ISSN 0101-2061 Ciência e Tecnologia de Alimentos Original Calcium bioavailability of raw and extruded amaranth grains Biodisponibilidade do cálcio do grão de amaranto antes e após extrusão Tania Aparecida FERREIRA1, José Alfredo Gomes ARÊAS2,* Abstract Calcium bioavailability of raw and extruded amaranth grains was assessed in a biological assay in rats. Rats were fed for 28 days on diets in which raw or extruded amaranth was the only calcium source, compared to a control diet with calcium carbonate. Calcium and phosphorous levels were determined in the rats’ serum during the experimental period and in the bones at the end of the experiment. Amaranth extrusion increased its calcium bioavailability, assessed by tibia and femur weights and calcium and phosphorous content of the bones. Apparent calcium absorption index, the force needed to break the bones and bone densitometry of both extruded and raw amaranth were the same, though different from the control group. The results show that amaranth can be a complementary source of dietary calcium the bioavailability of which is favorably modified by the extrusion process. Keywords: novel foods; densitometry; calcium absorption; rats; antinutritional factors. Resumo A biodisponibilidade do cálcio de amaranto antes e após extrusão foi avaliada em ensaio biológico. Ratos, alimentados por 28 dias com dietas em que amaranto, antes ou após extrusão, era a única fonte de cálcio da dieta, foram comparados com animais em dieta controle com teor próximo de cálcio, oferecido na forma de carbonato de cálcio. Cálcio e fósforo foram determinados no soro dos animais durante o período experimental e nos ossos, ao final do experimento. A extrusão do amaranto aumentou a biodisponibilidade do seu cálcio, quando avaliado pelo conteúdo de cálcio e fósforo da tíbia e fêmur dos animais, bem como pelo peso desses ossos. O índice aparente de absorção de cálcio, força para romper os ossos e densidade óssea foram iguais antes e após a extrusão do amaranto e diferentes do grupo controle. Os resultados mostram que o amaranto pode ser uma fonte complementar de cálcio e que sua biodisponibilidade aumenta com o processo de extrusão. Palavras-chave: novos alimentos; densitometria; absorção de cálcio; ratos; fatores antinutricionais. 1 Introduction The Andean crop amaranth (Amaranth caudatus L., and calcium for 2-5 year old children (INSTITUTE OF MEDICINE, other species: A. cruentus, A. hypocondriacus), is a pseudo 2002; KREBS, 2001). cereal that during the last twenty years has been the focus of Calcium deficiency is an important nutritional problem and attention in many scientific studies, chiefly on account of its its bioavailability is a fundamental aspect for human diet. The nutritional, functional, agricultural and technological potential objective of this work was therefore to determine the in vivo (BERGHOFER; SCHOENLECHNER, 2002; LEHMANN, 1996). calcium bioavailability of raw and extruded amaranth in rats Calcium and iron contents vary among cultivars of this crop after 28 days feeding. from 1300 to 2136 ppm in dry basis, (average 1870 ppm) and from 72.2 ppm to 116 ppm, (average 101 ppm), respectively (BECKER et al., 1981). This represents six times more calcium 2 Materials and methods and four times more iron than that observed in wheat grains. 2.1 Amaranth Amaranth is therefore a potential raw material to be introduced in human diet as a new iron and calcium source. Amaranth (Amaranthus caudatus L.) CAC-43A, Oscar Blanco variety, from the germ plasma bank of the Centro de Amaranth is not part of modern food consumption habits. Investigación en Cultivos Andinos (CICA) of the National Thus, a convenience food based on amaranth, similar to the ones University of San Antonio Abad del Cusco-Perú, was used in available to consumers, was produced by extrusion cooking this study. (CHÁVEZ-JÁUREGUI; SILVA; ARÊAS, 2000). This new product presented high commercial potential as a functional The amaranth grain was sieved and milled in a hammer food, as it is able to reduce blood cholesterol (PLATE; ARÊAS, mill (Marconi, São Paulo, Brazil). The flour thus produced was 2002), and provide high quality protein, high fiber and high defatted with n-hexane for 12 hours in a Soxhlet apparatus calcium contents for human diet. Consumption of a 30 g portion (residual lipid < 1%), and water was added to the desired of this product would meet practically 100% of the RDA for moisture and sealed in polyethylene bags for 48 hours at 5 °C. Recebido para publicação em 20/8/2008 Aceito para publicação em 26/6/2009 (003808) 1 Faculdade de Nutrição, Universidade Federal de Goiás, Rua 227, Qd. 68, s/no., Setor Leste Universitário, CP 131, CEP 74605-080, Goiânia - GO, Brasil 2 Departamento de Nutrição, Faculdade de Saúde Pública, Universidade de São Paulo – USP, Av. Dr. Arnaldo, 715, CEP 01246-904, São Paulo - SP, Brasil, E-mail: [email protected] *A quem a correspondência deve ser enviada 532 Ciênc. Tecnol. Aliment., Campinas, 30(2): 532-538, abr.-jun. 2010 Ferreira; Arêas 2.2 Extrusion Phosphorous determination Extrusion was performed with 15% feed moisture at Phosphorous was determined in the same samples for 150 °C in a laboratory scale single screw extruder of 20 mm calcium analyses, by conventional colorimetric method (FISKE; diameter and a length to diameter ratio equal to 20. The SUBBAROW, 1925) in a different aliquot of the same solutions extruder was divided into three independent heating zones. used for calcium determination. Screw compression ratio was 4:1 and screw speed 200 rpm. Die diameter was 3 mm and feeding was performed by gravity Biological experiments for calcium bioavailability through a hopper (CHÁVEZ-JÁUREGUI; SILVA; ARÊAS, 2000). After extrusion, the product was milled to produce flour The biological assay followed the guidelines of the Colégio‘ that had its dietary fiber, phytates and tannins, determined. Brasileiro de Experimentação Animal’ (Brazilian College for Animal Experiments) under supervision of a Veterinary professional and it was approved by the Goiás Federal University. Proximate composition The experiment was carried out using a completely randomized Proximate composition was determined by conventional block design with 32 newly-weaned rats, (Rattus norvergicus, var. methods: drying to constant weight at 105 °C for moisture, Wistar), in four groups of eight animals each, kept in individual calcination to constant weight at 550 °C for ash, defatting in glass cages. The animals were fed for 28 days on the experimental and Soxhlet apparatus with n-hexane for lipids, micro-kjeldahl for control diets with water provided ad libitum. They were kept organic nitrogen (N × 6.25 = protein) (INSTITUTO ADOLFO in individual cages in an animal house with air conditioning LUTZ, 1985). at 23 °C and equal daily light and dark periods of 12 hours. At the end of the experimental period, all animals were killed by guillotine by a Veterinary professional. The feces were collected Analysis of tannins daily in the last three days of experiment, dried at 105 °C, The tannins were extracted from 200 mg sample by 10 mL weighed and stored at 20 °C for further analysis. A “double- methanol for 20 minutes shaking followed by centrifugation decker” cage was designed to avoid urine contamination of the at 6.000 × g for 15 minutes. Tannins were determined in the collected feces. It consisted of two floors made of distinct mesh supernatant by the vanilla method (PRICE; VAN SCOYOE; size nets that allowed feces to pass through the first one, where BUTLER, 1978) using cathechin as standard and the results the animal was kept, whereas they were retained in a second one were calculated as cathechin equivalents. that was removed after the three-day collection period. Animal’s urine passed through both nets and was discarded. Before Analysis of phytates killing the animals fasted for 12 hours, the blood was collected, centrifuged at 3.000 × g for 20 minutes, and the serum stored at Phytates were analyzed by ferric chloride precipitation of –20 °C for further analysis. Right and left femurs and left tibias the phytates from water extracts of the samples, separation were removed from the animals, cleaned from adherent tissue and washing of this precipitate by centrifugation (6.000 × g) and stored either in foiled sheet at –20 °C, for breaking tests, (THOMPSON; ERDMAN, 1982) and phosphorous or in 70% ethanol solution at 10 °C, for bone densitometry and determination after hydrolysis (FISKE; SUBBAROW, 1925). calcium determination. A conversion factor of 3.5 was used to transform phosphorous contents in phytates (phosphorous corresponds to 28.2% in Diets mass of phytates). Calcium free saline mixture was prepared by removing calcium carbonate and used to produce the experimental diets Dietary fiber determination where the only calcium and protein (at 10% level) source were Soluble and insoluble fiber contents were determined by the the raw and extruded amaranth grains. Calcium content of enzymatic method as described (PROSKY et al., 1988). Enzymes this diet was 0.13 g per 100 g of diet. To keep an experimental used were amyloglucosidase A-9913, thermo resistant amylase diet with the same amount of nutrients, as compared to the A-3306 and protease P-3910, all provided by Sigma Aldrich control, the amount of protein, carbohydrate and lipids from Fine Chemicals (USA). raw and extruded amaranth were taken into account for its manufacturing. The control diet was then prepared based on the Calcium determination semi-purified diet AIN 93-G, according to National Research Council (NATIONAL RESEARCH COUNCIL, 1993), with Calcium was determined in diets and femurs. Calcium casein as protein source and calcium carbonate as the calcium analysis was carried out after wet digestion of the samples with source at the same concentration of the experimental diets, nitric acid and 30% hydrogen peroxide (5:1 v/v) mixture at 0.13 g per 100 g.