Enhancing plant protein functionality by using physical modification techniques

Abstract

Plant proteins have attracted attention recently owing to their sustainability, economic proposition, and health benefits. Plant proteins also have the potential to meet the protein demands of the rapidly growing world population in the future. However, their applications in the food industry are limited owing to their compact and aggregated structure, which reduces their solubility and hides the hydrophobic groups within the protein-folded conformation. These proteins require modifications to improve their solubility and expose the hydrophobic groups. Among the various types of modification approaches, physical modification approaches are favourable as they are considered safe, easy to use, and can be applied on a large scale. The focus of this review is to highlight and discuss the importance of plant proteins, the reasons for their modifications, their modifications by physical techniques, and their potential applications in the food industry.

‍Introduction

Proteins are essential nutrients which are required in sufficient amounts on a daily basis to maintain muscle mass and regulate various physiological functions in our bodies. Global protein demands are rising every year with the rapid increase in the world population. According to the UN Food and Agriculture Organization, a source of good quality protein (with a complete amino acids profile) will soon be the most challenging requirement to meet in ensuring food security.¹ The majority of protein sources have animal origins. According to a survey conducted in 2015, only around 30% of the protein intake in the US population comes from plant sources.² Therefore, there is huge potential for utilising plant proteins in the food industry. 

Additionally, in recent times, plants have become a favourite source of protein for consumers worldwide owing to their eco-friendly nature, sustainability, and economical cost. Also, they meet the dietary requirements of vegans and vegetarians.³ The global food industry is focusing on meat and dairy alternatives, which require plant proteins with high functional properties in order to stabilise multiphase systems, including emulsions (e.g., dressing, margarine, mayonnaise) and foams (e.g., ice cream, cappuccino and whipped cream). Compared to animal proteins, plant proteins have compact structures, which make them less soluble in aqueous solutions. These proteins require modifications to increase their solubility and enhance their functionalities.³

Various modification strategies for plant proteins have been proposed. Physical modification techniques are the most suitable as they are considered safe and easy to use. Additionally, they are cheap and can be applied on a commercial scale.⁴ Modified plant proteins have the potential to be used in the food industry as stabilisers or emulsifiers in food emulsions, cream and cheese production, protein-based drinks for athletes, and vegan and vegetarian-friendly burger patties.^4-5 The main goal of this review is to provide a comprehensive and concise overview of plant proteins, the mechanism of their modifications, the main types of physical modification techniques, and the potential applications of modified proteins in future foods.

Why do plant proteins need modification?

Plant proteins are normally extracted from different plant-based sources, including cereals, legumes, seeds, nuts, and others. During the extraction and separation process from raw material, protein molecules undergo modifications which alter the structure and functional properties, depending on the extraction method being employed. Dry and or wet fractionation methods are mainly used for protein extraction, depending upon the required protein purity. Dry fractionation produces protein concentrates with a purity of 45 – 80%. It involves milling of raw material followed by separation with air classification or electrostatic separation.⁶ It produces protein with a more intact structure. Wet fractionation, on the other hand, is suitable for obtaining high-purity protein isolates (≥ 90%).⁶ However, it employs harsh conditions such as high heat, strong acid and base, and a drying process, which denature proteins and alter their functionalities. 

The extraction process can induce significant changes in the native structure of proteins, which could result in the formation of aggregates, and reduce the protein solubility in aqueous solutions.⁷ The choice of extraction method is, therefore, critical in determining the solubility and applications of plant proteins. Optimisation of the extraction procedure is an important step in obtaining protein with maximum nativity and improving functionality. 

Several plant proteins in their native forms possess tightly packed conformations, due to the association of their subunits via covalent (disulfide) and non-covalent (hydrogen bonding, hydrophobic interactions, and electrostatic interactions).⁸ As a result, these plant proteins are weakly soluble in water, conferring a major disadvantage. Therefore, modifications are required to improve the solubility and functional properties of plant proteins.

Approaches to modifying plant proteins

Plant proteins from different sources vary in their chemistry and functional properties. There is generally no “one size fits all” approach to their modification. Before modifying a specific plant protein, it is essential to consider several factors: the protein source, its functional properties, the objectives of modification, and the underlying mechanism of modification to achieve the desired functional property. For instance, increasing the solubility of plant proteins may require reducing aggregate size, altering the pH and net charge, and or conjugation with more hydrophilic polymers.^9-10 Improving the emulsifying properties may require a modification technique that not only enhances solubility but also exposes more hydrophobic groups.¹¹ In summary, the choice of any modification technique is critical and depends on the desired functional properties in the protein. 

Modification techniques are of different types, including physical (in which physical force is being applied, such as high-pressure and heating), chemical (in which a suitable chemical is being used to induce modification), and / or biological (in which an enzyme is used for inducing modifications).¹⁰ In this review, we will briefly discuss the main types of physical modification techniques, how they modify the protein structure and possible applications of modified plant proteins in the food industry.  

Physical modification approaches

Thermal or heating treatment 

Thermal treatment, or heating, is one of the commonly used techniques to modify the structural and functional properties of plant proteins. Thermal treatment provides energy to break the covalent and non-covalent bonds within the protein structure and disrupt the tertiary structure of the protein. Further, the protein partially unfolds its structure, leading to an intermediate molten globule state with enhanced functionality (Fig. 1).¹⁰ However, heating protein at extremely high temperatures can cause irreversible changes and denature the protein, which has adverse effects on its functionality.³ Therefore, mild thermal treatment of plant proteins can be employed to alter the structure and improve their functional properties. It is reported in the literature that heating plant proteins improves their water binding ability, gelation behaviour, and foaming properties, while its effect on solubility might not be substantial.^12-14 

Ultrasonication

Ultrasonication is another popular physical method used to modify the structural properties of plant proteins.  It offers several advantages, such as being cheap, processing small amounts of samples, and requiring short processing time.^15-17 Ultrasonication is of two types: high-frequency, low-intensity ultrasound, which uses frequencies above 100 kHz and power < 1 W/cm² and low-frequency, high-intensity ultrasound, in which the frequency ranges from 16 – 100 kHz and power > 10 W/cm².¹⁸ 

High-intensity ultrasonication with a probe ultrasound processor is usually used to modify plant proteins. During processing, it generates acoustic waves which break down protein aggregates by the cavitation phenomenon. Gas bubbles are formed and collapse due to localised pressure differences, which release strong hydrodynamic shear forces and heat in the region of bubble collapse. The cavitation created during the ultrasound process breaks down the bigger aggregates into smaller aggregates, reduces particle size, and disrupts the non-covalent bonds (Fig. 2).¹⁸ It increases water and protein interactions and thus partially unfolds the protein structure, increasing protein solubility in aqueous dispersions.¹⁹ found that high-intensity ultrasound treatment increased pea protein solubility from 7.2 mg / L to 58.4 mg / L. Studies have also shown that emulsions prepared with plant proteins modified by ultrasonication have longer stability.^20-21

‍High-pressure homogenisation

High-pressure homogenisation (HPH) has earned much interest as a non-heating technique for modifying plant proteins and increasing their potential to be used as emulsifiers or stabilisers in the food sector.²² During the high-pressure homogenisation process, a protein dispersion is passed through a narrow nozzle under high pressure and turbulence. The shear stress and cavitation forces that occur break down bigger protein aggregates, and alter the structure and physicochemical properties of the protein.²³ In a recent study, plant proteins from plum seed, wolfberry, jujube seed, and hemp seed were treated with high-pressure homogenisation. Findings revealed that HPH significantly reduced the particle size and increased the solubility for all the protein suspensions. HPH also promoted the gelation behaviour of plant proteins.²⁴ 

Potential applications of modified plant proteins

Plant proteins are food grade, generally recognised as safe materials and possess both hydrophobic and hydrophilic properties, which make them suitable candidates for stabilising food emulsions.²⁵ An emulsion is a mixture of two immiscible liquids wherein one liquid is a dispersed phase, and another is continuous phase. Natural examples of emulsions are milk and butter, where milk is an oil-in-water emulsion (O / W) and butter is a water-in-oil emulsion (W / O), stabilised by milk proteins. 

Emulsions are thermodynamically unstable and can separate into aqueous and oil phases due to the high surface tension between the two immiscible phases.²⁶ Therefore, a suitable emulsifier or stabiliser is required to reduce the surface tension at the O / W interface, which can form a viscoelastic film around the droplets at the interfacial layer, providing mechanical stress and stabilisation against the phase separation. For instance, pea protein-based emulsions were prepared using high-pressure homogenisation and ultrasonication, and then the emulsions were processed at ultra-high temperatures (pre-heating at 95° C for 15 seconds and then heating at 140° C for 2 seconds). Emulsions prepared by both techniques showed stability at ultra-high temperatures, which suggests that plant protein-based emulsions can be used in the beverage industry.²⁷ 

Cheese is also a type of emulsion in which the protein, mainly casein, in the aqueous phase stabilises fat from milk. Traditional cheese manufacturing involves animal milk as the main ingredient, which is processed through a series of steps, including acidification, coagulation, curding and separation of whey, the addition of salt, and resting time for the cheese to ripen. One of the desired properties of cheese is its melting behaviour upon heating. Traditional cheese melts because non-covalent bonds between the casein proteins weaken or break and re-establish upon heating. Researchers have been focusing on developing plant protein-based cheese that mimics the melting properties of traditional cheese. For instance, cheese has been developed using pea protein isolate and its properties compared with commercially available animal protein-based cheese.²⁸ Pea protein formulations revealed good lubrication properties but did not show melting behaviour upon heating because of their covalently linked gel-like structure. The outcome was surprisingly different when zein, a protein from corn, was combined with pea protein, with a total protein content of 30% in the formulation. The melting behaviour was promising as the zein network mostly depends on non-covalent interactions. However, further research is required to improve the taste and sensory properties of plant protein-based cheese.

Plant proteins can also be used as a carrier for the delivery of bioactive compounds and nutraceuticals and for protecting them from harsh conditions such as pH, heat, and light. There are several review articles available in the literature on the potential applications of plant proteins as a suitable carrier for the delivery of bioactive compounds, essential oils, and drugs.^29-31

Conclusions

This review highlights the promising potential of plant proteins as an alternative to meat and dairy in the food sector. Most plant proteins exist in aggregated and compact structures, which affect their functional properties and limit their applications. Adopting a modification strategy can alter their structure and improve their functionalities. Physical modification techniques, including ultrasonication, high-pressure homogenisation, and controlled heating, are mainly used as they are considered safe. These modifications improve plant protein solubility, hydrophobicity, and gelation behaviour, making them suitable emulsifiers for the food industry. Modified plant proteins have been explored to replace animal proteins in the food industry, such as in food emulsions, cheese, and cream, and as a suitable protective material for delivering bioactive compounds. However, research gaps exist regarding their structural complexity, behaviour within food formulations, and the effect of modification techniques on the final product quality. Therefore, in-depth studies are required in these areas to enhance the functionality of plant proteins and enable them to play a pivotal role in the pursuit of sustainable and nutritious alternatives. 

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