**4.1 Enzymatic hydrolysis**

Application of enzymatic hydrolysis for modification of structural properties and improving functionality of legume proteins has been widely studied recently. Klost and Drusch [42] applied enzymatic hydrolysis for modification of solubility and interfacial properties of pea protein. Hydrolysis with trypsin up to 4% degree was reported to increase solubility from 30% to 60% at pH 4.0–6.0 due to the increased amount of terminal COO− and NH3+ groups. On the other hand, net surface charge and solubility were reported to decrease at pH 3.0 and 7.0 due to exposure of previously buried hydrophobic moieties leading to aggregation. Pea protein hydrolysates were observed to form emulsions with wider oil droplet size distributions which were not stable against creaming as a result of decreased net charge and lack of repulsion. Enzymatic hydrolysis was reported to positively affect the strength of interfacial films formed. In a recent study 11 different proteolytic enzymes were used for hydrolyzing pea protein isolate for improved functional and sensory properties [43]. Solubility of most of the hydrolysates at pH 4.5 was reported to be improved with decreasing peptide size and release of hydrophilic amino acids with hydrolysis. Among the hydrolysates studied, Esperase hydrolysates were reported to show the highest protein solubility whereas the highest foaming and emulsifying capacities were observed in Trypsin hydrolysates.

**97**

*Modification of Legume Proteins for Improved Functionality*

All hydrolysates were observed to have improved foaming capacity and stability compared to the native protein which was based on decreased peptide size and modification of surface hydrophobicity with hydrolysis. Among the sensory attributes evaluated, only bitterness was reported to change significantly after hydrolysis. Some of the hydrolysates were reported to have lower bitterness scores than that of the native protein. However, increased degree of hydrolysis resulted in increased bitterness. In another recent study, four different proteases were for used for investigation of combined effects of enzymatic hydrolysis and ultrafiltration treatment on faba bean protein functionality [41]. It was reported that hydrolysis with pepsin resulted in significant increases in solubility, foaming and oil holding capacities of faba bean protein. Fractionation with ultrafiltration was observed to allow for further improvements in foaming, oil holding and emulsifying capacities

Enzymatic cross-linking with transglutaminase is another biological approach used for improving protein functionality. Tang et al. [44] studied the effect of cross-linking on kidney bean protein isolate. The authors observed unfolding of the vicilin units and formation of higher molecular weight oligomers. Thermal stability of vicilin-rich kidney bean protein was reported to increase after the cross-linking treatment. However, the authors observed gradual decrease in solubility and emulsifying properties with increasing incubation time. Moreno et al. [46] compared the efficiency of cross-linking and thermal processing in gelation of pea protein. The main aim of the cross-linking treatment was to obtain improved gelling properties for various meat and seafood applications. It was reported that cross-linking resulted in polymerization of vicilin and legumin subunits forming new intermolecular protein complexes indicated by alterations in protein structure. Transglutaminase treatment was reported to improve conformational stability and flexibility of the gel network. Cross-linking with transglutaminase is generally applied to legume proteins for improving gelling properties. However, Glusac et al. [47] studied its effects on characteristics of emulsions stabilized by chickpea protein. Cross-linking treatment was reported to increase mean droplet size of the emulsion compared to the native protein and formed a gel-like structure. Emulsions were observed over a month and cross-linked chickpea protein-stabilized emulsions were indicated to show higher stability against phase separation compared to the

Although fermentation is a traditional process, its application for improving functional, nutritional and sensory properties of legume proteins and protein-rich flours has gained interest in the recent years. Cabuk et al. [48] investigated the effect of fermentation on properties of pea protein-enriched flour (~40 g/100 g protein). Fermentation was conducted for 11 h where the degree of hydrolysis was reported to reach 13.5%. Net surface charge at pH 4.0 was reported to increase at 1 h of fermentation and then decreased. Net surface charge at pH 7.0 and surface hydrophobicity at pH 4.0 were reported to increase. Solubility at pH 7.0 was reported to decrease from ~43% to 36% after 11 h of fermentation. The highest foaming capacity was reported for pea protein-enriched flour fermented for 5 h at pH 4.0. Emulsifying activity of pea protein-enriched flour was observed to decrease after 5 h whereas emulsion stability increased. It was concluded that functionality of fermented protein-enriched flours can be optimized depending on fermentation

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

of the peptides obtained.

emulsions stabilized by native protein.

**4.3 Fermentation**

**4.2 Cross-linking**

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

All hydrolysates were observed to have improved foaming capacity and stability compared to the native protein which was based on decreased peptide size and modification of surface hydrophobicity with hydrolysis. Among the sensory attributes evaluated, only bitterness was reported to change significantly after hydrolysis. Some of the hydrolysates were reported to have lower bitterness scores than that of the native protein. However, increased degree of hydrolysis resulted in increased bitterness. In another recent study, four different proteases were for used for investigation of combined effects of enzymatic hydrolysis and ultrafiltration treatment on faba bean protein functionality [41]. It was reported that hydrolysis with pepsin resulted in significant increases in solubility, foaming and oil holding capacities of faba bean protein. Fractionation with ultrafiltration was observed to allow for further improvements in foaming, oil holding and emulsifying capacities of the peptides obtained.

### **4.2 Cross-linking**

*Grain and Seed Proteins Functionality*

*Cross-linking with transglutaminase*

**Legume protein studied**

Kidney bean protein

Chickpea protein

enriched pea flour

Proteinenriched pea flour

*Fermentation* Protein-

**Treatment conditions**

Pea protein 10.5% protein, 0.3 M

Pea protein 17–23% protein, 5–7 U

5 U enzyme/g protein, 37 °C, 0–240 min

NaCl, 10 U enzyme, 40 °C, 30 min

enzyme/g protein, 40–90 °C, 30–60 min

200 U enzyme/g of protein, 37 °C, 60 min

*Lactobacillus plantarum* NRRL B-4496, 7 log CFU/g flour, 32 °C, 11 h

*Aspergillus niger* NRRL 334, *Aspergillus oryzae* NRRL 5590, 7 log CFU/g flour, 40 °C, 6 h

eight different microorganisms, 7 log CFU/g protein, 24 h

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

Lupin protein Fermentation with

**4.1 Enzymatic hydrolysis**

*DH: degree of hydrolysis.*

to the increased amount of terminal COO−

Application of enzymatic hydrolysis for modification of structural properties and improving functionality of legume proteins has been widely studied recently. Klost and Drusch [42] applied enzymatic hydrolysis for modification of solubility and interfacial properties of pea protein. Hydrolysis with trypsin up to 4% degree was reported to increase solubility from 30% to 60% at pH 4.0–6.0 due

net surface charge and solubility were reported to decrease at pH 3.0 and 7.0 due to exposure of previously buried hydrophobic moieties leading to aggregation. Pea protein hydrolysates were observed to form emulsions with wider oil droplet size distributions which were not stable against creaming as a result of decreased net charge and lack of repulsion. Enzymatic hydrolysis was reported to positively affect the strength of interfacial films formed. In a recent study 11 different proteolytic enzymes were used for hydrolyzing pea protein isolate for improved functional and sensory properties [43]. Solubility of most of the hydrolysates at pH 4.5 was reported to be improved with decreasing peptide size and release of hydrophilic amino acids with hydrolysis. Among the hydrolysates studied, Esperase hydrolysates were reported to show the highest protein solubility whereas the highest foaming and emulsifying capacities were observed in Trypsin hydrolysates.

and NH3+

groups. On the other hand,

**Effects observed on protein** 

Higher gel strength and more elastic gel formation

Higher gel strength and flexibility of the gel network

Formation of a gel-like emulsion, improved emulsion

Improved foaming capacity, emulsion stability, and oilholding capacity. Decreased foam stability and emulsifying

Decreased solubility and foaming properties. No significant changes in emulsifying properties. Improved water and oil binding properties

No significant changes in foaming activity and emulsifying capacity. Decreased solubility at pH 7.0 **Ref.**

[44]

[45]

[46]

[47]

[48]

[49]

[50]

**functionality**

stability

stability

activity

Decreased solubility, emulsifying activity and

**96**

*1*

**Table 3.**

Enzymatic cross-linking with transglutaminase is another biological approach used for improving protein functionality. Tang et al. [44] studied the effect of cross-linking on kidney bean protein isolate. The authors observed unfolding of the vicilin units and formation of higher molecular weight oligomers. Thermal stability of vicilin-rich kidney bean protein was reported to increase after the cross-linking treatment. However, the authors observed gradual decrease in solubility and emulsifying properties with increasing incubation time. Moreno et al. [46] compared the efficiency of cross-linking and thermal processing in gelation of pea protein. The main aim of the cross-linking treatment was to obtain improved gelling properties for various meat and seafood applications. It was reported that cross-linking resulted in polymerization of vicilin and legumin subunits forming new intermolecular protein complexes indicated by alterations in protein structure. Transglutaminase treatment was reported to improve conformational stability and flexibility of the gel network. Cross-linking with transglutaminase is generally applied to legume proteins for improving gelling properties. However, Glusac et al. [47] studied its effects on characteristics of emulsions stabilized by chickpea protein. Cross-linking treatment was reported to increase mean droplet size of the emulsion compared to the native protein and formed a gel-like structure. Emulsions were observed over a month and cross-linked chickpea protein-stabilized emulsions were indicated to show higher stability against phase separation compared to the emulsions stabilized by native protein.

### **4.3 Fermentation**

Although fermentation is a traditional process, its application for improving functional, nutritional and sensory properties of legume proteins and protein-rich flours has gained interest in the recent years. Cabuk et al. [48] investigated the effect of fermentation on properties of pea protein-enriched flour (~40 g/100 g protein). Fermentation was conducted for 11 h where the degree of hydrolysis was reported to reach 13.5%. Net surface charge at pH 4.0 was reported to increase at 1 h of fermentation and then decreased. Net surface charge at pH 7.0 and surface hydrophobicity at pH 4.0 were reported to increase. Solubility at pH 7.0 was reported to decrease from ~43% to 36% after 11 h of fermentation. The highest foaming capacity was reported for pea protein-enriched flour fermented for 5 h at pH 4.0. Emulsifying activity of pea protein-enriched flour was observed to decrease after 5 h whereas emulsion stability increased. It was concluded that functionality of fermented protein-enriched flours can be optimized depending on fermentation

conditions. In a follow-up study, fermentation was conducted with two different *Aspergillus* strains for 6 h and the degree of hydrolysis was reported to reach 10–11% [49]. The authors observed an increase in surface charge with increasing fermentation time whereas surface hydrophobicity was reported to decrease. Fermentation was indicated to result in negative effects on solubility and foaming properties where emulsifying properties were reported to remain unchanged. Decrease in solubility was attributed to increased protein–protein interactions and aggregation. On the other hand, water and oil binding properties were observed to be improved after fermentation. In another recent study, Schlegel et al. [50] used eight different microorganisms for fermenting lupin protein isolate. Fermentation was reported to result in no significant difference in solubility of lupin protein at pH 4.0; however, solubility at pH 7.0 decreased from ~64% to <42%. Emulsifying capacity of lupin protein was not affected by fermentation. On the other hand, foaming activity of fermented lupin protein was reported to be higher than that of native protein. Among the microorganisms studied, only two of them resulted in improved emulsifying capacity in the fermented lupin protein compared to the native protein. All microorganisms used for fermentation were found to decrease the bitterness score of lupin protein. *Lactobacillus brevis* was reported to be the most effective microorganism for improving the sensory profile as it was noted to decrease the intensity of undesired flavor markers.
