Modulating Digestibility and Mitigating Beany Flavor of Pea Protein

ABSTRACT

Pea (Pisum sativum) protein, as a pulse protein second only to soy protein, has shown increasing popularity in the plant-based food market. Numerous studies have shown the desirable techno-functionalities of pea protein for emulsifying, gelling, and foaming. Nevertheless, poor digestibility and unpleasant beany flavors of pea protein lower consumer acceptance. The current state of understanding on digestibility and beany attributes of pea proteins could help pave the way for pea protein-based food formulation with improved quality. This paper covers recently reported studies to modulate the digestibility and beany notes of pea protein by many processing techniques, and highlights the influence of non-protein components and food matrix structures while formulating pea protein-based food systems. Several modification techniques could alter the digestibility and improve the flavor of pea proteins by reorienting the protein structures or forming protein-based complexes. Precise tailoring the digestibility/flavor of pea proteins is promising based on current research, though further investigation is still required to reduce adverse effects or side products. In addition, more studies on simulated pea protein-based food systems are required to mimic the actual food comprising multi-components and specific structures to narrow the gap between lab-scale research and food industry applications.

KEYWORDS:

• Pea protein modulation
• gastrointestinal digestion
• beany flavor
• compositions
• structures
• pea protein-based food system

Introduction

Pulse proteins are receiving much attention among plant proteins as alternatives to dairy proteins due to their prominent sustainability, functional properties, and health benefits.^[1] Soy proteins are one of the most widely used pulse proteins, with a market value of USD 7.7 billion in 2022,^[2] while other emerging pulse proteins from peas, lentils, fava, and chickpeas show good development potential.^[3] Pea proteins, extracted from yellow peas (Pisum sativum), have become the second most popular plant protein followed by soy protein, with a market value of almost USD 2 billion in 2022 and a projected compound annual growth rate (CAGR) of 12.0% from 2023 to 2030.^[4] Pea protein could be an ideal alternative to soy protein due to its hypoallergenic, non-transgenic status and sufficient essential amino acid content.^[5–7]

Although the utilization of pea protein has gradually transitioned from marginal to the mainstream, it still faces many challenges in the food industry in terms of taste, texture, functionality, and nutritional properties.^[5] Indeed, the low solubility, beany flavor, and limited bioaccessibility/bioavailability of pea protein narrow its application as a functional ingredient or a nutritional supplement in the food system.^[8,9] Factors contributing to these negative consequences include the compact globular structure of pea proteins that interfere with functionality,^[10] and anti-nutritional factors such as trypsin inhibitors and lectins reduce bioaccessibility or bioavailability.^[11,12] Nevertheless, pea protein, which shows certain resistance to gastrointestinal digestion, seems to have the potential to be applied as a vehicle for sensitive nutrients and promote their absorption in the digestive system.^[13,14] Protein modification including physical, chemical, and biological methods enables to modulate bioaccessibility/bioavailability, and flavor profile without compromising functionality, by altering the protein structures to make them more applicable to specific food processing scenarios.^[15] Chemical modifications like phosphorylation, acylation, and glycation could selectively modulate functional groups on proteins to achieve customizable protein properties.^[16] Biological modifications like enzymatic hydrolysis could achieve tailor-made plant proteins with specific functionalities, though the high cost of enzymes is not conducive to scale-up production.^[17] Even though physical treatments are unable to achieve precise modulation like chemical and enzymatic modification, they are considered cost-effective with higher public acceptance and interest^[15] as more people are concerned about introducing extraneous or synthetic chemicals in food.^[1] Physical modifications with different processing effects include thermal (conventional, ohmic, microwave, extrusion, etc) and non-thermal (ultrasonication, high-pressure processing, etc.) methods.^[15] Different protein modification methods are purpose-oriented and closely related to their specific applications. Furthermore, fabricating protein-based products with different non-protein components (mainly carbohydrates, lipids, and salts) and various foodstuff forms (e.g. emulsions, gels, dehydrated matrix, etc.) can further alter protein conformation and change the digestibility or flavor characteristics.

Current reviews mostly focus on the improvement of techno-functionality like emulsifying, foaming and gelling properties of plant proteins,^[10,15,17] with only several focusing on the digestive performance of bioactive components in structured plant-based delivery systems.^[18] However, to the best of our knowledge, none of these reviews have covered targeted regulation strategies for in vitro digestibility of both pea protein and pea protein-based food systems. Moreover, although the removal of beany flavor in soy protein has been reviewed,^[8] the findings from research on mitigating beany flavor in pea protein have not been adequately covered. Several recent reviews mentioned the interactions of beany flavor components and pea proteins, as well as approaches to control the beany off-flavor.^[19,20] Nevertheless, the mechanisms for reducing the beany flavor of pea protein have not been systematically summarized, and the effects of other food components and food system structures on the beany flavor of pea protein-based food have never been discussed. To fill these gaps, this review systematically summarizes the latest research progress on pea protein modification methods, mainly relating to the adjustment of digestibility and mitigation of beany flavor, as well as the effects of compositions and structures of pea protein-based food matrix on gastrointestinal digestion and beany attributes. The findings could ideally provide future research ideas for pea protein-customized modulations and novel pea protein-based food formulations.

Pea protein fractions and structural characteristics

Peas contain 20–30% proteins and exist in the storage cells of pea cotyledon in the form of protein bodies (2–4 μm), which are protected by cell walls together with starch granules (5–30 μm).^[21] The major pea protein fractions are listed in Table 1. Pea proteins can be subdivided into salt-soluble globulins (50–60%) and water-soluble albumins (15–20%), the former has a larger molecular weight (MW) than the latter.^[24] Globulins are degraded in seed germination and provide nutrients for plant growth, while albumins act as metabolic and enzymatic proteins.^[30] The globulin fraction consists of legumin (11S), vicilin (7S) and minor convicilin (8S). Legumin has a hexameric structure with a high MW of 360–400 kDa, consisting of acidic subunits (α-chains) and basic subunits (β-chains) linked by disulfide bridges.^[31] Compared to legumin, vicilin and convicilin both possess trimeric structures with lower MW of 160–200 kDa and ~ 290 kDa, respectively. Albumin is rich in sulphurous amino acids, containing two major small MW components, namely pea albumin 1 (PA1a: 6 kDa; PA1b: 3 kDa) and pea albumin 2 (PA2: 26 kDa).^[26]

Table 1. The characteristics of major pea protein fractions.

Pea protein fraction	Protein type	Structure	Isoelectric point	Molecular weight	Reference
Globulin (50–60%)	Salt soluble	Complex globular	pH ~ 4.0–4.8	/	^[Citation22]
Legumin (11S)	/	Hexamer, acidic subunits (hydrophilic, located in the outer part), basic subunits (hydrophobic, located in the interior part)	/	360–400 kDa, composed of acidic (40 kDa) and basic (20 kDa) subunits	^[Citation23]
Vicilin (7S)	/	Trimer	/	160–200 kDa, consisting of 50 kDa and 30–35 kDa subunits	^[Citation24]
Convicilin (8S)-minor	/	Trimer (α-subunit of vicilin)	/	290 kDa in native form, ~70 kDa subunit	^[Citation25]
Albumin (2S, 15%−25%)	Water soluble	Tightly folded and non-helical globular structure	Pea albumin 2 (PA2, major): pH 5.2 Pea albumin 1 (minor): PA1a, pH 4.6; PA1b, pH 9.8	PA2: 26 kDa PA1: PA1a (6 kDa), PA1b (3 kDa)	^[Citation26–29]

Not applicable: /.

Different protein fractions possess various characteristics and structures, leading to differences in protein functionality. Legumin presents a good emulsifying capacity at neutral pH, while vicilin exhibits better emulsifying stability than legumin under an acidic environment except at around the isoelectric point (pH 4.0–4.8).^[32] PA2 plays an important role in emulsifying and foaming properties in albumins, especially near the isoelectric point of pea globulins (pH 4).^[33] The higher vicilin/legumin ratio of proteins is more likely to acquire better emulsifying properties.^[34] Moreover, albumins as enzymatic and metabolic proteins are related to lipoxygenase (LOX), which can influence the beany flavor of proteins derived from LOX-catalyzed oxidation.^[35,36] The pea proteins with rigid globular structures would show a higher energy barrier to alter their conformation than the highly flexible protein chains like caseins,^[10] which may affect bioaccessibility by burying the protein hydrolysis sites and reducing the accessibility of protease. Overall, the composition and structure of pea proteins are the main factors affecting techno-functionality, flavor, and nutritional properties.

Components related to digestibility and beany flavor in pea protein

Components associated with digestibility

The anti-nutrient factors (ANF) mixed in pea proteins, which can be classified into anti-nutritional proteins, glycosides, phenols and others, are the main components responsible for the digestive resistance of nutrients and micronutrients.^[37] In general, the impact of anti-nutrients on protein digestibility has the following aspects: a) reducing the biological activity of digestive enzymes; b) binding with proteins to prevent digestive enzymes from reaching specific digestive sites; c) interfering with the normal functioning of the digestive system and leading to metabolic disorders.^[38,39] The characteristics of the main anti-nutrients in pea proteins are summarized in Table 2.

Table 2. Main anti-nutrient factors in pea proteins.

Anti-nutrients	Structure/composition	Adverse impact	Thermostability^a	Reference
Anti-nutritional proteins
Trypsin inhibitors	A group of small serine protease enzyme inhibitors	Forming inactive complexes of trypsin and chymotrypsin	Heat labile	^[Citation40,Citation41]
Lectins (Phytohemagglutinin)	Glycoprotein: carbohydrate-binding proteins	Interacting with intestinal epithelial cells and changing the intestinal permeability; Resistant to the digestive enzymes	Heat labile	^[Citation38,Citation42]
Anti-nutritional glycosides
Saponins	Triterpene glycosides: sugars (glycone part) bond to one or more organic molecules (aglycone part)	Inhibiting digestive and metabolic enzymes such as trypsin and chymotrypsin	Heat stable	^[Citation37]
Anti-nutritional phenols
Tannins	Mainly condensed tannins: formed by the condensation of flavans, no sugar residues	Forming tannin-protein complexes by cross-linking the hydroxyl group of tannins and the carbonyl group of proteins; Interfering with protein hydrolysis	Heat stable	^[Citation37,Citation40]
Others
Phytic acid	A six-fold dihydrogen phosphate ester of inositol	Forming protein complexes directly or via a cation bridge, making proteins less susceptible to proteolytic attack	Highly heat stable	^[Citation40,Citation43]

^aThermostability of anti-nutrients.^[44]

Anti-nutrients can also be divided into heat-labile and heat-resistant groups, and the latter requires excessive heating conditions to inactivate, causing heat damage to other nutrients like minerals, and polyphenols in pea-based products. For example, heat-resistant phytic acid reduces protein digestibility by blocking the digestion sites of proteins, and a temperature below 100°C can hardly damage it.^[45] Although trypsin inhibitors are heat-labile, they remain at 10% activity after being heated at 100°C for 30 min due to the presence of disulfide bonds,^[46,47] which may still allow them to inactivate partial digestive enzymes and hinder protein digestion. To eliminate the negative consequences caused by thermal treatment, some non-thermal treatments (ultrasonication, high hydrostatic pressure),^[38] and biological treatments (germination, fermentation) are used to inactivate anti-nutrients, which will be discussed in detail in “Methods for modulating the digestion and beany flavor of pea protein.” Soaking and dehulling are conventional but effective methods for removing heat-stable anti-nutrients like tannins, saponins, and phytic acid.^[48–50]

Interestingly, some anti-nutrients can exert health benefits at low concentrations. For instance, low levels of lectins, saponins, tannins, and phytic acid have shown the potential to reduce glycaemic index, lower plasma cholesterol and triglyceride, and induce antioxidant/anti-inflammatory effects.^[37,51] The digestive resistance of some anti-nutrients (e.g. phytate) may slow the rate of gastric emptying,^[37] which could achieve intake reduction and weight loss. However, more research is required to determine the safe amount range for consumption or the correct proportion of various anti-nutrient factors.

Components associated with beany flavor

Beany flavor is an aromatic characteristic of beans and bean products, encompassing musty, earthy, dusty, sour aromatics, starchy, powdery feel, nutty and brown scent.^[52] The beany flavor components of pea protein can be either inherent in the pea seeds determined by their genotypes or developed during harvesting, processing, and storage from oxidation of unsaturated fatty acids.^[8,53,54] The oxidation process always induces unpleasant beany off-flavor and composes three main pathways, including enzymatic, automatic, and photosensitized oxidation. Among them, enzymatic oxidation induced by lipoxygenase-catalyzed degradation is the most prominent route for beany flavor component formation.^[55] Lipoxygenase (LOX, EC 1.13.11.12) is a non-heme iron-containing dioxygenase, a family of abundant and ubiquitous enzymes in legumes.^[56] LOX-2 and LOX-3 are two major lipoxygenase isoenzymes in mature pea seeds,^[57] with great specificity toward linoleic acid,^[58] the most abundant unsaturated fatty acid in peas, accounting for half of the total fat content.^[55] As shown in Fig. 1, linoleic acid is catalyzed into 9- and 13- hydroperoxyl derivatives in contact with lipoxygenase,^[55] and the 13-pathway is the dominant one.^[59] The hydroperoxyl derivatives are odorless but unstable, further degrading into short-chain aromatic compounds like aldehydes and alcohols under the action of hydroperoxide lyases (HPL) and alcohol dehydrogenase (ADH).^[8]

Figure 1. Lipoxygenase reaction with linoleic acid and region-specific catalytic sites (reprinted from^[55] with permission).

Among all the odor-active molecules listed in Table 3, the most diverse components are alcohols. 1-heptanol, 1-octanol and 3-methyl-1-butanol are inherent in peas, with 1-heptanol having the lowest odor threshold (3 ppb in water). Surprisingly, 3-methyl-1-butanol is not only the endogenous flavor component in peas whose concentration is affected by the genotype^[72] but also can be produced by lipoxygenase-initiated lipid oxidation.^[67] In addition, 1-pentanol, 1-hexanol, 1-nonanol, 1-octen-3-ol, hexanal and nonanal degraded from lipoxygenase-catalyzed linoleic oxidation have been reported to cause the beany off-flavor of pea ingredients.^[60,66] Among them, 1-octen-3-ol has a very low odor threshold (1 ppb in water) and can be further oxidized into 1-octen-3-one with a potent mushroom flavor.^[73] Hexanal is a non-beany volatile but important lipid oxidation marker with green and grassy aroma characteristics, and the mixture of hexanal/1-octen-3-one or hexanal/3-methyl-1-butanol, in a ratio 100: 1 shows an intense beany note.^[53,74,75] Pyrazines are either naturally occurring in peas like 2-isopropyl-3-methoxypyrazine^[53] or thermally derived (Maillard reaction) such as 2,6-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine^[76,77] belonging to alkyl pyrazines. Although the concentration of pyrazines inherent in peas is very limited, they are core components of beany aroma that contribute to the perceived green and earthy flavor due to the low odor threshold (2-Isopropyl-3-methoxypyrazine: 0.002 ppm in water).^[54] Pyrazines derived from the Maillard reaction endow samples with nutty and roasted characteristic flavors, which may mask or cover the beany flavor of peas.^[76] Furanoids such as 2-pentylfuran are often present in sprouted pea products, which may be associated with lipid autoxidation.^[68] While early research was focused on identifying the single aroma compounds contributing to beany flavor, the overall sensory representation of an aroma mixture is not a simple superposition of each compound.^[75] Although recent studies selected several markers belonging to the categories of alcohols, aldehydes, ketones, pyrazines, and furanoids for beany off-flavor characterization,^[64,69] the ratio of content among various flavor markers associated with overall flavor profile seems to be overlooked. More research is needed to clarify the contribution of individual beany flavor molecules in aromatic blends.

Table 3. Main beany flavor components in pea protein ingredients.

Flavor components	Aroma Characteristics	Origin	Odor threshold (ppb)^a	Reference
Alcohols
1-Pentanol	Beany	Lipoxygenase-catalyzed linoleic oxidation	120	^[Citation60,Citation61]
1-Hexanol	Lemon, grass, and green	Lipoxygenase-catalyzed linoleic oxidation	200	^[Citation62,Citation63]
1-Heptanol	Musty, herb	Inherent in peas	3	^[Citation64]
1-Octanol	Mushroom, vegetable, and green	Inherent in peas	42	^[Citation65]
1-Nonanol	Pea-like, vegetable, silt, and earthy	Lipoxygenase-catalyzed linoleic oxidation	50	^[Citation62,Citation66]
1-Octen-3-ol	Beany	Lipoxygenase-catalyzed linoleic/α-linolenic acid oxidation	1	^[Citation60,Citation61]
3-Methyl-1-butanol	Beany	Inherent in peas (Genotypes of peas); Lipoxygenase-catalyzed lipid oxidation	1000	^[Citation52,Citation53,Citation67]
Aldehydes
Hexanal	Green and grass	Lipoxygenase-catalyzed oxidation and/or free radical-initiated lipid oxidation (autoxidation)	4.5	^[Citation61]
Nonanal	Fatty, green, flora	Lipoxygenase-catalyzed linoleic oxidation	1	^[Citation66,Citation68]
Ketones
1-Octen-3-one	Beany, mushroom	Lipoxygenase-catalyzed oxidation and/or free radical-initiated lipid oxidation	0.005	^[Citation52]
Pyrazines
2-Isopropyl-3-methoxypyrazine	Green, grass, and earthy	Inherent in peas	0.002	^[Citation69]
Furanoids
2-Pentylfuran	Earthy, beany	Lipid autoxidation	6	^[Citation55,Citation70]

^aOdor threshold (ppb) in water.^{[52,55,65,71]}

Methods for modulating the digestion and beany flavor of pea protein

This section mainly summarizes the modification methods of pea protein to adjust digestibility based on their application purposes and mitigate beany flavor without compromising protein functionality in recent publications. It elucidates the mechanisms altering digestibility and beany flavor as summarized in Table 4 and Fig. 2, respectively, providing a guideline for targeted applications of pea proteins.

Figure 2. Mechanisms of mitigating beany odor by pea protein modifications, and potential adverse effects that need to be inhibited. Abbreviations in the figure: LOX: Lipoxygenase; HPP/HPH: high-pressure processing/homogenization; PEF: pulsed electric field; HIU: high- intensity ultrasonication; CP: cold plasma. The information collected from.^{[1,66,76,106–108,109]}

Table 4. Summary of the physical, chemical, and biological methods and related mechanisms for pea protein digestibility modulations.

Modulation methods	Digestion-related alteration	Modulation consequence/mechanism	Effect on digestibility			Reference
			+	−	±
Physical methods
Thermal	Protein unfolding; Hydrophobic group exposure	Increasing protease accessibility/digestion efficiency	√			^[Citation78]
	Flexible protein conformation; Loss of β-structures		√			^[Citation79]
	Particle size ↓, zeta potential ↑		√			^[Citation80]
	Heat-labile anti-nutrients ↓	Promoting or remaining enzyme activity/metabolism	√			^[Citation81]
	Inducing insoluble heat-aggregation/protein solubility ↓ (overheating)	Blocking the digestion of proteases; Inhibiting the activity of digestive enzymes		√		^[Citation82,Citation83]
Non-thermal	Protein aggregation ↓, hydrophobic and free sulfhydryl groups ↑ (HIU)	Increasing protease accessibility/digestion efficiency	√			^[Citation84]
	Maillard reaction products ↓ (CP)	Improving IVPDCAAS	√			^[Citation85]
	Anti-nutrients ↓ (HIU/HPP/CP), oligosaccharides ↓ (HPP)	Promoting or remaining enzyme activity/metabolism	√			^[Citation86–89]
	Immunoreaction/allergenicity ↓ (CP)	Limiting the identification of antibodies by the epitopes	√			^[Citation89]
	Cross-linking/disulfide bond ↑ (HIU/CP)	Blocking the digestion of proteases; Inhibiting the activity of digestive enzymes		√		^[Citation91,Citation92]
	Oxidization ↑ (CP)	/			√	^[Citation92]
Chemical methods
Glycation	Exposure of partial amino acid residues	Increasing protease accessibility/digestion efficiency	√			^[Citation93]
	Hiding amino acid residues (lysine)	Blocking the digestion of proteases; Inhibiting the activity of digestive enzymes		√		^[Citation94]
	The metabolic activity of gut microbiota ↑	Promoting or remaining enzyme activity/metabolism	√			^[Citation95]
	Advanced glycation end-products (AGE)	Causing metabolic problems and inflammatory responses		√		^[Citation96]
Acylation	Unfolding and altering the protein structure	/			√	^[Citation97]
Phosphorylation	Masking gastric digestive sites	Blocking the digestion of proteases; Inhibiting the activity of digestive enzymes		√		^[Citation98]
Biological methods
Proteolysis	Anti-nutrients ↓	Promoting or remaining enzyme activity/metabolism	√			^[Citation99]
	Digestible small molecule peptides and amino acids ↑	Increasing protease accessibility/digestion efficiency; Improving IVPDCAAS	√			^[Citation99]
	Hypoallergenic peptides separation	Limiting the identification of antibodies by the epitopes	√			^[Citation100]
Cross-linking (MTG)	Promoting intestinal digestion	Increasing protease accessibility/digestion efficiency	√			^[Citation101]
	High resistance to pepsin (dyspepsia), gastric emptying ↓	Blocking the digestion of proteases; Inhibiting the activity of digestive enzymes		√		^[Citation102]
Fermentation	Anti-nutrients ↓	Promoting or remaining enzyme activity/metabolism	√			^[Citation103]
	Sulfur amino acids ↓	Improving IVPDCAAS	√			^[Citation103]
Germination	Protease inhibitors ↓	Promoting or remaining enzyme activity/metabolism	√			^[Citation104]
	Biodegradation of limiting amino acids due to protein metabolism	Reducing IVPDCAAS		√		^[Citation104]
	Antioxidation ↓	/			√	^[Citation105]

Abbreviations in the figure: HIU: High-intensity ultrasonication; CP: Cold plasma; HPP: High-pressure processing; MTG: Microbial transglutaminase; IVPDCAAS: In vitro protein digestibility corrected amino acid score; “↑” suggests increase/facilitate, while “↓” indicates decrease/inhibit. “+” and “−” are associated with protein digestibility enhancement and mitigation, respectively, while the ambiguous effects are presented by “±”.

Physical methods

Physical modification consists of thermal and non-thermal treatments. Conventional thermal techniques rely on conduction and convection like water bath heating, and hot air/steam heating, while other thermal techniques convert electromagnetic energy (microwave, infrared, radio frequency processing), electric energy (ohmic processing) to thermal energy.^[110] Non-thermal treatments including ultrasonication, high-pressure, cold plasma, and pulsed electric field processing are applied to eliminate the possible adverse effects of heat effects and enhance the palatability, functionality, and nutritional values of proteins.^[85]

Conventional heating

Conventional heating is a very common strategy for the enhancement of digestibility and volatile flavor of pea proteins. Tang, Wichers and Hettinga^[78] applied water bath heating at 100°C for 30 min to pea protein solution and found that the degree of pea protein digestibility increased from ~52% to ~65% using an infant in vitro digestion model. It is proposed that heat treatment caused the soluble aggregation of pea proteins through disulfide bonds, while exposing more buried hydrophobic groups, resulting in a less tight protein conformation to enhance the protease accessibility.^[78] The inactivation of trypsin inhibitor is favorable to improving the digestibility of protein. Ma, Boye and Hu^[81] found that yellow peas cooked in boiling water for 30 min decreased the trypsin inhibitor activity by ~84% and increased the pea protein digestion by ~10% compared to untreated samples. However, the decrease of trypsin inhibitors does not necessarily promote protein digestion when heat aggregation dominates the protein structure.^[82] Therefore, it is necessary to consider the changes in protein structure and anti-nutrient composition simultaneously while evaluating protein digestibility and the heating conditions.

LOX can be thermally inactivated from 60°C,^[111] while it is quite heat stable below this temperature.^[53] Busto et al.^[57] indicated that the crude LOX extract mixed with proteins and starch residues had higher thermostability than the purified LOX due to the protective effect of other constituents. LOX is difficult to be completely removed during pea protein extraction, which even existed in the highly purified pea protein isolates,^[69] and it can be inferred that the LOX contained in extracted pea protein is more thermally stable compared to refined LOX. Few studies in the last 5 years have applied conventional heating to reduce LOX in peas, while some innovative thermal techniques are used for LOX inactivation, as mentioned in Section “Electromagnetic heating, electrical heating, and extrusion”. In addition, the newly formed odor-active molecules during heat treatment can inhibit the beany flavor. Bi et al.^[76] indicated that roasting imparted nutty and caramel-like flavor to peas because of the formation of pyrazines and pyranones, which can mask the beany notes in peas. Ultra-high temperature (UHT) processing (140°C for 6 s) enriched the flavor of pea protein beverage through lipid oxidation and Maillard reaction pathways, while reducing the beany flavor, as heating changed the composition and ratio of the aromatic components, altering the overall aroma profile.^[74]

Conventional thermal treatments are common and mature techniques. However, due to the low heat transfer efficiency, conventional heating often takes a long time to achieve the desired effects, thus some emerging heating methods are gradually used for protein modification to reduce energy consumption and improve the effectiveness of the treatment.

Electromagnetic heating, electrical heating, and extrusion

Microwave treatment is efficient volumetric heating, transmitting electromagnetic energy directly to a heating medium.^[112] Some studies have shown that microwave treatment improved protein digestibility, though the mechanism is ambiguous. Microwave-cooked yellow peas (1200 W for 25 min) increased the in vitro protein digestibility (IVPD) by~6%, resulting from the reduction of trypsin inhibitor activity and tannin contents and heat-induced interior part exposure of pea proteins.^[81] Sun et al.^[78] indicated that microwave treatment (cooking mode, 3 min) induced aggregation of pigeon pea protein via the formation of covalent disulfide bonds, while enhancing in vitro gastrointestinal digestion by reducing particle sizes and improving the protein-protease interactions due to partial denaturation. Although the above studies are not conducted solely on pea proteins, the possible mechanism by which the microwave technique improves the digestibility of legume proteins could be similar to that of pea proteins. Yen and Pratap-Singh^[83] applied a vacuum microwave technique (2 W/g microwave energy in 200 Torr vacuum level for 88 min) in peas which reduced the concentration of off-flavors, though they suspected that this technique would reduce protein digestibility due to a significant decrease in protein solubility. Similarly, Pratap-Singh et al.^[113] found that vacuum microwave reduced pea protein solubility, while a relatively low energy (100 W/g) treatment weakened the raw/beany and green/grassy aromas. It is worth noting that the pea protein slurry with high initial moisture content was susceptible to structural alteration by absorbing microwave energy and more active sites would be exposed than that with low initial moisture content,^[113] which may promote the formation of lipoprotein complexes to lower off-flavor concentration.^[114]

Other than microwave, infrared and radio frequency belonging to the electromagnetic spectrum are promising techniques for digestibility modulation and beany off-flavor removal of pea protein, though their application in pea protein is still limited. Infrared treatment is based on the medium absorption of infrared radiation in the wavelength range of 0.78–1000 μm for heating purposes.^[115,116] Laing^[117] reported that infrared heating at 120°C enhanced the IVPD by ~8% of yellow peas with 30% tempering moisture since infrared induced gelatinization and collapse of the starch granules, making the hydrolysis sites of pea proteins more accessible. Likewise, Heydari, Najib and Meda^[118] found that applying infrared (0.375 kW or 0.75 kW) to assist microwave heating (0.7 kW) and tempering at 50% (d.b.) moisture content efficiently modified protein in lentil flour. The partially denatured lentil protein enhanced the accessibility of susceptible sites to proteolysis, offsetting the adverse effects of protein aggregation on protein digestibility. In addition to microwave and infrared, radio frequency (RF) belongs to the electromagnetic spectrum with a frequency range of 1 MHz to 300 MHz,^[110] and three particular frequencies (13.56, 27.12, and 40.48 MHz) used across industrial, scientific, and medical fields, respectively.^[119] Zhang et al.^[120] applied RF heating (27.12 MHz) and effectively deactivated the LOX in peas within a few minutes, which is more efficient than conventional hot water heating. Additionally, RF heating can reduce anti-nutritional factors like trypsin inhibitors, tannins, saponins, and phytic acids efficiently, while the IVPD of samples was not necessarily changed due to limited alteration of protein conformation after treatment.^[86] Nevertheless, the non-uniform heating or edge overheating of RF is still an issue for this technique.^[110]

Ohmic heating (OH) is an electrical heating technique with high energy efficiency that utilizes the electrical conductivity of food mediums to achieve the heating process.^[121] No published data show that the OH was used for modulating the digestibility and flavor of pea protein, but available studies indicated that it enables pea protein isolate to change their secondary/tertiary structures^[122] and inactivates the undesired enzymes like peroxidase (POD) and LOX in vegetables.^[123] The above evidence suggests that OH could be a promising technique for pea protein digestibility and flavor adjustment. However, OH requires electrodes or electrolytes to be in direct contact with the food, and thus metal ion contamination is a concern.^[110]

Extrusion includes multiple unit operations such as mixing, heating, shaping, and cooling, and is a versatile technology to improve protein digestibility by deactivating anti-nutrients and altering the protein secondary structure.^[124] Beck, Knoerzer and Arcot^[79] reported that the low moisture extrusion process increased the proportion of β-turn and decreased the β-sheet of pea protein, which was favorable to protein digestibility since Carbonaro et al.^[125] demonstrated a linear increment of in vitro digestibility for the loss of β-structures in legume proteins. In addition, protein extrusion with appropriate mechanical energy input and texturization temperature affected the phase equilibrium and diffusion of beany flavor by forming large air chamber cavity structures, which could provide the evaporation driving force for volatile beany components.^[126,127] However, extrusion at high processing temperature (>155°C) or with intensive energy input (>609 kJ/kg) can increase the tightness of protein to hinder beany flavor evaporation, such that the extrusion conditions need to be well controlled.^[126]

Overall, these thermal techniques show the potential to modulate the digestibility and flavor of pea proteins, though more fundamental research is needed to elucidate the complex mechanism affected by multiple factors. Moreover, the moisture content of samples has a significant impact on the structural changes in pea proteins during heating, leading to the difference in digestibility and flavor of heat-modified proteins. Heating stresses can accelerate lipid and amino acid degradation to influence the aromatic profile and amino acid scores in peas,^[128] thus non-thermal techniques can be the alternative to alleviate the adverse heating effects.

Non-thermal techniques

Ultrasound exerts the cavitation effects (growth and collapse of gas bubbles) to cause differential pressure cycling, shear stress, and turbulence in liquid media, affecting protein structure with limited heat stress.^[129,130] High-intensity ultrasonication (HIU) with a frequency of 20−100 kHz has been widely reported in legume protein modification. de Oliveira et al.^[84] applied HIU (20 kHz, total energy input: 240 kJ) together with pH adjustment to break down protein aggregations and expose more hydrophobic and free sulfhydryl groups that are hidden in the core of the protein, retaining the digestibility of pea protein concentrates. Khatkar et al.^[131] indicated that ultrasonic-induced unfolding can improve the IVDP of soy protein. Referring to the HIU applied in soy protein could provide some inspiration for ultrasonic pea protein modification. It has been proven by Vanga, Wang and Raghavan^[87] that HIU assisted by thermal treatment (microwave heating) increased the IVDP of soymilk by ~11% compared to ultrasound without heating source, while the effect of non-thermal and thermal ultrasound on trypsin inhibitors reduction is comparable. Moreover, it was reported that HIU promoted transglutaminase-catalyzed cross-linking and reduced the pore sizes of soy protein hydrogels to facilitate encapsulation efficiency of bioactive compounds such as riboflavin, decreasing its release rate in the gastrointestinal phase to improve bioaccessibility.^[90] These results suggest that ultrasonic treatment can achieve targeted alteration of protein digestibility according to specific purposes. Zhang et al.^[132] indicated that the binding of beany flavor compounds and soy proteins could be destroyed by dual-frequency ultrasound (20 and 40 kHz) to facilitate the removal of undesirable flavors. Overall, although the current ultrasonic modification of pea protein focuses on improving techno-functionality like emulsification,^[133] it could be a promising technique for pea protein digestibility and flavor modulation, analogous to the results shown in ultrasound-modified soy protein.^[87,133,131]

High-pressure processing (HPP) or high hydrostatic pressure processing (HHP) is a technique in which the food matrix is subjected to 100–1000 MPa in a pressure-transmitting medium (e.g. water) for seconds to several minutes.^[134] It is well-accepted that HPP can improve legume protein digestibility by promoting the exposure of proteolytic groups and facilitating the accessibility of proteases.^[135] Laguna et al.^[136] demonstrated that HPP (holding at 600 MPa for 5 min) followed by re-heating (80°C for 30 min) improved the digestion rate of pea protein solution (5%, w/v) under matrix condition of pH 6.2 due to better solubility and the unfolding of globular pea protein subunits. Linsberger-Martin et al.^[88] found that trypsin inhibitors in peas were completely inactivated and phytic acid content was significantly reduced when treated with HHP at 600 MPa and 60°C regardless of time, and IVDP was enhanced by up to 4.3%. They also indicated that soaking peas under HHP (621 MPa) before cooking (98°C, 30 min) can prominently decrease the soaking time of peas from 12 h to 1 h and achieve a similar IVPD of 85.7%.^[88] It seems that high-pressure processing is usually combined with thermal treatment (up to 100°C) to achieve high effectiveness in increasing IVDP by inactivating anti-nutrients or changing protein conformation.^[86] It is still worth exploring the appropriate heating intensity to assist HPP or the reasonable sequence of heating and applying HPP in protein modulation. Moreover, HPP enables LOX to be inactivated at a lower temperature effectively compared to the sole thermal treatment,^[106] which may reduce the beany flavor formation due to thermal degradation. Overall, heating-assisted HPP shows the potential to achieve the desired quality of pea proteins with low thermal stress, for the processing of heat-labile pea protein-based products like probiotic yogurt.^[137]

The cold plasma (CP) technique uses energetic and reactive gases (mainly oxygen and nitrogen) that are excited by various energy power such as electrical, thermal, optical, or electromagnetic sources to achieve microbes/enzymes inactivation, and functional modification.^[92] CP-induced reactive oxygen and nitrogen species (ROS and RNS) could form disulfide-linked protein soluble aggregates and induce protein unfolding by disrupting non-covalent interactions.^[91] Considering the disulfide bond cross-linking may inhibit pepsin digestion^[138] and protein unfolding could expose more hydrolysis sites,^[76] the effect of the CP technique on the digestibility of pea protein remains uncertain. Other than protein structural alteration, another potential of cold plasma processing is reducing the Maillard reaction products induced by thermal treatment, which may decrease the protein digestibility corrected amino acid score due to the reduction of limiting essential amino acids.^[85] Moreover, the CP technique has been shown to mitigate the immunoreaction/allergenicity and anti-nutrients of plant proteins through reactive species-mediated linear epitope alteration or conformational changes.^[89] However, CP treatment can increase the possible oxidation of lipid and free sulfhydryl group, especially for long-term CP processing,^[92] which could enhance the beany flavor and generate disulfide bonds to induce a tight structure. Thus, optimizing processing parameters like treatment power/voltage, frequency, and exposure time of the CP technology is required to ensure that this technology can lead to the expected functionality, digestibility, and flavor of the protein.^[92]

Pulsed electric field (PEF) utilizes short and high voltage pulses (1–80 kV/cm) to modulate semi-solid or liquid food products with high electrical conductivity.^[85] PEF has been shown to modify the secondary/tertiary structures of proteins by interfering with the electrostatic interactions and inducing ionizations of chemical groups and intramolecular rearrangement.^[139] It is reported that LOX in pulses is sensitive to PEF processing and can be inactivated to various degrees depending on the processing parameters (pulse frequency and pulse width).^[140] Nevertheless, protein structure alteration does not necessarily lead to prominent impacts on protein functionality and digestibility,^[139] and LOX inactivation conditions may be ineffective due to changes in processing raw materials.^[141] Thus, the influence of PEF on the LOX activity and digestibility of pea proteins needs to be further studied.

In conclusion, many emerging non-thermal techniques are promising for adjusting the digestibility and enhancing the flavor of pea proteins. Nevertheless, there are still some uncertainties in their effects on the change of protein structures. In addition, research on the digestibility and flavor of pea protein using non-thermal physical techniques is far inferior to that of soy protein, so there is a call for more studies in this area targeted at pea proteins.

Chemical methods

Compared to physical modification, chemical modification methods are more goal-oriented and unambiguous for altering protein structures and achieving customizable properties. Functional groups can be selectively incorporated into proteins via the active residues, involved in forming covalent linkages and disturbing non-covalent forces like hydrogen bonds, electrostatic interactions, and hydrophobic interactions in the modulation process, which changes protein conformation to achieve desired protein properties.^[142,143] The modification sensitivity of amino acid side chains in proteins is greatly affected by the polarity and charge of adjacent residues, the nucleophilicity of the chemical reagent used, and external conditions (pH, temperature etc.).^[142,144] In this section, several typical chemical derivatizations (glycation, acylation, and phosphorylation) of pea proteins are discussed while other less common chemical modification methods are also mentioned.

Glycation

Glycation usually refers to the condensation of carbonyl and ε-amino groups at the initial stage of the Maillard reaction, happening between the proteins and carbohydrates (mainly reducing sugars or polysaccharides), which is featured by spontaneous nonenzymatic reaction and can be accelerated by thermal treatment or other physical assisted techniques like ultrasound.^[145,146] Glycation can not only enhance protein functionality but also endow food products with pleasant aroma flavors.^[1] Shen and Li^[97] and Tang et al.^[94] reported that pea protein glycated with guar gum and glucose, respectively, reduced the IVDP since glycation induced the blocking of lysine residues, decreasing the accessibility of proteases. However, it is also reported that thermal-induced glycation could partially unfold and expose more amino acid residues to increase the susceptibility to digestive enzymes and enhance protein digestibility.^[93] These diverse results may be caused by the discrepancy in the degree of glycation, the protein conformation changes, and the different molecular sizes of conjugated carbohydrates, which provide possibilities for food systems with various requirements for targeted/sustained release. Interestingly, Swiatecka et al.^[93] indicated that although the pea protein-glucose conjugates hindered the accessibility of gastric enzymes, they were beneficial to the growth and metabolic activity of gut microbiota such as lactobacilli and bifidobacteria. This study provides a new perspective on the role of glycated pea proteins in the gastrointestinal tract, though further research on the simulated human digestive system is still required.

As for the aroma flavor of glycated pea proteins, Zha et al.^[147] elucidated that glycating pea protein concentrates with gum Arabic (mass ratio of 1: 4) under controlled Maillard reaction (60°C, 79% relative humidity) for more than 3 days mitigated the beany odor significantly. The reduction of beany flavor can be explained by the release of the binding beany flavor components through the reorientation of the pea protein structure. Moreover, the formation of new characteristic flavor compounds like pyrazines and esters during the Maillard reaction reduced the overall beany perception.^[147] Zha et al.^[68] reached a similar conclusion by studying the aroma profile of glycated gum Arabic-pea protein hydrolysate via the controlled Maillard reaction. Furthermore, Lan et al.^[62] alleviated the beany flavor compounds (1-pentanol and 1-octen-3-ol) of pea protein isolates by applying solid dispersion-based spray drying and using gum Arabic and maltodextrin as carriers, which may be also relevant to glycation-induced structural changes and beany molecules evaporation of proteins during spray drying. In addition, Zha et al.^[148] applied pre-unfolding pea protein isolate (PPI) at alkaline condition (pH 10.0) to facilitate glycation with saccharides of diverse molecular mass and found that glucose and lactose lessened the beany odors of PPI to a greater extent than maltodextrins since the mono- and di-saccharide had higher grafted level on pea proteins. Furthermore, Zhao et al.^[150] found that PPI and xylo-oligosaccharide (XOS) conjugates produced by ultrasound and high-pressure homogenization-assisted Maillard reaction had higher antioxidant activity than thermal-induced conjugates since the former produced more Maillard reaction products as electron donors, which could reduce the beany-odor components derived from thermal lipid oxidation. These glycated pea proteins have also been shown to acquire enhanced solubility and emulsifying properties due to the unfolding of protein architecture and the increase in surface hydrophobicity and steric hindrance.^[66,149,148]

Overall, glycation via the Maillard reaction is regarded as “green chemistry” in protein modulation. Nevertheless, it is still a challenge to control the glycation degree and inhibit the advanced glycation end-products (AGEs) that are associated with metabolic problems and inflammatory responses.^[96]

Acylation

Acylation including acetylation and succinylation uses acetic anhydride or succinic anhydride to introduce acyl groups to a protein, aiming to change the protein conformation and acquire the desired functionality. Among all the amino acid residues, the ε-amino group of lysine residues shows high susceptibility to acylation reagents due to its low dissociation constant and high steric availability.^[150] Recent research from Shen and Li^[97] analyzed the acetylated and succinylated pea protein isolates with different anhydride-to-protein ratios (0.3 or 0.6, w/w) and found that acylation decreased the protein gastrointestinal digestibility compared to untreated proteins. Nevertheless, a much earlier study shows that acylated pea protein isolates achieved higher in vitro hydrolysis rates than untreated samples when digested by multienzymes (trypsin, chymotrypsin, and peptidase), especially for the pea protein isolates acetylated at 1 mmol anhydride/g protein.^[151] These contradictory conclusions may be attributed to the diverse acylation levels, leading to different unfolded and modified protein structures. Considering acylation may impair/strengthen protein-odorant bindings owing to the change of protein native structures, it could lead to the release or retention of some beany flavor compounds in pea proteins.^[1] Available studies on the aromatic flavor of acylated pea proteins are scarce, with further investigation needed to clarify the specific formation or change of flavor components after acylation.

Phosphorylation

Protein phosphorylation refers to the synthesis of phosphoproteins through the addition of acid phosphoryl groups to specific active groups including hydroxyl oxygen in serine, tyrosine, or threonine residue, and amino nitrogen in arginine guanidine group and imidazole nitrogen in histidine.^[139,152] It is recognized that phosphorylation can improve techno-functionalities like solubility, foaming, and thermal properties of pea protein,^[153,154] while the digestibility of phosphorylated pea protein remains unclear. The available research has shown that soy protein isolates (SPI) phosphorylated with sodium trimetaphosphate (STMP) improved the IVDP by~1.5%.^[155] In contrast, Liu et al.^[98] illustrated that SPI under phosphorylation using sodium tripolyphosphate reduced the IVDP by~3%, which in particular negatively affect gastric digestibility. The different results may be due to diverse phosphate reagents and concentrations, and various phosphorylation conditions (pH, temperature, and reaction time). The phosphorylated protein shows high resistance to gastric digestion,^[98] which may enable the proteins to act as a wall material to protect acid-sensitive nutrients through the gastric phase and improve bioaccessibility. In addition, Cui et al.^[153] successfully introduced phosphate groups from sodium hexametaphosphate (SHMP) into pea protein isolates (PPI) by conventional co-spray drying without using harsh reaction conditions or expensive enzymes. They concluded that phosphorylation altered the aroma profile by releasing some beany flavor compounds (1-heptanol) that were inherently bound to PPI, while it may enhance lipid oxidation during high-temperature spray drying. Thus, more studies are still required to figure out the optimal conditions that can overshadow potential negative influences like lipid oxidation to achieve better flavors.

Other chemical methods

Other than the aforementioned protein derivation methods, Hao et al.^[156] introduced polyphenols comprising epigallocatechin-3-gallate (EGCG), chlorogenic acid (CA), and resveratrol (RES) into pea protein isolate (PPI) solutions and obtained PPI-polyphenol complexes bound by non-covalent to facilitate the in vitro digestibility. The authors speculated that polyphenols make digestive enzymes more approachable to proteins by changing the secondary and tertiary structures of pea proteins.^[156] Furthermore, Wang et al.^[157] applied the pH-shift method to co-dissolve composites of pea and rice proteins bridged by secondary structure and found this method achieved limiting amino acids complementary and in vitro digestibility enhancement. These studies provide new insights to improve the bioaccessibility of pea protein with simple strategies. Moreover, alcohol washing with ethanol and isopropanol is an effective extraction method to mitigate the beany flavor and increase the IVDP from 79% to ~85%, though it may cause chemical residue and the loss of some amino acids.^[158] Cui et al.^[107] found that using solid dispersion co-spray drying for pea protein isolates preparation can promote the binding of beany flavor substances with cyclodextrin to entrap the beany flavor, while the physical mixing of the protein and cyclodextrin is not as effective. Interestingly, Gao et al.^[69] changed the alkaline extraction pH of pea protein isolates and decreased the beany flavor notes since LOX activity was pH-dependent. Krause et al.^[159] indicated that pea protein isolate had less beany odor than pea flour since the isoelectric point separation process inactivated LOX, promising for application in baked products. These results indicated that the protein extraction method is an important factor in shifting digestibility and flavor. Thus, more attention needs to be paid to developing efficient extraction methods or optimizing extraction conditions for protein properties modulation, considering the relevant research is insufficient in recent years.

Biological methods

Biological methods, usually including enzymatic hydrolysis (mainly proteolysis, and cross-linking by transglutaminase), microbial transformation (fermentation or biodegradation), and germination have received extensive attention in pea protein modification. Similar to chemical modification, biological approaches are specific to targeted substrates or reaction sites, while they have to be applied under controlled mild conditions to achieve desired effects and eliminate the occurrence of adverse reactions.^[1,160]

Enzymatic treatment

Enzymatic treatment is a technique with high specificity, and proteases and transglutaminase are commonly used enzymes for pea protein property improvement. Konieczny et al.^[99] reported that pea protein hydrolyzed with papain at 11.3% of hydrolysis degree increased the IVDP by ~ 6% and promoted in vitro protein digestibility corrected amino acid score (IVPDCAAS) by ~ 8% with increased methionine and cysteine (the limiting amino acids of pea protein). The studies indicated that the effect of enzymatic treatment on protein digestibility depended on proteases and degree of hydrolysis, while all pea proteins treated with enzymes (trypsin, savinase, and papain) remarkably reduced the activity of tannins, trypsin, and chymotrypsin inhibitors, regardless of hydrolysis degree.^[99] The pea protein treated with proteases improved digestibility due to the release of digestible short-chain peptides or small molecule amino acids after hydrolysis, and the removal or reduction of anti-nutrients.^[99] Considering the allergenicity of pea protein is also a big challenge in its metabolism, Ding et al.^[100] applied alcalase to hydrolyze pea protein and separated hypoallergenic peptides (ADLYNPR) with low binding ability to antigen-specific IgE and IgG1. Nevertheless, the effect of protease hydrolysis on the aromatic flavor in proteins is still unclear. On the one hand, it is believed that proteolysis can diminish the hydrophobic coupling of aroma elements. On the other hand, the protein hydrolysates expose more active sites and can adsorb more flavor components.^[1] Thus, using proteolysis to achieve precise release or adsorption of volatiles based on their diverse binding sites and susceptibility could be a promising study. Bitterness resulting from low-molecular-weight peptides with hydrophobic amino acids is an issue for proteolysis, while optimizing the hydrolysis degree and selecting non-bitter proteases or peptidase with de-bittering functions could lessen the unpleasant bitterness.^[161,162]

Other than proteolysis, Zhao et al.^[101] applied microbial transglutaminase (MTG) to crosslink pea protein and brown rice protein isolates by ε-(γ-Glutamyl)-Lysine bonds. Surprisingly, although the cross-linking bonds have high resistance to pepsin, the MTG-treated proteins show higher degrees of hydrolysis at the end of the digestion since enzymatic cross-linking endued protein blends with distinct structures that can be digested more efficiently than untreated counterparts in the intestinal phase.^[101] The pepsin resistance of transglutaminase-treated protein could slow down gastric emptying, reduce calorie intake, and play a role in losing weight. Furthermore, ultrasound and high-pressure processing can promote transglutaminase-induced cross-linking, changing the protein structures and digestive performance.^[1,90] While glutaminase is not common in pea protein modulation, it has been proven by Fang et al.^[163] that it improved the solubility of pea protein and reduced beany flavor, grittiness, and lumpiness through specific deamidation that mainly acts on glutamine and asparagine. However, the effects of cross-linked pea protein on the retention, release, or masking of beany flavor components still require more research.

Fermentation

Fermentation is an ancient technique with novel applications in improving the nutritional and organoleptic properties of pea proteins. Boroojeni, et al.^[102] documented that peas fermented with Bacillus licheniformis and Bacillus subtilis at 30°C for 48 h profoundly reduced the trypsin inhibitor, phytic acid, and raffinose, though the protein digestibility was not changed. By contrast, pea protein concentrate treated by Lactobacillus plantarum (32°C for 11 h) decreased the trypsin and chymotrypsin inhibitor activity and increased the IVDP by ~ 4%, while reducing the amino acid score due to the loss of sulfur amino acids.^[103] These results illustrated that fermentation is highly microorganism types-dependent. Pea protein fermentation (lactic acid bacteria) can also be performed after enzymatic pre-treatment (alcalase), which released more free amino acids and increased protein digestibility compared to fermentation alone since hydrolyzed proteins with lower molecular weight were more easily ingested by microorganisms.^[164] In addition, Xiang et al.^[65] removed beany off-flavor components (mainly aldehydes) by conducting biodegradation with Saccharomyces cerevisiae and Lactobacillus plantarum. It is believed that di-unsaturated aldehydes like (E, E)-2,4-decadienal were degraded to odor-less or non-volatile substances after fermentation, and more alcohols produced after fermentation occupied the binding sites of aldehydes with lower affinity to proteins.^[65,165] It is worth noting that yeasts can trigger the formation of esters with sweet and fruity flavors.^[109] Despite fermentation showing impressive results, the process of exploring and screening beneficial microorganisms and biosafety assessment is still tough work.

Germination

Germination (sprouting) is the natural growth process of plant seeds, during which endogenous enzymes can be activated to improve protein nutritional quality and flavor.^[166] Setia, et al.^[104] illustrated that germination improved the IVDP of pea seeds, which was associated with the reduction of protease inhibitors and weakening the binding of protein and starch after sprouting, while the IVPDCAAS significantly decreased in 3-day germinated peas due to the degradation of limited amino acids (threonine). Interestingly, germination reduced the antioxidant capacity of some pea seeds, while the total antioxidant capacity increased by 2−3 folds during in vitro gastrointestinal digestion since the increase of free amino nitrogen content after proteolysis enhanced the electron-donating capacity of proteins.^[105] During germination, the beany molecules originally trapped by macronutrients like proteins and starches may be released to increase the beany flavor due to the hydrolysis impact of amylase and protease.^[66] Moreover, the lipolysis, amino acids degradation, and LOX-catalyzed oxidation over the germination course may enhance beany notes.^[66,109] Even so, germination can alter the overall flavor of peas by enhancing aroma components (e.g. benzaldehyde) with sweet and fruity attributes.^[167] To conclude, germination is an effective technique for anti-nutrient degradation, while adding limited amino acids and antioxidants may be necessary to improve the overall amino acids score and lower the free radicals attributing to the oxidation and beany odor formation.^[109]

Pea protein-based food systems

When pea protein is applied to various food systems, the interaction between the protein and other food ingredients as well as diverse food structures can affect the digestibility and flavor attributes. This section provides new insights into the effects of non-protein compositions (carbohydrate, lipid, salt, water, etc.) and formulated structures (emulsions, gels, and dehydrated system) around pea protein-based food matrix on digestibility, with supplementary information on the influence of beany attributes. The potential applications and challenges of pea protein-based food systems are also discussed.

Interaction with major food components

Carbohydrates

Carbohydrates are composed of monosaccharides, oligosaccharides, and polysaccharides and are the main components of the food system. The interference of carbohydrates on the pea protein digestibility is mainly manifested by masking the active digestive sites of proteins, increasing the viscosity of the system, inhibiting the digestive enzymes, and thus hindering or delaying the digestive process. For instance, heating-induced glycation of pea proteins and glucose (reducing sugar) hid the digestive sites and impaired the protein digestibility, mainly in the gastric phase.^[94] Excepting glycation, complex coacervation can occur between oppositely-charged proteins and soluble polysaccharides (e.g. carrageenan, gum Arabic, and xanthan gum), affecting the protein digestibility behaviors by altering the non-covalent bindings and this is often used in targeted bioactive compounds retention and release.^[167] Moreover, gelatinized starch or soluble dietary fibers (e.g. pectin, inulin, β-glucans) can increase the viscosity of the food system, reducing the efficiency of digestive enzyme transportation and diffusion during digestion.^[18] Starch with high crystallinity like resistant starch or the complex formation between starch and other soluble polysaccharides could decrease the digestibility of the whole food system by reducing or even inhibiting the enzyme activity.^[18] Slow digestion can delay gastric emptying and increase satiety, which may be beneficial for body weight regulation.^[168] Thus, both fast and slow digestion plays an important role in nutrient intake, mainly depending on the customized requirements.

The special aroma profile of pea proteins and carbohydrates derived from the Maillard reaction under heating conditions was mentioned earlier (Section “Glycation”). Carbohydrates primarily affect the release/retention of flavor components in the pea protein-based system. Small sugar molecules like mono- or di-saccharides can bind with water, thus enriching flavors in the available water phase, which is conducive to their release during processing and indirectly affects the partition of flavors.^[169] Some oligosaccharides (cyclodextrins) and polysaccharides (starch, pectin, gums) form inclusion complexes with aromatic compounds.^[169] On the one side, this inclusion ability has the function of entrapping the beany flavor,^[107] diminishing the perception of beany flavor during consumption. On the other side, the desired flavors can be encapsulated or adsorbed by carbohydrates with complex structures (starch), negatively affecting the pleasant aromatic flavors to release, which is an issue that is often raised in carbohydrate-based fat replacers of low-lipid food.^[170] It is thus necessary to consider the discrepancy in binding sites of diverse flavor components to precisely preserve or release flavor components through carbohydrates.

Lipids

The effect of lipids on the gastrointestinal digestion of plant proteins is still vague. Guevara-Zambrano et al.^[171] formulated pea protein-based shakes without heat treatment and found that lipids had no significant impact on the digestion of protein in the gastrointestinal tract. Nevertheless, the proteins are prone to react with secondary metabolites from lipid oxidation during food processing, especially the processes involving heating, like generating Schiff bases between aldehydes (lipid oxidation products) and amino, and further participating in Amadori rearrangement, which hinders the binding sites of proteases and decreases the susceptibility of protein digestion.^[172] Chen et al.^[173] applied thermal extrusion technique (in the temperature range of 60°C~145°C) to produce pea protein extrudates and showed that fatty acids dramatically reduced the IVDP of proteins and decreased with the increased degree of fatty acid unsaturation, resulting from fatty acids and amino acids oxidation or the formation of protein-fatty acids aggregation. It is worth noting that pea protein combined with polysaccharides can achieve the target release of lipids. Guo et al.^[174] indicated that pectin and small molecule surfactants (rhamnolipid and tea saponin) effectively inhibited the aggregation of pea proteins when they together act as vehicles for hydrophobic nutrients transportation, improving the lipid phase digestion and promoted the bioaccessibility of β-carotene and curcumin. On the contrary, rational design of the gel interfaces between protein and polysaccharide is possible to reduce lipid digestion and decrease the risk of obesity or atherosclerosis.^[175] Therefore, understanding protein-carbohydrate-fat interactions to design customized food systems is promising, though there are some controversial results regarding the effects of protein-polysaccharide interfaces (including multilayer, particle, and covalent interfaces) on lipid digestion.^[175]

Lipids are precursors of desired and unpleasant flavors in the food system, which play an important role in flavor partition between the lipid and the aqueous phases.^[169,176] Although beany flavors are mainly derived from catalytic oxidation, autoxidation and photooxidation of lipids, lipid oxidation together with Maillard reaction and Strecker degradation is essential for the meaty aromas generation during processing,^[176] which is meaningful for plant meat development. Moreover, the volatility of some beany markers like 1-octen-3-ol decreased by emulsification of an oil/water system since lipids have a high affinity for non-polar/hydrophobic aromatic components to reduce the off-flavor diffusion.^[177]

Overall, lipid plays an important role in nutrient digestion and flavor alteration in the food system. Controlling nutrients targeted release and reducing the beany off-flavor while enhancing the characteristic flavors could be achieved by altering processing conditions and adjusting lipid droplet sizes and sources.

Water/Salts

Water is an important medium for food ingredients interaction. Different moisture content of the food system could induce distinct levels of pea protein aggregations through hydrophobic interaction, hydrogen and disulfide bonds, and secondary structure changes (β-sheets) during processing (e.g. extrusion and roasting), resulting in diverse digestibility performance.^[79,178,179] The salt ions from NaCl or CaCl₂ in the food system can change the surface hydrophobicity of proteins to hinder or promote the accessibility of proteases,^[180] and they can induce the protein-salt bridge to affect the release of components by electrostatic interaction. Guo, et al.^[14] suggested that calcium ions (Ca²⁺) at low concentrations (<24 mmol/L) penetrated the outer layer of high methoxyl pectin (HMP) and formed bridges between pea protein isolate and HMP, assembling a compact structure to enhance the retention and bioaccessibility of curcumin. Thus, salt ions-induced pea protein-polysaccharides can be a good vehicle to deliver hydrophobic bioactive components.

The addition of salts could make more aroma compounds transfer from the aqueous phase to the gaseous phase due to the salting-out effect.^[170] In addition, salts mainly affect the interaction of pea protein and flavor components in the food system, depending on the salt types and ionic strength. Wang and Arntfield^[181] summarized that non-chaotropic salts such as Na₂SO₄, NaCl at high concentrations facilitated the protein-ketone bindings by hydrophobic connections, while chaotropic salt (NaSCN) impeded the stability and enclosed hydrophobic regions of pea proteins so that impaired the flavor binding capacity. The authors also indicated that high concentrations (0.25–1 M) of univalent cations (Na⁺) had a stronger capacity to bind flavors than bivalent cations (Ca²⁺) at the same concentration.^[181] Moreover, Wang and Arntfield^[182] observed that ketones and esters were non-covalently bound to pea proteins (hydrophobic interactions, hydrogen bonds, and electrostatic interactions), while aldehydes and disulfide flavors were covalently bound (disulfide bonds). Although there is no available data about the enhanced binding of the specific beany odors by adding salt molecules, the above research provides a possibility to selectively modify the flavor components in the pea protein-based system by adding suitable salts with proper concentrations. However, more research is necessary to investigate whether the combination of pea protein and flavor components could affect the real perception of odor while preparing and consuming food products, by simulating the cooking and eating process.

The influence of food structures

Emulsions

In plant-based emulsion systems, pea protein is often adsorbed on the surface of oil droplets as a natural emulsifier, acting as a carrier for the transportation and protection of the bioactive ingredients in the gastrointestinal tract.^[18] Pea proteins, covalently or non-covalently bonded with polysaccharides/polyphenols, could be the vehicles of the oil phase to achieve the targeted or sustained release, resulting from the diverse emulsion structures with multi-scale composite interfacial layers.^[183,184] In brief, the digestion of the emulsion system is focused on the digestion of the water phase (mainly protein or carbohydrates) and the oil phase (hydrophobic bioactive ingredients or unsaturated fatty acids).

The most common structure in a pea protein-based food system is a single-layer oil-in-water emulsion, which is sensitive to digestive juices and could be easily destroyed by protease or lipase.^[175] The thinner surface layer and smaller droplet sizes of this conventional emulsion cause a faster rate of digestion of the coated oil phase.^[18] Nevertheless, Guevara-Zambrano et al.^[171] fabricated a plant-based shake with pea protein, sunflower oil, and soy lecithin, and indicated that the outer layer pea protein delayed the digestion rate of the coated lipids because the aggregation generated by the protein during gastric digestion could block lipase access to substrates. Aniya et al.^[185] applied water-in-oil-in-water (W/O/W) emulsion with pea protein isolate to encapsulate anthocyanins, finding that the pea protein-1% xanthan gum mixture as the outermost layer presented higher retention of anthocyanins than pea protein during gastrointestinal digestion. Sun et al.^[175] also suggested that the digestive behavior of emulsion was quite similar when only protein was used as an emulsifier in the outer layer, independent of whether this system is a binary- or mono-layer emulsion, while the supplement of polysaccharides may delay the digestion by hindering the accessibility of digestive enzymes. Likewise, complex coacervation structure fabricated by oppositely charged proteins and polysaccharides enables the lipid phase stabilized in double shells to be resistant to environmental stress such as pH, temperature, and process conditions.^[186] Pea protein-based Pickering emulsions have been widely reported, which can be applied in food products with high oil fraction (e.g. mayonnaise with 50 wt% oil content).^[187] It is believed that the combination of protein and polysaccharide can build a complementary colloidal particle interface with both hydrophilicity and hydrophobicity to achieve gastrointestinal stability.^[188] Overall, as shown in Fig. 3, the main mechanism of these protein-polysaccharides multi-scale interfaces of delaying oil phase release is to reduce protease accessibility, hinder bile salts from replacing emulsifiers, and lipase-colipase adsorption at oil droplets. Although pea protein often serves as a techno-functional ingredient (wall material) in emulsion systems, it can also be a good protein source in some plant-based emulsions such as plant milk. Thus, it is useful to probe into its final gastrointestinal fate, and the effects of undigested pea proteins on modulating gut microbiome and impacting human health are promising research topics.

Figure 3. Overview of the effects of emulsion and gel structures in pea protein-based matrix on protein/hydrophobic phase digestion and potential applications. I: monolayer emulsifiers (proteins) are replaced by bile salts and are accessible to lipid hydrolysis sites; II: colipase anchors lipase to the hydrophobic droplet interface; III: stable colloidal particles or multilayer structures hinders proteases accessibility, bile salts replacement and lipase-colipase adsorption. Interfacial structures: (a) single layer protein structure; (b) single protein gel; (c) protein colloidal particle; (d) protein-polysaccharide complex coacervation; (e) protein-polysaccharide covalent/non-covalent interactions; (f) protein-polysaccharide colloidal particle; (g1) protein-polysaccharide gel; (g2) protein-polysaccharide-ion gels (adapted from^[173,189] with permission).

The effect of emulsion structures on the seal or mitigation of unpleasant beany off-flavor is rarely reported, but inhibition of lipid phase oxidation by applying multi-scale pea protein-based emulsions seems to be beneficial to control the beany flavor formation during processing and storage. In particular, the interfacial layer composed of protein and polyphenols presents strong antioxidant capacity and good emulsifying stability.^[184]

Gels

Pea protein-based gel network could provide the desired texture for plant-based food products like yogurt, cheese, and meat. Pea protein aqueous solution or protein-based emulsion can be transformed into soft-solid-like hydrogels or emulsion gels by heating, acidification, fermentation, or enzymatic treatment.^[18,190] Heat-induced hydrogels formed only by pea protein are brittle/fragile with low hardness, which are unsuitable as bioactive compound carriers during gastrointestinal digestion since fragile gels can disintegrate quickly and expose a larger surface area to make enzymes accessible (Fig. 3 (b)).^[191] To obtain a more rigid gelation structure, Xu and Dumont^[192] applied polyelectrolyte complexation with chitosan to enhance the stability of pea protein – alginate/Ca²⁺ hydrogels in the intestinal phase, which showed some potential in nutrient delivery and controllable release (Fig. 3 (g2)). In addition, pea protein-involved hydrogels could be ideal protein nutritional supplements since the weak gel strength makes digestive enzymes approachable.^[191] Zhao et al.^[101] reported that the pea-rice protein hydrogels cross-linked by microbial transglutaminase altered the digestible sites of the protein, resulting in a higher level of protein hydrolysis in the intestinal digestion and suggesting higher bioaccessibility than the non-crosslinked equivalents. However, it is necessary to avoid the potential adverse effects of weak gel structure on the characteristic texture of the food systems.

Pickering emulsion can be seen as a fluid emulsion gel,^[193] which can achieve a controlled release of lipophilic nutrients. For instance, Qiao et al.^[194] fabricated pea protein nanoparticles stabilized Pickering emulsion gels by alkaline/heat treatment (pH 12, 70°C) and improved the bioaccessibility and curcumin stability in the intestinal phase due to the large surface area of small emulsion gel droplets after gastric digestion. Similarly, Shao and Tang^[13] applied microfluidization to develop a Pickering emulsion gel delivery system based on pea protein at acidic conditions (pH 3). As a result, the gel-like network achieved the intestine-targeted release for β-carotene. Research on other pea protein emulsion gel-based food systems (e.g. cheese, yogurt) is focused more on their mechanical properties like rheological and texture profiles,^[139,195] while few study is about digestibility, thus more investigation is required to fill this blank.

As for the beany flavor, some studies have shown that the pea protein-based gel system induced by microbial fermentation mainly using lactic acid bacteria or yeast can decrease the beany intensity and produce sweet or fruity flavors.^[108,196] Researchers often attribute these pleasant flavors to the metabolic transformation of food substrates or flavor components by microorganisms, while changes in the matrix structure caused by the food gelation also play an important role in the flavor release, retention, and perception.^[196,197] Interestingly, Wang and Arntfield^[198] discovered that the binding effects of aldehydes (including beany flavor marker hexanal) to pea proteins increased the strength of pea heat-induced protein gels, depending on the chain length and concentration of aldehydes. From another perspective, the binding of pea protein gels to aldehydes (the main category of beany flavor) may affect the flavor release and perception of the gel colloid system, though the authors did not explain it explicitly,^[198] which is worth considering for further research.

Dehydrated food system

Food dehydration process is popular in the plant-based food industry due to its superiority in food preservation and transportation. Besides removing moisture, dehydration can create unique structures and functionalities for dry matrices by different drying methods and modulating the precursors like emulsions and gels.^[199]

Porous dry matrices are appealing functional structures for product fabrication.^[199] Sun et al.^[200] reported that pea proteins were extruded at 130°C with the assistance of konjac gum (0.3%) and obtained textured pea proteins containing large air pores with good chewiness and hardness (Fig. 4 (b1)), which is expected to be used as ingredients for fibrous meat alternatives. In addition, the formation of air chamber cavities with thin wall layers in textured protein (Fig. 4 (a1)) could promote the beany flavor evaporation during extrusion to reduce the concentrations and perception of beany off-flavor, while thick cavity wall layers (Fig. 4(a2)) retaining the beany flavor.^[126] Nevertheless, retaining the digestibility of extruded proteins remains a challenge, as proteins may gain a dense structure during high-temperature extrusion, leading to protein aggregation and low availability and susceptibility of digestive enzymes, thus impairing protein digestibility, which was more prominent in the food system with more unsaturated fatty acids due to the high level of oxidative complexes.^[173]

Figure 4. Scanning electron microscopy (SEM) images of typical extrusion, spray-dried, and freeze-dried structures in legume protein-based systems. (A) Textured soy protein extruded under proper heating conditions had large air chambers with thin cavity wall structure (a1), while extrudates under excessive heating obtained small air chambers with thick cavity wall structure (a2) (scale bar = 500 μm); (B) pea protein isolate-konjac gum extrudates formed larger and more pore structure (b1) after extrusion compared with no konjac gum addition samples (b2) (scale bar = 50 μm). (C) Spray-dried probiotics (lactobacillus rhamnosus GG, LGG) encapsulated by pea protein isolate-pectin complex obtained smooth surface and small cavities (c1), while the freeze-dried encapsulated samples had sponge porous network (c2) (scale bar = 10 μm). (D) Spray-dried orange essential oil microparticles stabilized by pea protein concentrate and maltodextrin without visible pores on surface and had good oxidative stability (scale bar = 5 μm) (reprinted from^{[126,200–202]} with permission).

Other than extrudates, the porous structures of the spray-dried powder affect the encapsulation efficiency of the core materials. Francisco et al.^[201] used pea protein concentrate-maltodextrin as a matrix to encapsulate limonene essential oil by spray drying and indicated that microparticles with low porosity could be formed at a low protein concentration (2.4%) to prevent essential oil loss and oxidation (Fig. 4(D)). Moreover, it is reported that spray-dried microcapsules derived from pea protein isolate and sugar beet pectin complex coacervates can provide better protection for probiotics than freeze-dried ones in gastrointestinal digestion.^[202] As shown in Fig. 4(c1) and (c2), the sponge porous structure of freeze-dried samples, which was easy to be invaded by gastrointestinal digestive juices, while the spray-dried powder presenting a more intact and smoother surface with only a few cavities was more resistant.^[202] Nevertheless, the freeze-drying method could be a good option to improve the digestibility of dehydrated pea protein-based snacks for nutrient supply because its porous structure allows digestive enzymes to have adequate contact with hydrolysis sites.^[203]

Tunable porosity is a determining factor to modulate the digestibility and flavor of dehydrated food systems. Developing a model that can predict the porosity of the drying matrix could offer a more precise attempt to achieve the expected food quality.^[204]

Conclusions and future perspectives

In terms of digestibility and beany flavor of pea protein, there are identified research gaps in the precise modulation and interpretation of their mechanisms, and relevant research is still insufficient compared with those of milk proteins or even soy proteins. Understandably, the rate of protein digestion needs to align with the role it plays in the food system. Specifically, if pea protein is consumed as a nutritional supplement, fast digestibility, or slow digestibility with high degrees of hydrolysis may be required, while if the protein is used as a carrier for sensitive nutrients, delaying the protein digestibility to protect nutrients and fulfill targeted release in the gastrointestinal phase is ideal. Many trials for beany flavor adjustment like reducing lipid oxidation, mitigating/masking beany flavors, and generating pleasant aromatic compounds have been conducted. However, in most studies, beany molecules were analyzed independently, and the aromatic analysis of beany component mixtures was still unclear, though the blend of beany molecules is the actual scent contributor. Furthermore, the digestibility and beany flavor of pea protein are influenced by other food components (lipids, carbohydrates, salts, etc.) and the diverse structures of the food matrices in the food system, as the basis for the development and structural design of new pea protein-based products. Considering that the application of some protein modification technologies still shows uncertainties, and the research on actual food systems is lacking, the precise tailoring of digestibility and beany flavor of pea protein to meet their application requirements is still a challenge.

Most research on pea protein modification focuses on the post-treatment of extracted protein concentrate/isolate or protein extraction methods. The pre-treatment of raw materials for protein extraction deserves more investigation in terms of digestibility modification and flavor improvement since it has the potential to inactivate lipoxygenase by eliminating lipid oxidation during the extraction process and could alter digestibility by removing anti-nutrients and changing protein structure. The gastrointestinal digestibility of various natural/modified pea protein or pea protein-carbohydrate complexes, digestive rate (e.g. rapid, slow, resistant digestion), and targeted/sustained release characteristics could be defined so that different pea protein ingredients could be more accurately applied in the food system. More studies on beany flavor can focus on the effect of the concentration and (or) different proportions of the beany molecule mixtures, while also essential to building the odor thresholds for the blend of beany components. To cater for an emerging pea protein-based food market rapidly, research on simulated food systems with various components and specific structures is needed to mimic targeted pea protein-based products, and bridge the gap between lab-based results and real applications in the food industry.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Australian Research Council (ARC) Discovery Grant [DP200100642].