**3. Chemical modifications**

Structure and functionality of legume proteins are also modified by application of a variety of chemical methods including attachment of various molecules to protein structure via different pathways. Some recent reports on chemical methods applied to legume proteins for improved functionality are summarized in **Table 2**.

### **3.1 Attachment of low molecular weight molecules**

One of the most commonly applied methods for improving protein functionality is attachment of low molecular weight molecules to protein structure via different chemical pathways [3]. Yin et al. [33] reported that acetylation and succinylation treatments induced changes in the secondary and/or tertiary structures of kidney bean protein isolate and resulted in a decrease in isoelectric point and net surface charge at pH 7.0. Succinylation was reported to decrease surface hydrophobicity of kidney bean protein while acetylation increased hydrophobicity. Charoensuk et al. [25] indicated that the effect of succinylation treatment on mung bean protein isolate depended on the ratio of succinic anhydride to protein. A decrease in isoelectric point and net surface charge at pH 7.0 was observed as a result of succinylation whereas the amount of free sulfhydryl groups was not affected. Improved emulsifying activity after succinylation treatment was attributed to increased flexibility of succinylated protein. Shah et al. [27] hydrophobically modified pea

**93**

**Table 2.**

*Modification of Legume Proteins for Improved Functionality*

**Treatment conditions**

Acetylation (6–49%)

(58–90%)

Succinylation (9–40%)

modifications by *N*-substitutions

Maillard reaction with dextran

with gum Arabic

Maillard reaction with maltodextrin

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

Ultrasound pre-treatment and Maillard reaction with glucose

Ultrasound pre-treatment and Maillard reaction with glucose

Pea protein Maillard reaction

Lentil protein Succinylation

Pea protein Hydrophobical

**Effects observed on protein** 

Higher solubility at pH > 8.0. Lower solubility at pH 2.0–7.0. Improved water and oil absorption capacities, higher emulsion capacity, lower emulsion stability

Higher solubility at pH > 4.0. Lower solubility at lower pH. Increased water absorption capacity, viscosity, emulsifying activity and stability. Decreased oil absorption capacity, foaming capacity and stability.

Improved solubility and emulsifying activity

Improved solubility, foaming capacity, stability, emulsion stability, and water holding

Improved solubility, emulsifying

Improved solubility, emulsifying

shorter gelling time

activity and stability

activity and stability

Improved solubility and emulsifying properties

Improved solubility and emulsifying properties

capacity

Improved solubility, decreased gel strength and water holding

PEGylation Higher gelling strength and

capacity

**Ref.**

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

**functionality**

protein by *N*-substitutions using different reactants. Hydrophobical modifications applied induced changes in the secondary structure of pea protein which were observed with FTIR spectroscopy. Alterations in secondary structure were attributed to increased negative charge. Changes in thermal profile were based on partial denaturation of protein and aggregation. Solubility and water holding capacity of modified pea protein were observed to increase due to increased negative charge. Emulsion stability index was reported to increase as a result of increased charge, solubility and addition of hydrophobic groups to the protein molecule. Hydrophobically modified pea protein with improved functionality was indicated

to have a potential to be used as egg replacers in cake formulations.

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

*Attachment of low molecular weight molecules*

*Attachment of high molecular weight molecules* **Legume protein studied**

Chickpea protein

Mung bean protein

Black kidney bean protein

Mung bean protein

Mung bean protein

Black bean protein isolate

Soya bean protein


*Grain and Seed Proteins Functionality*

soluble hydrophobic aggregates at high shear.

**3.1 Attachment of low molecular weight molecules**

**3. Chemical modifications**

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

Structure and functionality of legume proteins are also modified by application

One of the most commonly applied methods for improving protein functionality is attachment of low molecular weight molecules to protein structure via different chemical pathways [3]. Yin et al. [33] reported that acetylation and succinylation treatments induced changes in the secondary and/or tertiary structures of kidney bean protein isolate and resulted in a decrease in isoelectric point and net surface charge at pH 7.0. Succinylation was reported to decrease surface hydrophobicity of kidney bean protein while acetylation increased hydrophobicity. Charoensuk et al. [25] indicated that the effect of succinylation treatment on mung bean

protein isolate depended on the ratio of succinic anhydride to protein. A decrease in isoelectric point and net surface charge at pH 7.0 was observed as a result of succinylation whereas the amount of free sulfhydryl groups was not affected. Improved emulsifying activity after succinylation treatment was attributed to increased flexibility of succinylated protein. Shah et al. [27] hydrophobically modified pea

of a variety of chemical methods including attachment of various molecules to protein structure via different pathways. Some recent reports on chemical methods applied to legume proteins for improved functionality are summarized in **Table 2**.

**92**

#### **Table 2.**

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

protein by *N*-substitutions using different reactants. Hydrophobical modifications applied induced changes in the secondary structure of pea protein which were observed with FTIR spectroscopy. Alterations in secondary structure were attributed to increased negative charge. Changes in thermal profile were based on partial denaturation of protein and aggregation. Solubility and water holding capacity of modified pea protein were observed to increase due to increased negative charge. Emulsion stability index was reported to increase as a result of increased charge, solubility and addition of hydrophobic groups to the protein molecule. Hydrophobically modified pea protein with improved functionality was indicated to have a potential to be used as egg replacers in cake formulations.

#### **3.2 Maillard reaction**

Forming protein-polysaccharide based conjugates through glycation or Maillard reaction has been indicated as a promising method for modification of protein functionality [34]. Zhou et al. [29] formed conjugates between mung bean protein isolate and dextran at 80–90 °C for changing durations of 1–6 h. Electrophoretic profile of mung bean protein showed that both vicilin and legumin subunits participated in the Maillard reaction. Conjugation was indicated to alter the secondary structure, decreasing the α-helix content due to heat-induced unfolding of the molecule and attachment of dextran. Fluorescence spectra were used as an indicator to monitor the changes in tertiary structure. The authors proposed that conjugation induced unfolding of mung bean protein up to a certain extent and increased the flexibility of protein structure. Solubility of the conjugates formed at 2–3 h was reported to be increased compared to mung bean protein due to the increased number of hydrophilic moieties introduced by the grafted dextran. However, a slight decrease in solubility was observed with increasing graft time due to formation of insoluble protein aggregates. Emulsifying activity and stability indices of conjugates followed a similar trend and first increased compared to the native mung bean protein and then decreased with increasing graft time. Initial increase in emulsifying activity and stability indices was attributed to improved solubility and flexibility due to conjugation. The possible mechanism behind impaired emulsifying properties observed with increasing graft time was explained by heat induced protein aggregation and reduction of interfacial activity of mung bean protein with increased attachment of dextran. The authors reported that conjugates formed at 80 °C with lower glycosylation degrees and browning showed better functionality compared to the conjugates formed at 90 °C.

Wang et al. [28] investigated the effect of ultrasound treatment on conjugation of mung bean protein isolate and glucose. Similar to the study of Zhou et al. [29], the authors reported that Maillard reaction resulted in changes in the secondary structure of mung bean protein. Furthermore, ultrasound-treated conjugates were reported to have a less compact tertiary structure compared to the heat-treated conjugates and the native protein. Application of ultrasound treatment in Maillard reaction was indicated to form conjugates with a higher degree of glycosylation and improved solubility. Higher solubility observed in ultrasound-treated conjugates was attributed to two factors: breaking of insoluble aggregates and addition of more hydrophilic groups due to enhanced conjugation with the ultrasonication treatment. Similarly, ultrasound-treated conjugates showed better emulsifying activity and stability compared to heat-treated conjugates due to dispersion of aggregates and improved mobility of the protein molecule. Jin et al. [30] also investigated the effect of ultrasound treatment on conjugation of black bean protein isolate and glucose via Maillard reaction. Ultrasound treatment was reported the increase the reaction rate indicated with a higher degree of glycation at a shorter time. FTIR profile of the samples indicated that ultrasound-treated conjugates lost more ordered secondary structure (α-helix and β-sheet content) compared to the heat-treated conjugates. Alterations in protein structure resulting in increased unordered structure content were reported to improve the flexibility of the molecule and hence, emulsifying properties. Changes in fluorescence spectra indicated that the Maillard reaction resulted in alterations in the tertiary structure and ultrasonication treatment further increased the extent of these changes. Moreover, ultrasound-treated conjugates were reported to show higher surface hydrophobicity, improved solubility, emulsifying activity and stability indices compared to the heat-treated conjugates and the native protein. In another recent study, Zha et al. [31] formed conjugates between pea protein and gum Arabic and monitored the changes in functional properties

**95**

*Modification of Legume Proteins for Improved Functionality*

and flavor profile of pea protein. Pea protein-gum Arabic conjugates were reported

Functional properties of legume proteins can be improved via various biological methods including enzymatic hydrolysis, cross linking and fermentation. A brief summary of the findings of recent studies focusing on biological modification of

**Effects observed on protein** 

Decreased interfacial tension. Decreased emulsifying activity

Improved solubility. Decreased interfacial tension. Decreased emulsifying activity and

Improved solubility, oil absorption and foaming capacities. Decreased emulsifying activity

**Ref.**

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

**functionality**

and stability

stability

properties

Improved solubility. Hydrolysates with lower DH showed better emulsion

Improved solubility and oilholding capacity. Decreased emulsifying activity

Alcalase hydrolysates showed higher emulsion stability

Improved solubility, foaming capacity, oil holding capacity

Improved solubility. Samples at 2% DH showed better emulsion properties

Improved solubility. Hydrolysates obtained with trypsin showed the highest foaming and emulsifying

capacities

**Treatment conditions**

trypsin (4–20% DH1

Hydrolysis with Flavourzyme (1–10%

Hydrolysis with Alcalase (4–15% DH)

Hydrolysis with Alcalase and Flavourzyme (12–50% DH)

Hydrolysis with pancreatin (5–34%

Hydrolysis with pepsin and Alcalase (24–28% DH)

Hydrolysis with different proteases (2–16% DH)

trypsin (2–4% DH)

different proteases (2–10% DH)

DH)

Pea protein Hydrolysis with

Pea protein Hydrolysis with

DH)

)

to show improved solubility compared to the native pea protein. Incubation time was indicated as an important factor affecting the solubility of conjugates. Emulsion forming and stabilizing ability was determined by measuring droplet size. Emulsions stabilized by pea protein-gum Arabic conjugates were reported to have smaller droplets compared to emulsions stabilized by pea protein. Conjugatestabilized emulsions showed the highest stability against environmental factors including temperature, pH and ionic strength when incubation time was kept at 3 days. The undesired (beany or grassy) flavor markers in pea protein were reported to decrease significantly after 1 day of incubation. Increasing the incubation time

was reported to improve the flavor profile of the conjugates further.

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

**4. Biological modifications**

legume proteins is presented in **Table 3**.

**Legume protein studied**

Chickpea protein

Chickpea protein

Chickpea protein

Bambara bean protein

Black bean protein

Faba bean protein

*Hydrolysis* Lentil protein Hydrolysis with

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

and flavor profile of pea protein. Pea protein-gum Arabic conjugates were reported to show improved solubility compared to the native pea protein. Incubation time was indicated as an important factor affecting the solubility of conjugates. Emulsion forming and stabilizing ability was determined by measuring droplet size. Emulsions stabilized by pea protein-gum Arabic conjugates were reported to have smaller droplets compared to emulsions stabilized by pea protein. Conjugatestabilized emulsions showed the highest stability against environmental factors including temperature, pH and ionic strength when incubation time was kept at 3 days. The undesired (beany or grassy) flavor markers in pea protein were reported to decrease significantly after 1 day of incubation. Increasing the incubation time was reported to improve the flavor profile of the conjugates further.
