid residues, thus resulting in the loss of nutritional and biological value of the released peptides [ 8 ]. Enzymatic proteolysis is a better alternative for hydrolyzing proteins due to the enzyme’s specificity, but it can be expensive, time consuming, and require the use of acids and bases for pH control [ 84 ]. In the case of fermentation, it offers the advantage of removing hyper-allergic or antinutritional factors, but the costs are also relatively high [ 8 ]. Moreover, to produce protein hydrolyzates and bioactive peptides, efficient and scalable methods should be used to avoid the use of harmful chemicals and solvents, costly enzymes, and long processing times [ 21 ]. Therefore, in recent years, novel technologies such as ultrasounds, microwave-assisted processing, HHP, PEF, and SW hydrolysis are being explored to enhance the production of bioactive peptides [83] (Figure 1). In addition to extracting proteins, microwave radiation is capable of improving proteolysis and releasing low molecular weight peptides when combined with enzymatic hydrolysis, thereby increasing the bioactivities of the resultant hydrolyzates. Thus, in comparison to thermal processing, MA-hydrolysis has significantly increased the dipeptidyl peptidase IV (DPP-IV) and ACE inhibitory activities of cricket (Gryllodes sigillatus) protein hydrolyzates [ 85 ], and the antioxidant capacity of chia (Salvia hispanica) protein hydrolyzates [ 86 ]. Similarly, this combination has been used to prepare bioactive peptides from bovine serum albumin, ginkgo (Ginkgo biloba) nuts [ 87 ], milk protein concentrate [ 88 ], bighead carp (Aristichthys nobilis) [ 89 ], fish protein [ 90 ], trout frame (Oncorhynchus mykiss) [ 91 ], and collagen sea cucumber (Acaudina molpadioides) [ 92 ]. Microwave radiation facilitates the exposure of cleavage sites of the protein to the enzyme action; thus, the hydrolysis efficiency, processing time, and reproducibility are generally improved by this technology. Other advantages of microwave processing over conventional methods include its simple handling and low cost [93]. Processes 2021,9, 1626 16 of 24 different formulations even at low concentrations. In this way, efficient delivery systems can be designed. Varying supercritical processes parameters (temperature, pressure, flow rate ratios, nozzle diameter and depressurization rate, among others) the particle size, and morphology can be effectively controlled. Furthermore, low temperatures favour the handling of heat-sensitive substances, and circumventing the use of organic solvents, or the easy removal when they are used, is another key advantage of SC-CO 2 technology applied to the formation of bioactive protein-based microparticles. 4.2. Structural and Conformational Modifications Resulting from SC-CO2Treatments Proteins and their products are used as ingredients in several food applications (dairy and bakery products, infant foods, and beverages) with the objective of enhancing nutritional value, creating emulsification, foaming, and antioxidant barriers, among others [ 148 ]. Since the functionality of food proteins are related to their structures, the structural modification of proteins can lead to new or improved functionalities [ 149 ]. In this respect, SC-CO 2 treatment was described as a green method to achieve protein modification [ 150 ]. SC-CO 2 offers many advantages in comparison with thermal processing methods, such as minimizing the alterations and quality of food [ 151 ]. Several studies have been reported, using SC-CO 2 for the inactivation of microorganisms and enzymes in liquid foods [ 152 ]. Despite many reports concerning the physical modification of polysaccharides under SC-CO 2 processing [ 153 ], fewer studies can be found concerning the effects of SC-CO 2 treatment on protein quality and functionality. For example, Zhong et al. analysed the effect of SC-CO 2 treatment on the rheological properties of whey protein [ 154 ]. The authors concluded that temperature, pressure, holding time, protein concentration, and pH were the main operating parameters influencing the conformational, structural, and functionalities of whey protein isolate (WPI). Later, Xu et al. conducted a complete analysis of the physical, conformational, and structural properties of WPI treated with SC-CO 2 in comparison with the thermal treatment of WPI solution [ 150 ]. Increased turbidity of WPI treated with SC-CO 2 (50–60 ◦ C at 20 MPa for 1 h) suggested more intensive WPI denaturation in comparison with thermal processing. Furthermore, dynamic light scattering measurements showed higher aggregates and a maximum mean particle size when WPI was processed with SC-CO 2 at 60 ◦ C, indicating the partial aggregation of the dimer into the polymers. An analysis of the fluorescence emission spectra of proteins suggested changes in the polarity of the protein residue’s microenvironment from a less polar to a more polar. The authors evidenced secondary and tertiary protein structure changes induced by SC-CO 2 treatment, resulting in physicochemical and functional properties of proteins. The mechanisms of protein denaturalization due to SC-CO 2 treatment is actually a major topic of research due to its importance in cold sterilization methods. The complexity of the process has limited the possibility of attaining microstructural information related with microbial and/or enzyme inactivation by SC-CO 2 . In a recent work, Monhemi and Dolatabadi used molecular dynamics simulation of SC-CO 2 pasteurization to conclude that the denaturation of proteins (myoglobin and lysozyme as models) is produced at the CO 2 /water interface [ 155 ]. The protein migrates from a pure aqueous phase to the CO 2 /water interface, releasing the hydrophobic cores to the CO 2 phase and the hydrophilic residues to the aqueous phase and then the protein is denatured to a flat and extended conformation. Several other works have been recently reported, aiming to explain the molecular mechanisms of protein denaturalization in SC-CO 2 [ 156 , 157 ], or describing novel food applications of SC-CO 2 enzyme inactivation. For example, Podrepšek et al. applied an SC-CO 2 treatment to inactivate the polyphenol oxidase enzyme in order to extend shelf life of a coarse-ground flour from whole wheat (graham flour) while preserving, at the same time, its high quality [ 158 ]. The effect of pressure on protein concentration and enzyme activity was significant, evidencing a 35% decrease in polyphenol oxidase enzyme activity after SC-CO 2 exposure at 300 bar for 24 h. Furthermore, the quality of the flour remained almost the same or even improved, indicating that the SC-CO 2 -treated Processes 2021,9, 1626 17 of 24 graham flour is still suitable for use in the bakery industry. Also, SC-CO 2 + ethanol extraction of yellow pea was found to enhance organoleptic attributes of the edible seed, reducing most chemical constituents, and lowering certain functionality qualities, such as pasting viscosities. Scanning electron micrographs indicated that protein-rich particles were adhered to the surface of starch granules of pea flour extracted with SC-CO 2 , and this change may be responsible for the different functionality observed [157]. Thermal extrusion has been effectively used to improve the techno-functional properties of food proteins. The combination of temperature and shear weakens the structure of proteins and the extrusion process helps to partially unfold and denature proteins to modify their functionalities. SC-CO 2 extrusion is an effective technique to produce conformational changes in proteins, providing several advantages in comparison to steam extrusion cooking, such as low-temperature processing, formation of a uniform and porous product, and pH control. For instance, the use of milk protein concentrate (MPC) in extruded products is still a challenge because of its sensitivity to high temperature and shear. For example, Yoon et al. have demonstrated the improved physicochemical properties of MPC extruded with SC-CO 2 in comparison with steam extrusion [ 158 ]. Also, Gopirajah et al. applied SC-CO 2 extrusion to improve emulsifying properties of MPC, obtaining a better emulsion activity index, reduced creaming index, and similar viscosity as compared to commercial sodium caseinate [159]. Ding et al. showed that SC-CO 2 treatment can be a novel method to improve the foam ability of egg white protein [ 160 ]. At 9 MPa SC-CO 2 treatments, the protein foaming capacity increased 3.6 times in comparison with the original egg white. The protein electrostatic force was reduced due to the pH change and their particle size decreased. Accordingly, these effects resulted in an increase of protein hydrophobicity and viscosity. 5. Conclusions Protein extraction is commonly considered to be a crucial step in contributing to the final greenness of the complete protein analysis process. In recent years, important advances have been achieved with the design, development, and application of novel methodological approaches for extracting high quality proteins at high yields. These new techniques also reduce the use of hazardous chemicals and solvents, and decrease the temperature and time conditions of the extraction process. Emerging eco-innovative extraction technologies are becoming a promising alternative to conventional methods for producing safe and nutritive proteins with preserved techno-functional properties. Furthermore, the combination of some of these methodologies with enzymatic hydrolysis has been demonstrated to improve the yield and biological properties of protein-derived peptides with the capability to be incorporated into functional foods and nutraceuticals. As presented in this review, although many advantages have been associated with the use of these technologies at a laboratory scale, the research is still in its infancy, thus further studies demonstrating their economical and environment suitability at an industrial scale are required. Author Contributions: Conceptualization, T.F. and B.H.-L.; writing—original draft preparation, G.F.-O., T.F. and B.H.-L.; writing—review and editing, T.F. and B.H.-L. funding acquisition, B.H.-L. 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