Plant Foods for Human Nutrition 36: (1987) Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht -- Printed in the Netherlands

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1 Plant Foods for Human Nutrition 36: (1987) Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht -- Printed in the Netherlands The nutritive value of amaranth grain (Amaranthus caudatus) 2. As a supplement to cereals BIRTHE PEDERSEN, L, HALLGREN, INGE HANSEN* and B.O. EGGUM* Carlsberg Research Laboratory, Department of Biotechnology, 10 Gamle Carlsbergvej, DK-2500 Valby, Denmark * National Institute of Animal Science, Department of Animal Physiology and Biochemistry, 25 Rolighedsvej, DK-1958 Frederiksberg C, Denmark (Received April 9, 1986; accepted August 18, 1986) Key words: amaranth grain, protein and energy utilization, protein complementation, maize, wheat, sorghum Abstract. The purpose of this investigation was to study the nutritional value of grain amaranth (Amaranthus caudatus) as a supplement to cereals, and to determine the effect of heat on nutritional quality of cereal/amaranth mixtures. Maize, wheat and low-tannin sorghum blends containing 0%, I0%, 25% and 50% of popped amaranth flour were studied in balance experiments with growing rats. The popped amaranth was very high in lysine (5.19 g/16 g N), but lysine was still the first limiting amino acid in the blends. However, amino acid scores were raised significantly by amaranth substitution, and the tryptophan deficiency of maize protein was alleviated by addition of amaranth. True protein digestibility was, in general, not affected by amaranth substitution, but the biological value increased considerably with increasing levels of inclusion. Amaranth substitution also resulted in remarkable increases in weight gain of the rats. The energy digestibility of the different blends was quite similar and there was only little variation in the amount of digestible energy of the mixtures. Cooking produced a very small but significant reduction in protein and energy digestibility. However, the effects of cooking were minor and probably without practical importance. In conclusion, addition of amaranth to cereal flours improved protein quality without affecting energy utilization. Amaranth seems to be an effective source of protein to combine with cereal proteins. Introduction Amaranths are hardy, fast growing, cereal-like plants; and the role of amaranth as an undere ploited plant with promising economic value has recently been recognized [13, 14]. Amaranth is still cultivated as a minor food crop in Central and South America, and parts of Asia and Africa. Traditional recipes for the preparation of amaranth seeds vary among cultures. In Latin America and in the Himalayas, amaranth flour is used to produce a variety of fiat breads. The grain is also commonly popped and consumed as is or used as an ingredient or confection. Correspondence should be addressed to Dr Birthe Pedersen, Carlsberg Research Laboratory, Dept of Biotechnology, 10 G1. Carlsbergvej, DK-2500 Valby, Denmark.

2 326 Amaranth grain contains about 15% of protein [15]. The amino acid profile makes it an attractive protein source and, because of its high lysine content, it tends to be viewed as a protein that, if consumed along with other cereals would provide a 'balanced' protein [18]. As a consequence, there has been an increasing interest in using amaranth flour in blends with wheat or maize. Amaranth flour blends can be used for making tortillas, breads, cookies, pasta and marzipan [16, 17]. Breads containing 10 to 15% of amaranth flour have been described as being nutty, pleasant-tasting, and generally preferred over white bread [18]. PER values have been reported to be improved in tortillas made from maize-amaranth flour [16] and in wheat bread containing 20% of amaranth flour [17]. However, in general, there is a tack of experimental data on the nutritional quality of amaranth flour blends and products made from these blends. The purpose of the present investigation was to study the nutritional value of grain amaranth as a supplement to cereals, and to determine the effect of heat on nutritional quality of cereal/amaranth mixtures. Maize, wheat and sorghum blends, containing up to 50% of popped amaranth flour, were studied in balance experiments with growing rats. Sorghum was included in the study because amaranth grows well under dry conditions where sorghum is also known to thrive. Material and methods Materials Soft Danish winter wheat (Kraka), maize (U.S. No. 3) and a low-tannin sorghum (Dabar) from Sudan were milled into flour in a hammer mill (100 LU, Alpine) equipped with a perplex rotor and a 0.5 mm mesh screen. A pale seeded variety of Amaranthus caudatus (CAC-064) was obtained from Peru. The amaranth seeds were popped and milled as described previously [15]. Cereal/amaranth mixtures containing 0%, 10%, 25% and 50% of amaranth flour on a dry matter basis were prepared. To evaluate the effect of cooking, the cereal/amaranth mixtures were suspended in 4.5 parts (by weight) of boiling water and placed on a boiling water-bath for 30 rain. After heating, the samples were cooled, freeze-dried and hammer milled. Animals and diets The experimental procedure has been described by Eggum [7]. Groups of five male Wistar rats weighing approximately 70 g were used in balance experiments with a preliminary period of 4 days and a collection period of 5 days. The rats were weighed at the beginning of the experiment and divided into groups so that the mean weights of the groups differed by no more than 0.7 g, The rats were housed individually in Plexiglass metabolic

3 327 cages in a controlled environment. Temperature and relative humidity were held at 25 C and 50%, respectively. Lighting was controlled by alternating 12-h periods of light and darkness. Each animal received 10 g dietary dry matter and 150 mg N daily throughout the study. Distilled water was supplied ad libitum. Body weight and dietary intake were recorded. Urine and faeces were collected separately during the balance period. The faeces were lyophitized and ground into a fine powder prior to analyses. The diets were formulated to contain 1.5% of dietary nitrogen. An N-free mixture consisting of (g/kg dry matter) autoclaved potato starch 806.0, sucrose 90.0, cellulose powder 52.0, and soya-bean oil 52.0, was used to adjust the dietary nitrogen content. All diets contained on dry basis 40.0 g/kg of a mineral mixture (6) and 16.0 g/kg of a vitamin mixture [6]. True protein digestibility (TD), biological value (BV) and net protein utilization (NPU = TD BV) were measured. Corrections were made for endogenous excretion of N in faeces (1.7 mg N/g dry matter consumed/ rat) and in urine (76 mg N/5 days/rat) as described by Eggum [7]. Utilizable protein in the mixtures (UP = NPU Protein content) was calculated. Estimation of digestible energy (DE) was based on analysis of diets and faeces. Digestible energy of the cereal/amaranth mixtures was estimated by calculation, and corrections were made for the digestibility of energy in the N-free mixture. In a preliminary experiment DE of the N-free mixture was found to be 91.7% Analytical methods AOAC methods [2] were used for determination of dry matter (No ), ash (No ) and nitrogen (No ). Energy was determined by bomb calorimetry (IKA-C 400, Janke and Kunkel). Fat [19], sugar [11], starch [6] and total dietary fibre [1] were also determined. Amino acid analyses were conducted according to Mason et al. [12], and tryptophan was determined by HPLC and fluorescence spectrophotometry after hydrolysis with sodium hydroxide [4]. The data were subjected to one-way and two-way analysis of variance and differences between groups were identified by Tukey's HSD test [10]. Regression analysis were performed on treatment means. Results The proximate composition of maize, wheat, sorghum and popped ama-" ranth is listed in Table 1 and the amino acid composition in Table 2. Compared with cereal grains, popped amaranth was very high in lysine and low in leucine. Amino acid scores of maize, wheat, sorghum and amaranth were 54, 51, 37 and 74, respectively. Amino acid scores of the

4 328 Table 1. Chemical composition (% dry basis) of maize, wheat, sorghum and amaranth Maize Wheat Sorghum Amaranth Ash Protein (N x 6.25) Fat Sugar Starch Dietary fibre blends increased considerably with increasing levels of amaranth, and at the highest level of substitution, scores were comparable to the amino acid score of amaranth alone (Table 3). Based on the FAO Provisional Amino Acid Scoring Pattern [9], the limiting amino acid in popped amaranth was leucine followed by valine and threonine. In the cereals and all the blends, lysine was the first limiting amino acid. Threonine was the second limiting amino acid, except in maize and maize/amaranth flour (90/10) in which the second limiting amino acid was tryptophan. Protein content of the popped amaranth seeds was 14.5% on a dry basis, which is higher than in the cereal grains. Consequently, protein content of the blends rose with increasing levels of substitution (Table 3). Food intake did not vary among groups of rats fed wheat blends, however, in rats fed maize or sorghum blends, food intake increased significantly (P < 0.05) with increasing levels of amaranth in the mixtures Table 2. Amino acid content (g/16g N) of maize, wheat, sorghum and amaranth Maize Wheat Sorghum Amaranth Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine c Leucine ~ Lysine 2.96 a a 5.19 Methionine 1.99! Phenylalanine Proline Serine Threonine 3.28 c 2.56 b 2.71 b 3.31 c Tryptophan 0.70 b Tyrosine Valine b a First limiting amino acid. b Second limiting amino acid. c Third limiting amino acid. a- Based on FAO 1973 reference scoring pattern [9].

5 Table 3, Protein content (dry basis), N ratio and Amino Acid Score of cereal/amaranth mixtures Cereal/amaranth weight ratio (dry basis) 100/0 90/10 75/25 50/50 Maize: Protein (N x 6.25)% Cereal/amaranth N ratio 100/0 86/14 67/33 41/59 Amino Acid Score" Wheat: Protein (N x 6.25) % I Cereal/amaranth N ratio I00/0 89/11 73/27 47/53 Amino Acid Score ~ Sorghum: Protein (N x 6.25) % Cereal/amaranth N ratio 100/0 89/11 72/28 46/54 Amino Acid Score" Based on FAO 1973 reference scoring pattern [9]. 329 Table 4. Food intake (dry basis), weight gain and food conversion ratio in rats fed cereal/ amaranth mixtures Cereal/amaranth Uncooked Cooked weight ratio (dry basis) 100/0 90/10 75/25 50/50 100/0 90/10 75/25 50/50 Maize: Food intake per 9 days (g) 85.5 b 88.0 ~b 89.8 ~ 89.4" 88.2 ab 87.4 ab 89.1 ab 89.5" Weight gain per 9 days (g) 14.6 ab* 14.8 a~ 16.9 ~b~ 18.9 a 12.2 ~ 13.2 b~ 16.0 ab~ 17.9 ab Food conversion ratio 5,9 ~b 6,0 ~b 5.3 ~b 4.8 b 7.8" 6.4 ~b 5,7 ab 5,28 Wheat: Food intake per 9 days (g) Weight gain per 9 days (g) 12.9 ~ 13.4 b~ 16.0 a~ 17.P b 11.5 c 14.4 b~ 16.9 ~b 19.9 a Food conversion ratio 6.8 ab 6.5 ~b 5.7 ~ 5.3 b~ 7.8 ~ 6.0 ~ 5.3 ~ 4.4 ~ Sorghum: Food intake per 9 days (g) 72,9 b 81.9 ~b 81.5 ab 84.9 ~b 79.5 ab 76.0 ab 80.2 ab 8g.4 a Weight gain per 9 days (g) 9,0 b 12.0 "b 13.& 16,& 9.2 b 10.5 ~b 12.4 "b 16.4 ~ Food conversion ratio 8.4 b~ 6.9 ~ 6.4 "~ 5.3" 8.6 ~ 8.1 "b~ 6.7 ~b~ 5,5 ab ~-~Mean values with unlike superscript letters in the same row were significantly different (P < 0.05). SDpooi~d: Maize: Food intake = 1.8, Weight gain = 2.4, FCR = 1.2. Wheat: Food intake = 3.4, Weight gain = 2.3, FCR = 1.0. Sorghum: Food intake = 7.0, Weight gain = 2,9, FCR = 1.5.

6 330 Table 5. Protein utilization in rats fed cereal/amaranth mixtures Cereal/amaranth Uncooked Cooked weight ratio (dry basis) 100/0 90/10 75/25 50/50 100/0 90/10 75/25 50/50 Maize: True protein digestibiiity 96.3 b 94.4 b Biological value 64.7 ~ 65.5 a Net protein utilization 62.4 ~b 61.8 ~u Utilizable protein (g/100g, dry basis) 6.2 ~ 6.5 ~ Wheat: True protein digestibility 91.7 ab 93.0" Biological value 62.1 a 64.F d Net protein utilization 57.0 ~d 59.6 Ca Utilizable protein (g/100g, dry basis) b~ Sorghum: True protein digestibility 94.4 b 95.0 b Biological value 54.9 ~ 54.6 ~ Net protein utilization 51.8 a 51.8 a Utilizable protein (g/100g, dry basis) 93.0 "b 90.8" 90.3" 89.@ 90.2 ~ 90.3 ~ 71.2 ~ 77.7 d 66.8 "b 68.8 ~ 74.2 ~ 78.3 d 66.3 ~ " 61.7 ~b 66.9 ~ 70.7 ~ 7.3 b 8.6 c 6.0 a 6.4 ~ 7.4 b 8.6 ~ 90.7 ~b 89.9 ab 89,3 b 88,8 b 89,0 b 89,5 ab 67.4 ~ 74.4 ab 62,4 d 68,0 ~d 69.7 "~ 75,7 a 61.1 c 66.9 ab 55,6 d 60.3 a 62.0 b~ 67.7 ~ a 7.3 ~ 8.0 t~ 8.4 b 9.3 a 93.4 b 91.8 ab 91.7 ab a 87.4 a 63.2 ab 71,3 b 54,0 a 64.1 ab 67.7 b 70.3 b 59.0 ~c a 53.0 ab 58.3 abc 61.5 ~ 6.5 ab 6.6 abc 7.8 cd " 6,8 abe 7.7 b~d 8.4 ae ~-~ Mean values with unlike superscript letters in the same row were significantly different (P < 0.05). SDpooJ~d: Maize: TD = 1.6, BV = 2.6, NPU = 3.0, UP = 0.3. Wheat: TD = 1.8, BV = 3.2, NPU = 2.6, UP = 0.4. Sorghum: TD = 2.9, BV = 4.7, NPU = 4:2, UP = 0.5. (Table 4). The effect was most marked in rats consuming sorghum based diets. Weight gain of rats also increased significantly (P < 0.001) with increasing proportions of amaranth in the blends, and the weight gain of rats fed sorghum/amaranth flour (50/50) was almost twice of that of rats fed sorghum alone. Addition of popped amaranth to the cereal flours also resulted in significantly (P < 0.05) reduced food conversion ratios (g dry matter eaten/g weight gain). Cooking parameters. had no effect on any of these True protein digestibility of wheat and sorghum mixtures was unaffec- ted by the level of amaranth in the blends, but the biological value and net protein utilization increased substantially (P < 0.001) with increasing levels of substitution (Table 5). Cooking of these mixtures had no effect on BV or NPU but caused a moderate but significant (P < 0.001) reduction in TD. In the maize blends, addition of amaranth flour also resulted in a marked increase in BV and NPU. At the highest levels of substitution, TD was reduced in the uncooked maize mixtures, In general, cooking resulted in lower TD values and higher BV values of the maize blends, NPU on the other hand, was unaffected by heat. For all three cereals, the amount of utilizable protein in the blends increased significantly (P < 0.001) with

7 331 Table 6. Energy utilization in rats fed cereal/amaranth mixtures Cereal/amaranth Uncooked Cooked weight ratio (dry basis) 100/0 90/10 75/25 50/50 100/0 90/10 75/25 50/50 Maize: Digestible energy(%) 88.9 ~ 88.4 ab 88.1 ab 86.7 a~ 85.5 ~ 86.2 t* 86.5 at* 86.3 t~ Digestible energy (kj/g, dry basis) 16.8 ~ 16.8 ~ 16.7 ~ b 16.4 ~b 16.4 "b 16,6 ab Wheat: Digestible energy(%) 88.P b 89.5 a 87.8 "b~ 87.8 at* 86.4 b 86.4 ~ 85.8 c 86.4 ~ Digestible energy (kj/g, dry basis) 16.1 b~d 16.5 ab 16.3 abc 16.6 ~ 15,8 ~ 15.9 ~ 16.0 ~a 16.3 ~b Sorghum." Digestible energy (%) 90.3 ~ 90.6" 89.8 ~ 88.8 ~b 86.9 ~ 85.8 ~ 87.I b~ 85.8 Digestible energy (kj/g, dry basis) 17.1Y b 17.P IT.P 17.0 ~b ~ 16.5 ~ 16.4 ~ a-c Mean values with unlike superscript letters in the same row were significantly different (P < 0.05). SDpool0d: Maize: DE% = 1.2, DE(kJ/g) = 0.2. Wheat: DE% = 1.0, DE(kJ/ g) = 0.2. Sorghum: DE% = 1.3, DE(kJ/g) = 0.2. increasing amaranth flour levels. UP was similar in cooked and uncooked mixtures. For all three types of cereal blends, the digestibility of energy (DE %) was unaffected by the level of amaranth whereas heat produced a relatively small but significant (P < 0.001) reduction in energy digestibility (Table 6). Cooking also caused a very small reduction (P < 0.001) in the amount of digestible energy (k J/g) of the blends. The gross energy content of maize, wheat, sorghum and popped amaranth was 18.9, 18.3, 18.8 and 19.4 kj/g, respectively. The energy content of the wheat blends tended to increase slightly with increasing levels of substitution and for the wheat mixtures the content of digestible energy was raised (P < 0.001) by inclusion of amaranth. For all treatments, weight gain of rats was correlated to NPU (r = 0.92, P < 0.001) and to lysine content of the mixtures (r = 0.95, P < 0.001). BV of the blends was closely correlated to the concentration of lysine (r = 0.95, P < 0.001). There was also a strong relationship between BV and amino acid scores (r = 0.95, P < 0.001). Discussion Although some differences exist among and within species, the literature shows general agreement in gross proximate composition and amino acid profile of amaranth seeds [18]. The amino acid composition of the popped amaranth used in the present study was in close agreement with values reported earlier [18]. Lysine was present at a level about twice of that in the cereal grains, and although popping may have reduced the quantity of

8 332 lysine slightly, popping seems to have no major effect on protein quality or N digestibility [5, 15]. However, in spite of the high lysine content of the popped amaranth, lysine was still the first limiting amino acid in the cereal blends containing up to 50% of amaranth flour. Nevertheless, amino acid profiles of the cereal blends were markedly improved and amino acid scores were raised significantly by amaranth substitution. Especially the unfavorable amino acid composition of sorghum was greatly improved by amaranth inclusion, and amino acid scores rose from only 37 in sorghum flour to 68 at the highest level of inclusion. Also, the tryptophan deficiency of maize protein was alleviated by addition of amaranth. At the 25% level of substitution, trytophan was only the fourth limiting amino acid, and at the highest level of inclusion the content of tryptophan exceeded that of the FAO amino acid scoring pattern [9]. As indicated by the amino acid data, the biological value increased considerably with increasing levels of amaranth in the blends. The increase in BV was most marked for the sorghum mixtures in which the biological value rose from 54% to 71% in the blend containing 50% of amaranth. Yet, even at the highest level of substitution, the biological value of the cereal blends was much lower than the 86% found for the popped amaranth in a previous study [15]. This very high biological value of the popped amaranth protein, as well as the consistency between amino acid scores and BV values of the blends found in the present study, strongly indicate that the availability of essential amino acids in amaranth protein is high. By contrast, in cereal grains protein utilization often seems to be limited by low lysine availabilities [7]. In general, true protein digestibility of the blends was not affected by amaranth substitution. This is to be expected as the true protein digestibility (88%) of the popped amaranth is only slightly lower than that of the cereal grains [15]. As protein digestibility was essentially unchanged by amaranth substitution net protein utilization of the blends increased, as a result of the rise in the biological value, with increasing levels of amaranth. Because of the increase in NPU as well as the rise in protein content, the quantity of utilizable protein in the maize/amaranth mixture (50/50) was 40% higher than in the maize flour itself. For wheat and sorghum, the amount of utilizable protein was raised by 25% and 35%, respectively, at the highest level of substitution. In consistency with the N balance results, amaranth substitution resulted in remarkable increases in weight gain of the rats and concomitant reductions in food conversion ratios. The increase in food intake with increasing amounts of amaranth in the diets, was most marked for the groups of the rats fed the sorghum based mixtures. The increased food intake, probably related to the improved protein quality of the diet, strongly indicates that palatability of the popped amaranth is high. Popping has been reported to improve feed consumption in rats [5].

9 333 Probably due to a relatively high level of fat, the energy content of the popped amaranth was slightly higher than that of the cereal grains. The energy content of the different blends varied only little and the digestibility of the energy, ranging from 87% to 91% in the uncooked mixtures and from 86% to 87% in the cooked blends, was also quite similar. Consequently, there was only little variation in the amount of digestible energy of the mixtures. Cooking did produce a very small but significant reduction in energy digestibility, which is probably due to formation of enzyme-resistant starch [3, 6]. Protein digestibility of the cooked blends was also slightly inferior to that of the uncooked mixtures. In the maize blends, a decrease in protein digestibility occured concomitantly with an increase in biological value. Thus, the N balance results obtained for maize reflect a shift towards increased faecal N excretion and decreased urinary excretion. Similar changes in N excretion pattern, as a result of cooking, have been reported previously for rice and sorghum [3, 8]. However, the mechanisms involved are probably different. Cooking had no effect on weight gain of rats, food conversion ratios, net protein utilization or the amount of utilizable protein in the blends. The reduction in energy digestibility was, in general, less than 4%. Thus, the effects of cooking observed in this study seem to be without practical importance. However, the effects of heat on digestibility might have been more pronounced if a high-tannin sorghum and not a low tannin variety had been used [3]. In conclusion, our results are in accordance with those of Sfinchez- Marroquin [16, 17] who reported PER of maize and wheat products to be improved by amaranth substitution. Our study has shown that addition of amaranth to cereal flours improves protein quality without affecting energy utilization, and amaranth seems to be an effective source of protein to combine with cereal proteins. References 1. Asp NG, Johansson CG, Hallmer H, Siljestr6m M (1983) Rapid enzymatic assay of insoluble and soluble dietary fibre. J Agric Food Chem 31: Association of Official Agricultural Chemists (1980) Official methods of analysis, 13th edn. Washington DC: AOAC 3. Bach knudsen KE, Munck L (1985) Dietary fibre contents and compositions of sorghum and sorghum-based foods. J Cereal Sci 3: Bech-Andersen S (1986) Tryptophan determination in feedstuffs (Personal Communication) 5. Betschart AA, Irving DW, Shepherd AD, Saanders RM (1981) Amaranthus Cruentus: Milling characteristics, distribution of nutrients within seed components, and the effects of temperature on nutritional quality. J Food Sci 46: Bj6rck I, Nyman M, Pedersen B, Siljestr6m M, Asp NG, Eggum BO (1986) On the digestibility of starch in wheat bread -- Studies in vitro and in vivo. J Cereal Sci 4:I Eggum BO (1973) A study of certain factors influencing protein utilization in rats and pigs. Copenhagen: Beretn 406 National Institute of Animal Science

10 Eggum BO, Monowar L, Bach Knudsen KE, Munck L, Axtell J (1983) Nutritional quality of sorghum and sorghum foods from Sudan. J Cereal Sci 1: Food and Agriculture Organization (1973) Energy and protein requirements. Nutr Rep Ser No 52. Rome: FAO 10. Gill JL (1978) Design and analyses of experiments in the animal and medical sciences, Vol 1. Iowa: The Iowa State University Press 11. MacRae JC, Armstrong DG (1968) Enzyme method for determination of s-linked glucose polymers in biological materials. J Sci Food Agric 19: Mason VC, Bech-Andersen S, Rudemo M (1980) Hydrolysate preparation for amino acid determination in feed constituents. Z Tierphysiol Tierern/ihrg u Futtermittelkde 43: National Academy of Science (1975) Underexploited tropical plants with promising economic value. Washington DC: NAS 14. National Academy of Science (1984) Amaranth: Modern prospects for an ancient crop. Washington DC: NAS 15. Pedersen B, Kalinowski LS, Eggum BO (1987) The nutritive value of amaranth grain (Amaranthus caudatus). 1. Protein and minerals of raw and processed grain. (Qualitas Plantarum): Plant Foods for Human Nutrition 36: S~inchez-Marroquin A (1980) Potencialidad agro-industrial del amaranto. Mexico: Centro de Estudios Econdmicos y Sociales del Tercer Mundo 17. S~inchez-Marroquin A, Domingo MV, Maya S, Saldafia C (1985) Amaranth flour blends and fractions for baking applications. J Food Sci 50: Saunders RM, Becker R (1984) Amaranthus: A potential food and feed resource. In: Pomeranz Y (ed) Advances in Cereal Science and Technology, Vol VI. Minnesota: AACC, pp Stoldt W (1952) Vorschlag zur Vereinheitlichung der Fettbestimmung in Lebensmitteln. Fette und Seifen 54: