The effect of popping and fermentation on protein quality of three different varieties of amaranth grains cultivated in Ethiopia was evaluated. Total lysine content of the grains was higher than that of commonly available cereals but close to that of legumes. Methionine and cysteine contents in the grains were also higher than that found in cereal and legume proteins. Percentage of total indispensable amino acids, excluding tryptophan, was 43% - 49%, which was higher than WHO reference pattern (31%). Popping resulted in 36% and 37% reduction in total lysine and cysteine contents, respectively, whereas fermentation reduced cysteine, lysine and methionine contents by 16%, 20% and 20%, respectively. From the free amino acids, histidine was the major indispensable amino acid but threonine was not detected. During popping, all free amino acids, except threonine, were reduced. On the other hand, fermentation significantly increased (p < 0.01) most amino acids except arginine, which was significantly decreased (p < 0.01), and tyrosine and glutamic acid, for which no change was observed. Popping decreased in vitro protein digestibility (IVPD) by 8.3% - 17.1% while fermentation increased IVPD by 4.8% - 7.5%. Substitution of amaranth for wheat and/or maize during complementary food formulation could contribute much to the daily requirements of indispensable amino acids of young children.
The genus Amaranthus belongs to the family Amaranthaceae. It has a huge biodiversity and several of them are cultivated as leafy vegetables, grains, and ornamental plants while others are weeds [
Amaranth was neglected from the food table for many decades after the arrival of the Spanish conquistadors in Latin America as it was used in ceremonial dishes associated with human sacrifice by the Aztecs [
Concerning the nutritional quality, protein content of amaranth grains is higher than that of most common cereals [
Other processing techniques such as boiling, microwave cooking, and autoclaving also affect protein digesti- bility. According to El-Adawy et al. (2002) [
In developing countries like Ethiopia, complementary foods for young children are mainly prepared from mixes of cereals and legumes. It is known that among essential amino acids, legumes are rich sources of lysine but lack methionine and cysteine, while cereals are rich in these two amino acids but are poor sources of lysine. Mixing legumes and cereals during complementary food formulation is a common strategy to meet essential amino acid requirements. However, a too high proportion of legumes sometimes compromise the acceptability of the product and, in addition, legumes contain high levels of antinutritional factors. It is thus advisable to reduce the proportion of legumes in complementary food formulation.
The aim of the present study was, therefore, to evaluate the protein quality of three different varieties of ama- ranth grains cultivated in Ethiopia in the raw state and after popping or fermentation by analyzing the contents of free and total amino acids and in vitro protein digestibility of amaranth grain porridge to assess its potential application as an ingredient for complementary food formulation.
Three different varieties of amaranth grains, white, red and brown in color, were purchased from six farmers in Chat Kebelle, Bench Majji Zone, in the Southern Nations, Nationalities and Peoples region, Ethiopia. Composite samples of each variety prepared from the grains of the six origins were sorted, cleaned and washed to remove immature seeds, sand and soil. The washed seeds were sun dried, milled and sieved in a 0.425 mm sieve. The flour was stored at 4˚C until further analysis.
Citrate buffer pH 2.2 and ninhydrin reagent were purchased from Biochrom (France). Sodium hydroxide (BioXtra, ≥98% acidimetric), pellets (anhydrous), L-Norleucine (N8513 suitable for amino acid analysis), aminobutyric acid (A2129; ≥99%), methanesulfonic acid (M4141; 4 M with 0.2% (w/v) tryptamine) were purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA). Amino acid standards (AA-S18; analytical standard), hydrochloric acid 0.1 N were purchased from Fluka (Fluka Chemicals, Buchs, Switzerland). All reagents and chemicals used were of analytical grade.
Sun dried seeds were placed in a hot clay pan for 10 - 15 seconds until they popped. The popped grains were milled to pass through a 0.425 mm sieve and stored in polyethylene bags at 4˚C.
Natural fermentation was carried out according to the method described in [
Amaranth gruels were prepared using the method described in [
The nitrogen content of all Samples was determined using Kjeldhal method and a conversion factor of 5.85 was used [
1) Extraction procedure
a) Free amino acids
Free amino acids were analyzed following the method used by Moore et al. (1958) [
b) Total amino acids
Samples (10 - 20 mg) of dried flour were weighed in a Schlenk tube and 50 µl of 25 µM Norleucine and 450 µL of 4 M methanesulfonic acid were added. The tube was flushed with nitrogen, closed and heated at 150˚C for 2 h. After cooling, 450 µL of 4 M NaOH was added to the hydrolysate, which was diluted to 5 ml with a loading buffer (citrate buffer at pH 2.2). All extractions were performed in triplicate.
2) Amino acid analysis
Sample extracts for free and total amino acid analysis were filtered using a 0.45 µm membrane filter and injected into the amino acid analyzer (Biochrom 30+, Biochrom, France), using a lithium cation exchange resin column and ninhydrin as detection compound. All total and free amino acid contents were determined except total tryptophan, which is the most fragile amino acid and is destroyed by the extraction procedure. Amino acid standards were also run in a similar way as the samples.
In vitro protein digestibility was determined according to the method of Akeson and Stahmanna (1964) [
The amino acid score (AAS) is the ratio of the amino acid content in the protein of a food/diet to the content of the same amino acid in the requirement pattern. The score determines the effectiveness with which absorbed dietary nitrogen can meet the indispensable amino acid requirements at the safe level of protein intake. This is thus a measure of the actual amounts of individual amino acids in a food with respect to the need for this amino acid. And the quality of the protein will finally depend on the indispensable amino acid for which the AAS is the lowest. However, AAS does not account for the digestibility of the protein. Therefore, another scale called the protein digestibility corrected amino acid score (PDCAAS) was adopted by WHO/FAO (2007) [
From the amino acid data, intakes of indispensable amino acids were calculated for comparison with the WHO estimated needs [
All measurements were done in triplicate and statistical analyses were performed using the software Statgra- phics plus 5.1 (Statpoint, Warrenton, USA). Two-way ANOVA was applied to the experimental data and means were separated using Fischer’s least significant difference tests with a probability p < 0.05.
The amount of almost all essential amino acids in the raw samples was above the new FAO/WHO standard pattern for all age groups [
The lysine content of raw amaranth ranged from 65 to 74 mg∙g−1, protein which is close to the lysine contents in legumes (70 - 75 mg∙g−1 protein) including chickpeas, cowpeas and lentils but twice higher than that found in many other cereals, such as barley, wheat, rice, rye and maize: 22 - 37 mg∙g−1 protein. Methionine and cysteine contents were ranged from 24 to 29 and 43 to 51 mg∙g−1 protein, respectively, which were higher than that found in the legumes and cereals mentioned above [
The percentage of indispensable amino acids, excluding tryptophan, tryptophan, was highest in raw white
Protein (g/100g DM) | White amaranth | Red amaranth | Brown amaranth | Standard pattern¶ | |||
---|---|---|---|---|---|---|---|
13.88 ± 0.16 | 15.15 ± 0.08 | 15.53 ± 0.24 | |||||
Amino acid | Total | Free | Total | Free | Total | Free | |
Cysteine* | 50.7 ± 8.3 | 0.9 ± 0.0 | 42.7 ± 3.0 | 1.3 ± 0.2 | 45.5 ± 4.3 | 0.7 ± 0.1 | |
Histidine* | 37.2 ± 3.1 | 1.4 ± 0.1 | 33.9 ± 2.5 | 1.5 ± 0.0 | 34.8 ± 2.0 | 1.2 ± 0.1 | 18.0 |
Isoleucine* | 39.4 ± 3.0 | 0.4 ± 0.1 | 37.2 ± 2.6 | 0.4 ± 0.1 | 34.9 ± 2.4 | 0.3 ± 0.1 | 31.0 |
Leucine* | 67.1 ± 2.9 | 0.4 ± 0.0 | 60.8 ± 2.6 | 0.4 ± 0.0 | 59.5 ± 0.2 | 0.2 ± 0.0 | 63.0 |
Lysine* | 73.9 ± 2.3 | 1.2 ± 0.0 | 66.7 ± 7.2 | 1.1 ± 0.0 | 65.3 ± 1.6 | 0.9 ± 0.0 | 52.0 |
Methionine* | 28.8 ± 0.5 | 0.4 ± 0.1 | 23.8 ± 3.4 | 0.5 ± 0.2 | 25.8 ± 0.7 | 0.3 ± 0.1 | |
Phenylalanine* | 46.5 ± 3.6 | 0.2 ± 0.0 | 42.6 ± 1.4 | 0.2 ± 0.0 | 40.4 ± 3.5 | 0.1 ± 0.0 | |
Threonine* | 40.3 ± 1.9 | 0.0 ± 0.0 | 35.0 ± 3.4 | 0.0 ± 0.0 | 36.7 ± 1.1 | 0.0 ± 0.0 | 27.0 |
Tryptophan* | ND | 1.0 ± 0.0 | ND | 1.4 ± 0.1 | ND | 0.7 ± 0.0 | 7.4 |
Valine* | 48.3 ± 1.2 | 0.7 ± 0.1 | 42.0 ± 4.6 | 0.7 ± 0.1 | 41.3 ± 2.7 | 0.5 ± 0.1 | 42.0 |
Alanine | 44.0 ± 2.0 | 1.1 ± 0.0 | 38.2 ± 5.8 | 0.5 ± 0.1 | 39.0 ± 1.5 | 1.0 ± 0.0 | |
Arginine | 109.8 ± 8.0 | 1.6 ± 0.0 | 102.2 ± 10.0 | 3.6 ± 0.3 | 100.6 ± 4.0 | 1.0 ± 0.0 | |
Aspartic acid | 94.4 ± 5.2 | 1.1 ± 0.0 | 86.0 ± 9.3 | 2.2 ± 0.1 | 89.4 ± 10.0 | 1.2 ± 0.0 | |
Glutamic acid | 203.5 ± 14.0 | 2.9 ± 0.0 | 183.5 ± 15.0 | 4.6 ± 0.3 | 181.8 ± 3.0 | 1.8 ± 0.0 | |
Glycine | 100.1 ± 4.0 | 1.3 ± 0.0 | 89.1 ± 6.4 | 0.4 ± 0.1 | 100.0 ± 4.0 | 1.1 ± 0.0 | |
Proline | 135.7 ± 25.6 | 2.4 ± 0.0 | 125.0 ± 30.0 | 3.7 ± 0.2 | 118.4 ± 17.4 | 1.5 ± 0.0 | |
Serine | 65.5 ± 4.0 | 1.3 ± 0.2 | 58.5 ± 4.8 | 2.1 ± 0.1 | 68.9 ± 1.6 | 0.6 ± 0.0 | |
Tyrosine | 52.9 ± 6.0 | 0.5 ± 0.0 | 45.8 ± 2.7 | 0.7 ± 0.0 | 49.7 ± 3.4 | 0.4 ± 0.0 | |
AAA§ | 99.4 ± 6.3 | 88.4 ± 2.6 | 90.1 ± 3.4 | 46.0 | |||
SAA¥ | 79.5 ± 7.9 | 66.5 ± 5.9 | 71.3 ± 4.9 | 26.0 | |||
NH3 | 78.3 ± 6.1 | 3.2 ± 0.0 | 70.0 ± 14.3 | 2.0 ± 0.1 | 76.6 ± 12.2 | 1.4 ± 0.1 |
†Values are means of results obtained in triplicate ± SD expressed in mg∙g−1 protein and means followed by different letters in the same row are significantly different at p < 0.05. §AAA: aromatic amino acids (phenylalanine and tyrosine). ¥SAA: sulfur amino acids (cysteine and methionine). ND: not determined. *Indispensable amino acids [
amaranth (49%) followed by 43% in both raw red and brown amaranth. These values are higher than the reference protein pattern for 1 - 2 years old children (31%). The amaranth amino acid profile thus provides a good balance of total indispensable amino acids, and some of the limiting amino acids, especially lysine in cereals and methionine and cysteine in legumes, could be complemented by amaranth.
The free amino acid content of the three varieties of raw amaranth is listed in
Popping decreased the amount of six amino acids including three indispensable amino acids: lysine, methionine, and cysteine. The decrease was highest for lysine and cysteine and reached 36% and 37% reduction, respec- tively (
In the present study, fermentation also led to a significant decrease in the contents of the same six amino acids as that of popping (
Popping and fermentation led to highly significant changes in free amino acid contents (p < 0.01) (
Amino acid | p-value | Processing | ||||
---|---|---|---|---|---|---|
Raw | Popped | Fermented | ||||
Mean ± SD | Mean ± SD | % variation | Mean ± SD | % variation | ||
Cysteine | 0.0023* | 46.3 ± 6.0 a | 29.4 ± 8.2 c | −37 | 38.9 ± 15. 0b | −16 |
Histidine | 0.8097 | 35.3 ± 2.7 | 35.0 ± 1.3 | - | 34.6 ± 2.8 | - |
Isoleucine | 0.1014 | 37.2 ± 3.0 | 33.7 ± 4.7 | - | 35.4 ± 3.2 | - |
Leucine | 0.4034 | 62.5 ± 4.0 | 60.3 ± 4.3 | - | 60.8 ± 5.0 | - |
Lysine | 0.0000* | 68.6 ± 5.6 a | 44.0 ± 10.1 c | −36 | 55.0 ± 16.8 b | −20 |
Methionine | 0.0062* | 26.1 ± 2.8 a | 23.0 ± 4.1 b | −12 | 20.8 ± 5.1 b | −20 |
Phenylalanine | 0.3940 | 43.2 ± 2.8 | 41.4 ± 3.3 | - | 41.4 ± 4.5 | - |
Threonine | 0.0836 | 37.3 ± 3.1 | 35.1 ± 2.0 | - | 35.4 ± 3.3 | - |
Tyrosine | 0.1473 | 49.5 ± 4.8 | 48.5 ± 2.7 | - | 45.7 ± 4.8 | - |
Valine | 0.0832 | 43.9 ± 4.3 | 40.2 ± 5.5 | - | 42.2 ± 3.8 | - |
Alanine | 0.3488 | 40.4 ± 4.2 | 38.7 ± 2.6 | - | 39.8 ± 2.6 | - |
Arginine | 0.0003* | 104.2 ± 8.0 a | 88.6 ± 11.9 b | −15 | 83.2 ± 15.1 b | −20 |
Aspartic acid | 0.0191* | 89.9 ± 6.5 a | 84.8 ± 2.7 b | −6 | 83.1 ± 5.5 b | −8 |
Glutamic acid | 0.0711 | 189.6 ± 15.0 | 183.6 ± 6.8 | - | 176.3 ± 13.3 | - |
Glycine | 0.6262 | 96.4 ± 7.1 | 93.8 ± 3.7 | - | 95.4 ± 7.3 a | - |
Proline | 0.0584 | 126.4 ± 23.0 | 112.9 ± 15.5 | - | 106.5 ± 8.4 | - |
Serine | 0.0049* | 64.3 ± 5.6 a | 59.1 ± 3.2 b | −8 | 57.2 ± 3.7 b | −11 |
Ammonia | 0.1330 | 75.0 ± 10.6 | 71.9 ± 9.5 | - | 80.7 ± 10.3 | - |
†Values are means of results obtained in triplicate ± SD for the three amaranth varieties. *The effect of processing is significant (p < 0.05) and means followed by different letters in the same row are significantly different at p < 0.05.
Amino acid | p-value | Processing | ||||
---|---|---|---|---|---|---|
Raw | Popped | Fermented | ||||
Mean ± SD | Mean ± SD | % variation | Mean ± SD | % variation | ||
Arginine | p < 0.01 | 2.0 ± 1.2 a | 0.8 ± 0.6 b | −60 | 0.3 ± 0.1 c | −85 |
Cysteine | 1.0 ± 0.3 b | 0.5 ± 0.1 c | −50 | 3.7 ± 0.7 a | 270 | |
Histidine | 1.4 ± 0.2 b | 0.4 ± 0.1 c | −71 | 4.6 ± 2.6 a | 229 | |
Isoleucine | 0.4 ± 0.1 b | 0.1 ± 0.1 c | −75 | 2.8 ± 0.5 a | 600 | |
Leucine | 0.3 ± 0.1 b | 0.1 ± 0.1 c | −67 | 6.5 ± 1.4 a | 2067 | |
Lysine | 1.1 ± 0.2 b | 0.7 ± 0.2 c | −36 | 2.0 ± 0.6 a | 82 | |
Methionine | 0.4 ± 0.2 b | 0.1 ± 0.1 c | −75 | 2.6 ± 0.5 a | 550 | |
Phenylalanine | 0.2 ± 0.1 b | 0.0 ± 0.0 c | −100 | 3.5 ± 0.8 a | 1650 | |
Threonine | 0.0 ± 0.0 b | 0.0 ± 0.0 b | - | 0.2 ± 0.0 a | 100 | |
Tryptophan | 1.0 ± 0.3 b | 0.1 ± 0.1 c | −90 | 2.1 ± 0.7a | 110 | |
Tyrosine | 0.5 ± 0.1 a | 0.0 ± 0.1 b | −100 | 0.5 ± 0.4 a | - | |
Valine | 0.6 ± 0.1 b | 0.3 ± 0.2 c | −50 | 4.0 ± 0.7 a | 344 | |
Alanine | 0.9 ± 0.3 b | 0.5 ± 0.3 c | −44 | 5.2 ± 1.2 a | 478 | |
Aspartic acid | 1.5 ± 0.5 b | 0.5 ± 0.2 c | −67 | 2.3 ± 0.5 a | 53 | |
Glutamic acid | 3.1 ± 1.2 a | 0.6 ± 0.4 b | −81 | 3.2 ± 1.7 a | - | |
Glycine | 0.9 ± 0.4 b | 0.8 ± 0.5 c | −11 | 3.6 ± 1.2 a | 300 | |
Ornithine | 0.2 ± 0.0 b | 0.1 ± 0.0 c | −50 | 2.1 ± 1.0 a | 950 | |
Proline | 2.5 ± 1.0 a | 0.4 ± 0.3 c | −84 | 2.4 ± 1.3 b | 4 | |
Serine | 1.3 ± 0.7 b | 0.3 ± 0.2 c | −77 | 1.7 ± 0.3 a | 31 | |
Ammonia | 2.2 ± 0.8 b | 3.2 ± 0.6 c | −46 | 14.1 ± 2.3 a | 541 |
†Values are means of results obtained in triplicate ± SD expressed for the three amaranth varieties. Means followed by different letters in the same row are significantly different at p < 0.01.
Popping led to a significant decrease in almost all free amino acid contents (
Conversely, fermentation increased the amount of almost all free amino acids, except tyrosine, glutamic acid and proline, which remained unchanged, and arginine, which strongly decreased (
In vitro protein digestibility (IVPD) is shown in
fiber and polyphenols (data not shown), which could bind the protein and reduce its susceptibility to enzymatic attack [
Both popping and fermentation significantly affected, (p < 0.05), the IVPD (
In addition, hydrolysis of proteins by microorganisms during fermentation, evidenced by the increase in free amino acids, could increase IVPD. Studies by El Hag et al. (2002) [
For all the indispensable amino acids, the PDCAAS, which takes both the amino acid score and protein diges- tibility into account, was highest for the white followed by the red and brown variety (
Amino acid | White amaranth | Red amaranth | Brown amaranth | ||||||
---|---|---|---|---|---|---|---|---|---|
Raw | Popped | Fermented | Raw | Popped | Fermented | Raw | Popped | Fermented | |
AAA§ | 178 | 135 | 174 | 149 | 138 | 159 | 139 | 125 | 133 |
Histidine | 170 | 128 | 170 | 146 | 143 | 162 | 138 | 126 | 139 |
Isoleucine | 105 | 81 | 107 | 93 | 70 | 94 | 80 | 70 | 79 |
Leucine | 88 | 68 | 89 | 75 | 66 | 79 | 67 | 61 | 69 |
Lysine | 117 | 68 | 110 | 100 | 66 | 104 | 89 | 40 | 49 |
SAA§ | 252 | 157 | 248 | 198 | 134 | 224 | 195 | 119 | 99 |
Threonine | 123 | 94 | 122 | 101 | 88 | 110 | 97 | 83 | 90 |
Valine | 95 | 74 | 95 | 78 | 60 | 82 | 70 | 60 | 71 |
*The scoring pattern for indispensable amino acids was considered for children aged 1 - 2 years [
Amino acid | Amount of amino acid (mg/100g DM) | Requirement$ | Percentage contribution (%)¥ | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Wheat† | Maize† | Amaranth | Wheat | Maize | Amaranth | ||||||
White | Red | Brown | White | Red | Brown | ||||||
AAA | 764 | 795 | 1379 | 1340 | 1400 | 458 - 476 | 51 - 53 | 53 - 56 | 92 - 96 | 90 - 93 | 94 - 98 |
His | 274 | 244 | 516 | 514 | 540 | 171 - 178 | 49 - 51 | 44 - 46 | 92 - 96 | 92 - 97 | 98 - 102 |
Ileu | 441 | 325 | 547 | 563 | 542 | 279 - 321 | 44 - 51 | 32 - 37 | 54 - 62 | 56 - 64 | 54 - 62 |
Leu | 811 | 1129 | 932 | 921 | 925 | 566 - 642 | 40 - 46 | 56 - 64 | 46 - 53 | 46 - 52 | 46 - 53 |
Lys | 334 | 244 | 1026 | 1011 | 1014 | 497 - 535 | 20 - 22 | 15 - 16 | 61 - 66 | 61 - 65 | 60 - 65 |
SAA | 418 | 316 | 1103 | 1008 | 1108 | 241 - 262 | 51 - 56 | 39 - 42 | 136 - 148 | 124 - 135 | 135 - 147 |
Thr | 346 | 334 | 559 | 531 | 570 | 264 - 273 | 40 - 42 | 39 - 41 | 65 - 67 | 62 - 64 | 67 - 70 |
Val | 523 | 433 | 671 | 636 | 641 | 380 - 428 | 39 - 44 | 32 - 36 | 50 - 56 | 48 - 54 | 48 - 54 |
*Assuming daily consumption of 50 g of complementary food on a dry weight basis formulated with 64% of cereals or amaranth. †Shewry et al. (2007) [
(
All three varieties of amaranth investigated in this study can be considered as a potential source of protein. Due to its high essential amino acid contents, amaranth could potentially substitute other more common cereals used in complementary food formulation for young children and thus reduce the proportion of legumes. Although popping improved the sensorial attributes of amaranth porridge, it reduced protein quality through the loss of heat labile amino acids. On the other hand, fermentation was better than popping in maintaining the amino acid profile with its added advantage of improving the protein digestibility although it resulted in a marked decrease in lysine content.
The authors are grateful to Addis Ababa University, SIDA SAREC Swedish government, and Institut de Recherche pour le Development (IRD), Montpellier, France for supporting the study.