Campus Monterrey
School of Engineering and Sciences
Development and characterization of a high hydrosoluble food ingredient using extruded whole chickpea flour and sequential Alcalase® and -
amylase treatment
A thesis presented by
Robinzon Silvestre de León
Submitted to the
School of Engineering and Sciences
in partial fulfillment of the requirements for the degree of
Master of Science In Biotechnology
Monterrey Nuevo León, June 15th, 2020
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To my family for all their unconditional confidence and support.
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I would like to express my deepest gratitude to all my advisors for their patience and their encouragement through this work. Also, I want to thank to all my professors and colleagues from Centro de Biotecnología FEMSA who were involved in the development of my research project.
Finally, I would like to express my specially gratitude to the Tecnológico de Monterrey for their support on tuition as well as CONACyT (CVU: 927539) for master scholarship .
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extruded whole chickpea flour and sequential Alcalase® and α-amylase treatment
by
Robinzon Silvestre de León Abstract
Chickpea is an adequate source of proteins and starch which can be used to develop new nutritious and functional food products such as vegetable beverages. However, in order to use chickpea to develop a functional, healthy and nutritional beverage, its processing is needed to improve the digestibility and increase the quantity of soluble components into an aqueous system.
Therefore, in the present research work, extrusion of whole chickpea and sequential hydrolyses with Alcalase® and α-amylase were evaluated to develop a high soluble chickpea-based food ingredient. The thermoplastic extrusion process was carried out varying processing moisture (15.6% or 22.55%), final barrel temperature (143 °C or 150 °C) and screw speed (450 rpm, 580 rpm, or 700 rpm) to generate three SME inputs (127.95 Wh/kg, 161.58 Wh/kg, and 199.13 Wh/kg).
After extrusion, flours were hydrolyzed with Alcalase® and α-amylase in order to maximize soluble compounds after hydration. In general, extrusion did not affect chemical composition, but caused structural modifications that influenced changes in functional properties and modified in vitro protein and starch digestibilities. Extruded chickpea flours presented higher content of soluble proteins and increased starch hydrolysis after Alcalase® and α-amylase treatment, respectively.
It was found that extrusion treatment of chickpea with a SME input of 127.95 Wh/kg produced at 22.5% processing moisture, 150 °C of final temperature and 580 rpm of screw speed in combination with the later Alcalase®/α-amylase treatments achieved the highest release of both soluble proteins (70%) and soluble solids (62%) and the highest degree of starch hydrolysis (84%).
These results were used to transform whole chickpea flour into a valuable soluble food ingredient by means of a combination of extrusion and sequential Alcalase®/α-amylase treatment. This soluble food ingredient was freeze dried, milled and characterized in terms of chemical composition and protein quality. It was found that the resulting powder had 53.7%, 20.2% and 3.6% of reducing sugars, proteins, and fat contents, respectively. The soluble powder had an in vitro protein digestibility of 83.1%, a PDCAAS value of 0.831 and it did not present any limiting amino acids which suggest that this product had the potential to be used to develop instant chickpea beverages with an excellent nutrimental quality.
Keywords: Chickpea, Extrusion, Enzymatic hydrolysis, In vitro digestibility, Soluble solids, Soluble proteins.
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Fig. 1. Pasting properties of raw and extruded chickpea. RW (continuous line), raw chickpea flour;
EA (small dashed line), chickpea extrudate with a SME input of 161.58 Wh/kg at 15.6%
processing moisture, 143 °C and 450 rpm; EB (large dashed line), chickpea extrudate with a SME input of 199.13 Wh/kg at 15.6% processing moisture, 150 °C and 700 rpm; EC (dotted line), chickpea extrudate with a SME input of 127.95 Wh/kg at 22.5% processing moisture, 150 °C and 580 rpm. Each line is the result of triplicate experiments. ...38 Fig. 2. Scanning electron micrographs for RW and extruded chickpea at 300X magnification. a) RW, b) EA, c) EB and d) EC. RW, raw chickpea flour; EA, chickpea extrudate with a SME input of 161.58 Wh/kg at 15.6% processing moisture, 143 °C and 450 rpm; EB, chickpea extrudate with a SME input of 199.13 Wh/kg at 15.6% processing moisture, 150 °C and 700 rpm; EC, chickpea with a SME input of 127.95 Wh/kg at 22.5% processing moisture, 150 °C and 580 rpm. ...41 Fig. 3. Kinetics of protein hydrolysis with Alcalase® for RW and extruded chickpea estimated with the free amino nitrogen (FAN) assay. (■) RW, raw chickpea flour; (●) EA, chickpea extrudate with a SME input of 161.58 Wh/kg at 15.6 processing moisture, 143 °C and 450 rpm; (♦) EB, chickpea extrudate with a SME input of 199.13 Wh/kg at 15.6% processing moisture, 150 °C and 700 rpm; (▲) EC, chickpea extrudate with a SME input of 127.95 Wh/kg at 22.5% processing moisture, 150 °C and 580 rpm. The error bars are standard deviations from triplicate experiments.
The kinetic constant k (FAN min-1) was calculated adjusting data to a zero-order reaction ( [ ] = ). Kinetic constants (k) with different letters are significantly different at P < 0.05.
...43 Fig. 4. Degree of starch hydrolysis by α-amylase for raw and extruded chickpea expressed as dextrose equivalents (DE). (■) RW , raw chickpea flour; (●) EA, chickpea extrudate with a SME input of 161.58 Wh/kg at 15.6 processing moisture, 143 °C and 450 rpm; (♦) EB, chickpea extrudate with a SME input of 199.13 Wh/kg at 15.6% processing moisture, 150 °C and 700 rpm;
(▲) EC, chickpea extrudate with a SME input of 127.95 W h/kg at 22.5% processing moisture, 150 °C and 580 rpm. The error bars are standard deviation from triplicate experiments. Values of the starch hydrolysis were adjusted to a first-order reaction [ ] = ∞(1 − exp(− ), where DE (%), DE∞(%) and k (min-1) are dextrose equivalent at each time, the maximum dextrose equivalent and kinetic constant, respectively. Kinetic constants (k) with different letters are significantly different at P < 0.05. ...44
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Table 1. Chemical composition of raw and extruded chickpea flours. ...33
Table 2. Particle size distribution of raw and extruded chickpea flours. ...34
Table 3. Functional properties of raw and extruded chickpea flours. ...36
Table 4. In vitro protein and starch digestibilities of raw and extruded chickpea flours. ...37
Table 5. Effect of different extrusion conditions on FTIR intensity ratios for protein and starch structures of chickpea flours. ...39
Table 6. Comparison of soluble solids (SS) and protein solubility (SP) from RW and extrudates digested with Alcalase® and α-amylase in an aqueous suspension with 12% of dry flour. ...46
Table 7. Distribution of protein, fat, and solids in the water soluble and insoluble fractions obtained by enzymatic hydrolysis of RW and EC flours. ...49
Table 8. Chemical characterization of the different fractions obtained by enzymatic hydrolysis of RW and EC. ...51
Table 9. Amino acid profile, IVPD, and PDCAAS of RW, EC, and their soluble and insoluble fractions obtained after enzymatic treatment. ...54
Table A. 1. A. 1 Abbreviations ...60
Table A. 2. Acronyms ...60
Table A. 3. Variables and symbols ...61
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Contenido
1. Introduction ...1
1.1 Background and motivation ...1
1.2 General Objective ...3
1.3 Specific objectives ...3
1.4 Hypothesis ...4
2. Theoretical Framework ...5
2.1 Chickpea grain ...5
2.1.1 Chemical composition ...6
2.1.2 Health benefits ...10
2.1.3 Applications of chickpea ...11
2.2 Extrusion technology ...14
2.2.1 Effect of extrusion on food chemical components ...15
2.3 Enzymatic treatments ...19
2.3.1 Amylolytic treatments ...20
2.3.2 Proteolytic treatment ...21
3 Materials and methods ...24
3.1 Raw materials...24
3.2 Evaluation of the effect of extrusion conditions on chemical composition, functional properties and susceptibility to hydrolysis of whole chickpea flour ...24
3.2.1 Extrusion treatments ...24
3.2.2 Chemical composition and particle size distribution ...25
3.2.3 Functional properties ...25
3.2.4 In vitro protein digestibility ...26
3.2.5 In vitro starch digestibility ...26
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3.2.7 FTIR analysis ...27
3.2.8 SEM analysis ...27
3.2.9 Enzymatic protein hydrolysis ...27
3.2.10 Enzymatic starch hydrolysis ...28
3.2.11 Total soluble solids and protein solubility ...28
3.3 Preparation and characterization of a soluble food ingredient using sequential Alcalase® and α-amylase on extruded chickpea flour ...29
3.3.1 Preparation of soluble and insoluble fractions ...29
3.1.1 Distribution of proteins, fat, and solids...30
3.1.2 Chemical characterization ...30
3.1.3 Protein quality ...30
3.2 Statistical analysis ...31
4 Results and discussions...32
4.1 Evaluation of the effect of extrusion conditions on chemical composition, functional properties and susceptibility to hydrolysis of whole chickpea flour ...32
4.1.1 Chemical composition and particle size distribution ...32
4.1.2 Functional properties ...34
4.1.3 In vitro protein and starch digestibilities...36
4.1.4 Pasting properties ...37
4.1.5 FTIR analysis ...39
4.1.6 SEM analysis ...40
4.1.7 Enzymatic protein hydrolysis ...42
4.1.8 Enzymatic starch hydrolysis ...43
4.1.9 Total soluble solids and protein solubility ...45
4.2 Preparation and characterization of a soluble food ingredient using sequential Alcalase® and α-amylase on extruded chickpea flour ...48
4.2.1 Distribution of proteins, fat, and solids...48
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4.2.3 Protein quality ...53
Chapter 5 ...57
5 Conclusions ...57
Chapter 6 ...59
6 Recommendations and future work ...59
Appendix A ...60
Abbreviations and acronyms ...60
Apéndice B ...61
Variables and Symbols ...61
Bibliography ...62
Published papers ...77
Curriculum Vitae ...78
Chapter 1
1. Introduction
1.1 Background and motivation
Chickpea is the third most important legume around the world with a global annual production in 2018 of 17.21 million tons, and Mexico is currently the eighth place in terms of production (FAOSTAT, 2018). The demand for this particular legume is expected to increase because it represents a suitable choice to develop nutritious and gluten-free foods (Shaabani et al., 2018).
Chickpea has good protein (~22%) and dietary fiber (~16%) contents, and a high amount of starch (~50%) with comparatively low glycemic index (Asif et al., 2013; Wood and Grusak, 2007). The chemical composition of chickpea makes this legume a concentrated and inexpensive source of proteins, with an adequate balance of essential amino acids that is complementary to a cereal - based diet (Asif et al., 2013; Wood and Grusak, 2007). Additionally, chickpea contain a high proportion of resistant starch (up to 35%) which is not digested by the human digestive system and acts as a potent prebiotic (Wood and Grusak, 2007).
The consumption of chickpea and chickpea based products is increasing due to its nutritional properties (Shaabani et al., 2018; Wood and Grusak, 2007). Recent studies have incorporated protein isolates and flours from chickpea into formulations in order to improve functional and physicochemical characteristics of breads (Collar and Armero, 2018; Shaabani et al., 2018) and infant foods (Malunga et al., 2014). However, even though chickpeas have good protein and starch content, they are difficult to digest, require long cooking time and present problems related to palatability (Chavan et al., 1987). Cooking, germination, fermentation and roasting are suitable methods which have been studied in order to overcome these problems and enhance functional properties (Baik and Han, 2012; El-Adawy, 2002). Nevertheless, there is a need of searching
other simple, low-cost and environment friendly processing techniques to transform chickpea into a nutritious and functional ingredient.
In recent years, extrusion technologies have been widely used to process cereals, pseudocereals and legumes into novel ingredients or foods. Particularly, thermoplastic extrusion, which uses high temperatures and mechanical shear to cook and plasticize food materials, triggers relevant chemical reactions mainly in the starch and protein moieties (Moscicki, 2011). Extrusion can effectively modify food materials to yield intermediate ingredients (precooked flours, texturized proteins) or ready-to-eat finished products like expanded snacks and other extruded breakfast cereals (Yovchev et al., 2017). This technology has the advantages of using short times and being an economical and sustainable continuous process (Wani et al., 2019). The effect on chemical composition and physical characteristics due to high temperature, high shear force and variable moisture during extrusion have been studied mainly in cereals and other legumes like soybean (Fallahi et al., 2016; Martínez et al., 2014; Sharma and Gujral, 2013). However, there are few studies regarding extrusion of chickpea flour (Raza et al., 2019; Wang et al., 2019; Wani et al., 2019) and these studies are focused on evaluating the effect of processing conditions on chemical composition, gelatinization, water holding capacity, solubility index, and in vitro protein and starch digestibilities. Moreover, these authors have stated that extruded chickpea have the potential of being used in food industry, but additional processing is needed. However, there are not studies of evaluation the effect of combining extrusion and other processing on chickpea or other legume flour.
The use of enzymatic treatments has proved been an adequate alternative to improve the content of soluble proteins and carbohydrates of legumes. Enzymatic hydrolysis has been used in protein isolates and cooked chickpea to improve protein solubility and starch degradation (Ghribi et al., 2015; Malunga et al., 2014). Treatment with α-amylase has improved the in vitro digestion of starch in pea, kidney bean, and cowpea gelatinized (Hoover and Zhou, 2003). Moreover, it has
demonstrated that the degree of hydrolysis by amylases in lentils is higher when their starch granules have been previously gelatinized (Chung et al., 2009). Proteases such as Alcalase®, which is an inexpensive enzyme, have been used to increase the release of small peptides from black gram and kidney bean protein extracts (Kasera et al., 2015). This enzyme has also been used to improve the amount of bioactive peptides in common bean (de Souza Rocha et al., 2015) and rice milk (Ngamsuk et al., 2020). The use of amylolytic and proteolytic enzymes increased the amount of soluble sugars and improved the solubility of proteins and solids (Ghribi et al., 2015;
Xu et al., 2014). Furthermore, it is has been stated that thermoplastic extrusion could increase the susceptibility of starch and protein to be hydrolyzed (Martínez et al., 2014; Zheng et al., 2006).
Only a few studies have combined amylolytic treatment with extrusion in wheat and rice flours (Wu et al., 2020; Xu et al., 2015, 2014) focusing in the evaluation of starch gelatinization, fermentation efficiency and functional properties. However, to our knowledge, there are no available studies about the effect of extrusion process to improve the efficiency of subsequent proteolytic and amylolytic treatment on legume flours such as chickpea with the aim of increasing both starch and protein hydrolyses into an aqueous fraction, which is the intent of this thesis.
1.2 General Objective
To evaluate the interaction of twin-screw extrusion and the use of sequential Alcalase® and α- amylase treatments to transform whole chickpea flour into a high hydrosoluble food ingredient with adequate content of protein and carbohydrates.
1.3 Specific objectives
· To generate three extruded flours from whole chickpea using different specific mechanical energy (SME) inputs to study the effect of extrusion conditions (temperature, screws speed and processing moisture) on chemical composition, particle size distribution,
functional characteristics, pasting properties and in vitro protein and starch digestibilities of the three extrudates.
· To study the effect of extrusion conditions on structural modifications of starch and proteins in the three extruded flours through SEM and FTIR.
· To evaluate the effect of extrusion conditions on kinetics of protein and starch hydrolyses of the three extrudates by Alcalase® and α-amylase using free amino nitrogen (FAN) procedure and reducing sugars, respectively.
· To identify adequate times for protein and starch hydrolyses and perform sequential Alcalase® and α-amylase treatments in the non-extruded flours and the three extrudates to assess their effect on the generation of soluble solids and proteins.
· To characterize a high hydrosoluble ingredient prepared using sequential Alcalase® and α-amylase treatments on the extruded whole chickpea flour with higher in vitro digestibility and susceptibility to hydrolysis.
1.4 Hypothesis
The extrusion process with subsequent Alcalase® and α-amylase treatment could increase the solubility of proteins and carbohydrates contained in whole chickpea flour and generate a valuable soluble ingredient with the potential to be used for developing a vegetable chickpea beverage.
Chapter 2
2. Theoretical Framework
2.1 Chickpea grain
Chickpea is one of the most ancient domesticated grains along with wheat, lentil, barley, pea, rye flax and vetch (Abbo et al., 2003). It belongs to the genus Cicer which is a member of the family Leguminosae, subfamily Papilionoideae and the monogeneric tribe Cicerae Alef. The tribe Cicerae includes many species, but the single cultivated species are classified as Cicer arietinum
(Millan et al., 2015; Wood and Grusak, 2007). C. arietinum is divided into desi and kabuli cultivars, which are different mainly in shape and color. Kabuli chickpea has large and cream-colored seeds while desi type has small and dark seeds (Millan et al., 2015).
Chickpea is the third most important legume around the world. It represents around the 17% of global legume cultivation. The larger producer is India with a 11.38 million of tons in 2018 which represents 66% respect to total chickpea production, followed by Australia and Turkey with a 5.8% and 3.7%, respectively. Some countries in America are among the top ten in chickpea global production. United States has the 6th place in chickpea cultivation worldwide, followed by Mexico (8th) and Canada (10th). The chickpea production in Mexico is about 352 thousand tons (FAOSTAT, 2018). In general, the Asian countries harvest chickpea for their population consumption and have low production per hectare. On the other hand, Australia, United States, Mexico and Canada harvest chickpea for exportation and they have high production per hectare (Rawal and Kalamvrezos, 2018). There is a current trend in chickpea consumption in the world due to improvement in production methods, but also because it represents a suitable and low- cost source of proteins for underdeveloped countries (Merga and Haji, 2019). Moreover, the
changing in dietary habits also is driving an increase of chickpea consumption because it is a gluten-free grain rich in proteins, starch and dietary fiber (Shaabani et al., 2018).
2.1.1 Chemical composition
The chemical composition of chickpea present great range of variability. This variability depends on cultivar type, environmental factors and agronomic practices. In general, the main macro and micronutrients are present in the cotyledons, but the seed coat contains most of the dietary fiber and calcium (Chavan et al., 1987). Whole chickpea is known to have high content of carbohydrates (52-71%) and proteins (13-29%), but also it has good content of dietary fiber (11- 23%) and fat (3-9%) (Asif et al., 2013; Wood and Grusak, 2007). Moreover, many compounds in chickpea have bioactive properties, placing it as an accessible crop with nutritional and nutraceutical properties (Bar-El Dadon et al., 2017; Jukanti et al., 2012).
Carbohydrates
The main storage polysaccharide in chickpea is starch which is in the range of 30 to 57% and it constituted the main dietary energy source when this legume is consumed by humans. The amylose content in chickpea starch ranges from 32 to 46% (Chavan et al., 1987). Additionally, within all the starch, the 35% is resistant starch which is not digested by the human digestive system and can serve as prebiotic (Topping and Clifton, 2001). Chickpea also contains pectic compounds, hemicellulose, cellulose and lignin, which are called dietary fiber. In general, desi types contain higher dietary fiber (up to 23%) than kabuli types (up to 17%) (Wood and Grusak, 2007).
Chickpea is also rich in oligosaccharides with α-galactosidic linkages. Due to the fact that humans do not synthetize α-galactosidases, this type of oligosaccharides are not hydrolyzed by human intestine where they can be fermented causing flatulence. However, because of this property, galactooligosaccharides are considered as prebiotics (Topping and Clifton, 2001; Xiaoli et al.,
2008). The main galactooligosaccharides presented in chickpea seeds are ciceritol, raffinose, stachyose and verbascose (Sánchez-Mata et al., 1999). The content of oligosaccharides in chickpea is up to 8.3% and ciceritol is the most abundant of these type of compounds (up to 5%) (Xiaoli et al., 2008). In addition to oligosaccharides, a few quantities of free monosaccharides and disaccharides are present in chickpea. Glucose, fructose, sucrose and maltose are present most commonly in 0.7%, 0.3%, 2.0% and 0.6%, respectively (Gangola et al., 2014).
Proteins
The main storage proteins that have been found in chickpea are globulins, albumins, and glutelins which represent 57%, 18%, and 12% of the total extractable proteins, respectively. Globulins are soluble in salt-water solution, albumins are soluble in water, and glutelins are soluble in diluted acids or bases (Chavan et al., 1987; Roy et al., 2010). The globulin fraction is deficient in sulphur amino acids (such as methionine, cysteine and tryptophan), while the glutelin and albumin fractions have high content of those amino acids. All chickpea protein fractions are rich in lysine and arginine, which make chickpea amino acids complementary to those in cereal proteins (Duranti, 2006). However, chickpea proteins are difficult to digest in comparison to animal proteins.
Khan et al. (1979) and Milán-Carrillo et al. (2000) have reported values of true protein digestibility (TD) for chickpea of 89%, while the red meat and milk have TD values up to 99% and 95%, respectively (Sarwar et al., 1989). On the other hand, chickpea proteins has higher TD values than mung beans (85%), kidney bean (81%), lentil (84%), barley (84%), and oat (84%) (Eggum and Jacobsen, 1976; Khan et al., 1979; Sarwar et al., 1989; Stone et al., 2019). The low digestibility of proteins is related to the presence of antinutritional factors but also it is linked to the fact that the globulins present in chickpea form a very compact structure which is resistant to be digested (Millan et al., 2015). Furthermore, chickpea proteins have an important nutraceutical characteristic. The 97% of the chickpea globulins are formed by legumin which is a protein with
six units. The enzymatic hydrolysis of this type of protein release small peptides which are known to have antioxidant capacity and the property of reducing blood pressure (Yust et al., 2003).
Lipids
Chickpea has higher content of fat (up to 9%) compared to other legumes such as lentil, black bean, pea, mung bean, an faba bean, but the profile of lipids is mostly composed by polyunsaturated fatty acids (Asif et al., 2013; Sparvoli et al., 2015). In relation to the total amount of fat, chickpea has around 66%, 19% and 15% of polyunsaturated, mono-unsaturated and saturated fatty acids, respectively. Most fatty acids (more than 80%) in chickpea are linoleic and oleic acids which are effective in lowering serum cholesterol (Hu et al., 2001; Jukanti et al., 2012).
Furthermore, chickpea contains some phytosterols in small quantities, which are known to have antioxidant capacity (Padhi et al., 2017). The main phytosterols in chickpea are sitosterol (up to 0.16%) and campesterol (up to 0.02%) (Oomah et al., 2011).
The different lipids present in chickpea grains are important since they have effects on the nutraceutical properties of chickpea-based foods. However, the lipids in chickpea are also responsible for its undesirable flavor and unpalatability (Wood and Grusak, 2007). Moreover, lipids can be degraded by means of oxidation and interact with proteins and carbohydrates which reduce their digestibility and modify their functional properties (Chavan et al., 1987).
Vitamins and minerals
Chickpea contains small quantities of micronutrients such as vitamins and minerals. Ascorbic acid is the main water-soluble vitamin with a content up to 60 µg/g. Chickpea is also rich in niacin (up to 15.4 µg/g), pyridoxine (up to 5.4 µg/g) and folic acid (1.50-5.57 µg/g). However, the majority of water-soluble vitamins are destroyed by processing or storage conditions (Oomah et al., 2011;
Wood and Grusak, 2007). In the case of lipid-soluble vitamins, chickpea contains vitamin E (up to 137 µg/g) and vitamin K (up to 137 µg/g). Among all vitamin E forms, chickpea is particularly
rich in tocopherols (up to 124 µg/g). Chickpea also contains β-carotene (up to 0.49 µg/g) that is converted into vitamin A (Jukanti et al., 2012). All lipid-soluble vitamins present in chickpea have health benefits and are more resistance to degradation in comparison to water-soluble vitamins (Wood and Grusak, 2007). In the case of minerals, chickpea contains about 2.91 mg/g of K, 1.68 mg/g of P, 1.38 mg/g of Mg, 1.6 mg/g of Ca 72 µg/g of Fe and 34 µg/g of Zn. Other minerals are present in traces, but one of the most nutritionally important is selenium (up to 0.083 µg/g) due to its antitumoral activity (Oomah et al., 2011).
Antinutritional factors
Despite having high contents of starch, proteins and dietary fiber, chickpea also have some antinutritional compounds which generally interfere with the proper assimilation of proteins and starch. The main antinutritional compounds presented in chickpea are amylase inhibitors, protease inhibitors, tannins, phytic acids, and saponins. It has been reported that chickpea contains up to 13.19 mg/g and 15.7 mg/g of chymotrypsin and trypsin inhibitors, respectively.
These inhibitors belong to the Kunitz and Bowman-Birk inhibitor families which binds competitively to chymotrypsin and trypsin in the digestive system. The overall effect of these protease inhibitors is to reduce the digestion of the proteins in chickpea grains (Wood and Grusak, 2007). Chickpea also contains amylase inhibitors (up to 50 inhibitory activity units/g) which act mainly in pancreatic amylases and reduce starch hydrolysis (Chavan et al., 1987; Wood and Grusak, 2007). Phytic acid content in chickpea has been reported to be up to 1.8 mg/g and its salts (phytate phosphorus) are the major source of phosphorus in this legume. Phytic acid binds to minerals, proteins, and starch forming insoluble complexes that reduce the mineral bioavailability and the protein and starch digestibility (Jukanti et al., 2012; Wood and Grusak, 2007). Moreover, chickpea also contains condensed tannins (up to 0.3%) and saponins (up to 5.6) which along with lipids give to chickpea its characteristic flavor. Tannins also bind with proteins and reduce their digestibility and their solubility, while saponins have haemolytic activity (Millan et al., 2015).
2.1.2 Health benefits
The chemical composition of chickpea do not only determines its nutritional value but is also implicated in many physiological benefits due to the presence of a variety of compounds that have been correlated to cause effects on many health conditions such as obesity, cancer, and coronary diseases. The most common of such bioactive compounds present in chickpea are resistant starch and dietary fiber. Resistant starch is not transformed into glucose in the digestive system which makes chickpea a carbohydrate source with low glycemic index. Foods with low glycemic index are an adequate source of energy for patients with diabetes (Wood and Grusak, 2007). In the same sense, the dietary fiber in chickpea give the sensation of fullness, which reduces the food intake and helps to control obesity. Moreover, dietary fiber has been related to reduce the risk of cardiovascular diseases by decreasing the level of cholesterol and try-acyl glycerol (Gupta et al., 2017).
In addition to carbohydrates, proteins can also contribute to some health benefits. However, they need to be hydrolyzed since peptides are the compounds with bioactivity. In that sense, Yust et al. (2003) extracted the legumin fraction from chickpea globulins and hydrolyzed it with Alcalase®.
This process generated six small peptides with the capacity of inhibiting angiotensin I-converting enzyme (ACE). This enzyme plays a major role in regulation of blood pressure and its inhibition is correlated to a reduction in hypertension. Additionally, albumin fraction has also been hydrolyzed to produce some small peptides (1.2-1.4 kDa). These peptides are composed mainly by histidine and proline which provide them with a strong antioxidant capacity (Xue et al., 2015).
The health benefits associated with resistant starch, dietary fiber, and proteins are important in chickpea since this legume has important amounts of those compounds. However, other compounds presented in small quantities in chickpea also play an important role due to their antioxidant and antitumoral properties. The most important of those compounds present in
chickpea are the phenolic compounds. Chickpea contains a concentration of total phenolic compounds in the range of 1.01 to 2.55 mg/g (Gupta et al., 2017). The main phenolic compounds in chickpea are biochanin A, formononetin, daidzein, and genistein (Jukanti et al., 2012). Due to the importance of phenolic compounds in human health, many efforts have been done to improve their availability in chickpea seeds. Guardado-Félix et al. (2017) found that the availability of isoflavonoid compounds increased up to 83% in chickpea grains after germination with previous soaking in selenium enriched medium. This study also shown that the antioxidant capacity of the isoflavonoid compounds was increased by a 33% in comparison to non-treated chickpea grains.
Based on these results, further studies were done by Guardado-Félix et al. (2018) in order to assess the antitumoral capacity of the extracted isoflavonoids. They found that germinated chickpea with high level of selenium and content of isoflavonoids reduce the proliferation of colon cancer cells (Guardado-Félix et al., 2018) .
2.1.3 Applications of chickpea
The previous sections showed that chickpea is an important nutritious legume and it also have a variety of bioactive compounds. However, chickpea present problems regarding digestibility due to the presence of resistance starch, tannins, and enzyme inhibitors. Many methods have been proposed to overcome these problems and to extend the utilization of chickpea in developing food products. These processes include germination, cooking, roasting, and fermentation (Wood and Grusak, 2007). Among these techniques, cooking is the main method used to process chickpea.
Cooking increases starch, protein, and dietary fiber contents of the chickpea grains. This increment in the content of such compounds is a concentration effect since minerals, phenolics, water-soluble vitamins, sucrose and oligosaccharides diffuse into the cooking water (Wang et al., 2010). Another common process to transform chickpea into a valuable food product is roasting.
Roasting can be performed up to 350°C and can produce several changes in chickpea grain. For
example, the roasted chickpea grains are more expanded and crispier than raw chickpea grains and they also are less hard. In addition, water holding capacity, oil binding capacity and phenolic contents of chickpeas increase as temperature increases during the roasting process (Baik and Han, 2012; Jogihalli et al., 2017). However, roasting of chickpea reduces the water solubility of chickpea flour since proteins are denatured and aggregated due to the high temperature (Jogihalli et al., 2017).
Other important method to increase the potential applications of chickpea in food industry is fermentation. Fermentation of chickpea flour using Rhizopus oligosporus has been studied by Baik and Han (2012) and Xiao et al. (2014), while fermentation with Cordyceps militaris was studied by Reyes-Moreno et al. (2004). These authors concluded that fermentation increases protein digestibility and available lysine in comparison to unfermented chickpeas. Moreover, fermentation increases phenolic content in chickpea flour which enhances its antioxidant capacity but reduces starch and oligosaccharides content.
The previous methods reduce in some degree the nutraceutical quality of chickpeas. Among other methods, germination is a widely used method to process chickpea grain since it does not affect the nutraceutical quality of chickpeas and it is performed under mild conditions. Germination of chickpeas increases the amount of proteins, reduces the amount of total starch and increases protein digestibility (Ghavidel and Prakash, 2007). Also, the water holding capacity is higher for flours from germinated seeds, while the cooking time is reduced (Haileslassie et al., 2019; Xu et al., 2019). Moreover, a recent study shows that germination of chickpea in selenium enriched medium increases in large extent the total phenolic compounds of chickpea grains and hence many of their physiological activities (Guardado-Félix et al., 2017).
The chosen method to process chickpea depends on the final use or application. In India, the most used processes are cooking and roasting. These processes allow to consume chickpeas
directly, or to develop different meals for different purposes alone or in combination with wheat and barley. United Sates and Mexico produce chickpea mainly for exportation, and only few amounts of chickpea are cooked and directly consumed in salads or stews. However, research to develop new food products using chickpea flours or chickpea protein concentrates and isolates has been done in recent years. Shaabani et al. (2018) used millet flour and chickpea protein isolate to prepare a gluten-free muffin. These authors concluded that chickpea isolates in combination with transglutaminase and xanthan gum can produce millet muffins with adequate consistency and crust color. Moreover, Mohammed, Ahmed, & Senge (2014) prepared bread using chickpea and wheat flours. They found that substitution of 10% of the wheat flour with chickpea flour in the formulation for bread preparation increased the water absorption and stability of the subsequent prepared dough and had no effect on color crust of the final prepared bread.
In other study, Collar & Armero (2018) proved that addition of chickpea to wheat flour can produce a bread with high antioxidant capacity.
Besides bread preparation, some authors have incorporated chickpea extracts into beverage- type products to improve its physical properties and to add a vegetable protein source. For instance, Aguilar-Raymundo & Vélez-Ruiz (2019) studied the effect of partial substitution of milk with aqueous chickpea extract on the physical properties and acceptance of a common yogurt.
They concluded that adding chickpea extract to a yogurt formulation produced a food system with similar acceptance to a control yogurt. However, the syneresis of the yogurt increased with the concentration of chickpea extracts. In other study, Li et al. (2016) where able to prepare a fermented vegetable milk with good acceptance and high content of γ-amino butyric acid.
However, there are many problems regarding the preparation of an adequate chickpea beverage.
The most important one is the stability since only a small proportion of proteins and carbohydrates from chickpea are solubilized in a chickpea beverage and the solubility of such compounds is reduced when the beverage is cooled (Wang et al., 2018).
2.2 Extrusion technology
The previous section described processing methods to transform chickpea into a valuable ingredient for food industry. However, cooking, fermentation, and gemination need long time to be carried out. Moreover, cooking and roasting require considerable amounts of energy consumption. Therefore, there is a need of finding sustainable processing methods which use short times and less energy in order to widen the range of applications of chickpea grains. In recent years, extrusion technologies have been widely used for processing of cereals, pseudocereals and legumes and the production of novel ingredients or foods. Particularly, thermoplastic extrusion, which is a high-temperature process that plasticizes and cooks moistened, expansive, starchy and/or high protein food materials in a barrel by a combination of moisture, pressure, temperature and mechanical shear, resulting in diverse chemical reactions (Moscicki, 2011).
Extrusion can effectively modify food materials to yield intermediate ingredients (precooked flours, texturized proteins) or ready-to-eat finished products like expanded snacks and other extruded breakfast cereals (Yovchev et al., 2017). This technology has the advantages of using short times and being an economical and sustainable continuous process (Wani et al., 2019). Extrusion cooking improves digestibility in legumes by eliminating or reducing the content of phytic acid, lectins, tannins, protease inhibitors and α-amylase inhibitors (Alonso et al., 2000). Moreover, the texture and functionality of flours have been extensively modified using extrusion since it promotes the gelatinization and degradation of starch, solubilization of dietary fiber, denaturation of proteins and inactivation of antinutritional factors (Martínez et al., 2014).
Extrusion process is a very popular technology due to its advantages over other thermal processing. These advantages include ease automation, versatility, high productivity, low cost and production of food with diverse shapes (Faraj et al., 2004) . It has been reported that
advances in extrusion technology have allowed the design of low-cost extruders that are already available for food processing in developing countries (Dansby and Bovell-Benkamin, 1997).
However, despite increased use of extrusion technology, extrusion process is a complex system where a variety of parameters need to be monitored in order to create a food product with desirable physical properties and adequate quality (Chen et al., 2010) .
The different effects of extrusion on food materials are generally described in terms of system parameters. The system parameters include the specific mechanical energy input, the thermal input and the residence time of the material into the extruder. System parameters are also known as process response parameters because they depend on other variables called process parameters (Pansawat et al., 2008). The process parameters include screw speed, processing moisture, barrel temperature, screw configuration and type of material to extrude. Both system and process parameters are correlated to the changes in the final product (product parameters) such as texture, color, degree of gelatinization, solubility and viscosity (Brümmer et al., 2002;
Pansawat et al., 2008). The next section describes the structural and chemical changes that extrusion causes on the component of foods, mainly in cereals and legumes.
2.2.1 Effect of extrusion on food chemical components
Starch
Extrusion causes a variety of changes in starch structure that include fragmentation, gelatinization, retrogradation and expansion. The high temperature combined with high shear causes the partial destruction of the crystallinity of starch granules with low content of amylose. In case of starches with high content of amylose, the crystallinity is reduced only if the processing moisture is high (>30%) (Ye et al., 2017). During extrusion with high processing moisture, the water enters through the amorphous state of the starch which losses its integrity by swelling. On the contrary, if the processing moisture is low, then the integrity of the starch granule is difficult to change, but it can
be achieved using high mechanical stress and temperature inside the extruders (Lai and Kokini, 1991). This destruction of the starch granule integrity causes gelatinization of the starch as well as increases viscosity of the melt into the extruder. Then, the gelatinized starch mass can be forced to pass through a die and leave the extruder which causes water evaporation due to pressure differences. This process allow the production of a high expanded and fragile product used mainly for elaboration of rice, corn, and wheat snacks (Camire et al., 1990; Moscicki, 2011).
The extruded starch tends to have both higher water absorption capacity and water solubility index in comparison to raw starch. The increment of water absorption capacity is the result from the rupture of hydrogen bonds in the starch structures which lead to exposure of additional sites for water interaction, while the increment in water solubility index occurs due to dextrinization of amylose chains from the starch (Ye et al., 2017). Extrusion also has important effects on starch digestibility. The starch digestibility is generally increased by extrusion conditions. This increment is attributed to destruction of amylase inhibitors and the transformation of resistant starch fractions into digestible fractions. However, it has been shown that severe extrusion conditions with low processing moisture causes the formation of amylose-lipid complexes which are resistant to enzymatic digestion (Liu et al., 2017).
Proteins
In the same way than starch, proteins go through many structural changes when they are processed by extrusion. The main effect of extrusion on protein concentrates or on materials with high content of proteins is the texturization which can be used to produce meat analogs from legume proteins. Texturization of proteins is usually performed using high shear forces at temperature of 140-180 °C and processing moistures of 10-40% which create a highly wet mass.
When it leaves the extruder, the water evaporates and passes through the mass which creates a complex protein network with many pores and with higher elasticity and water holding capacity (Camire et al., 1990). However, when a mixture of proteins and starch is extruded, the
texturization of proteins that can be reached is generally poor, and more processing moisture is needed to get and adequate texturized protein since both starch and proteins compete for water uptake (Samuelsen et al., 2013).
The texturization is not the only effect of extrusion on protein fractions. The thermal conditions denature proteins and expose new hydrophilic sites which promotes aggregation of proteins (Camire et al., 1990). Moreover, extrusion also causes an increment of β-sheet protein conformation in the protein matrix (Withana-Gamage et al., 2011).The aggregation of proteins and the increment of β-sheet conformation reduce the water solubility of proteins. However, the denaturation of proteins along with the mechanical stress into the extruder destroy protease inhibitors, reduce condensed tannins, and expose other protein chemical sites that are more accessible to be degraded by enzyme. This improvement of susceptibility of proteins to be hydrolyzed also generates an increment of protein digestibility (Alonso et al., 2000).
Dietary fiber
Dietary fiber is also affected by extrusion processes. Extrusion generally does not change the total dietary fiber, but it causes an interconversion of insoluble fiber fraction into soluble fiber fractions (Faraj et al., 2004). The redistribution of fiber fraction occurred because mechanical stress breaks some parts of the insoluble fraction into smaller fragments which are easier to degrade. This is important since soluble fiber have many health benefits (Camire et al., 1990).
Moreover, the fragmentation of dietary fiber molecules reduces their viscosity and particle size distribution (Robin et al., 2012).
Nevertheless, the presence of dietary fiber in some food materials can interfere with functionality of other compounds achieved by extrusion process. For instance, dietary fiber reduces expansion of food products because when the extrudates go out to the die, the water is retained inside the fibrous fractions rather than evaporating. The reduction in expansion is correlated with an
increase of hardness of extrudes products. Moreover, some fragments of dietary fiber can interact with proteins and lead the formation of a resistant fraction which is difficult to digest (Robin et al., 2012).
Lipids
Extrusion causes some changes in the lipids profile. The heat and moisture used in the extrusion process increase the amount of free fatty acids due to degradation of triglycerides. The free fatty acids are not toxic for human consumption, but they are more prone to oxidation which affect the flavor of extruded products (Camire, 2001). Moreover, extrusion can change the conformational structure of polyunsaturated fatty acids from cis to trans forms which destroy its health benefits.
Another important effect of extrusion on lipids is the formation of lipid -protein complexes and amylose-lipid complexes. These complexes make difficult the quantification of crude fat which is the reason why some extrudates had apparent lower lipids than unextruded product (Camire et al., 1990; Singh et al., 2007). In addition to that, lipids act as a lubricant inside the extruder which reduce the mechanical stress on starch granules and also reduce the thermal energy input. This fact can reduce the degree of starch gelatinization (Lin et al., 1997).
Severe extrusion conditions (high screw speed) can be used to extract oil form some vegetable sources. The mechanical stress during extrusion break the spheresomes and release the oil into the matrix inside the extruder. This released oil can be recuperated before the material leave the extruder. Furthermore, using relative high temperatures, the viscosity of oils is reduced which enhances the amount of oil that can be recovered when the extrudate leave the die of the extruder (Uitterhaegen and Evon, 2017).
Vitamins, minerals and other compounds
Extrusion not only affect macronutrients, but also causes changes in the structure and content of micronutrients. For example, vitamins are lost during the extrusion process mainly due to heat
and mechanical stress. Folic acid, ascorbic acid, vitamin A and vitamin E are the most sensible to extrusion conditions. On the other hand, niacin, biotin, and vitamin of B group are quite stable.
However, it is possible to avoid vitamin loss by reducing residence time and water content inside the extruder (Camire et al., 1990; Riaz et al., 2009).
On the other hand, in general, minerals are not affected by extrusion process. However, minerals in some legumes are more available after extrusion because the temperature and shear forces destroy tannins and phytic acid. Also, it is possible to fortify extruded food with iron by small fragments that are released from the screw (Singh et al., 2007).
In the case of phenolic compounds, phenolic acids such as ferulic, syringic and coumaric acids increase after extrusion cooking. This happens because mechanical stress causes rupture of cell tissues which make phenolic acids more available. However, isoflavonoids are very sensible to extrusion conditions, mainly anthocyanin. Loss of isoflavonoids can be reduced by controlling barrel temperature and residence time (Brennan et al., 2011).
2.3 Enzymatic treatments
Enzymes have been used in food industry for long time. Many products are not conceived now without the use of enzymes. Enzymes are used for production of syrups, clarification of juices, production of lactose-free milk, brewing, and baking (James and Simpson, 1996). A variety of enzymes are used in food industry, but the most commonly used for cereal and legume processing are amylases and proteases (Fernandes and Carvalho, 2016; Poutanen, 2020).
Amylases are used in starchy foods and glucose polymers to increase sweetness as well as to increase nutritional value by improving starch digestibility and increasing soluble sugars. In the same way, proteases are used to change functionality of proteins, preparation of protein hydrolysates, improvement of digestibility and release of bioactive peptides (Fernandes and Carvalho, 2016).
In general, additional processing methods are applied in cereal and legumes before performing any enzymatic treatment (Cuadrado and Pedrosa, 2017; Poutanen, 2020) . These processing methods that include cooking, roasting, milling, or germination are required to increase the susceptibility of the food material to be hydrolyzed. For instance, germination of wheat grains does not only improves their nutritional profile but also increases the degree of starch hydrolysis (Poutanen, 2020). In the same way, milling enhance the digestibility of sorghum and helps enzymes to hydrolyze it (Al-Rabadi et al., 2012). Cooking and roasting are also suitable thermal processing methods that enhance the enzymatic hydrolysis of protein and starch in peanuts and lupins (Cuadrado and Pedrosa, 2017). The following sections show some examples of studies where amylolytic and proteolytic treatment are used in cereals and legumes in order to increase their water solubility and how pretreatment of flours with other physical or chemical methods can increase their susceptibility to be hydrolyzed by enzymes.
2.3.1 Amylolytic treatments
Amylases have been used in a wide range of studies to increase the degree of hydrolysis of many cereals and legumes. For instance, Arasaratnam and Balasubramaniam (1993) evaluated the efficiency of α-amylase and β-amylase to hydrolyze dry and wet milled raw corn flour. They found that the amylolytic treatment hydrolyzed more efficiently dry milled corn starch compared to wet milled corn starch. However, in that study, amylases required at least 3 h to reach a high efficiency of hydrolysis.
Therefore, pretreatment of cereal flours with other processing methods before using amylolytic treatments is necessary to increase the rate of starch hydrolysis (Ye et al., 2017). Taking this into consideration, Xu et al. (2014) studied the effect of combining extrusion and α-amylase treatment on functional properties and fermentation efficiency of different rice flours. They found that the solubility of the flour treated with a combination of extrusion and enzymatic treatment is higher in
comparison to extruded flours or. This increment in solubility of the flour is associated to fragmentation of starch which then increased significantly the fermentation efficiency for further Chinese rice wine production.
In the case of legumes, it is known that they have lower degree of hydrolysis than cereals. For instance, Zhang et al. (2016) and Hoover & Zhou (2003) stated that pinto bean, wrinkle pea, horse gram, and chickpea starches are more resistant to hydrolysis by α-amylase than corn starch. In other study, Zhou et al. (2004) evaluated the degradation of different starches from legume sources using pancreatic α-amylase. They concluded that the extent of hydrolysis was greater for black bean and lentil in comparison to pinto bean and wrinkle pea. Moreover, they showed that the structure of starch chains influenced the extent of hydrolysis for each starch legume. However, it is known that the degree of hydrolysis of starch from legumes cannot be performed beyond certain point due to the presence of amylase inhibitors and resistant starch. Therefore, if the aim is to increase the amount of hydrolyzed starch in a legume, it is mandatory to combine amylolytic treatment with heat processing. Among several processing methods, extrusion cooking in combination with thermostable α-amylase in cereals or other starch sources has the potential of increasing degree of starch hydrolysis compared to other thermal processing methods (Govindasamy et al., 1997; Wu et al., 2020; Xu et al., 2014). Moreover, as mentioned in previous sections, extrusion reduce expenses and time consumption.
2.3.2 Proteolytic treatment
Proteases are a group of enzymes that hydrolyze peptide bonds. The proteases are divided into endopeptidases or exopeptidases. This classification considers if the proteases cleaved their substrates in the middle of the polypeptide chain or near the end of the polypeptide chain.
Exopeptidases are further divided into aminopeptidases or carboxypeptidases depending if they act on the n-terminus or the c-terminus. The selection of a certain type of protease depend on
the specific application and solubility of the final product. For example, when high degree of hydrolysis and protein solubility are need, then Alcalase® and Flavourzyme® are the most effective ones (Tavano, 2013).
Several studies have shown the advantage of using Alcalase® or Flavourzyme® to modify the functionality and solubility of vegetable proteins from cereals and legumes. For instance, protein corn hydrolysates were prepared using Alcalase® and Flavourzyme® by Kong, Zhou, & Qian (2007). They found that Alcalase® generated a corn protein hydrolysate with higher degree of hydrolysis and soluble proteins than Flavourzyme®. In the same way, Clemente et al. (2000) proved that a chickpea protein isolate treated with Alcalase® was more soluble than the counterpart treated with Flavourzyme®. However, they also found that a combination of Alcalase® and Flavourzyme® is even more efficient. Moreover, beyond degree of hydrolysis, it has been proved that Alcalase® is one of the best proteases to reduce allergenicity and to increase the number of bioactive peptides in Phaseolus vulgaris. In addition, Alcalase® produces hydrolysates with more acceptability in terms of taste (Tavano, 2013).
Furthermore, processing of cereal or legume proteins before being treated with Alcalase®
increases the degree of hydrolysis. In that sense, germination of soybean grains and subsequent hydrolysis of their proteins using Alcalase® increased the degree of hydrolysis and protein solubility (Yang et al., 2017). In other study, Zheng et al. (2006) showed that the degree of hydrolysis was higher in extruded corn protein hydrolysates in comparison to the non-extruded counterpart. In the same way, Surówka et al. (2004) proved that hydrolysis of extruded soybean concentrates significantly increased its protein solubility.
Despite the promising increment of susceptibility to hydrolysis and protein solubility by means of extrusion process in protein legume concentrates, there are not available studies about the effect of combining extrusion process and proteolytic treatment in whole legumes. Moreover, since
chickpea contains high amount of both protein and starch, the extrusion of whole chickpea could increase its susceptibility to be hydrolyzed by enzymes. Therefore, the combination of extrusion followed by a sequential amylolytic and enzymatic treatment could be used as possible processing methods to transform whole chickpea into a high hydrosoluble ingredient with the potential to develop a beverage-type product with adequate nutritional value.
Chapter 3
3
Materials and methods
3.1 Raw materials
White chickpea grains type Kabuli were purchased at Granos de Sinaloa S.A. de C.V. Once the grain was cleaned to remove foreign material, 20 kg were taken from different areas of the bulk in order to obtain a representative sample from the material. The whole chickpea grains were ground with a blade mill (Thomas Model 5 Wiley® Mill, Swedesboro, NJ) equipped with a 2 mm screen. The resulting whole flour was packed in plastic bags and stored at room temperature for further analyses and labeled as RW. Protein hydrolysis was carried out using the non-specific serine endopeptidase Alcalase® 2.4L (Novozymes) with a specific activity of 2.4 Anson units/mL.
Thermostable α-amylase (Thermozyme® L340) with a specific activity of 340 000 modified Wohlgemuth units/mL and provided by ENMEX was used to conduct starch hydrolysis tests.
3.2 Evaluation of the effect of extrusion conditions on chemical composition, functional properties and susceptibility to hydrolysis of whole chickpea flour
3.2.1 Extrusion treatments
Extrusion trials were conducted with a co-rotating twin-screw extruder (BTSM-30, Bühler AG, Uzwil, Switzerland) equipped with screws of 30 mm diameter. The screw configuration was selected to cause high shear stress for expanded products as described by Cortés-Ceballos, Pérez-Carrillo, & Serna-Saldívar (2015). The feedstock flowrate was kept constant at 31.95 kg/h.
Preliminary trials were carried out in order to set the range of extrusion conditions. Results revealed that conditions for expanded products using high screw speed, relative high barrel temperature, high specific mechanical energy (SME) and adequate processing moisture are
needed to transform chickpea flour into a more soluble flour. After conducting preliminary tests, the three following extrusion treatments were selected: EA, chickpea flour extruded with a SME input of 161.58 Wh/kg produced at 15.6% processing moisture, 143 °C and 450 rpm; EB, chickpea flour extruded with a SME input of 199.13 Wh/kg at 15.6% processing moisture, 150 °C and 700 rpm; and EC, chickpea flour extruded with a SME input of 127.95 Wh/kg at 22.5% processing moisture, 150 °C and 580 rpm. The moisture level of each treatment was adjusted inside the extruder barrel. The necessary water volume to reach the selected moisture level was supplied by the extruder automated liquid feeder to the flow of chickpea flour fed to the extruder barrel.
SME consumed during extrusion was obtained from the operation panel. All extrudates were dried at 70 °C for 12 h, ground with a blade mill (Thomas Model 5 Wiley® Mill, Swedesboro, New Jersey) equipped with a 2 mm screen and flours packed in plastic bags were stored at room temperature for further analysis.
3.2.2 Chemical composition and particle size distribution
Moisture, total protein, crude fat, total starch and ash contents of RW, EA, EB and EC were determined by official methods 925.10, 978.02, 920.85, 996.11 and 923.03, respectively (AOAC, 2000). Soluble and insoluble dietary fiber determined with the official method 991.43 (AOAC, 2000). The analyses were performed in triplicate. Particle size distribution of RW and the three different ground extrudates were carried out in triplicate using the methodology of Schwarz, Barr, Joyce, Power, & Horsley (2002) using a nest of U. S. standard sieves Nos. 20 (841 µm), 35 (500 µm), 60 (250 µm), 100 (149 µm) and a collection pan.
3.2.3 Functional properties
Water holding capacity (WHC) was determined according to the methodology of Regenstein &
Regenstein (1984) whereas the water solubility index (WSI) was calculated with the method of
Wani et al. (2019). Foaming capacity (FC) was determined according to (Martínez et al., 2014) with slight modifications: a suspension of 30 mL with protein content of 1% was whipped with an Ultra Turrax (IKA T18 basic, Wilmington, NC) at 14,000 rpm for 1 min and the foam volume was recorded after 30 s to calculate FC. Protein dispersibility index (PDI) was estimated in distilled water according to (Fallahi et al., 2016) using 1 g of flour dry weight basis (dwb). Analyses were performed in triplicate.
3.2.4 In vitro protein digestibility
In vitro protein digestibility (IVPD) was estimated in triplicate with the multienzyme technique of
Hsu, Vavak, Satterlee, & Miller (1977) in which the pH drop after 10 min incubation was proportional to protein digestibility. The percentage of IVPD was calculated using (1):
= 210.46 − (1)
3.2.5 In vitro starch digestibility
Slowly digestible (SDS), rapidly digestible (RDS) and resistant (RS) starch fractions were determined in duplicate according to the methodology of Gourineni, Stewart, Skorge, & Sekula (2017).
3.2.6 Pasting properties
Pasting properties of the flours were evaluated in a Rapid Visco Analyzer (RVA 1170, Newport Scientific, Warriewood, Australia) according with the methodology of Honců et al. (2016) with some modifications: a suspension of 20% of solids was used and stirred at 160 rpm. The heating- cooling program was performed as follows: hold at 25 °C for 3 min, heat to 95 °C at 10°C/min, hold at 95 °C for 3 min, cool to 25 °C at -15°C/min, hold at 25 °C for 4 min. Analyses were performed in duplicate.
3.2.7 FTIR analysis
Fourier-Transform Infrared (FTIR) Spectrophotometer (Spectrum One, Perkin Elmer) equipped with a universal Attenuated Total Reflectance (ATR) was used to study the changes in starch and protein secondary structures caused by the extrusion treatments. Spectra were collected in duplicate from 4,000 to 800 cm-1 using 10 scans at a resolution of 4 cm-1. In order to compare the results, each spectrogram was subject to normalization, baseline correction and Fourier self - deconvolution with a K-factor of 1.5 as recommended by Withana-Gamage, Wanasundara, Pietrasik, & Shand (2011). The intensity ratios of 995 cm-1/1,022 cm-1 and 1,045 cm-1/1,022 cm-
1 related to degradation and modification of starch (Warren et al., 2016) were calculated from the spectra of all flours. Also, the intensity peaks of the protein secondary structures α-helix (1,654 cm-1) and β-sheet (1,638 cm-1) (Beck et al., 2017) were used to calculate the α-helix/β-sheet ratios.
3.2.8 SEM analysis
The microstructure and degree of damage of each flour were analyzed using Scanning Electron Microscopy (EVO MA 25 SEM Zeiss, Germany). The samples were mounted on a conductive adhesive and coated with gold on an aluminum platform. All examinations were made with an acceleration voltage of 10 kV and photographed at 300X.
3.2.9 Enzymatic protein hydrolysis
Hydrolysis of proteins by Alcalase® was carried out for RW and extrudates flours. Suspensions of 3 g of flour (dry basis) with 25 mL of water were prepared into 50 mL polypropylene tubes and placed into a hot water bath at 60 °C. A tube with 25 mL of suspension was prepared for each time of hydrolysis. The enzymatic reaction started when 200 µL of Alcalase® 2.4L were added and was stopped immersing the tubes with samples in a boiling water bath for 10 min. The degree of protein hydrolysis was determined by measuring free amino nitrogen (FAN) with the ninhydrin
colorimetric method (Lie, 1973). The first 30 min of the kinetics of protein hydrolysis was adjusted to a zero-order reaction described by (2) where kP (FAN min-1) was the kinetic constant and [FAN]
was the concentration of free amino nitrogen.
[ ]⁄ = (2)
3.2.10 Enzymatic starch hydrolysis
Starch hydrolysis was carried out for RW and extrudates flours using α-amylase. Suspensions of 3 g of flour (dry basis) with 25 mL of water were prepared into 50 mL polypropylene tubes and placed in a hot water bath at 95 °C. A tube with 25 mL of suspension was prepared for each time of hydrolysis. The enzymatic reaction started when 35 µL of thermostable α-amylase (Thermozyme® L340) were added and was stopped by adjusting the pH to 3 with 12 M HCl.
Reducing sugars (expressed in g of glucose) produced by enzymatic hydrolysis were measured using the DNS method (Miller, 1959). The degree of starch hydrolysis was expressed as dextrose equivalent (DE) using equation (3).
= ( )
ℎ ℎ ( ) × 0.9 × 100% (3)
The first 20 min of the kinetics of starch hydrolysis was adjusted to a first-order reaction described by equation (4) where kS (min-1) was the kinetic constant, [DE] was the dextrose equivalents and DE∞ was the maximum DE reached.
[ ]⁄ = ∞[1 − exp(− )] (4)
3.2.11 Total soluble solids and protein solubility
Total soluble solids (SS) and soluble protein (SP) were assessed in sample suspensions to evaluate the susceptibility of the extruded chickpea flours to the enzymatic hydrolyses.
Suspensions containing 12% of dry flour were prepared with 25 mL of distilled water into polypropylene tubes. Enzymatic treatment was carried out as follows: first, each tube was equilibrated at 60 °C into a water bath and 200 µL of Alcalase® 2.4 L were added; then, after 30 min of reaction, the temperature was increased to 90 °C and 35 µL of α-amylase (Thermozyme®
L340) were added to react during 20 min. After the enzymatic treatment, tubes were immediately cooled using an ice bath to reach room temperature (22 °C) and centrifuged at 12,000 rpm for 20 min. Resulting supernatants were separated for further SS and SP determinations. The hydrolysis times for Alcalase® and α-amylase were chosen based on results from individual protein and starch hydrolyses. SS were assessed by placing 5 g of supernatant into an aluminum dish and evaporating the solvent at 100 °C in an oven during 24 h. Values of SS were expressed as percentage of dry solids in the supernatant in relationship to the total dry flour weight. SP were evaluated by measuring the total crude protein in the supernatant by the Kjeldahl method and expressed as percentage related to the total protein in the dry flour.
3.3 Preparation and characterization of a soluble food ingredient using sequential Alcalase® and α-amylase on extruded chickpea flour
3.3.1 Preparation of soluble and insoluble fractions
Susceptibility to hydrolysis by Alcalase® and α-amylase is directly related to DE, SS and SP values reached in each sample. Based on the results from these parameters, the EC flour was selected to prepare and characterize a hydrosoluble chickpea-based ingredient with high soluble solids using the following procedure in duplicate: a suspension containing 12% of solids was prepared with 1 L of distilled water into a glass container. The suspension was heated on a hot plate until it reached 60°C with constant agitation. Then, 8 mL of Alcalase® 2.4L was added into the suspension. After 30 min, the temperature of the mixture was raised to 90°C and then, 1.4 mL of thermostable -amylase (Thermolyze® L340) was added. After 20 min of amylolytic
treatment, the mixture was cooled at room temperature (24 °C) and centrifuged at 12 000 rpm for 20 min. Supernatants and pellets were separated, freezed-dried and milled with a coffee grinder. Each dry fraction was packed into plastic bag and stored at room temperature for further analysis. This procedure was also applied to RW for comparison purposes. The supernatants or soluble fractions from RW and EC were labeled as RW-S and EC-S, respectively. In the same way, the pellets or insoluble fractions were labelled as RW-P and EC-P.
3.1.1 Distribution of proteins, fat, and solids
The distribution of solids, proteins and fat in each fraction (RW-S, RW-P, EC-S and EC-P) were calculated by relating the grams of each component with its initial content in RW or EC. These results were expressed in grams per 100 grams of initial content of each component.
3.1.2 Chemical characterization
Moisture, total protein, and crude fat for soluble and insoluble fractions were determined using the official methods mentioned in section 3.2.2. FAN and reducing sugars of the soluble and insoluble fractions from RW and EC, where extracted in distilled water and assayed using the methods described in sections 3.2.9 and 3.2.10, respectively. The analyses were performed in duplicate.
3.1.3 Protein quality
The protein quality is expressed in terms of amino acid profile, IVPD values and protein digestibility corrected amino acid scores (PDCASS). The amino acid profiles of RW, EC and each fraction were determined with the AOAC method 982.30 (AOAC, 2000). The IVPD values for soluble and insoluble fractions was estimated in duplicates as described in section 3.2.4.
PDCAAS were calculated according to WHO/FAO/UNU (2007) using equations (5) and (6).
= (5)
= × (6)
A truncated value of 1.0 was used for the limiting amino acid score when it was greater than 1.0.
3.2 Statistical analysis
Before carrying analysis of variance (ANOVA), data were first tested for normality and homoscedasticity. Data were analyzed by one-way ANOVA and means were compared with Tukey’s test at 95 of confidence (P < 0.05). Experimental data were assessed using the Minitab 19 Statistical Software.
