**3. Amaranth protein**

Amaranth (*Amaranthus* spp.) seeds contain ∼13–15% protein with major fractions of albumin, globulin, and glutelin [4, 10]. Tömösközi et al. [11] investigated functional properties of amaranth protein in model systems and used casein and soy protein isolate as reference proteins for comparison. Amaranth protein isolate (80% protein) was obtained from defatted amaranth flour from two different varieties with alkaline extraction followed by the isoelectric precipitation method. Following extraction, amaranth protein isolate was separated into fractions based on the Osborne-type fractionation method. Fractions of albumin, globulin, and glutelintype alkali-soluble residual proteins were obtained and tested for functionality. The authors observed similarities between protein profiles of soy and amaranth. However, emulsifying and foaming properties of amaranth protein and derived fractions were found to be relatively poor compared to casein and soy protein. Among the amaranth protein fractions, solubilities of albumin and globulin fractions were reported to be significantly higher than that of the residue protein. It was concluded that optimization of protein extraction and enzymatic or chemical modification of protein structure may be required for effective utilization of amaranth protein preparations as food ingredients in various end-product applications [11].

Figueroa-González et al. [12] investigated the effects of pH-shifting and ultrasonication treatments on the structure, physicochemical, and foaming properties of amaranth protein. Amaranth protein isolate (83% protein) was obtained from defatted amaranth flour with alkaline extraction-isoelectric precipitation method. Amaranth protein dispersions were prepared in distilled water (30 mg/mL, pH 7.0) and protein was modified by five different treatments—pH-shifting at pH 2.0 and 12.0, sonication (750 W) for 10 min at an amplitude of 50%, and pH-shifting (at pH 2.0 and 12.0) followed by sonication. After the modification treatments, amaranth protein dispersions were dried at 35°C for 45 h in the oven to avoid protein denaturation. Alkaline pH-shifting followed by sonication was reported to result in a significant decrease in the hydrodynamic diameter of amaranth protein. On the other hand, hydrodynamic diameter of protein was observed to increase after the acidic pH-shifting treatment. The isoelectric point of amaranth protein increased from 4.0 to 4.2 after the alkaline pH-shifting treatment and to 4.5 after the combined alkaline pH-shifting and ultrasound treatments. However, ultrasound treatment alone was reported to decrease the isoelectric point of amaranth protein to 3.5. Alkaline pHshifting and ultrasound treatments were reported to induce changes in the secondary structure fractions of amaranth protein. Moreover, both pH-shifting treatments and combination of pH-shifting and ultrasound treatments resulted in changes in the sulfhydryl groups and disulfide bonds of amaranth protein. Both pH-shifting treatments were reported to improve the solubility of amaranth protein, where the highest protein solubility was observed in the sample treated with a combination of alkaline pH-shifting and ultrasound. The foaming capacity and stability of amaranth protein were reported to increase significantly after all treatments except for the acidic pH-shifting treatment. Moreover, treatments applied were indicated to improve the *in vitro* digestibility of amaranth protein that was attributed to the modifications in protein structure, which lead to increased accessibility to digestive enzymes [12].

Das et al. [13] investigated the effects of pH treatment and the extraction pH on the physicochemical and functional properties of amaranth protein isolate. Amaranth protein isolate was obtained from defatted amaranth flour with alkaline extraction at different pH values (9.0, 10.0, 11.0, and 12.0) followed by isoelectric precipitation

(pH 4.5). Amaranth protein isolates were subjected to pH treatments at pH 3.0–9.0 and tested for functionality. The protein content of amaranth proteins extracted at different pH values changed between 56 and 85%, where the isolate obtained at pH 9.0 showed the highest purity, solubility, and particle size but the lowest yield. The authors reported that maintaining the extraction and treatment pH values at 9.0 resulted in significant improvements in functional properties including solubility, water and oil binding capacities, emulsifying and foaming properties. Extraction and treatment at pH 9.0 were also indicated to result in better thermal properties and improved gelation characteristics. Moreover, emulsifying, foaming, and gelation properties of amaranth protein isolates were reported to be affected by the particle size, wettability, and solubility [13].

Enzymatic modification is recently applied as a useful tool to improve the functionality and biological activity of amaranth protein. Kamal et al. [14] prepared amaranth protein hydrolysates using bromelain, chymotrypsin, and pronase E enzymes at three different hydrolysis durations (2, 4, and 6 h). Bioactive peptides were identified by LC-MS-QToF analysis. Amaranth protein hydrolysates were reported to contain bioactive peptides with inhibitory properties against enzymatic markers linked with hypertension and diabetes [14].

In another recent study, Karimi et al. [15] investigated the effects of using selective hydrolyzed protein of amaranth on sourdough fermentation, bread quality, and shelf life. Amaranth protein isolate was obtained from defatted amaranth flour using the alkaline extraction-isoelectric precipitation method. Protein hydrolysates were prepared with Alcalase® treatment for 3 h. The authors reported that amaranth protein hydrolysates increased the growth of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* significantly in sourdough compared to amaranth protein isolate and amaranth flour. The use of amaranth protein hydrolysates in the sourdough formulation was indicated to result in changes in dough pH and total titratable acidity after fermentation, as well as changes in specific volume, water activity, and textural characteristics of bread. Sensory properties and shelf life of bread were reported to be improved by the addition of amaranth protein hydrolysates to sourdough [15].

Kiwicha (*Amaranthus caudatus*) is a less known pseudocereal that is indicated as a good source of protein (∼13–19%) with a balanced amino acid profile [16]. The physicochemical and functional properties of kiwicha proteins still remain to be explored. There are only few recent reports in the literature on the biological activities of peptides obtained from kiwicha proteins with enzymatic hydrolysis [17–19].

#### **4. Chia protein**

The protein content of chia (*Salvia hispanica* L.) seeds changes between ∼19 and 23%, and the major protein fractions are reported as globulins, albumins, glutelins, and prolamins [20]. Segura-Campos [21] characterized chia seed proteins. Protein fractions of albumins, globulins, prolamins, and glutelins were obtained based on the Osborne-type fractionation method. The main protein fraction in chia was reported to be glutelin that showed the highest water absorption and holding capacities. Significant differences were observed in emulsifying and foaming properties of fractions obtained from chia protein [21].

Julio et al. [22] prepared different protein fractions of albumins, globulins, glutelins, and prolamins from chia protein-rich fraction of chia seeds, a by-product of chia oil extraction process. The solubility profile of chia protein-rich fraction, globulins,

*Proteins from Pseudocereal Grains DOI: http://dx.doi.org/10.5772/intechopen.102504*

and prolamins was observed to be similar and made a peak at pH 9.0. On the other hand, maximum solubility was observed at pH 5.0 for glutelin and albumin fractions. Detailed emulsion characterization tests including destabilization kinetics and particle size distributions revealed that globulin fraction resulted in the most stable emulsion systems. The authors reported that higher pH values resulted in improved stability in emulsions stabilized with globulins, glutelins, and chia protein-rich fraction [22].

Urbizo-Reyes et al. [23] prepared chia protein hydrolysates with ultrasound treatment followed by microwave-assisted hydrolysis. For this purpose, chia seed mucilage and chia seed oil were extracted from the seeds prior to protein hydrolysis. Chia protein hydrolysates were prepared using Alcalase® or sequential hydrolysis with Alcalase® and Flavourzyme®. Enzymatic hydrolysis reaction was conducted using a conventional or microwave-assisted system. Chia protein hydrolysates obtained using sequential hydrolysis with microwave treatment were reported to show significantly higher *in vitro* antioxidant activity. Hydrolysates were also indicated to show antidiabetic and antihypertensive activities. Microwave treatment during hydrolysis was reported to improve the solubility profile, emulsifying and foaming properties of hydrolysates [23].

### **5. Teff protein**

Teff (*Eragrostis tef*) contains ∼13–21% protein with major fractions of glutelin, albumin, and prolamins [24]. Compared to commonly studied proteins from pseudocereal grains, including quinoa, amaranth, and chia, teff proteins remain to be explored in terms of functionality. Gebru et al. [24] studied the variations in amino acid profile and protein composition of white and brown teff seeds during protein extraction. Three different methods were used to obtain fractions of albumins, globulins, prolamins, and glutelins from teff seed flour. White and brown teff seeds were indicated to undergo different changes during protein extraction. Moreover, the essential amino acid content of brown teff seeds was reported to be significantly higher than that of white seeds. Extraction with tert-butanol was indicated to increase prolamin yield compared to extraction with ethanol. Glutelin was reported to be the major protein fraction in both seeds, with white seeds containing higher amounts of glutelin compared to brown seeds. The electrophoretic profile of storage proteins was observed to be different indicating the genetic variations between white and brown teff seeds [24].

Teff flour is widely used in formulations of gluten-free bread and bakery products. Adebowale et al. [25] compared the characteristics of protein fractions in three different teff types with sorghum with the main focus on bread-making quality. The major protein fraction in teff was reported to be prolamin. Aqueous alcohol-soluble protein fraction was indicated to be rich in glutamine and leucine. The authors suggested that differences in the electrophoretic profile of proteins indicated that teff prolamin is less polymerized compared to sorghum prolamin. Functional properties of teff prolamins useful in bread making were attributed to the differences in thermal profile, lower polymerization, and hydrophobicity [25].

#### **6. Buckwheat protein**

Common buckwheat (*Fagopyrum esculentum*) and tartary buckwheat (*Fagopyrum tataricum*) are indicated as good sources of protein (8–18% protein) with a balanced

amino acid profile [3, 26]. The major storage protein in buckwheat seeds is reported to be 13S globulin [27] and the main protein fractions were reported as albumin, globulin, and glutelin [3]. Functional and bioactive properties of buckwheat protein were recently reviewed by Jin et al. [3]. Tomotake et al. [28] compared the physicochemical and functional properties of buckwheat protein with soy protein isolate and casein. Buckwheat protein was obtained from buckwheat flour using the alkaline extractionisoelectric precipitation method. Solubility of buckwheat protein was significantly higher than that of soy protein at pH 2.0–10.0, but lower compared to casein at pH 7.0–10.0. The stability of emulsions stabilized by buckwheat protein was observed to be lower compared to soy protein and casein-stabilized emulsions at pH 7.0–10.0. Moreover, the water holding capacity of buckwheat protein was found to be lower than that of soy protein [28].

Xue et al. [29] investigated the effects of high-intensity ultrasound treatment and Maillard reaction on structural, interfacial, and emulsifying properties of buckwheat protein. Buckwheat protein isolate was prepared from defatted buckwheat flour with alkaline extraction method followed by isoelectric precipitation. Buckwheat protein isolate-dextran conjugates were prepared via Maillard reaction combined with ultrasound treatment. The secondary and tertiary structures and surface hydrophobicity of buckwheat protein isolate-dextran conjugates obtained with ultrasonication were observed to be different than those of conjugates obtained with classical heating. As a result of the modifications in protein structure, emulsifying properties and surface activity of conjugates obtained with ultrasonication were reported to be improved compared to classical heating [29].

In another recent study, Wu et al. [30] investigated the effect of extraction pH on structure, functional properties, and digestibility of tartary buckwheat protein. Protein isolates were prepared from defatted tartary buckwheat flour using alkaline extraction at different pH values (pH 7.0–13.0) followed by isoelectric precipitation. Tartary buckwheat flour and protein isolates were separated into albumin, globulin, prolamin, and glutenin fractions based on Osborne-type protein fractionation. Protein extraction at alkaline conditions was reported to increase protein extraction yield. Increased extraction pH was indicated to decrease the albumin content of tartary buckwheat protein isolate while glutenin content increased. The solubility of isolates extracted at pH > 12.0 was observed to decrease. On the other hand, emulsion stability increased at the same conditions that were attributed to increased surface hydrophobicity. The differences observed in *in vitro* digestibility of tartary buckwheat protein isolates obtained at different pH values were related to the modifications in protein structure. The highest digestive rate was observed in isolates obtained at pH 7.0 and 8.0 [30].

In addition to functional properties, buckwheat protein and derived bioactive peptides are reported to show various biological properties, including cholesterollowering activity, blood pressure controlling enzyme inhibitory activity, antimicrobial and antioxidant activities that suggest the potential use of buckwheat protein and peptides as functional food ingredients [3].

#### **7. Cañihua protein**

Cañihua (*Chenopodium pallidicaule* Aellen), is a less known pseudocereal, contains ∼14–19% protein and a balanced essential amino acid profile that are comparable to other commonly known pseudocereals, such as quinoa and amaranth [31].

#### *Proteins from Pseudocereal Grains DOI: http://dx.doi.org/10.5772/intechopen.102504*

The major protein fractions in cañihua are reported as albumin and globulin [32]. Betalleluz-Pallardel et al. [33] used response surface methodology for optimization of protein extraction conditions from defatted cañihua grain meal aiming maximized protein extraction yield. Simultaneous effects of pH (7.0–11.0), temperature (25–60°C), solvent:meal ratio (10:1–40:1), time (10–60 min), and NaCl concentration (0–2 M) on protein extraction yield were investigated. Optimum protein extraction conditions were determined as pH 10.0, 21°C, 37:1 (v/w) solvent:meal ratio, and 5 min of extraction time, which resulted in ∼80% protein extraction yield [33].

Enzymatic hydrolysis was applied to cañihua protein for obtaining peptides with biological activities. Chirinos et al. [34] derived hydrolysates and peptides from cañihua protein concentrate. Protein concentrate (79% protein) was obtained from defatted cañihua meal with alkaline extraction-isoelectric precipitation method. Cañihua protein concentrate was subjected to enzymatic hydrolysis with Alcalase®, Neutrase®, and Flavourzyme® at 50°C up to 240 min. The hydrolysates obtained were purified via ultrafiltration and size exclusion chromatography to obtain three peptide fractions. The authors reported that cañihua protein can be considered as a good source of bioactive peptides with antioxidant and angiotensin-I converting enzyme (ACE) inhibitory activities. Specifically, cañihua protein hydrolysate obtained with Neutrase®-Alcalase® sequential hydrolysis for 180 min was indicated to show good *in vitro* bioactivity. Two of the purified peptide fractions composed of 3–11 amino acids were indicated to show good *in vitro* antioxidant and antihypertensive activity [34].

In another recent study, Moscoso-Mujica et al. [35] also applied enzymatic hydrolysis to cañihua protein. Cañihua flour was obtained from the seeds of two different varieties (Ramis and Cupi-Sayhua) and defatted prior to protein extraction. Protein fractions of albumins, 7S globulins, 11S globulins, and glutelins were obtained based on solubility differences and subjected to sequential hydrolysis with Alcalase® and pepsin-pancreatin. Hydrolysates with varying degrees of hydrolysis were obtained and tested for antimicrobial activity against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*. The authors reported that among the 216 hydrolysates obtained, only 28 showed significant antimicrobial activity. It was suggested that antimicrobial peptides obtained from fractions of globulins and glutelins can potentially be utilized as novel nutraceutical ingredients [35].

## **8. Conclusion**

Pseudocereals are indicated as good protein sources with a balanced amino acid profile. Nutritional composition and protein characteristics of pseudocereal grains change depending on the seed variety and growing conditions. Moreover, the methods used for protein extraction and fractionation affect protein structure, composition, and hence, functionality. Enzymatic hydrolysis has been shown to be a useful tool for obtaining peptides from pseudocereal proteins with biological activities, including antioxidant, antimicrobial, and antihypertensive properties. Proteins and peptides from pseudocereal grains can be potentially utilized as ingredients in innovative product formulations due to their nutritional quality, functional properties, and biological activities. More research is needed to investigate the effects of pseudocereal proteins on end-product quality to elucidate the potential and increase the utilization of pseudocereal proteins as food ingredients.
