**2. Physical modifications**

Functional properties of legume proteins can be modified by application of a variety of physical methods including thermal treatment, ultrasonication, and high pressure. A brief summary of the findings of recent studies focusing on investigation of the effects of physical methods on functionality of legume proteins is presented in **Table 1**.

#### **2.1 Thermal treatment**

Thermal treatment is commonly applied to legume proteins for modification of structure and functionality. Tang et al. [5] applied heat treatment to kidney, red bean and mung bean protein isolates at 95 °C for 30 min and studied its effects on structural properties and functionality of the proteins. The authors observed heat-induced protein denaturation which was evident by the modifications in the secondary and tertiary protein structures. Heat treatment was indicated to increase the surface hydrophobicity of bean proteins due to unraveling of previously buried hydrophobic moieties. Although the authors observed protein denaturation and significant increase in hydrophobicity, solubility of heat-treated bean proteins was reported to increase as well. This finding was attributed to the possible increase in charged residues on protein surface due to denaturation and partial unfolding of the molecule. Emulsifying activity of heat-treated bean proteins was also reported to increase compared to the native proteins due to increased hydrophobicity and solubility after heat treatment. Peng et al. [8] investigated the effect of heat treatment (95 °C, 30 min) on surface hydrophobicity, interfacial properties, emulsifying activity and stability of pea proteins. It was reported that heat treatment increased surface hydrophobicity due to unraveling of protein structure and exposure of hydrophobic groups buried inside the molecule. Application of heat treatment was reported to result in slightly reduced interfacial tension and significantly higher percentage of adsorbed proteins at the interface compared to the native protein. Heat-treated pea protein was able to form emulsions with significantly smaller droplets and higher stability against creaming due to higher adsorption of proteins at the oil–water interface. Chao and Aluko [9] applied heat treatment to pea protein isolate at a wider temperature range (50–100 °C, 30 min) for modification of emulsifying and foaming properties. The authors observed changes in protein conformation and increased hydrophobicity which were attributed to protein unfolding. Those changes in protein structure were reported to lead to protein aggregation induced by thermal treatment up to 100 °C. Emulsion formation and stabilization by pea proteins were reported to improve at pH 7.0, which was indicated by reduced oil droplet size compared to the native protein. However, average oil droplet size of heat treated pea proteinstabilized emulsions were reported to be larger than that of the native protein-stabilized emulsions at pH 5.0. Foaming properties of pea protein were observed to be negatively affected by heat treatment regardless of the pH applied. In another recent study, Bühler et al. [7] applied dry heating to faba bean protein concentrate in an air oven (75–175 °C, 1 h) for modification of water holding capacity. The authors used

**89**

**Table 1.**

*Modification of Legume Proteins for Improved Functionality*

**Legume protein studied**

Red kidney bean protein

Faba bean protein

*Ultrasonication* Black bean

*High pressure* Kidney bean

protein

Yellow field pea protein

*Other techniques* Pea protein Controlled shear at

protein

Soybean β-conglycinin and glycinin

Faba bean protein

Red kidney bean, red bean and mung bean proteins

**Treatment conditions**

**Effects observed on protein** 

**Ref.**

[5]

[6]

[7]

[8]

[9]

[11]

[12]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

**functionality**

emulsifying activity

emulsifying and foaming activities with moderate heating (15–30 min)

capacity, decreased solubility

oil–water interface, improved creaming stability, increased

Improved emulsifying activity at pH 7.0, decreased foaming properties regardless of pH

Improved solubility, emulsifying activity, and

Higher adsorption at the oil–water interface, improved foaming properties

Improved solubility, emulsifying, foaming, water holding and gelling properties

Improved water holding capacity, foaming, and emulsifying properties, increased viscosity

foaming properties

50–150 MPa

stability

emulsifying and foaming properties with increasing pressure up to 100 MPa, improved gelling properties at

Improved solubility and heat

Increased water and fat binding capacities, improved solubility

Higher adsorption at the oil–water interface, improved emulsifying properties

Improved solubility [10]

Improved foaming properties [13]

95 °C, 30 min Improved solubility and

95 °C, 15–120 min Improved solubility,

75–175 °C, 60 min Increased water-holding

viscosity

stability

200–600 MPa, 5 min Improved emulsifying and

Pea protein 95 °C, 30 min Higher adsorption at the

Pea protein 50–100 °C, 30 min,

Pea protein 20 kHz, 30–90%

Chickpea protein 20 kHz, 300 W,

pH 3.0–7.0

20 kHz, 150–450 W, 12–24 min

20 kHz, 400 W, 5–40 min

20 kHz, 50–75% amplitude, 15–30 min

amplitude, 30 min

5–20 min

200–600 MPa, 15 min

100–1500 s−1

pressure plasma

pressure plasma

*Summary of recent studies on physical modification of legume proteins for improved functionality.*

Pea protein Cold atmospheric

Grass pea protein Cold atmospheric

Lentil protein 25–150 MPa Improved solubility,

*DOI: http://dx.doi.org/10.5772/intechopen.96274*

*Thermal treatment*


*Grain and Seed Proteins Functionality*

**2. Physical modifications**

presented in **Table 1**.

**2.1 Thermal treatment**

Recently, Sharif et al. [3] reviewed the modification methods applied to legume proteins for improvement of emulsifying properties for encapsulation applications. Ge et al. [4] discussed the modifications applied specifically to pea protein for improved functionality. This chapter presents an overview from a broader perspective of the most commonly used methods for modification of functional properties

Functional properties of legume proteins can be modified by application of a variety of physical methods including thermal treatment, ultrasonication, and high pressure. A brief summary of the findings of recent studies focusing on investigation of the effects of physical methods on functionality of legume proteins is

Thermal treatment is commonly applied to legume proteins for modification of structure and functionality. Tang et al. [5] applied heat treatment to kidney, red bean and mung bean protein isolates at 95 °C for 30 min and studied its effects on structural properties and functionality of the proteins. The authors observed heat-induced protein denaturation which was evident by the modifications in the secondary and tertiary protein structures. Heat treatment was indicated to increase the surface hydrophobicity of bean proteins due to unraveling of previously buried hydrophobic moieties. Although the authors observed protein denaturation and significant increase in hydrophobicity, solubility of heat-treated bean proteins was reported to increase as well. This finding was attributed to the possible increase in charged residues on protein surface due to denaturation and partial unfolding of the molecule. Emulsifying activity of heat-treated bean proteins was also reported to increase compared to the native proteins due to increased hydrophobicity and solubility after heat treatment. Peng et al. [8] investigated the effect of heat treatment (95 °C, 30 min) on surface hydrophobicity, interfacial properties, emulsifying activity and stability of pea proteins. It was reported that heat treatment increased surface hydrophobicity due to unraveling of protein structure and exposure of hydrophobic groups buried inside the molecule. Application of heat treatment was reported to result in slightly reduced interfacial tension and significantly higher percentage of adsorbed proteins at the interface compared to the native protein. Heat-treated pea protein was able to form emulsions with significantly smaller droplets and higher stability against creaming due to higher adsorption of proteins at the oil–water interface. Chao and Aluko [9] applied heat treatment to pea protein isolate at a wider temperature range (50–100 °C, 30 min) for modification of emulsifying and foaming properties. The authors observed changes in protein conformation and increased hydrophobicity which were attributed to protein unfolding. Those changes in protein structure were reported to lead to protein aggregation induced by thermal treatment up to 100 °C. Emulsion formation and stabilization by pea proteins were reported to improve at pH 7.0, which was indicated by reduced oil droplet size compared to the native protein. However, average oil droplet size of heat treated pea proteinstabilized emulsions were reported to be larger than that of the native protein-stabilized emulsions at pH 5.0. Foaming properties of pea protein were observed to be negatively affected by heat treatment regardless of the pH applied. In another recent study, Bühler et al. [7] applied dry heating to faba bean protein concentrate in an air oven (75–175 °C, 1 h) for modification of water holding capacity. The authors used

of legume proteins, with a major focus on the most recent studies.

**88**

#### **Table 1.**

*Summary of recent studies on physical modification of legume proteins for improved functionality.*

soy protein concentrate as a reference. Heat treatment was reported to induce partial denaturation, which in turn increased hydrophobicity and resulted in decreased protein solubility. On the contrary, water holding capacity of faba bean protein was observed to increase and became comparable to soy protein.

#### **2.2 Ultrasonication**

Ultrasonication has been recently investigated as a novel tool for modifying the structure and improving functionality of legume proteins. Gharibzahedi and Smith [21] recently reviewed the current applications and challenges of this technology for legume proteins. Jiang et al. [10] applied low-frequency (20 kHz) ultrasonication to black bean protein isolate. Primary structure of black bean protein was reported to remain unchanged after the ultrasonication treatment indicated by the electrophoresis profile. On the other hand, secondary structure of black bean protein was observed to change after ultrasonication where the proportion of α-helix decreased and β-sheet increased. Ultrasonication-related alterations in tertiary structure were monitored by the changes in fluorescence spectra. Average particle size of ultrasound-treated black bean protein was reported to be larger compared to that of native protein which was attributed to formation of aggregates. Net surface charge of ultrasonicated black bean protein was observed to change depending on the power applied. Surface hydrophobicity of black bean protein was also reported to increase after the ultrasonication treatment. Similar to heat treatment applications, ultrasonication is indicated to result in unfolding of the protein molecule up to a certain degree and increase hydrophobicity. However, surface hydrophobicity was reported to decrease with a further increase in the ultrasonication power due to aggregation of protein molecules which in turn enclose the hydrophobic sections of the protein. Ultrasonication treatment was reported to result in an increase in solubility of black bean protein, directly related to ultrasonication power and treatment time which was based on increased interaction between protein molecules and water. The authors concluded that the modifications on protein functionality could be optimized based on the ultrasonication conditions. Martinez-Velasco et al. [12] studied the effect of ultrasound treatment on surface characteristics and foaming properties of faba bean protein isolate. The authors optimized the parameters of ultrasonication treatment and reported that an amplitude of 73% and duration of 17 min resulted in lower interfacial tension, higher solubility, higher adsorption at the interface, smaller bubble diameter, and higher foam stability. In a similar study, Xiong et al. [13] investigated the effect of ultrasound treatment on structural characteristics and foaming ability and stability of pea protein isolate. Ultrasonication was reported to induce unfolding of the protein molecule up to a certain degree and hence increased hydrophobicity. These changes combined with decreased particle size were reported to result in decreased surface tension, improved foaming ability and stability after the treatment. In a recent study, Wang et al. [14] applied ultrasound treatment to chickpea protein isolate and investigated the changes in interfacial, physicochemical, and gelling properties. The authors observed increase in solubility, foaming capacity, emulsifying activity, and gel strength as a result of the ultrasonication treatment. Improved gelling properties of the ultrasonicated chickpea protein were attributed to the changes observed in hydrophobicity and particle size as a result of the ultrasonication treatment.

#### **2.3 High pressure**

Potential of high pressure application to modify the structure and functionality of legume proteins has been recently investigated by various researchers. Chao et al. [16]

**91**

**2.4 Other techniques**

*Modification of Legume Proteins for Improved Functionality*

lentil protein was observed to form weak gel-like structures.

Other physical techniques including cold plasma and controlled shear have also been applied to legume proteins for modification of their structural and functional properties. In a recent study, Mehr and Koocheki [20] applied atmospheric cold

applied high hydrostatic pressure (200–600 MPa) to yellow field pea protein isolate and determined its effects on physicochemical characteristics and functional properties of the protein. Gel electrophoresis patterns were observed to indicate aggregation due to increased interaction between protein molecules after high pressure application. High pressure treatment was reported to induce changes in the tertiary structure identified by fluorescence spectroscopy. The extent of structural changes depended on the level of pressure applied. The highest level of denaturation and unfolding of the molecule was observed at 600 MPa pressure. Despite those changes, protein solubility was found to remain unchanged. At the same time, emulsifying properties of yellow field pea protein were reported to be improved after the high hydrostatic pressure treatment. Mean droplet size of emulsions stabilized by high pressuretreated yellow field pea protein was reported to be significantly smaller compared to that of emulsions stabilized by untreated protein. Formation of smaller oil droplets was attributed to increased interactions between oil droplets and unraveled hydrophobic groups of high pressure-treated protein. Effect of high pressure treatment on emulsion stability was found to change depending on protein concentration and pH. Specifically, at relatively lower protein concentrations, pH 3.0 and 5.0, emulsion stability was reported to decrease as a result of high pressure treatment which was attributed to protein aggregation decreasing the ability of the protein to form strong interfacial membranes. On the other hand, at pH 7.0, stability of pressure-treated protein-stabilized emulsions was reported to be higher compared to that of untreated protein due to increased surface charge. Foaming properties of yellow field pea protein isolate were found to be improved with high pressure treatment at all protein concentrations and pH values studied. Sim et al. [22] compared the efficacy of high pressure treatment (250–550 MPa) with heat treatment (95 °C, 15 min) with respect to gel forming properties of pea protein concentrate. The authors indicated that high pressure treatment changed the tertiary and quaternary structure of pea protein which resulted in protein denaturation, aggregation, and network formation. The extent of these changes increased with increasing pressure level. Pea protein subjected to heat treatment formed a gel at a lower concentration (12 g/100 g) compared to the pea protein subjected to high pressure at 250 MPa for 15 min (16 g/100 g). Moreover, heat-treated pea protein was reported to show greater gel strength compared to high pressure-treated protein. Ahmed et al. [15] applied high pressure treatment (200–600 MPa) to kidney bean protein isolate and determined the changes in protein structure and functionality. Investigation of thermal properties of kidney bean protein revealed that high pressure treatment resulted in unraveling of protein structure. Protein unfolding and changes in tertiary structure due to high pressure application were also verified by the shifts observed in Fourier transfer infrared (FTIR) spectroscopy profile. The magnitude of those changes increased with increasing pressure level. High pressure-treated pea protein was reported to show higher thermal denaturation temperature, improved water holding capacity, foaming, and emulsifying properties when the treatment was applied at above 600 MPa. In a recent study, Saricaoglu [17] applied high pressure (25–150 MPa) homogenization for improving functional properties of lentil protein isolate. The author reported that an increase in pressure from 50 to 150 MPa resulted in unraveling of the protein structure and improved solubility, emulsifying, and foaming properties of lentil protein. However, high pressure-treated

*DOI: http://dx.doi.org/10.5772/intechopen.96274*

#### *Modification of Legume Proteins for Improved Functionality DOI: http://dx.doi.org/10.5772/intechopen.96274*

*Grain and Seed Proteins Functionality*

**2.2 Ultrasonication**

soy protein concentrate as a reference. Heat treatment was reported to induce partial denaturation, which in turn increased hydrophobicity and resulted in decreased protein solubility. On the contrary, water holding capacity of faba bean protein was

Ultrasonication has been recently investigated as a novel tool for modifying the structure and improving functionality of legume proteins. Gharibzahedi and Smith [21] recently reviewed the current applications and challenges of this technology for legume proteins. Jiang et al. [10] applied low-frequency (20 kHz) ultrasonication to black bean protein isolate. Primary structure of black bean protein was reported to remain unchanged after the ultrasonication treatment indicated by the electrophoresis profile. On the other hand, secondary structure of black bean protein was observed to change after ultrasonication where the proportion of α-helix decreased and β-sheet increased. Ultrasonication-related alterations in tertiary structure were monitored by the changes in fluorescence spectra. Average particle size of ultrasound-treated black bean protein was reported to be larger compared to that of native protein which was attributed to formation of aggregates. Net surface charge of ultrasonicated black bean protein was observed to change depending on the power applied. Surface hydrophobicity of black bean protein was also reported to increase after the ultrasonication treatment. Similar to heat treatment applications, ultrasonication is indicated to result in unfolding of the protein molecule up to a certain degree and increase hydrophobicity. However, surface hydrophobicity was reported to decrease with a further increase in the ultrasonication power due to aggregation of protein molecules which in turn enclose the hydrophobic sections of the protein. Ultrasonication treatment was reported to result in an increase in solubility of black bean protein, directly related to ultrasonication power and treatment time which was based on increased interaction between protein molecules and water. The authors concluded that the modifications on protein functionality could be optimized based on the ultrasonication conditions. Martinez-Velasco et al. [12] studied the effect of ultrasound treatment on surface characteristics and foaming properties of faba bean protein isolate. The authors optimized the parameters of ultrasonication treatment and reported that an amplitude of 73% and duration of 17 min resulted in lower interfacial tension, higher solubility, higher adsorption at the interface, smaller bubble diameter, and higher foam stability. In a similar study, Xiong et al. [13] investigated the effect of ultrasound treatment on structural characteristics and foaming ability and stability of pea protein isolate. Ultrasonication was reported to induce unfolding of the protein molecule up to a certain degree and hence increased hydrophobicity. These changes combined with decreased particle size were reported to result in decreased surface tension, improved foaming ability and stability after the treatment. In a recent study, Wang et al. [14] applied ultrasound treatment to chickpea protein isolate and investigated the changes in interfacial, physicochemical, and gelling properties. The authors observed increase in solubility, foaming capacity, emulsifying activity, and gel strength as a result of the ultrasonication treatment. Improved gelling properties of the ultrasonicated chickpea protein were attributed to the changes observed in hydrophobicity and

observed to increase and became comparable to soy protein.

particle size as a result of the ultrasonication treatment.

Potential of high pressure application to modify the structure and functionality of legume proteins has been recently investigated by various researchers. Chao et al. [16]

**90**

**2.3 High pressure**

applied high hydrostatic pressure (200–600 MPa) to yellow field pea protein isolate and determined its effects on physicochemical characteristics and functional properties of the protein. Gel electrophoresis patterns were observed to indicate aggregation due to increased interaction between protein molecules after high pressure application. High pressure treatment was reported to induce changes in the tertiary structure identified by fluorescence spectroscopy. The extent of structural changes depended on the level of pressure applied. The highest level of denaturation and unfolding of the molecule was observed at 600 MPa pressure. Despite those changes, protein solubility was found to remain unchanged. At the same time, emulsifying properties of yellow field pea protein were reported to be improved after the high hydrostatic pressure treatment. Mean droplet size of emulsions stabilized by high pressuretreated yellow field pea protein was reported to be significantly smaller compared to that of emulsions stabilized by untreated protein. Formation of smaller oil droplets was attributed to increased interactions between oil droplets and unraveled hydrophobic groups of high pressure-treated protein. Effect of high pressure treatment on emulsion stability was found to change depending on protein concentration and pH. Specifically, at relatively lower protein concentrations, pH 3.0 and 5.0, emulsion stability was reported to decrease as a result of high pressure treatment which was attributed to protein aggregation decreasing the ability of the protein to form strong interfacial membranes. On the other hand, at pH 7.0, stability of pressure-treated protein-stabilized emulsions was reported to be higher compared to that of untreated protein due to increased surface charge. Foaming properties of yellow field pea protein isolate were found to be improved with high pressure treatment at all protein concentrations and pH values studied. Sim et al. [22] compared the efficacy of high pressure treatment (250–550 MPa) with heat treatment (95 °C, 15 min) with respect to gel forming properties of pea protein concentrate. The authors indicated that high pressure treatment changed the tertiary and quaternary structure of pea protein which resulted in protein denaturation, aggregation, and network formation. The extent of these changes increased with increasing pressure level. Pea protein subjected to heat treatment formed a gel at a lower concentration (12 g/100 g) compared to the pea protein subjected to high pressure at 250 MPa for 15 min (16 g/100 g). Moreover, heat-treated pea protein was reported to show greater gel strength compared to high pressure-treated protein. Ahmed et al. [15] applied high pressure treatment (200–600 MPa) to kidney bean protein isolate and determined the changes in protein structure and functionality. Investigation of thermal properties of kidney bean protein revealed that high pressure treatment resulted in unraveling of protein structure. Protein unfolding and changes in tertiary structure due to high pressure application were also verified by the shifts observed in Fourier transfer infrared (FTIR) spectroscopy profile. The magnitude of those changes increased with increasing pressure level. High pressure-treated pea protein was reported to show higher thermal denaturation temperature, improved water holding capacity, foaming, and emulsifying properties when the treatment was applied at above 600 MPa. In a recent study, Saricaoglu [17] applied high pressure (25–150 MPa) homogenization for improving functional properties of lentil protein isolate. The author reported that an increase in pressure from 50 to 150 MPa resulted in unraveling of the protein structure and improved solubility, emulsifying, and foaming properties of lentil protein. However, high pressure-treated lentil protein was observed to form weak gel-like structures.

#### **2.4 Other techniques**

Other physical techniques including cold plasma and controlled shear have also been applied to legume proteins for modification of their structural and functional properties. In a recent study, Mehr and Koocheki [20] applied atmospheric cold

plasma to Grass pea protein isolate and monitored the changes in protein structure and emulsifying properties. The authors reported that the extent of changes in protein structure depended on the level of voltage applied and duration of application. An initial increase in voltage and treatment time was indicated to result in an increase in the content of carbonyl groups which was determined to monitor the interaction between the amino acid side chains and reactive chemical species of plasma. On the other hand, the amount of free sulfhydryl groups was observed to decrease with the plasma treatment which indicated the structural modification of Grass pea protein. At the optimized treatment conditions globulins were dissociated, increasing the absorption rate of protein into the oil–water interface. Cold plasma treatment applied was indicated to alter the protein structure on secondary and tertiary levels. Ability of the plasma treated protein to decrease the interfacial tension was reported to be affected by the treatment conditions in such a way that lower voltage values resulted in lower interfacial tension. On the contrary, emulsion stabilized with the protein treated with higher voltage was observed to have smaller oil droplets and show higher stability against creaming. Bussler et al. [19] compared the efficiency of thermal treatment with cold plasma treatment for improvement of functionality of grain pea proteins. Plasma treatment was reported to induce changes in the tertiary or quaternary structure which in turn resulted in increased solubility, water and fat binding capacities. It was concluded that the plasma treatment applied could potentially be used as an alternative non-thermal method for improving protein functionality. In a recent study, Bogahawaththa et al. [18] applied controlled shear (100 or 1500 s−1) to pea protein isolate and determined the changes in protein solubility and heat stability during heating at 90 °C for 5 min. It was reported that pea protein subjected to shear at 1500 s−1 showed significantly higher solubility and heat stability compared to the protein subjected to shear at 100 s−1 which was based on the changes in secondary structure and formation of soluble hydrophobic aggregates at high shear.
