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Amaranth: Potential Source for Flour Enrichment
C H A P T E R
10 Amaranth: Potential Source for Flour Enrichment Narpinder Singh*, Prabhjeet Singh†, Khetan Shevkani‡, and Amardeep Singh Virdi* *Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India † Department of Biotechnology, Guru Nanak Dev University, Amritsar, India ‡ Department of Applied Agriculture, Central University of Punjab, Bathinda, India
O U T L I N E Introduction
123
Transgenic Applications
132
Grain Characteristics
124
Technological Issues
132
Grain Composition
124
Summary Points
133
Grain Protein Characteristics
127
Acknowledgments
133
Nutraceutical Properties of Amaranth Proteins
130
References
133
Food and Nonfood Applications
130
Further Reading
135
List of Abbreviations AmA API CaMV cDNA GI kDa PAGE SDS
amaranth albumin amaranth protein isolate cauliflower mosaic virus complementary DNA glycemic index kilo Dalton polyacrylamide gel electrophoresis sodium dodecyl sulfate
INTRODUCTION Amaranth (Amaranthus), a traditional Mexican plant is a cosmopolitan genus of herbs with approximately 60 plant species, the majority of which are wild.1 It was cultivated 5000–7000 years ago in areas of Mexico, but the cultivation was discontinued in the early 15th century until the late 1970s, when it was rediscovered and utilized as a new economic crop.2,3 Amaranthus plants have inflorescences and foliage in colors ranging from purple to red to gold. It is a dicotyledonous plant and is also considered as a pseudocereal because of its properties and characteristics.4 Amaranth is generally cultivated in arid zones, where commercial crops cannot be grown. Furthermore, amaranth is also known for its high nutritive value and several nutraceutical health-benefiting effects.5–7
Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00010-1
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© 2019 Elsevier Inc. All rights reserved.
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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT
The Amaranthus plant has a good capacity to produce a high biomass and is used to produce grains, leafy vegetables, and ornamentals. Several species of amaranth are often considered as weeds. Amaranthus cruentus and Amaranthus hypochondriacus are the two species that are primarily cultivated for grain, whereas Amaranthus blitum, Amaranthus dubius, Amaranthus tricolor, Amaranthus lividus, and Amaranthus spinosus are used as vegetables. Amaranthus tricolor and Amaranthus caudatus are also grown for ornamental and decorative purposes. Amaranthus viridis, Amaranthus retroflexus, Amaranthus hybridus, Amaranthus gracilis, Amaranthus gangeticus, Amaranthus paniculatus, and Amaranthus graecizans are wild types of the plant. Its leaves are a potential alternative source of betalains because of betacyanin pigments, and they also show anticancer activity.
GRAIN CHARACTERISTICS Amaranth grains are nearly spherical and about 1 mm in diameter, and they vary in color from creamish yellow to reddish and have unique compositions of protein, carbohydrates, and lipids. A. hypochondriacus produces creamish yellow grains, while A. caudatus has red grains (Fig. 1). Hunter color L*, a*, and b* values are around 62–68, 5.5–6.7, and 21.2–23.7, respectively, for grains of A. hypochondriacus, versus 49–51, 13–13.8, and 10.6–13.2, respectively, for A. caudatus. These two species also differ in grain size. A. hypochondriacus grains were larger and showed greater thousand kernel weight, varying between 0.62 and 0.88 g, as compared to A. caudatus grains, with thousand grain weight ranging between 0.46 and 0.70 g.8 Amaranth seeds have circular embryos or germ, which surrounds the starch-rich perisperm and, together with the seed coat, represent the bran fraction, which is relatively rich in fat and protein.9 The bran fraction is proportionally higher in amaranth seeds in comparison to common cereals, such as maize and wheat, which explains the higher levels of protein and fat present in these seeds.9
GRAIN COMPOSITION Amaranth is a good source of starch, proteins, lipids, dietary fiber, and minerals. On a dry basis, amaranth grains contain 12.5%–15.5% proteins, 73.7%–77.0% carbohydrates, 7.1%–8.0% lipids and 3.0%–3.5% minerals,10 while the dietary fiber content ranges from 19.5%–49.3%.11 Starch is the most abundant carbohydrate in amaranth grain. Amaranth grain has approximately 62%–65% starch, which is made up of amylose and amylopectin. Amylose is essentially a linear polymer of glucose, while amylopectin is highly branched, consisting of a main chain of (1–4)-linked α-D-glucose, along with short chains of (1–6)-α-D-glucose-linked branches. Amaranth starch has low amylose content, ranging from 2% to 12% depending on the genotype. Amylopectin is the most abundant component of amaranth starch. Amaranth starch may contain 90%–98% amylopectin, which is composed of 1700 amylopectin molecules on average,12 which in turn exhibit smooth polymodal chain length distribution, with the peak maxima at degree of polymerization (DP) around 11–12.13 In addition to amylose and amylopectin, amaranth starch granules contain 0.16%–0.28% bound lipids.14 Although starch granule lipids are present as minor components, they have a great effect on starch properties and functionality.15 Amaranth starch granules have diameters ranging between 0.5 and 2.5 μm, which is similar to rice but smaller than that found in the starches of other cereal grains. A comparison of amaranth starch granules with those of wheat, rice, and potato is illustrated in Fig. 2. Amaranth starch has polygonal-shaped granules and displays an A-type X-ray pattern (Fig. 2), which is similar to wheat, rice, and maize starches. Amaranth starch granules are mostly small and show unimodal size distribution; however, the granular size and distribution vary widely among cultivars and species.13 FIG. 1 Grains of two different amaranth species.
A. hypocondriacus
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A. caudatus
125
GRAIN COMPOSITION
FIG. 2 Scanning electron micrographs of different starches.
FIG. 3 X-ray diffractions (XRDs) of wheat and amaranth starch. From Singh, N. Unpublished data.
Relative intensity
Wheat
Amaranth
5
10
15
20
25
30
2(°)
Amaranth starch shows greater crystallinity than wheat starch, with strong reflections at 2°θ ¼ 15.1 degrees, 17.2 degrees, 18.1 degrees, and 23.2 degrees (Fig. 3). An additional peak at 2°θ ¼ 20.0 degrees is usually present, indicating the presence of amylose-lipid complexes. Amaranth starch shows intercultivar variability in crystallinity. Its starch has pasting temperature and gelatinization temperature ranges (To–Tp) between 69°C–72°C and 60°C–77°C, respectively. The difference in pasting behavior among starches from various genotypes has been observed due to differences in amylose content and crystallinity, as well as the presence or absence of amylose-lipid complexes. The pasting curve of starch separated from two genotypes of A. hypocondriacus is illustrated in Fig. 4. In comparison to cereal, pulse, tuber, and root starches, amaranth starch generally produces more stable pastes (low breakdown) owing to its small size and low amylose content.3
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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT
100
3000
80
2000
60
1500 40
IC-540860
1000
Temperature (°C)
Viscosity (cP)
2500
RMA-22
20
500 0
0 0
3
6
9
12
Time (min)
FIG. 4 Pasting curves of starch separated from different amaranth genotypes. From Singh N. Unpublished data. TABLE 1
GIs of Various Foods
Food
GI
Amaranth grain (raw)a
87
a
94
White bread
a
Amaranth grain (popped)
101
a
Amaranth grain (roasted)
106
a
Amaranth grain (flaked)
106
Amaranth grain (extruded)a
91
Amaranth grain (popped)b
97
Pearl barleyb
25
Sweet cornb
53
White riceb
64
Brown riceb
55
Parboiled riceb
47
b
48
Bulgur wheat Corn flakes
b
84
Puffed wheat
b
74
b
70
Wheat bread
b
69
Whole-meal bread b
28
Lentils
b
18
Soybean
Baked beans (canned)
b
48
a Capriles et al.18 [GI (predicted), determined using the equation 39.71 + 0.549 (hydrolysis index) of Goni et al.58]. b Foster-Powell et al.59; the reference food is glucose.
Amaranth grain is a high-glycemic food. Amaranth starch granules digested faster within the first hour than normal cornstarch.16 The rapid digestion of amaranth starch was attributed to its small size, low amylose content, and high tendency to lose its crystalline and granular starch structure completely during heating. The glycemic index (GI) of amaranth is compared with that of other cereal grains and foods in Table 1. The GI defines the carbohydrates present 2. FLOURS AND BREADS
GRAIN PROTEIN CHARACTERISTICS
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in various foods on the basis of the postprandial level of blood glucose.17 The relationship between the rate of in vitro digestion and the glycemic response to food is well known. Raw amaranth seeds had rapidly digestible starch (RDS) content of 30.7% (dry weight basis) and a predicted GI of 87.2.18 Amaranth grains are enriched in various minerals, such as calcium, phosphorus, iron, potassium, and zinc, and vitamins E and B complexes, all of which are concentrated in the bran and germ portions of the grain. Amaranth contains higher amounts of riboflavin and ascorbic acid than cereal and is a good source of vitamin E.7 It also is a rich source of polyphenols (flavonoids), with relatively high antioxidant activity. Caffeic acid, p-hydroxybenzoic acid, and ferulic acid are the main phenolic compounds found in amaranth grains.19 The presence of polyphenols such as rutin (4.0–10.2 mg/g flour) and nicotiflorin (7.2–4.8 mg/g flour) in A. hypochondriacus varieties grown in the Mexican Highlands also has been reported.20 The calcium, magnesium, and oxalate concentrations in the grains of 30 amaranth genotypes of A. cruentus, A. hybridus, and A. hypochondriacus have been studied.21 Calcium and magnesium concentrations in the grains ranged between 134 and 370 mg/100 g and 230 and 387 mg/100 g, respectively, whereas the oxalate content ranged between 178 and 278 mg/100 g. Although dietary oxalate is a potential risk factor for kidney stone development and reduces the availability of calcium and magnesium, most of the oxalates in the amaranth grains are in insoluble form; as a result, the absorption may be low. However, this needs to be confirmed by bioavailability investigation. The dietary fiber and lipid content in the amaranth grain ranged between 8% and 17% and 3.0% and 10.5%, respectively. Although amaranth grain contains higher lipids than most of the cereals, the composition of its oil is quite similar to that of cereals, being high in unsaturated fatty acids (approximately 77%). Amaranth oil contains mainly linoleic acid and also contains tocotrienols that are associated with cholesterol-lowering activity in mammalian systems.22 Amaranth grain oil contains a significant amount (up to 8%) of squalene, exhibiting anticarcinogenic and hypocholesterolemic effects.23,24 Therefore, amaranth oil has the potential of replacing other squalene sources (e.g., shark and whale, which are endangered species).
GRAIN PROTEIN CHARACTERISTICS Cereals are normally deficient in lysine and tryptophan, whereas legume proteins show a deficiency of sulfurcontaining amino acids (namely, cysteine and methionine). Amaranth proteins, on the contrary, contain significant amounts of both sulfur-containing amino acids and lysine. Amaranth grains have higher protein (11%–17%) than most cereals. Amaranth is an appropriate grain for people who are allergic to gluten. The germ and endosperm of amaranth grain contain 65% and 35% of protein against an average of 15% and 85% in most cereals, respectively. The amino acid composition of various amaranth protein fractions is given in Table 2. Albumins and globulins are relatively rich in lysine and valine, while glutenins are high in leucine, threonine, and histidine. Aside from the amino acid composition, the protein quality also depends on bioavailability or digestibility. Protein digestibility, available lysine, net protein utilization, and protein efficiency ratio, which are indicators of protein nutritional quality, are substantially higher for amaranth protein than for cereal grains.25 Therefore, amaranth proteins are a promising food ingredient, capable of complementing and supplementing cereal or legume proteins.25 The protein digestibility corrected amino acid score of amaranth whole-meal flour is higher (0.64) than those of wheat (0.40) and oats (0.57).26 An average protein digestibility of 74.2% for raw amaranth whole-meal flour was reported.26 Thermal processing improves protein digestibility due to the opening of carbohydrate-protein complexes, the inactivation of antinutritional factors such as trypsin inhibitors, or both.26 Apart from their nutritional properties, amaranth proteins also possess functional properties that play important roles in formulation and processing.27 Amaranth protein isolate (API) improved the quality attributes (i.e., crust color, appearance, volume, height, springiness, and cohesiveness) and produced gluten-free muffins comparable to those with gluten.28 Contrary to legumes and cereals, where grain proteins generally serve as storage molecules for the growing plantlets, amaranth grain consists of the highest amount of albumins, which are usually biologically active. According to the Osborne classification, amaranth grain consists of three major fractions [namely, albumins (51%), globulins (16%), and glutelins (24%)] and a minor fraction (i.e., alcohol-soluble fraction or prolamine between 1.4% and 2.0%),29,30 whereas legume grain contains salt-soluble globulins as the major storage protein fraction. Based on size exclusion chromatographic analysis, it was reported that globulins were prominent proteins in kidney beans and field peas, while amaranth proteins contained both globulins and albumins as major fractions.31 Cereals such as maize and wheat, on the contrary, contain alcohol-soluble prolamins as the major storage proteins.32 The characterization of grain proteins of amaranth has been carried out using different techniques of extraction and electrophoresis.29,32–34 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of amaranth proteins 2. FLOURS AND BREADS
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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT
TABLE 2 Amino Acid Composition of Amaranth (A. hypochondriacus L.) Protein Fractions Protein Fractions Amino Acid
Meal
Albumins
Globulins
Prolamins
Glutelins
Isoleucinea
–
3.7
4.2
6.2
5.8
Leucinea
–
5.7
5.7
5.7
10.5
Lysinea
–
7.6
6.7
4.2
4.6
Methioninea
–
4.1
3.4
7.4
3.1
Cystinea
–
5.9
3.9
6.5
6.2
Phenylalaninea
–
5.1
5.0
9.0
6.8
Tyrosinea
–
3.3
4.3
4.0
3.8
–
3.9
4.1
3.2
8.6
–
4.5
4.7
2.7
3.8
–
2.5
1.1
1.1
4.7
–
5.1
4.0
4.7
3.6
–
8.1
9.5
9.4
2.7
–
6.2
8.7
6.2
6.1
–
17.5
17.3
13.4
13.2
Glycine
–
6.2
6.6
4.4
4.9
a
Proline
–
3.7
3.9
4.7
4.6
Serine
a
–
4.8
4.9
5.1
5.3
Serine
b
7.3
6.4
7.7
8.0
9.0
10.7
10.5
13.9
10.7
10.3
3.0
2.3
2.3
1.8
2.4
7.3
8.9
9.3
6.8
8.5
5.1
3.4
4.0
7.2
5.4
6.6
6.2
5.4
8.6
6.3
5.7
5.0
4.0
4.5
5.9
1.9
2.9
2.8
3.0
3.0
5.9
4.0
5.0
4.5
5.0
3.9
3.5
4.0
4.5
5.0
6.2
5.5
6.0
10.0
8.0
3.4
3.0
2.0
3.9
4.3
5.7
6.6
7.0
6.7
4.2
a
Threonine a
Valine
a
Histidine a
Alanine
a
Arginine
a
Aspartic acid
a
Glutamic acid a
b
Glycine
b
Histidine
b
Arginine
b
Threonine b
Alanine
b
Proline
b
Tyrosine b
Valine
b
Isoleucine b
Leucine
Phenylalanine b
Lysine
b
a
Expressed as g of amino acids/100 g of crude protein. Expressed in percent in moles. From Barba de la Rosa AP, Gueguen J, Paredes-Lopez O, Viroben G. Fractionation procedures, electrophoretic characterization and amino acid composition of amaranth seed protein. J Agric Food Chem 1992;40:931–936; Segura-Nieto M, Vaazquez-sanchez N, Rubio-Velazqez H, Olguin-Martin LE, Rodriguez-Nester CE, HerreraEstrella L. Characterization of amaranth (Amaranthus hypocondriacus) seed proteins. J Agric Food Chem 1992;40:1553–1558.
b
reveals a wide range in the molecular weight of polypeptide subunits among varieties and species (Fig. 5). On the basis of differential extraction, the amaranth albumin (AmA) was classified as albumin 1 and albumin 2.35 Albumin 1 is extractable with water, saline solution, or both, whereas albumin 2 is extractable with water after the removal of albumin 1 and globulin with saline solution. Albumin 2 has amarantin as the major protein component.30 The subunit size of albumin proteins varied from 10 to 37 kDa,29,33 with low molecular subunits being more abundant.36 Barbara de la
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97.4 66.0
43.0
110 96 91 73 61 56 53 43
(A)
IC540862
IC540860
IC540839
IC467901
IC042311–7
IC042254–5
IC042265–2
IC095341
52 43.0
42 40 38 33
29.0
28
26 24
26 23
23
22
20
20.0
18 17 16 14.3
IC38312
66.0
34 31
20.0
VL–0344
97.4
38
29.0
RMA–30
RMA–22
PRA–3
PRA–2
Annapurna
Marker
IC540869
IC540828
Amaranthus hypochondriacus
IC467902
IC423448
IC423393
IC363742
IC258399
IC38181
IC38165
Marker
Amaranthus caudatus
10
20 16
14.3
15 14
(B)
FIG. 5 SDS-PAGE (12% resolving gel) analysis (under reducing conditions) of (A) A. caudatus and (B) A. hypochondriacus protein isolates. From Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. Thesis submitted to Guru Nanak Dev University, India, 2014.
Rosa et al.,20 however, differentiated the albumin fraction into two groups of proteins corresponding to around 18 kDa and between 40 and 80 kDa. The proteins of around 18 kDa were termed methionine-rich proteins due to their high methionine content (between 16% and 18%).37 Determination of sedimentation coefficient by centrifugation has also been widely used to characterize the proteins. On the basis of sedimentation coefficients, the amaranth seed globulins are categorized into 10S and 12.7S, as compared to 7/8S and 11/12S for the legume seed globulins. The electrophoretic behavior of 10S and 12.7S amaranth globulin fractions on denaturing gel was observed to be similar to that of 7S and 11S storage proteins of legumes; hence, they are referred to as 7S and 11S, respectively.34 The higher sedimentation coefficients of amaranth globulins, as observed on linear sucrose gradients, suggested that these proteins contain polypeptides with higher molecular weight than those present in 7S and 11S from pea globulins.36 The 7S and 11S amaranth seed globulins also differed in their solubility in salt solution, with the former being extractable with 0.1 M and the latter with 0.8 M of sodium chloride (NaCl).20 The 7S globulin fraction of amaranth grain was characterized by the presence of a main band of 38 kDa and lacked disulfide bridges, whereas the 11S-like globulins consisted of both acidic (35–38 kDa) and basic polypeptides (22–25 kDa). These results were in agreement with earlier studies finding that globulins consisted of polypeptides of heterogenous sizes, as demonstrated by SDS-PAGE analysis.36 However, these observations contradicted the findings of Gorinstein et al.,29 which reported that the globulin was composed of polypeptides of only 14–18 kDa. Martinez et al.,30 proposed that both 7S and 11S globulins correspond to one type of globulin, whereas polymerized globulins (albumin 2) and glutelins correspond to two other types of globulins. This notion, however, needs to be supported by establishing sequence homology and by proving a common genetic origin of globulin, albumin 2, and glutelin. The most important component of globulins is amarantin, which alone constitutes 90% of the total globulins and about 19% of the total grain protein.38 Amarantin is a homohexameric molecule of approximately 300–400 kDa, comprised of subunits of about 59 kDa. Each of these subunits consists of an acidic polypeptide of 34–36 kDa and a basic polypeptide of 22–24 kDa linked by disulfide bonds. The additional subunit of 54 kDa present in amarantin has been proposed to act as an inducer of polymerization.30
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The amaranth glutelin showed high similarity with 11S globulins and was comprised of three major polypeptide groups of 22–25 kDa, 35–38 kDa, and around 55 kDa.39 It is likely that both glutelins and 11S globulins may belong to the same structural gene family. The differences in composition of alcohol-soluble proteins have been reported in various studies. Gorinstein et al.29 observed only low-molecular-weight subunits of 10–20 kDa via SDS-PAGE analysis of the alcohol-soluble proteins, whereas Barba de la Rosa et al.33 reported the presence of subunits with both low and high molecular mass. Furthermore, great similarity between the electrophoretic patterns of reduced prolamine and glutelins was also observed in the latter study. The lack of consistency in the composition of various protein fractions of amaranth grain, which is evident from the literature, may be due to the various procedures used in the extraction and analyses. In view of the nutritional importance of the amaranth, it is imperative that a systematic study should be undertaken to analyze the proteome of amaranth leaves and grains by employing the latest techniques of proteomics. This will enable the identification and characterization of nutritionally important proteins, the genes for which can then be cloned and expressed heterologously in other crops for enhancing their nutritive value.
NUTRACEUTICAL PROPERTIES OF AMARANTH PROTEINS The albumins and globulins are rich in lysine and valine, whereas prolamins have comparatively higher content of methionine and cystine. Glutelins, on the other hand, contain higher levels of leucine, threonine, and histidine. Compared to legume grain albumins, which contain several antinutritional factors, the AmA fraction is considered safe. The AmA fraction is comparable with egg-white proteins and can be used as an egg substitute in various products. The 11S globulin fraction is rich in peptides of angiotensin-converting enzyme (ACE) inhibitor, whereas the glutelin fraction contains antihypertensive activity, as well as the anticarcinogenic lunasinlike peptide,40 thus signifying its nutraceutical properties. Amaranth proteins also exhibit antioxidant activity, which increases after gastrointestinal digestion41 and potential antitumor properties through putative mechanism of action.42 In addition, they affect the metabolism of liver lipids and have a hypotriglyceridemic effect in rats.24 Simulated gastrointestinal digestion of APIs resulted in the identification of several bioactive peptides, which showed very high antithrombotic activity. These bioactive peptides belong to 11S globulin and agglutinin fractions of amaranth.43 An amaranth peptide (SSEDIKE) attenuated the expression of the CCL20 (Caco-2 cells transfected with a luciferase reporter under the control of the CCL20 promoter) gene at the transcript level in colonic epithelial cells. Thus, the antiinflammatory effect of amaranth protein hydrolysate was demonstrated at molecular levels.44 Milk allergens are a major cause of intestinal inflammation and are considered as allergens. Oral administration of the amaranth-charged peptide (SSEDIKE) resulted in the inhibition of the allergen response in a mouse model system.44 Delgado et al.45 demonstrated that the simulated gastrointestinal digestion of Amaranthus mantegazzianus proteins yielded bioactive peptides (i.e., AWEEREQGSR, YLAGKPQQEH, IYIEQGNGITGM, and TEVWDSNEQ), which showed potential antioxidant activity and belongs to the 11S globulin protein family. Furthermore, amaranth bioactive cationic (HVIKPPSRA and KFNRPETT) and neutral (GDRFQDQHQ) peptides were also shown to exhibit the in vivo inhibition of Cu+2/H2O2-induced LDL oxidation.46
FOOD AND NONFOOD APPLICATIONS The unique small starch granule size and composition has been suggested to be responsible for unique gelatinization and freeze/thaw characteristics, which could be exploited in the development of products by the food industry.47 Amaranth starch can be used in many food preparations, such as custards, pastes, and salad, and nonfood applications such as cosmetics, biodegradable films, paper coatings, and laundry starch. The modified A. viridis starches also have the potential to partially replace fat in fat-rich foods such as mayonnaise and salad cream.48 Amaranth flour is used as a thickener in gravies, soups, and stews. Sprouted amaranth is used in salads. Defatted amaranth flours may be preferred over full-fat flours, as they have better flavor stability and can be marketed as low-fat or low-calorie products. In addition, extracted oil can be used for other purposes and these flours have improved functionality.10 However, the improvement in the functional properties of flours depends on protein characteristics, which in turn vary based on the cultivars.10,27 The cooking of amaranth improves its digestibility and absorption of nutrients. Amaranth flour lacks the gluten proteins present in wheat, and hence it is not suitable for bread-making. A blend of amaranth flour with wheat meal or flour is used in the preparation of an unleavened flatbread known as chapatis in India and tortillas in Latin America. Amaranth flour is also used to prepare biscuits, muffins, pancakes, pastas, flatbreads, extruded products, and other foodstuffs. In India, the grains are most commonly used in the form of candy known as laddoos. 2. FLOURS AND BREADS
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131
Waxy and normal maize starch granules were transformed into starch nanocrystals, and protein films were prepared after blending with amaranth protein formulations.49 One study demonstrated that nanocomposite films exhibited improved water vapor permeability, water uptake, surface hydrophobicity, mechanical behavior, and delayed weight loss in soil than that of normal protein films. It was proposed that the good dispersion of the nanoreinforcements in the protein matrix is due to the formation of disulfide bonds among waxy maize nanocrystals, as well as hydrogen bonding between normal maize nanocrystals and protein matrix.49 Amaranth proteins compared well with pulse (kidney bean and field pea) proteins in terms of edible/biodegradable film-forming31 and gluten-free muffinmaking (Fig. 6) properties.28 Furthermore, API edible films prepared from different ultra-high-pressure treatments demonstrated uniform surface and more opaque characteristics, along with improved mechanical properties, lower water solubility, and water vapor permeability, as compared to nontreated protein dispersions.50,51 Encapsulation of folic acid into API-pullulan electrospun fibers resulted in photoprotection of folic acid.52 It has been reported that APIs at acidic pH (2.0) show improved foam stability over those with alkaline pH.53 Increased unfolding, greater flexibility, and the net charge on amaranth proteins under acidic conditions were attributed to
FIG. 6 Crusts (I) and crumb cross sections (II) of muffins prepared from (A) cornstarch; (B) cornstarch with wheat gluten; (C) cornstarch with kidney bean protein isolate; (D) cornstarch with field pea protein isolate; and (E) cornstarch with API. The starch was replaced with calculated amount of proteins in order to get a protein content of 10%. From Shevkani K, Singh N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Int J Food Sci Tech 2014;49:2237–2244.
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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT
the higher foam stability of proteins. These findings thus demonstrated that the viscoelasticity and flexibility of foams were influenced by the solubility of protein isolates at different ionic strengths and pHs of solubilizing solvents.53 Suarez and Añón54 also demonstrated that APIs solubilized at low pH (2.0) adsorbed in an oil/water interface twice as quickly as APIs prepared at pH 6.3 and allowed in the formation of oil-in-water emulsions. These findings indicated the treatment of APIs at various ionic and pH conditions may be helpful in the improvement of texture and sensory properties of food products with improved nutraceutical values. In comparison to amaranth grain, vegetable amaranth has received less attention. Vegetable amaranth is used as a delicacy or a food staple in many parts of the world. Amaranthus leaves are used as a vegetable in the northern states of India. However, its use is limited to saag, a dish prepared by cooking mustard leaves with garlic, ginger, green chilies, and salt. Vegetable amaranth tastes better than spinach and is substantially higher in calcium, iron, and phosphorous.
TRANSGENIC APPLICATIONS Improving the balance of essential amino acids in important crop plants remains one of the major objectives of plant breeders. Transgenic technology presents an attractive alternative for improving the nutritional quality of grain proteins. Heterologous transgenic expression of storage protein genes with higher levels of limiting amino acids has been reported. Transgenic expression of high levels of a particular amino acid may affect the normal physiology of seed development adversely or produce seeds with a biased amino acid composition. Therefore, expressing a gene for a heterologous protein with a balanced amino acid composition is a better alternative. A gene of a 35-kDa albumin protein (AmA1), which is expressed during early to mid-maturation stages of embryogenesis in the amaranth seed was cloned.55 The amino acid composition of this protein conforms to the World Health Organization (WHO)–recommended values for a highly nutritional protein because it is rich in various essential amino acids. Potato is the most important noncereal crop in terms of total global food production; therefore, transgenic expression of this gene in the tubers of this crop has been achieved. Heterologous expression of AmA1 under constitutive [Cauliflower mosaic virus (CaMV) 35S promoter] and tuber-specific promoter (granule-bound starch synthase) in potato resulted in significant enhancement in total protein content, with an increase in essential amino acids.56 Furthermore, the growth and production of tubers in transgenic plants were also higher than for control plants. The maize, which is a staple food in many countries but lacks essential amino acid in the grains, has also been targeted for heterologous expression of amaranth proteins to enhance the nutritional quality of its protein. Complementary DNA (cDNA) of an 11S globulin storage protein, amarantin, which has a high content of essential amino acids, was expressed in maize under the CaMV 35S promoter and an endosperm-specific promoter (rice glutelin-1).57 Heterologous expression of this gene resulted in an increase of 18% in lysine, 28% in sulfur-containing amino acids, and 36% in isoleucine, as well as a 32% increase in total seed protein. Furthermore, the heterologously expressed protein was digested by simulated gastric and intestinal fluids, thus confirming the biodigestibility of the transgenic protein. These studies, therefore, validate the potential of using various amaranth genes for supplementing and complementing the proteins in both cereal and noncereal staple crops. However, detailed studies on the analysis of proteomes of amaranth species needs to be carried out to identify the candidate genes, which can be employed for transgenic improvement. Furthermore, the generation of mutants in amaranth is also required to determine the role of specific proteins in the growth and development of the plant so that appropriate improvement of the germplasm can be undertaken through conventional breeding strategies.
TECHNOLOGICAL ISSUES The presence of diversity in composition among amaranth genotypes necessitates in-depth characterization of biochemical constituents for its specific applications in the food industry. The smaller granules in amaranth starch, which resemble the size of fat globules in cow’s milk, can be exploited to mimic fat in a number of food products. Some of the genotypes have higher polyphenols with higher antioxidant activity, which also could be utilized in the development of new products. Amaranth grain has the potential to develop various food products for people suffering from celiac disease, a disorder that makes the body intolerant to gluten proteins.
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SUMMARY POINTS • Amaranth grain is a good source of dietary fiber and has high GI. It is low in resistant starch (RS), and its starch has uniquely small granules with low tendency toward retrogradation. • Grain amaranth has higher protein than most of the cereal grains and is an appropriate food for people who are allergic to gluten. • Amaranth grain oil is quite similar to that of cereals, being high in unsaturated fatty acids, and it contains mainly linoleic acid. Its oil also contains tocotrienols, which are associated with cholesterol-lowering activity in mammalian systems. Amaranthus grain oil also contains a significant amount of squalene, which has anticarcinogenic and hypocholesterolemic effects. • Amaranth grain proteins are mainly composed of three major fractions (namely, albumins, globulins, and glutelins) with little or no storage of prolamin. Amarantin is the most important component of globulins and constitutes 90% of the total globulins and about 19% of the total grain protein. • Heterologous expression of the amarantin gene (AmA1) in potato results in significant enhancement in total protein content, with an increase in essential amino acids as well. • Amaranth is also a good source of minerals such as iron, magnesium, phosphorus, copper, and manganese. Its unique composition also makes it an attractive food complement and supplement.
Acknowledgments The financial assistance to NS from the Department of Science and Technology, Ministry of Science and Technology, Government of India, is acknowledged.
References 1. Stallknecht GE, Schulz-Schaeffer JR. Amaranth rediscovered. In: Janick J, Simon JE, editors. New crops. New York: John Wiley & Sons; 1993. p. 211–8. 2. Cai YZ, Corke H, Wu HX. Amaranth. In: Wrigley CW, Corke H, Walker CE, editors. Encyclopedia of grain science. Vol. 1. Oxford: Elsevier; 2004. p. 1–10. 3. Zhu F. Structures, physicochemical properties, and applications of amaranth starch. Crit Rev Food Sci Nutr 2017;57:313–25. 4. Breene WM. Food uses of grain amaranth. Cereal Foods World 1991;36:426–30. 5. Plate AY, Ar^eas JA. Cholesterol-lowering effect of extruded amaranth (Amaranthus caudatus L.) in hypercholesterolemic rabbits. Food Chem 2002;76:1–6. 6. Mendonca S, Saldiva PH, Cruz RJ, Ar^eas JA. Amaranth protein presents cholesterol-lowering effect. Food Chem 2009;116:738–42. 7. Rastogi A, Shukla S. Amaranth: a new millennium crop of nutraceutical values. Crit Rev Food Sci Nutr 2013;53:109–25. 8. Kaur S, Singh N, Rana JC. Amaranthus hypochondriacus and Amaranthus caudatus germplasm: characteristics of plants, grain and flours. Food Chem 2010;123:1227–34. 9. Bressani R. Composition and nutritional properties of amaranth. In: Peredes-Lopez O, editor. Amaranth biology, chemistry and technology. Boca Raton: CRC Press; 1994. p. 185–205. 10. Shevkani K, Singh N, Kaur A, Rana JC. Physicochemical, pasting, and functional properties of amaranth seed flours: effects of lipids removal. J Food Sci 2014;79:C1271–7. 11. Pedersen B, Knudsen KB, Eggum BO. The nutritive value of amaranth grain (Amaranthus caudatus). Plant Foods Hum Nutr 1990;40:61–71. 12. Wilhelm E, Aberle T, Burchard W, Landers R. Peculiarities of aqueous amaranth starch suspensions. Biomacromolecules 2002;3:17–26. 13. Singh N, Kaur S, Kaur A, Isono N, Ichihashi Y, Noda T, Rana JC. Structural, thermal, and rheological properties of Amaranthus hypochondriacus and Amaranthus caudatus starches. Starch 2014;66:457–67. 14. Hoover R, Sinnott AW, Perera C. Physicochemical characterization of starches from Amaranthus cruentus grains. Starch-St€ arke 1998;50:456–63. 15. Shevkani K, Singh N, Bajaj R, Kaur A. Wheat starch production, structure, functionality and applications—a review. Int J Food Sci Technol 2017;52:38–58. 16. Konishi Y, Nojima H, Okuno K, Asaoka M, Fuwa H. Characterization of starch granules from waxy, nonwaxy, and hybrid seeds of Amaranthus hypochondriacus L. Agric Biol Chem 1985;49:1965–71. 17. Jenkins AL. The glycemic index: looking back 25 years. Cereal Foods World 2007;52:50–3. 18. Capriles VD, Coelho KD, Guerra-Matias AC, Areas JA. Effects of processing methods on amaranth starch digestibility and predicted glycemic index. J Food Sci 2008;73:H160–4. 19. Klimczak I, Malecka M, Pacholek B. Antioxidant activity of ethanolic extracts of amaranth seeds. Nahrung 2002;46:184–6. 20. Barba de la Rosa AP, Fomsgaard IS, Laursen B, Mortensen AG, Olvera-Martınez L, Silva-Sanchez C, Mendoza-Herrera A, Gonzalez-Castaneda J, De Leon-Rodrıguez A. Amaranth (Amaranthus hypochondriacus) as an alternative crop for sustainable food production: phenolic acids and flavonoids with potential impact on its nutraceutical quality. J Cereal Sci 2009;49:117–21. 21. Gelinas B, Seguin P. Oxalate in grain amaranth. J Agric Food Chem 2007;55:4789–94. 22. Becker R. Preparation, composition and nutritional implications of amaranth seed oil. Cereal Foods World 1989;36:426–9.
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23. Sun H, Wiesenborn D, Tostenson K, Gillespie J, Rayas-Duarte P. Fractionation of squalene from amaranth seed oil. J Am Oil Chem Soc 1997;74:413–8. 24. Escudero NL, Zirulnik F, Gomez NN, Mucciarelli SI, Mucciarelli SI, Gimenez MS. Influence of a protein concentrate from Amaranthus cruentus seeds on lipid metabolism. Exp Biol Med 2006;231:50–9. 25. Guzman-Maldonado SH, Paredes-Lopez O. Biotechnology for the improvement of nutritional quality of food crop plants. In: Paredes-Lo’pez O, editor. Molecular biotechnology for plant food production. Lancaster: Technomic Publishing; 1999. p. 553–620. 26. Bejosano FP, Corke H. Protein quality evaluation of Amaranthus wholemeal flours and protein concentrates. J Sci Food Agric 1998;76:100–6. 27. Shevkani K, Singh N, Rana JC, Kaur A. Relationship between physicochemical and functional properties of amaranth (Amaranthus hypochondriacus) protein isolates. Int J Food Sci Technol 2014;49:541–50. 28. Shevkani K, Singh N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Int J Food Sci Technol 2014;49:2237–44. 29. Gorinstein S, Moshe R, Greene LJ, Arruda P. Evaluation of four amaranthus species through protein electrophoretical patterns and their amino acid composition. J Agric Food Chem 1991;51:851–4. 30. Martinez EN, Castellani OF, Anon MC. Common molecular features among amaranth storage proteins. J Agric Food Chem 1997;46:4849–53. 31. Shevkani K, Singh N. Relationship between protein characteristics and film-forming properties of kidney bean, field pea and amaranth protein isolates. Int J Food Sci Technol 2014;50:1033–43. 32. Gorinstein S, Denue IA, Arruda P. Alcohol soluble and total proteins from amaranth seeds and their comparison with other cereals. J Agric Food Chem 1991;39:848–50. 33. Barba de la Rosa AP, Gueguen J, Paredes-Lopez O, Viroben G. Fractionation procedures, electrophoretic characterization and amino acid composition of amaranth seed protein. J Agric Food Chem 1992;40:931–6. 34. Barba de la Rosa AP, Paredes-Lopez O, Gueguen J. Characterization of amaranth globulins by ultracentrifugation and chromatographic techniques. J Agric Food Chem 1992;40:937–40. 35. Konishi Y, Horikawa K, Oku Y, Azumaya J, Nakatani N. Extraction of two albumin fractions from amaranth grains: comparison of some physicochemical properties and putative localization in the grains. J Agric Biol Chem 1991;55:1745–50. 36. Segura-Nieto M, Vaazquez-sanchez N, Rubio-Velazqez H, Olguin-Martin LE, Rodriguez-Nester CE, Herrera-Estrella L. Characterization of amaranth (Amaranthus hypocondriacus) seed proteins. J Agric Food Chem 1992;40:1553–8. 37. Segura-Nieto M, Barba-de-la-Rosa AP, Paredes-Lopez O. Biochemistry of amaranth proteins. In: Paredes-Lopez O, editor. Amaranth biology, chemistry and technology. Boca Raton: CRC Press; 1994. p. 75–106. 38. Romero-Zepada H, Paredes-Lopez O. Isolation and characterization of amarantin, the 11S amaranth seed globulin. J Agric Food Chem 1996;19:329–39. 39. Abugoch LE, Martinez EN, Anon MC. Influence of the extracting solvent upon the structural properties of amaranth (Amaranthus hypochondriacus) glutelin. J Agric Food Chem 2003;51:460–5. 40. Silva-Sanchez C, Barba de la Rosa AP, Leon-Galvan F, de Lumen BO, De Leon-Rodrıguez A, Gonzalez de Mejıa E. Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed storage proteins. J Agric Chem 2008;56:1233–40. 41. Delgado MCO, Tironi VA, Añón MC. Antioxidant activity of amaranth protein or their hydrolysates under simulated gastrointestinal digestion. LWT Food Sci Technol 2011;44:1752–60. 42. Barrio DA, Anon MC. Potential antitumor properties of a protein isolate obtained from the seeds of Amaranthus mantegazzianus. Eur J Nutr 2010;49:73–82. 43. Sabbione AC, Nardo AE, Añón MC, Scilingo A. Amaranth peptides with antithrombotic activity released by simulated gastrointestinal digestion. J Funct Foods 2016;20:204–14. 44. Moronta J, Smaldini PL, Fossati CA, Añon MC, Docena GH. The anti-inflammatory SSEDIKE peptide from Amaranth seeds modulates IgEmediated food allergy. J Funct Foods 2016;25:579–87. 45. Delgado MCO, Nardo A, Pavlovic M, Rogniaux H, Añón MC, Tironi VA. Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins. Food Chem 2016;197:1160–7. 46. Fillería SFG, Tironi VA. Prevention of in vitro oxidation of low density lipoproteins (LDL) by amaranth peptides released by gastrointestinal digestion. J Funct Foods 2017;34:197–206. 47. Becker R, Wheeler EL, Lorenz K, Stafford AE, Grosjean OK, Betschart AA. A compositional study of amaranth grain. J Food Sci 1981;46:1175–8. 48. Fasuan TO, Gbadamosi SO, Akanbi CT. Modification of amaranth (Amaranthus viridis) starch, identification of functional groups, and its potentials as fat replacer. J Food Biochem 2018; https://doi.org/10.1111/jfbc.12537. In press. 49. Condes MC, Añón MC, Mauri AN, Dufresne A. Amaranth protein films reinforced with maize starch nanocrystals. Food Hydrocoll 2015;47:146–57. 50. Condes MC, Añón MC, Mauri AN. Amaranth protein films prepared with high-pressure treated proteins. J Food Eng 2015;166:38–44. 51. Condes MC, Añón MC, Dufresne A, Mauri AN. Composite and nanocomposite films based on amaranth biopolymers. Food Hydrocoll 2018;74:159–67. 52. Aceituno-Medina M, Mendoza S, Lagaron JM, López-Rubio A. Photoprotection of folic acid upon encapsulation in food-grade amaranth (Amaranthus hypochondriacus L.) protein isolate—pullulan electrospun fibers. LWT Food Sci Technol 2015;62:970–5. 53. Bolontrade AJ, Scilingo AA, Añón MC. Amaranth proteins foaming properties: film rheology and foam stability-part 2. Colloids Surf B: Biointerfaces 2016;141:643–50. 54. Suarez SE, Añón MC. Comparative behaviour of solutions and dispersions of amaranth proteins on their emulsifying properties. Food Hydrocoll 2018;74:115–23. 55. Raina A, Datta A. Molecular-cloning of a gene encoding a seed-specific protein with nutritionally balanced amino-acid-composition from Amaranthus. Proc Natl Acad Sci U S A 1992;89:1774–8. 56. Chakraborty S, Chakraborty N, Datta A. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proc Natl Acad Sci U S A 2000;97:3724–9.
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57. Rascón-Cruz Q, Sinagawa-García S, Osuna-Castro JA, Paredes-López O. Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize. Theo App Gene 2004;108:335–42. 58. Goni I, Garcia-Alonso A, Saura-Calixto F. A starch hydrolysis procedure to estimate glycemic index. Nutr Res 1997;17:427–37. 59. Foster-Powell K, Holts S, Brand-Miller JC. International table of glycemic index and glycemic load values. Am J Clin Nutr 2002;76:5–56.
Further Reading 60. Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. thesis submitted to Guru Nanak Dev University, India; 2014.
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10 Amaranth: Potential Source for Flour Enrichment Narpinder Singh*, Prabhjeet Singh†, Khetan Shevkani‡, and Amardeep Singh Virdi* *Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India † Department of Biotechnology, Guru Nanak Dev University, Amritsar, India ‡ Department of Applied Agriculture, Central University of Punjab, Bathinda, India
O U T L I N E Introduction
123
Transgenic Applications
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Grain Characteristics
124
Technological Issues
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Grain Composition
124
Summary Points
133
Grain Protein Characteristics
127
Acknowledgments
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Nutraceutical Properties of Amaranth Proteins
130
References
133
Food and Nonfood Applications
130
Further Reading
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List of Abbreviations AmA API CaMV cDNA GI kDa PAGE SDS
amaranth albumin amaranth protein isolate cauliflower mosaic virus complementary DNA glycemic index kilo Dalton polyacrylamide gel electrophoresis sodium dodecyl sulfate
INTRODUCTION Amaranth (Amaranthus), a traditional Mexican plant is a cosmopolitan genus of herbs with approximately 60 plant species, the majority of which are wild.1 It was cultivated 5000–7000 years ago in areas of Mexico, but the cultivation was discontinued in the early 15th century until the late 1970s, when it was rediscovered and utilized as a new economic crop.2,3 Amaranthus plants have inflorescences and foliage in colors ranging from purple to red to gold. It is a dicotyledonous plant and is also considered as a pseudocereal because of its properties and characteristics.4 Amaranth is generally cultivated in arid zones, where commercial crops cannot be grown. Furthermore, amaranth is also known for its high nutritive value and several nutraceutical health-benefiting effects.5–7
Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00010-1
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© 2019 Elsevier Inc. All rights reserved.
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The Amaranthus plant has a good capacity to produce a high biomass and is used to produce grains, leafy vegetables, and ornamentals. Several species of amaranth are often considered as weeds. Amaranthus cruentus and Amaranthus hypochondriacus are the two species that are primarily cultivated for grain, whereas Amaranthus blitum, Amaranthus dubius, Amaranthus tricolor, Amaranthus lividus, and Amaranthus spinosus are used as vegetables. Amaranthus tricolor and Amaranthus caudatus are also grown for ornamental and decorative purposes. Amaranthus viridis, Amaranthus retroflexus, Amaranthus hybridus, Amaranthus gracilis, Amaranthus gangeticus, Amaranthus paniculatus, and Amaranthus graecizans are wild types of the plant. Its leaves are a potential alternative source of betalains because of betacyanin pigments, and they also show anticancer activity.
GRAIN CHARACTERISTICS Amaranth grains are nearly spherical and about 1 mm in diameter, and they vary in color from creamish yellow to reddish and have unique compositions of protein, carbohydrates, and lipids. A. hypochondriacus produces creamish yellow grains, while A. caudatus has red grains (Fig. 1). Hunter color L*, a*, and b* values are around 62–68, 5.5–6.7, and 21.2–23.7, respectively, for grains of A. hypochondriacus, versus 49–51, 13–13.8, and 10.6–13.2, respectively, for A. caudatus. These two species also differ in grain size. A. hypochondriacus grains were larger and showed greater thousand kernel weight, varying between 0.62 and 0.88 g, as compared to A. caudatus grains, with thousand grain weight ranging between 0.46 and 0.70 g.8 Amaranth seeds have circular embryos or germ, which surrounds the starch-rich perisperm and, together with the seed coat, represent the bran fraction, which is relatively rich in fat and protein.9 The bran fraction is proportionally higher in amaranth seeds in comparison to common cereals, such as maize and wheat, which explains the higher levels of protein and fat present in these seeds.9
GRAIN COMPOSITION Amaranth is a good source of starch, proteins, lipids, dietary fiber, and minerals. On a dry basis, amaranth grains contain 12.5%–15.5% proteins, 73.7%–77.0% carbohydrates, 7.1%–8.0% lipids and 3.0%–3.5% minerals,10 while the dietary fiber content ranges from 19.5%–49.3%.11 Starch is the most abundant carbohydrate in amaranth grain. Amaranth grain has approximately 62%–65% starch, which is made up of amylose and amylopectin. Amylose is essentially a linear polymer of glucose, while amylopectin is highly branched, consisting of a main chain of (1–4)-linked α-D-glucose, along with short chains of (1–6)-α-D-glucose-linked branches. Amaranth starch has low amylose content, ranging from 2% to 12% depending on the genotype. Amylopectin is the most abundant component of amaranth starch. Amaranth starch may contain 90%–98% amylopectin, which is composed of 1700 amylopectin molecules on average,12 which in turn exhibit smooth polymodal chain length distribution, with the peak maxima at degree of polymerization (DP) around 11–12.13 In addition to amylose and amylopectin, amaranth starch granules contain 0.16%–0.28% bound lipids.14 Although starch granule lipids are present as minor components, they have a great effect on starch properties and functionality.15 Amaranth starch granules have diameters ranging between 0.5 and 2.5 μm, which is similar to rice but smaller than that found in the starches of other cereal grains. A comparison of amaranth starch granules with those of wheat, rice, and potato is illustrated in Fig. 2. Amaranth starch has polygonal-shaped granules and displays an A-type X-ray pattern (Fig. 2), which is similar to wheat, rice, and maize starches. Amaranth starch granules are mostly small and show unimodal size distribution; however, the granular size and distribution vary widely among cultivars and species.13 FIG. 1 Grains of two different amaranth species.
A. hypocondriacus
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A. caudatus
125
GRAIN COMPOSITION
FIG. 2 Scanning electron micrographs of different starches.
FIG. 3 X-ray diffractions (XRDs) of wheat and amaranth starch. From Singh, N. Unpublished data.
Relative intensity
Wheat
Amaranth
5
10
15
20
25
30
2(°)
Amaranth starch shows greater crystallinity than wheat starch, with strong reflections at 2°θ ¼ 15.1 degrees, 17.2 degrees, 18.1 degrees, and 23.2 degrees (Fig. 3). An additional peak at 2°θ ¼ 20.0 degrees is usually present, indicating the presence of amylose-lipid complexes. Amaranth starch shows intercultivar variability in crystallinity. Its starch has pasting temperature and gelatinization temperature ranges (To–Tp) between 69°C–72°C and 60°C–77°C, respectively. The difference in pasting behavior among starches from various genotypes has been observed due to differences in amylose content and crystallinity, as well as the presence or absence of amylose-lipid complexes. The pasting curve of starch separated from two genotypes of A. hypocondriacus is illustrated in Fig. 4. In comparison to cereal, pulse, tuber, and root starches, amaranth starch generally produces more stable pastes (low breakdown) owing to its small size and low amylose content.3
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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT
100
3000
80
2000
60
1500 40
IC-540860
1000
Temperature (°C)
Viscosity (cP)
2500
RMA-22
20
500 0
0 0
3
6
9
12
Time (min)
FIG. 4 Pasting curves of starch separated from different amaranth genotypes. From Singh N. Unpublished data. TABLE 1
GIs of Various Foods
Food
GI
Amaranth grain (raw)a
87
a
94
White bread
a
Amaranth grain (popped)
101
a
Amaranth grain (roasted)
106
a
Amaranth grain (flaked)
106
Amaranth grain (extruded)a
91
Amaranth grain (popped)b
97
Pearl barleyb
25
Sweet cornb
53
White riceb
64
Brown riceb
55
Parboiled riceb
47
b
48
Bulgur wheat Corn flakes
b
84
Puffed wheat
b
74
b
70
Wheat bread
b
69
Whole-meal bread b
28
Lentils
b
18
Soybean
Baked beans (canned)
b
48
a Capriles et al.18 [GI (predicted), determined using the equation 39.71 + 0.549 (hydrolysis index) of Goni et al.58]. b Foster-Powell et al.59; the reference food is glucose.
Amaranth grain is a high-glycemic food. Amaranth starch granules digested faster within the first hour than normal cornstarch.16 The rapid digestion of amaranth starch was attributed to its small size, low amylose content, and high tendency to lose its crystalline and granular starch structure completely during heating. The glycemic index (GI) of amaranth is compared with that of other cereal grains and foods in Table 1. The GI defines the carbohydrates present 2. FLOURS AND BREADS
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in various foods on the basis of the postprandial level of blood glucose.17 The relationship between the rate of in vitro digestion and the glycemic response to food is well known. Raw amaranth seeds had rapidly digestible starch (RDS) content of 30.7% (dry weight basis) and a predicted GI of 87.2.18 Amaranth grains are enriched in various minerals, such as calcium, phosphorus, iron, potassium, and zinc, and vitamins E and B complexes, all of which are concentrated in the bran and germ portions of the grain. Amaranth contains higher amounts of riboflavin and ascorbic acid than cereal and is a good source of vitamin E.7 It also is a rich source of polyphenols (flavonoids), with relatively high antioxidant activity. Caffeic acid, p-hydroxybenzoic acid, and ferulic acid are the main phenolic compounds found in amaranth grains.19 The presence of polyphenols such as rutin (4.0–10.2 mg/g flour) and nicotiflorin (7.2–4.8 mg/g flour) in A. hypochondriacus varieties grown in the Mexican Highlands also has been reported.20 The calcium, magnesium, and oxalate concentrations in the grains of 30 amaranth genotypes of A. cruentus, A. hybridus, and A. hypochondriacus have been studied.21 Calcium and magnesium concentrations in the grains ranged between 134 and 370 mg/100 g and 230 and 387 mg/100 g, respectively, whereas the oxalate content ranged between 178 and 278 mg/100 g. Although dietary oxalate is a potential risk factor for kidney stone development and reduces the availability of calcium and magnesium, most of the oxalates in the amaranth grains are in insoluble form; as a result, the absorption may be low. However, this needs to be confirmed by bioavailability investigation. The dietary fiber and lipid content in the amaranth grain ranged between 8% and 17% and 3.0% and 10.5%, respectively. Although amaranth grain contains higher lipids than most of the cereals, the composition of its oil is quite similar to that of cereals, being high in unsaturated fatty acids (approximately 77%). Amaranth oil contains mainly linoleic acid and also contains tocotrienols that are associated with cholesterol-lowering activity in mammalian systems.22 Amaranth grain oil contains a significant amount (up to 8%) of squalene, exhibiting anticarcinogenic and hypocholesterolemic effects.23,24 Therefore, amaranth oil has the potential of replacing other squalene sources (e.g., shark and whale, which are endangered species).
GRAIN PROTEIN CHARACTERISTICS Cereals are normally deficient in lysine and tryptophan, whereas legume proteins show a deficiency of sulfurcontaining amino acids (namely, cysteine and methionine). Amaranth proteins, on the contrary, contain significant amounts of both sulfur-containing amino acids and lysine. Amaranth grains have higher protein (11%–17%) than most cereals. Amaranth is an appropriate grain for people who are allergic to gluten. The germ and endosperm of amaranth grain contain 65% and 35% of protein against an average of 15% and 85% in most cereals, respectively. The amino acid composition of various amaranth protein fractions is given in Table 2. Albumins and globulins are relatively rich in lysine and valine, while glutenins are high in leucine, threonine, and histidine. Aside from the amino acid composition, the protein quality also depends on bioavailability or digestibility. Protein digestibility, available lysine, net protein utilization, and protein efficiency ratio, which are indicators of protein nutritional quality, are substantially higher for amaranth protein than for cereal grains.25 Therefore, amaranth proteins are a promising food ingredient, capable of complementing and supplementing cereal or legume proteins.25 The protein digestibility corrected amino acid score of amaranth whole-meal flour is higher (0.64) than those of wheat (0.40) and oats (0.57).26 An average protein digestibility of 74.2% for raw amaranth whole-meal flour was reported.26 Thermal processing improves protein digestibility due to the opening of carbohydrate-protein complexes, the inactivation of antinutritional factors such as trypsin inhibitors, or both.26 Apart from their nutritional properties, amaranth proteins also possess functional properties that play important roles in formulation and processing.27 Amaranth protein isolate (API) improved the quality attributes (i.e., crust color, appearance, volume, height, springiness, and cohesiveness) and produced gluten-free muffins comparable to those with gluten.28 Contrary to legumes and cereals, where grain proteins generally serve as storage molecules for the growing plantlets, amaranth grain consists of the highest amount of albumins, which are usually biologically active. According to the Osborne classification, amaranth grain consists of three major fractions [namely, albumins (51%), globulins (16%), and glutelins (24%)] and a minor fraction (i.e., alcohol-soluble fraction or prolamine between 1.4% and 2.0%),29,30 whereas legume grain contains salt-soluble globulins as the major storage protein fraction. Based on size exclusion chromatographic analysis, it was reported that globulins were prominent proteins in kidney beans and field peas, while amaranth proteins contained both globulins and albumins as major fractions.31 Cereals such as maize and wheat, on the contrary, contain alcohol-soluble prolamins as the major storage proteins.32 The characterization of grain proteins of amaranth has been carried out using different techniques of extraction and electrophoresis.29,32–34 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of amaranth proteins 2. FLOURS AND BREADS
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TABLE 2 Amino Acid Composition of Amaranth (A. hypochondriacus L.) Protein Fractions Protein Fractions Amino Acid
Meal
Albumins
Globulins
Prolamins
Glutelins
Isoleucinea
–
3.7
4.2
6.2
5.8
Leucinea
–
5.7
5.7
5.7
10.5
Lysinea
–
7.6
6.7
4.2
4.6
Methioninea
–
4.1
3.4
7.4
3.1
Cystinea
–
5.9
3.9
6.5
6.2
Phenylalaninea
–
5.1
5.0
9.0
6.8
Tyrosinea
–
3.3
4.3
4.0
3.8
–
3.9
4.1
3.2
8.6
–
4.5
4.7
2.7
3.8
–
2.5
1.1
1.1
4.7
–
5.1
4.0
4.7
3.6
–
8.1
9.5
9.4
2.7
–
6.2
8.7
6.2
6.1
–
17.5
17.3
13.4
13.2
Glycine
–
6.2
6.6
4.4
4.9
a
Proline
–
3.7
3.9
4.7
4.6
Serine
a
–
4.8
4.9
5.1
5.3
Serine
b
7.3
6.4
7.7
8.0
9.0
10.7
10.5
13.9
10.7
10.3
3.0
2.3
2.3
1.8
2.4
7.3
8.9
9.3
6.8
8.5
5.1
3.4
4.0
7.2
5.4
6.6
6.2
5.4
8.6
6.3
5.7
5.0
4.0
4.5
5.9
1.9
2.9
2.8
3.0
3.0
5.9
4.0
5.0
4.5
5.0
3.9
3.5
4.0
4.5
5.0
6.2
5.5
6.0
10.0
8.0
3.4
3.0
2.0
3.9
4.3
5.7
6.6
7.0
6.7
4.2
a
Threonine a
Valine
a
Histidine a
Alanine
a
Arginine
a
Aspartic acid
a
Glutamic acid a
b
Glycine
b
Histidine
b
Arginine
b
Threonine b
Alanine
b
Proline
b
Tyrosine b
Valine
b
Isoleucine b
Leucine
Phenylalanine b
Lysine
b
a
Expressed as g of amino acids/100 g of crude protein. Expressed in percent in moles. From Barba de la Rosa AP, Gueguen J, Paredes-Lopez O, Viroben G. Fractionation procedures, electrophoretic characterization and amino acid composition of amaranth seed protein. J Agric Food Chem 1992;40:931–936; Segura-Nieto M, Vaazquez-sanchez N, Rubio-Velazqez H, Olguin-Martin LE, Rodriguez-Nester CE, HerreraEstrella L. Characterization of amaranth (Amaranthus hypocondriacus) seed proteins. J Agric Food Chem 1992;40:1553–1558.
b
reveals a wide range in the molecular weight of polypeptide subunits among varieties and species (Fig. 5). On the basis of differential extraction, the amaranth albumin (AmA) was classified as albumin 1 and albumin 2.35 Albumin 1 is extractable with water, saline solution, or both, whereas albumin 2 is extractable with water after the removal of albumin 1 and globulin with saline solution. Albumin 2 has amarantin as the major protein component.30 The subunit size of albumin proteins varied from 10 to 37 kDa,29,33 with low molecular subunits being more abundant.36 Barbara de la
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97.4 66.0
43.0
110 96 91 73 61 56 53 43
(A)
IC540862
IC540860
IC540839
IC467901
IC042311–7
IC042254–5
IC042265–2
IC095341
52 43.0
42 40 38 33
29.0
28
26 24
26 23
23
22
20
20.0
18 17 16 14.3
IC38312
66.0
34 31
20.0
VL–0344
97.4
38
29.0
RMA–30
RMA–22
PRA–3
PRA–2
Annapurna
Marker
IC540869
IC540828
Amaranthus hypochondriacus
IC467902
IC423448
IC423393
IC363742
IC258399
IC38181
IC38165
Marker
Amaranthus caudatus
10
20 16
14.3
15 14
(B)
FIG. 5 SDS-PAGE (12% resolving gel) analysis (under reducing conditions) of (A) A. caudatus and (B) A. hypochondriacus protein isolates. From Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. Thesis submitted to Guru Nanak Dev University, India, 2014.
Rosa et al.,20 however, differentiated the albumin fraction into two groups of proteins corresponding to around 18 kDa and between 40 and 80 kDa. The proteins of around 18 kDa were termed methionine-rich proteins due to their high methionine content (between 16% and 18%).37 Determination of sedimentation coefficient by centrifugation has also been widely used to characterize the proteins. On the basis of sedimentation coefficients, the amaranth seed globulins are categorized into 10S and 12.7S, as compared to 7/8S and 11/12S for the legume seed globulins. The electrophoretic behavior of 10S and 12.7S amaranth globulin fractions on denaturing gel was observed to be similar to that of 7S and 11S storage proteins of legumes; hence, they are referred to as 7S and 11S, respectively.34 The higher sedimentation coefficients of amaranth globulins, as observed on linear sucrose gradients, suggested that these proteins contain polypeptides with higher molecular weight than those present in 7S and 11S from pea globulins.36 The 7S and 11S amaranth seed globulins also differed in their solubility in salt solution, with the former being extractable with 0.1 M and the latter with 0.8 M of sodium chloride (NaCl).20 The 7S globulin fraction of amaranth grain was characterized by the presence of a main band of 38 kDa and lacked disulfide bridges, whereas the 11S-like globulins consisted of both acidic (35–38 kDa) and basic polypeptides (22–25 kDa). These results were in agreement with earlier studies finding that globulins consisted of polypeptides of heterogenous sizes, as demonstrated by SDS-PAGE analysis.36 However, these observations contradicted the findings of Gorinstein et al.,29 which reported that the globulin was composed of polypeptides of only 14–18 kDa. Martinez et al.,30 proposed that both 7S and 11S globulins correspond to one type of globulin, whereas polymerized globulins (albumin 2) and glutelins correspond to two other types of globulins. This notion, however, needs to be supported by establishing sequence homology and by proving a common genetic origin of globulin, albumin 2, and glutelin. The most important component of globulins is amarantin, which alone constitutes 90% of the total globulins and about 19% of the total grain protein.38 Amarantin is a homohexameric molecule of approximately 300–400 kDa, comprised of subunits of about 59 kDa. Each of these subunits consists of an acidic polypeptide of 34–36 kDa and a basic polypeptide of 22–24 kDa linked by disulfide bonds. The additional subunit of 54 kDa present in amarantin has been proposed to act as an inducer of polymerization.30
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The amaranth glutelin showed high similarity with 11S globulins and was comprised of three major polypeptide groups of 22–25 kDa, 35–38 kDa, and around 55 kDa.39 It is likely that both glutelins and 11S globulins may belong to the same structural gene family. The differences in composition of alcohol-soluble proteins have been reported in various studies. Gorinstein et al.29 observed only low-molecular-weight subunits of 10–20 kDa via SDS-PAGE analysis of the alcohol-soluble proteins, whereas Barba de la Rosa et al.33 reported the presence of subunits with both low and high molecular mass. Furthermore, great similarity between the electrophoretic patterns of reduced prolamine and glutelins was also observed in the latter study. The lack of consistency in the composition of various protein fractions of amaranth grain, which is evident from the literature, may be due to the various procedures used in the extraction and analyses. In view of the nutritional importance of the amaranth, it is imperative that a systematic study should be undertaken to analyze the proteome of amaranth leaves and grains by employing the latest techniques of proteomics. This will enable the identification and characterization of nutritionally important proteins, the genes for which can then be cloned and expressed heterologously in other crops for enhancing their nutritive value.
NUTRACEUTICAL PROPERTIES OF AMARANTH PROTEINS The albumins and globulins are rich in lysine and valine, whereas prolamins have comparatively higher content of methionine and cystine. Glutelins, on the other hand, contain higher levels of leucine, threonine, and histidine. Compared to legume grain albumins, which contain several antinutritional factors, the AmA fraction is considered safe. The AmA fraction is comparable with egg-white proteins and can be used as an egg substitute in various products. The 11S globulin fraction is rich in peptides of angiotensin-converting enzyme (ACE) inhibitor, whereas the glutelin fraction contains antihypertensive activity, as well as the anticarcinogenic lunasinlike peptide,40 thus signifying its nutraceutical properties. Amaranth proteins also exhibit antioxidant activity, which increases after gastrointestinal digestion41 and potential antitumor properties through putative mechanism of action.42 In addition, they affect the metabolism of liver lipids and have a hypotriglyceridemic effect in rats.24 Simulated gastrointestinal digestion of APIs resulted in the identification of several bioactive peptides, which showed very high antithrombotic activity. These bioactive peptides belong to 11S globulin and agglutinin fractions of amaranth.43 An amaranth peptide (SSEDIKE) attenuated the expression of the CCL20 (Caco-2 cells transfected with a luciferase reporter under the control of the CCL20 promoter) gene at the transcript level in colonic epithelial cells. Thus, the antiinflammatory effect of amaranth protein hydrolysate was demonstrated at molecular levels.44 Milk allergens are a major cause of intestinal inflammation and are considered as allergens. Oral administration of the amaranth-charged peptide (SSEDIKE) resulted in the inhibition of the allergen response in a mouse model system.44 Delgado et al.45 demonstrated that the simulated gastrointestinal digestion of Amaranthus mantegazzianus proteins yielded bioactive peptides (i.e., AWEEREQGSR, YLAGKPQQEH, IYIEQGNGITGM, and TEVWDSNEQ), which showed potential antioxidant activity and belongs to the 11S globulin protein family. Furthermore, amaranth bioactive cationic (HVIKPPSRA and KFNRPETT) and neutral (GDRFQDQHQ) peptides were also shown to exhibit the in vivo inhibition of Cu+2/H2O2-induced LDL oxidation.46
FOOD AND NONFOOD APPLICATIONS The unique small starch granule size and composition has been suggested to be responsible for unique gelatinization and freeze/thaw characteristics, which could be exploited in the development of products by the food industry.47 Amaranth starch can be used in many food preparations, such as custards, pastes, and salad, and nonfood applications such as cosmetics, biodegradable films, paper coatings, and laundry starch. The modified A. viridis starches also have the potential to partially replace fat in fat-rich foods such as mayonnaise and salad cream.48 Amaranth flour is used as a thickener in gravies, soups, and stews. Sprouted amaranth is used in salads. Defatted amaranth flours may be preferred over full-fat flours, as they have better flavor stability and can be marketed as low-fat or low-calorie products. In addition, extracted oil can be used for other purposes and these flours have improved functionality.10 However, the improvement in the functional properties of flours depends on protein characteristics, which in turn vary based on the cultivars.10,27 The cooking of amaranth improves its digestibility and absorption of nutrients. Amaranth flour lacks the gluten proteins present in wheat, and hence it is not suitable for bread-making. A blend of amaranth flour with wheat meal or flour is used in the preparation of an unleavened flatbread known as chapatis in India and tortillas in Latin America. Amaranth flour is also used to prepare biscuits, muffins, pancakes, pastas, flatbreads, extruded products, and other foodstuffs. In India, the grains are most commonly used in the form of candy known as laddoos. 2. FLOURS AND BREADS
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Waxy and normal maize starch granules were transformed into starch nanocrystals, and protein films were prepared after blending with amaranth protein formulations.49 One study demonstrated that nanocomposite films exhibited improved water vapor permeability, water uptake, surface hydrophobicity, mechanical behavior, and delayed weight loss in soil than that of normal protein films. It was proposed that the good dispersion of the nanoreinforcements in the protein matrix is due to the formation of disulfide bonds among waxy maize nanocrystals, as well as hydrogen bonding between normal maize nanocrystals and protein matrix.49 Amaranth proteins compared well with pulse (kidney bean and field pea) proteins in terms of edible/biodegradable film-forming31 and gluten-free muffinmaking (Fig. 6) properties.28 Furthermore, API edible films prepared from different ultra-high-pressure treatments demonstrated uniform surface and more opaque characteristics, along with improved mechanical properties, lower water solubility, and water vapor permeability, as compared to nontreated protein dispersions.50,51 Encapsulation of folic acid into API-pullulan electrospun fibers resulted in photoprotection of folic acid.52 It has been reported that APIs at acidic pH (2.0) show improved foam stability over those with alkaline pH.53 Increased unfolding, greater flexibility, and the net charge on amaranth proteins under acidic conditions were attributed to
FIG. 6 Crusts (I) and crumb cross sections (II) of muffins prepared from (A) cornstarch; (B) cornstarch with wheat gluten; (C) cornstarch with kidney bean protein isolate; (D) cornstarch with field pea protein isolate; and (E) cornstarch with API. The starch was replaced with calculated amount of proteins in order to get a protein content of 10%. From Shevkani K, Singh N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Int J Food Sci Tech 2014;49:2237–2244.
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the higher foam stability of proteins. These findings thus demonstrated that the viscoelasticity and flexibility of foams were influenced by the solubility of protein isolates at different ionic strengths and pHs of solubilizing solvents.53 Suarez and Añón54 also demonstrated that APIs solubilized at low pH (2.0) adsorbed in an oil/water interface twice as quickly as APIs prepared at pH 6.3 and allowed in the formation of oil-in-water emulsions. These findings indicated the treatment of APIs at various ionic and pH conditions may be helpful in the improvement of texture and sensory properties of food products with improved nutraceutical values. In comparison to amaranth grain, vegetable amaranth has received less attention. Vegetable amaranth is used as a delicacy or a food staple in many parts of the world. Amaranthus leaves are used as a vegetable in the northern states of India. However, its use is limited to saag, a dish prepared by cooking mustard leaves with garlic, ginger, green chilies, and salt. Vegetable amaranth tastes better than spinach and is substantially higher in calcium, iron, and phosphorous.
TRANSGENIC APPLICATIONS Improving the balance of essential amino acids in important crop plants remains one of the major objectives of plant breeders. Transgenic technology presents an attractive alternative for improving the nutritional quality of grain proteins. Heterologous transgenic expression of storage protein genes with higher levels of limiting amino acids has been reported. Transgenic expression of high levels of a particular amino acid may affect the normal physiology of seed development adversely or produce seeds with a biased amino acid composition. Therefore, expressing a gene for a heterologous protein with a balanced amino acid composition is a better alternative. A gene of a 35-kDa albumin protein (AmA1), which is expressed during early to mid-maturation stages of embryogenesis in the amaranth seed was cloned.55 The amino acid composition of this protein conforms to the World Health Organization (WHO)–recommended values for a highly nutritional protein because it is rich in various essential amino acids. Potato is the most important noncereal crop in terms of total global food production; therefore, transgenic expression of this gene in the tubers of this crop has been achieved. Heterologous expression of AmA1 under constitutive [Cauliflower mosaic virus (CaMV) 35S promoter] and tuber-specific promoter (granule-bound starch synthase) in potato resulted in significant enhancement in total protein content, with an increase in essential amino acids.56 Furthermore, the growth and production of tubers in transgenic plants were also higher than for control plants. The maize, which is a staple food in many countries but lacks essential amino acid in the grains, has also been targeted for heterologous expression of amaranth proteins to enhance the nutritional quality of its protein. Complementary DNA (cDNA) of an 11S globulin storage protein, amarantin, which has a high content of essential amino acids, was expressed in maize under the CaMV 35S promoter and an endosperm-specific promoter (rice glutelin-1).57 Heterologous expression of this gene resulted in an increase of 18% in lysine, 28% in sulfur-containing amino acids, and 36% in isoleucine, as well as a 32% increase in total seed protein. Furthermore, the heterologously expressed protein was digested by simulated gastric and intestinal fluids, thus confirming the biodigestibility of the transgenic protein. These studies, therefore, validate the potential of using various amaranth genes for supplementing and complementing the proteins in both cereal and noncereal staple crops. However, detailed studies on the analysis of proteomes of amaranth species needs to be carried out to identify the candidate genes, which can be employed for transgenic improvement. Furthermore, the generation of mutants in amaranth is also required to determine the role of specific proteins in the growth and development of the plant so that appropriate improvement of the germplasm can be undertaken through conventional breeding strategies.
TECHNOLOGICAL ISSUES The presence of diversity in composition among amaranth genotypes necessitates in-depth characterization of biochemical constituents for its specific applications in the food industry. The smaller granules in amaranth starch, which resemble the size of fat globules in cow’s milk, can be exploited to mimic fat in a number of food products. Some of the genotypes have higher polyphenols with higher antioxidant activity, which also could be utilized in the development of new products. Amaranth grain has the potential to develop various food products for people suffering from celiac disease, a disorder that makes the body intolerant to gluten proteins.
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SUMMARY POINTS • Amaranth grain is a good source of dietary fiber and has high GI. It is low in resistant starch (RS), and its starch has uniquely small granules with low tendency toward retrogradation. • Grain amaranth has higher protein than most of the cereal grains and is an appropriate food for people who are allergic to gluten. • Amaranth grain oil is quite similar to that of cereals, being high in unsaturated fatty acids, and it contains mainly linoleic acid. Its oil also contains tocotrienols, which are associated with cholesterol-lowering activity in mammalian systems. Amaranthus grain oil also contains a significant amount of squalene, which has anticarcinogenic and hypocholesterolemic effects. • Amaranth grain proteins are mainly composed of three major fractions (namely, albumins, globulins, and glutelins) with little or no storage of prolamin. Amarantin is the most important component of globulins and constitutes 90% of the total globulins and about 19% of the total grain protein. • Heterologous expression of the amarantin gene (AmA1) in potato results in significant enhancement in total protein content, with an increase in essential amino acids as well. • Amaranth is also a good source of minerals such as iron, magnesium, phosphorus, copper, and manganese. Its unique composition also makes it an attractive food complement and supplement.
Acknowledgments The financial assistance to NS from the Department of Science and Technology, Ministry of Science and Technology, Government of India, is acknowledged.
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FURTHER READING
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57. Rascón-Cruz Q, Sinagawa-García S, Osuna-Castro JA, Paredes-López O. Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize. Theo App Gene 2004;108:335–42. 58. Goni I, Garcia-Alonso A, Saura-Calixto F. A starch hydrolysis procedure to estimate glycemic index. Nutr Res 1997;17:427–37. 59. Foster-Powell K, Holts S, Brand-Miller JC. International table of glycemic index and glycemic load values. Am J Clin Nutr 2002;76:5–56.
Further Reading 60. Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. thesis submitted to Guru Nanak Dev University, India; 2014.
2. FLOURS AND BREADS