**4. Seed storage proteins**

Apart from protein comprising a major part of the seed, the other constituents include 1.5–2% fat, minerals, vitamins, polyphenols, oxalates, saponins and phytic acid [32–34]. Starch and dietary fibre account for 60 percent of carbohydrate content and rest include non-starch part of carbohydrates comprising sucrose, cellulose, and oligosaccharides (**Figure 1**) [34, 36]. Protein and the starch fraction of seed show high variations, whereas the other components remain comparatively constant [18]. Pea proteins are classified based on Osborne fractionation [37] into two different categories, i.e., globulins soluble in salt and albumins soluble in water which collectively account for 80% of the pea seed protein. Young embryos after germination of seed obtain nitrogen from globulins and some of

**109**

**Figure 2.**

**Figure 1.**

*The average composition of pea seeds [35].*

*Pea Seed Proteins: A Nutritional and Nutraceutical Update*

the albumins which are also known as storage proteins. Globulins are further divided into two categories based on coefficients of sedimentation, i.e., legumin (11S fraction), vicilin and convicilin (7S fraction) as shown in **Figure 2**. The two classes differ from each other in structure and molecular weight. Legumin has a molecular mass ranging from 300 to 400 kDa and hexameric protein form. There are three polypeptide families of legumin, and sequence similarities differentiate them into various groups. The LegA polypeptide comprises of legA, legB, legA2, legC, and legE, LegJ polypeptide comprises leg J, legK, legL and legM whereas LegS is single member of family [39, 40]. The LegA and LegJ families comprise an apparent subdivision with the molecular mass of 65 kDa, and on the other hand, the apparent subdivision of LegS has \*) kDa molecular mass. Only a single peptide of legumin is imported to the endoplasmic reticulum and removed during translation. Ultimately, trimers of legumin peptide are formed and moved to the

*Size of subunits of pea proteins, including the cleavage site of (a) Legumin (b) Vicilin (c) Convicilin [38].*

*DOI: http://dx.doi.org/10.5772/intechopen.95323*

*Pea Seed Proteins: A Nutritional and Nutraceutical Update DOI: http://dx.doi.org/10.5772/intechopen.95323*

the albumins which are also known as storage proteins. Globulins are further divided into two categories based on coefficients of sedimentation, i.e., legumin (11S fraction), vicilin and convicilin (7S fraction) as shown in **Figure 2**. The two classes differ from each other in structure and molecular weight. Legumin has a molecular mass ranging from 300 to 400 kDa and hexameric protein form. There are three polypeptide families of legumin, and sequence similarities differentiate them into various groups. The LegA polypeptide comprises of legA, legB, legA2, legC, and legE, LegJ polypeptide comprises leg J, legK, legL and legM whereas LegS is single member of family [39, 40]. The LegA and LegJ families comprise an apparent subdivision with the molecular mass of 65 kDa, and on the other hand, the apparent subdivision of LegS has \*) kDa molecular mass. Only a single peptide of legumin is imported to the endoplasmic reticulum and removed during translation. Ultimately, trimers of legumin peptide are formed and moved to the

**Figure 1.**

*Grain and Seed Proteins Functionality*

**Amino acids cv.** 

*Non-essential amino acids*

*Essential amino acids*

**Table 2.**

*ucero*

**cv.**  *ramrod*

**cv.**  *agra*

Asp 10.39 10.08 9.98 10.87 10.55 10.69 10.58 Glu 17.09 16.49 15.43 15.07 16.19 15.96 16.16 Ser 4.89 4.80 4.77 4.23 4.16 4.05 4.25 Gly 8.16 8.26 7.85 4.11 4.0 3.98 3.92 Arg 5.76 4.93 4.12 9.36 8.60 9.68 8.32 Ala 5.17 6.35 5.75 4.19 3.88 3.83 3.79 Pro 3.62 3.64 3.52 3.77 3.57 3.64 3.63

His 1.07 1.13 1.03 2.22 2.16 2.18 2.16 Val 3.85 3.89 3.61 4.72 4.29 4.34 4.32 Met 0.65 0.70 0.70 5.0 1.08 1.05 0.99 Cys 0.30 0.37 0.39 2.01 2.03 1.9 1.67 Ile 3.51 2.64 2.52 4.23 3.86 3.77 3.9 Leu 5.72 6.51 7.01 7.11 6.45 6.33 6.55 Phe 5.07 5.06 4.59 4.87 4.59 4.33 4.56 Tyr 3.98 3.76 3.77 2.79 3.18 2.87 3.18 Lys 18.34 19.69 17.03 6.93 6.55 6.39 6.63 Thr 3.04 4.22 6.92 3.45 3.64 3.34 3.53 Trp 0.02 0.02 0.02 n.a. n.a. n.a. n.a.

*cv. terno*

*cv. Xantos* *cv.suit cv.*

*achat*

The muscle development and growth in human body is dependent on postprandial essential amino acid availability particularly leucine [30]. AA composition, essential AA content and anti-nutritional factors regulate the availability of essential AA [31]. Thus, variation in AA composition particularly in essential AA are desirable for improving AA profile of pea proteins. Natural variation among varieties for AA profile is present as depicted in **Table 2**. Wide crosses and mutants can be searched for more desirable AA profile of pea proteins. Furthermore, introgression approach can be deployed for improvement of existing germplasm using a natural variation.

Apart from protein comprising a major part of the seed, the other constituents include 1.5–2% fat, minerals, vitamins, polyphenols, oxalates, saponins and phytic acid [32–34]. Starch and dietary fibre account for 60 percent of carbohydrate content and rest include non-starch part of carbohydrates comprising sucrose, cellulose, and oligosaccharides (**Figure 1**) [34, 36]. Protein and the starch fraction of seed show high variations, whereas the other components remain comparatively constant [18]. Pea proteins are classified based on Osborne fractionation [37] into two different categories, i.e., globulins soluble in salt and albumins soluble in water which collectively account for 80% of the pea seed protein. Young embryos after germination of seed obtain nitrogen from globulins and some of

**108**

**4. Seed storage proteins**

*Amino acid profile of different pea cultivars [7, 25].*

*The average composition of pea seeds [35].*

**Figure 2.**

*Size of subunits of pea proteins, including the cleavage site of (a) Legumin (b) Vicilin (c) Convicilin [38].*

pre-vacuolar compartment [41]. Furthermore, the peptides are processed into basic and acidic polypeptides of 20 and 40 kDa with the help of vacuole processing enzyme and the two peptides are linked by disulphide bridge. A complete protein structure is assembled from trimers to hexamers. The molecular weight of vicilin is 47–50 kDa and it forms trimers of 150 kDa molecular mass [42]. Only some vicilins undergo cleavage at post translational level [43]. Vicilin contains two cleavage regions which are separately processed. Three fragments of 13 kDa (â), 20 kDa (R) and 16 kDa (γ) are obtained by cleavage in both regions. Two fragments of 25 kDa (â + γ) and 20 kDa (R) are obtained, if site A is cleaved and two fragments of 16 kDa (γ) and 36 kDa (R + â) are obtained if site B is cleaved [43, 44]. Noncovalent bonds held processed peptides [40, 44]. Glycosylation takes place near to C terminus of γ subunit of vicilin polypepetides as they are glycosylated [45]. Trimers of 210 kDa molecular mass are formed by convicilin protein having a molecular mass of 70 kDa. Heteromeric trimers comprising convicilin and vicilin polypeptides also occur [2, 46]. Elimination of single peptide is only reported post translational modification in the case of convicilin and glycosylation is absent [47]. Convicilin and vicilin show sequence similarity of amino acids at C terminus whereas N terminal being highly charged have different sequences between two polpeptides [48, 49]. Based on isoform, sequence similarity occurs between 122 and 166 amino acid residues. Physiochemical properties of globulins are different, owing to variations in molecular weight and structure.

The water-soluble albumin proteins have 5–80 kDa molecular mass and consist of enzymes and anti-nutritional factors such as amylase inhibitors, lectins and protease inhibitors [32]. Further two classes are obtained in albumins, i.e., albumin protein with two polypeptides having 25 kDa molecular weight and another with 6 kDa molecular weight [46]. Minor portions include prolamins which are soluble in diluted alcohol and glutenins, which are soluble in diluted acid [32]. The protein structure can be altered by external factors such as temperature, pH and salts during the extraction process resulting in different surface features and conformations.

The globulin protein classes, i.e., vicilin and legumin in different concentrations, can make good gels, whereas convicilin is known to hinder gel formation [50]. The food industry needs raw material with desirable composition of globulin in peas like high levels of vicilin and legumin or low levels of convicilin [40]. Further, gel making property not only depends on the composition of globulins but also matter of isoforms of isolate [51, 52]. The genetic variation in the composition of globulins and decreased levels of anti-nutrients in albumin fraction of pea proteins are desirable material for development of new varieties using breeding techniques. Natural variation is reported in case of the protein content of pea and its composition, which can be used in breeding programs [53–55]. The r locus in the pea genome is known to control the starch synthesis, which shows pleiotropism with protein content and its composition [56, 57]. With the advancement of techniques for elucidating in planta processing of proteins, there will be more clues for the controlled composition of proteins using genome editing techniques.

#### **5. Seed crude protein determination in pea**

#### **5.1 Protein isolate extraction methods**

Alkaline extraction/isoelectric precipitation (AE/IEP) – This method utilizes the high solubility of pea proteins in alkaline conditions and their minimal solubility at isoelectric point (pI) at pH between 4 to 5 [32]. This method is the most common for legume protein extraction, and it takes advantage of similar solubility characters

**111**

*Pea Seed Proteins: A Nutritional and Nutraceutical Update*

for legumin and vicilin [33, 58]. The de-fatted flour of legume (with or without seed coat) is dispersed in water and pH is adjusted to an alkaline range using NaOH, KOH or Ca(OH)2, and further left for 30–180 mins for maximizing protein solubility [32, 33]. Without de-fatting process, the protein-lipid interaction limits the solubility of protein leading to decrease in the isolated yield, and the temperature can be increased to 50–60°C to aid solubilization [59, 60]. The protein denaturation can be limited by avoiding the higher temperatures. The mixture is further centrifuged, and supernatant is collected, and isoelectric pH is adjusted using HCl or H2SO4. The precipitated protein is collected after centrifugation and washed, neutralized, and dried by drum or freeze drying [32, 33]. The isolate yield can be increased up to 80–94% by optimal processing conditions and the conditions used in a process can affect the purity, yield and functionality of the isolate [58]. Hoang [58] evaluated that the extraction pH and flour: water ratio were most critical factors. The flour: water ratios of 1:5 to 1:20 (w/v) was reported [32] but Hoang [58] stated that the increase in concentration gradient between the solid and liquid phase in low ratio slurry can increase solubility. Although high alkalinity increases the isolate solubility and yield of protein, but the pH 11 and above are basically associated with increase in swelling of starch, leading to contamination of starch in isolate product [58]. Alkaline Extraction is also responsible for the adverse chemical reactions like the conversion of serine and cysteine residues to lysinoalanine compounds (nephrotoxic), decreased proteins bioavailability, and racemization of amino acids [61, 62]. The processes employing high alkaline pH, high temperature is associated with high yield of isolate, but there is high susceptibility of denaturation of isolate [61, 63]. The particle size of flour and solubilizing agent used can also affect the yield of isolate. The optimum particle size for flour is 100–150 μm and it was reported that NaOH and KOH generate more yield in comparison to Ca(OH)2 [64]. Also, there was protein loss of 6.2% from discarded supernatant from this extraction method [58] and in place of IEP, ultrafiltration (UF) or diafiltration membranes with specific molecular weight cutoffs can be utilized for isolating proteins of interest from the supernatant [32]. The efficiency of extraction can be improved by alteration in the molecular weight cutoffs, membrane type, concentration, and volume of the filtrate and addition of diafiltration to UF techniques [65]. The albumin proteins can be recovered by controlling these factors and further result in enhancing yield of isolate and alteration in isolate functionality leading to reduction in effluent losses. The use of UF can provide milder conditions for extracted proteins, so that their functionality can be enhanced and it gives higher

Boye et al. [65] also confirmed that there were slightly higher protein levels in UF than the IEP process. Membrane filtration is also effective in reduction of antinutritional compounds in isolate [65]. Taherian et al. [67] conducted a study for functional properties of commercial and membrane-processed yellow pea protein isolates. The use of UF results in reduction of phytic acid upto 28–68% and possess improved functionality (e.g., solubility, rheology, foaming and emulsification) for commercially available isolates. The solubility of the commercial protein isolates was reported as ~20% vs. ~80% by using UF/diafiltration at pH 2.0. Fuhrmeister and Meuser [68] found the enhanced solubility, emulsifying, foaming and fatholding properties by UF recovery of proteins from wrinkled pea relative to heat,

SE has advantage of the salting-in and out phenomenon of proteins which is followed by desalting for lowering the ionic strength of protein environment [32, 69]. In this process, the flour is stirred in salt solution of ionic strength

*DOI: http://dx.doi.org/10.5772/intechopen.95323*

yields in comparison to IEP [66].

acid, and heat/acid precipitation.

**5.2 Salt extraction (SE) and micellization**

#### *Pea Seed Proteins: A Nutritional and Nutraceutical Update DOI: http://dx.doi.org/10.5772/intechopen.95323*

*Grain and Seed Proteins Functionality*

pre-vacuolar compartment [41]. Furthermore, the peptides are processed into basic and acidic polypeptides of 20 and 40 kDa with the help of vacuole processing enzyme and the two peptides are linked by disulphide bridge. A complete protein structure is assembled from trimers to hexamers. The molecular weight of vicilin is 47–50 kDa and it forms trimers of 150 kDa molecular mass [42]. Only some vicilins undergo cleavage at post translational level [43]. Vicilin contains two cleavage regions which are separately processed. Three fragments of 13 kDa (â), 20 kDa (R) and 16 kDa (γ) are obtained by cleavage in both regions. Two fragments of 25 kDa (â + γ) and 20 kDa (R) are obtained, if site A is cleaved and two fragments of 16 kDa (γ) and 36 kDa (R + â) are obtained if site B is cleaved [43, 44]. Noncovalent bonds held processed peptides [40, 44]. Glycosylation takes place near to C terminus of γ subunit of vicilin polypepetides as they are glycosylated [45]. Trimers of 210 kDa molecular mass are formed by convicilin protein having a molecular mass of 70 kDa. Heteromeric trimers comprising convicilin and vicilin polypeptides also occur [2, 46]. Elimination of single peptide is only reported post translational modification in the case of convicilin and glycosylation is absent [47]. Convicilin and vicilin show sequence similarity of amino acids at C terminus whereas N terminal being highly charged have different sequences between two polpeptides [48, 49]. Based on isoform, sequence similarity occurs between 122 and 166 amino acid residues. Physiochemical properties of globulins are different,

The water-soluble albumin proteins have 5–80 kDa molecular mass and consist of enzymes and anti-nutritional factors such as amylase inhibitors, lectins and protease inhibitors [32]. Further two classes are obtained in albumins, i.e., albumin protein with two polypeptides having 25 kDa molecular weight and another with 6 kDa molecular weight [46]. Minor portions include prolamins which are soluble in diluted alcohol and glutenins, which are soluble in diluted acid [32]. The protein structure can be altered by external factors such as temperature, pH and salts during the extraction process resulting in different surface features and conformations. The globulin protein classes, i.e., vicilin and legumin in different concentrations, can make good gels, whereas convicilin is known to hinder gel formation [50]. The food industry needs raw material with desirable composition of globulin in peas like high levels of vicilin and legumin or low levels of convicilin [40]. Further, gel making property not only depends on the composition of globulins but also matter of isoforms of isolate [51, 52]. The genetic variation in the composition of globulins and decreased levels of anti-nutrients in albumin fraction of pea proteins are desirable material for development of new varieties using breeding techniques. Natural variation is reported in case of the protein content of pea and its composition, which can be used in breeding programs [53–55]. The r locus in the pea genome is known to control the starch synthesis, which shows pleiotropism with protein content and its composition [56, 57]. With the advancement of techniques for elucidating in planta processing of proteins, there will be more clues for the controlled

Alkaline extraction/isoelectric precipitation (AE/IEP) – This method utilizes the high solubility of pea proteins in alkaline conditions and their minimal solubility at isoelectric point (pI) at pH between 4 to 5 [32]. This method is the most common for legume protein extraction, and it takes advantage of similar solubility characters

owing to variations in molecular weight and structure.

composition of proteins using genome editing techniques.

**5. Seed crude protein determination in pea**

**5.1 Protein isolate extraction methods**

**110**

for legumin and vicilin [33, 58]. The de-fatted flour of legume (with or without seed coat) is dispersed in water and pH is adjusted to an alkaline range using NaOH, KOH or Ca(OH)2, and further left for 30–180 mins for maximizing protein solubility [32, 33]. Without de-fatting process, the protein-lipid interaction limits the solubility of protein leading to decrease in the isolated yield, and the temperature can be increased to 50–60°C to aid solubilization [59, 60]. The protein denaturation can be limited by avoiding the higher temperatures. The mixture is further centrifuged, and supernatant is collected, and isoelectric pH is adjusted using HCl or H2SO4. The precipitated protein is collected after centrifugation and washed, neutralized, and dried by drum or freeze drying [32, 33]. The isolate yield can be increased up to 80–94% by optimal processing conditions and the conditions used in a process can affect the purity, yield and functionality of the isolate [58]. Hoang [58] evaluated that the extraction pH and flour: water ratio were most critical factors. The flour: water ratios of 1:5 to 1:20 (w/v) was reported [32] but Hoang [58] stated that the increase in concentration gradient between the solid and liquid phase in low ratio slurry can increase solubility. Although high alkalinity increases the isolate solubility and yield of protein, but the pH 11 and above are basically associated with increase in swelling of starch, leading to contamination of starch in isolate product [58]. Alkaline Extraction is also responsible for the adverse chemical reactions like the conversion of serine and cysteine residues to lysinoalanine compounds (nephrotoxic), decreased proteins bioavailability, and racemization of amino acids [61, 62]. The processes employing high alkaline pH, high temperature is associated with high yield of isolate, but there is high susceptibility of denaturation of isolate [61, 63]. The particle size of flour and solubilizing agent used can also affect the yield of isolate. The optimum particle size for flour is 100–150 μm and it was reported that NaOH and KOH generate more yield in comparison to Ca(OH)2 [64]. Also, there was protein loss of 6.2% from discarded supernatant from this extraction method [58] and in place of IEP, ultrafiltration (UF) or diafiltration membranes with specific molecular weight cutoffs can be utilized for isolating proteins of interest from the supernatant [32]. The efficiency of extraction can be improved by alteration in the molecular weight cutoffs, membrane type, concentration, and volume of the filtrate and addition of diafiltration to UF techniques [65]. The albumin proteins can be recovered by controlling these factors and further result in enhancing yield of isolate and alteration in isolate functionality leading to reduction in effluent losses. The use of UF can provide milder conditions for extracted proteins, so that their functionality can be enhanced and it gives higher yields in comparison to IEP [66].

Boye et al. [65] also confirmed that there were slightly higher protein levels in UF than the IEP process. Membrane filtration is also effective in reduction of antinutritional compounds in isolate [65]. Taherian et al. [67] conducted a study for functional properties of commercial and membrane-processed yellow pea protein isolates. The use of UF results in reduction of phytic acid upto 28–68% and possess improved functionality (e.g., solubility, rheology, foaming and emulsification) for commercially available isolates. The solubility of the commercial protein isolates was reported as ~20% vs. ~80% by using UF/diafiltration at pH 2.0. Fuhrmeister and Meuser [68] found the enhanced solubility, emulsifying, foaming and fatholding properties by UF recovery of proteins from wrinkled pea relative to heat, acid, and heat/acid precipitation.

#### **5.2 Salt extraction (SE) and micellization**

SE has advantage of the salting-in and out phenomenon of proteins which is followed by desalting for lowering the ionic strength of protein environment [32, 69]. In this process, the flour is stirred in salt solution of ionic strength

(1:10 (w/v) ratio) for 10–60 mins and further followed by removal of insoluble matter by settling, screening, decanting, filtering or centrifugation. The supernatant is desalted and dried [32, 69, 70]. The choice or concentration of salts is selected according to salting-in and salting-out characteristics of the protein and any unwanted proteins, respectively because the proteins precipitate at an array of ionic strengths [71, 72]. The salting-in of proteins generally occurs at ionic strength (between 0.1 to 1 M) [60] and the other factors include interactions of salt and sample components and ensuring the use of food-grade salts [69, 73]. The major advantage for this technique is that extreme level of acidic or alkaline pH alongwith elevated temperature is not required. The extraction occurs at pH level of 5.5–6.5, but Crevieu et al. [74] reported slightly alkaline pH for increasing protein solubility [69]. The pH can be maintained by the addition of acid or base or a salt solution with buffering capacity can be used. The supernatant with extract of high-salt protein should have a protein concentration of 15 to 100 mg/mL [69] and many methods have been used for decreasing its ionic strength.

In the process of micellization, protein precipitation is induced by adding cold water at a ratio of 1:3 to 1:10 (v/v) of high-salt protein extract to water [69, 75]. The solubilized proteins can be adjusted to low ionic strength by the dilution of protein solution through different dissociation reactions which forms loosely associated and low molecular weight aggregates. After reaching a specific concentration of protein, the aggregates can re-associate into low molecular weight species, known as micelles [69]. The arrangement of micelles is as thermodynamical spheres with minimum interfacial energy by giving exposure to polar moieties in outer aqueous environment and hydrophobic moieties towards the center. The proteins possesing more surface hydrophobicity have more protein–protein interactions and are also more successful for creating large and uniform aggregates [69]. The diluted solution can be left to stand for certain time for increasing micelle formation. This is followed by centrifugation and further the pellet is dried, and the high salt aqueous solution is discarded [32, 69]. Mwasaru et al. [75] reported that after using 0.25 M NaCl solution at pH value of 6.5 and 6 hours of micellization standing time, the protein extractability for pigeon pea and cowpea was yielded a 40.2% and 36.7%, respectively and these values were further compared to alkaline-extracted samples at pH value of 10.5 and 8.5, respectively, where the yields increased with respect to alkalinity. Gueguen [36] evaluated that 95% yield can be attained using micellization method.

#### **5.3 Dialysis**

The another commonly used method for desalting is dialysis. It is the process of membrane separation driven by a potential gradient for diffusing water and other solutes with low molecular weight like, salt and this process carried out using semipermeable membrane [72]. Gueguen et al. [70] and Crevieu et al. [74] used pea protein membranes with cutoffs of 8000 Da and 12,000–14,000 Da, respectively. The diffusion requires time for causing equilibrium on both sides and is complete when the potential gradient becomes negligible [72]. The changes in fresh, precooled liquid against which the sample is dialyzed helps in ensuring that very low concentrations of solutes remain in the sample. Gueguen et al. [70] cited a process of 130 hours which requires five changes of water of 20 times the extract volume. Crevieu et al. [74] dialyzed solution of globulin against two changes of 10 times the extract volume of ammonium carbonate, that requires 70 hours and results in a yield of 66.8%. Dialysis can also be used for separation of gloulin and fractions. According to the protein classification of Osborne, the dialyzed sample is centrifugated and it results in dissolved albumin fractions in supernatant and precipitated fractions of globulin in the pellet [70]. The phenolic compounds present in pea can

**113**

*Pea Seed Proteins: A Nutritional and Nutraceutical Update*

be reduced by additional steps during processing, like the use of alcohol washes and charcoal filters. The cross linkage of proteins can be improved by antioxidant activity of phenolic compounds which can negatively affect protein digestibility and enzymatic activity, leading to undesirable color and flavor compounds within

The application of bioactive ingredients (hydrophobic, hydrophilic compounds,

There are extrusion techniques which include low-moisture extrusion (LME, 40%) and high-moisture extrusion (HME, >40%), these techniques are widely used in commercial food production. LME is generally used for preparation of snacks and HME is used basically for meat analogue preparation. The research of pea protein based extruded products is very common nowadays and many researchers reported that pea protein was used in different starches like rice starch [77–79] wheat starch [80] and corn grits [81] for preparing protein-fortified extruded snacks by LME, and the results concluded that pea protein-fortified extruded products exhibits high content of protein and possess balanced amino acid profile in

There are many studies which report that by the addition of pea protein in cereal products can improve the nutritional value of the product because pea protein provides the essential amino acids and improve the texture of cereal product [4, 82–85]. The plant protein can be used as substitute for animal protein for meeting nutritional need of lacto-vegetarians and thus can make the food healthier. Several researchers are working on partly or fully substitution of dairy proteins with pea protein and the

Based on the literature reviewed in this chapter, we think that analogous research and advancement on pea proteins would be required if any significant boost in pea protein utilization is envisaged. While pea protein isolates have usually been discussed in the research literature as relatively mundane, you will find very few sensory analysis information to help the claim. The main limitation on the sales of pea protein meals components is the trouble in fighting with the well-established, versatile soy protein items which dominate the meals protein market. Soy proteins are already available for a very long time, and research by the main producing businesses has resulted in several tailored items for programs. Pea concentrates and flours are generally referred to as having a terrible taste (beany, bitter). The incorporation of pea concentrates and flours into meals products such as bread, is usually restricted by flavour problems. This truth is insignificant within the

minerals, and probiotics) is less due to their instability, less bioavailability, and unsuitable flavors in the food system. So, encapsulation can be a promising technique for solving these problems related to bioactive ingredients. Nowadays, there is an increase in research for pea protein as encapsulating materials, because of its health benefits, nil genetic modifications, and hypoallergenic issues [76]. As many researchers have recognized the importance of natural polymers for preparing biodegradable packaging and since pea protein acts as a biodegradable and biocompatible natural polymer, it can be used for producing biodegradable films. It can provide promising possibility for the application of pea proteins for making

*DOI: http://dx.doi.org/10.5772/intechopen.95323*

**6. Food applications of pea proteins**

comparison to pure extrudates of starch.

**7. Conclusion and future prospects**

biodegradable films in industrial-scale food production.

impact on taste and structure of these products [86–90].

the food product.

*Grain and Seed Proteins Functionality*

(1:10 (w/v) ratio) for 10–60 mins and further followed by removal of insoluble matter by settling, screening, decanting, filtering or centrifugation. The supernatant is desalted and dried [32, 69, 70]. The choice or concentration of salts is selected according to salting-in and salting-out characteristics of the protein and any unwanted proteins, respectively because the proteins precipitate at an array of ionic strengths [71, 72]. The salting-in of proteins generally occurs at ionic strength (between 0.1 to 1 M) [60] and the other factors include interactions of salt and sample components and ensuring the use of food-grade salts [69, 73]. The major advantage for this technique is that extreme level of acidic or alkaline pH alongwith elevated temperature is not required. The extraction occurs at pH level of 5.5–6.5, but Crevieu et al. [74] reported slightly alkaline pH for increasing protein solubility [69]. The pH can be maintained by the addition of acid or base or a salt solution with buffering capacity can be used. The supernatant with extract of high-salt protein should have a protein concentration of 15 to 100 mg/mL [69] and

In the process of micellization, protein precipitation is induced by adding cold water at a ratio of 1:3 to 1:10 (v/v) of high-salt protein extract to water [69, 75]. The solubilized proteins can be adjusted to low ionic strength by the dilution of protein solution through different dissociation reactions which forms loosely associated and low molecular weight aggregates. After reaching a specific concentration of protein, the aggregates can re-associate into low molecular weight species, known as micelles [69]. The arrangement of micelles is as thermodynamical spheres with minimum interfacial energy by giving exposure to polar moieties in outer aqueous environment and hydrophobic moieties towards the center. The proteins possesing more surface hydrophobicity have more protein–protein interactions and are also more successful for creating large and uniform aggregates [69]. The diluted solution can be left to stand for certain time for increasing micelle formation. This is followed by centrifugation and further the pellet is dried, and the high salt aqueous solution is discarded [32, 69]. Mwasaru et al. [75] reported that after using 0.25 M NaCl solution at pH value of 6.5 and 6 hours of micellization standing time, the protein extractability for pigeon pea and cowpea was yielded a 40.2% and 36.7%, respectively and these values were further compared to alkaline-extracted samples at pH value of 10.5 and 8.5, respectively, where the yields increased with respect to alkalinity. Gueguen [36]

many methods have been used for decreasing its ionic strength.

evaluated that 95% yield can be attained using micellization method.

The another commonly used method for desalting is dialysis. It is the process of membrane separation driven by a potential gradient for diffusing water and other solutes with low molecular weight like, salt and this process carried out using semipermeable membrane [72]. Gueguen et al. [70] and Crevieu et al. [74] used pea protein membranes with cutoffs of 8000 Da and 12,000–14,000 Da, respectively. The diffusion requires time for causing equilibrium on both sides and is complete when the potential gradient becomes negligible [72]. The changes in fresh, precooled liquid against which the sample is dialyzed helps in ensuring that very low concentrations of solutes remain in the sample. Gueguen et al. [70] cited a process of 130 hours which requires five changes of water of 20 times the extract volume. Crevieu et al. [74] dialyzed solution of globulin against two changes of 10 times the extract volume of ammonium carbonate, that requires 70 hours and results in a yield of 66.8%. Dialysis can also be used for separation of gloulin and fractions. According to the protein classification of Osborne, the dialyzed sample is centrifugated and it results in dissolved albumin fractions in supernatant and precipitated fractions of globulin in the pellet [70]. The phenolic compounds present in pea can

**112**

**5.3 Dialysis**

be reduced by additional steps during processing, like the use of alcohol washes and charcoal filters. The cross linkage of proteins can be improved by antioxidant activity of phenolic compounds which can negatively affect protein digestibility and enzymatic activity, leading to undesirable color and flavor compounds within the food product.
