Protein evaluation of four oat (Avena sativa L.) cultivars adapted for cultivation in the south of Brazil

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1 Plant Foods for Human Nutrition 53: , Kluwer Academic Publishers. Printed in the Netherlands. Protein evaluation of four oat (Avena sativa L.) cultivars adapted for cultivation in the south of Brazil I. PEDÓ, V.C. SGARBIERI and L.C. GUTKOSKI Instituto de Tecnologia de Alimentos, Av. Brasil 2880, Caixa Postal 139, Campinas, SP, CEP , Brasil Received 26 March 1998; accepted in revised form 28 August 1998 Abstract. Four oat cultivars adapted for soil and climate conditions in the southern region of Brazil were evaluated for protein nutritive value. Evaluations were done both in vitro and in vivo. In vitro evaluation was done by essential amino acid profile, available lysine, amino acid scoring, and protein digestibility corrected amino acid-scoring (PDCAAS). Nitrogen balance indices and PER were determined in vivo with rats. In all four cultivars (UFP-15, UFP-16, CTC-03, UFRGS-14), lysine was the most limiting amino acid. Available lysine, amino acid score and PDCAAS were highest for cultivar UFRGS-14 and lowest for CTC-03. When compared to casein, only nitrogen retention for UFRGS-14 did not differ statistically (p >0.05); all other indices of protein quality were inferior to casein for the oat cultivars. The oat cultivars tended to be identical among themselves, except for apparent protein digestibility which was significantly higher in the UFRGS-14 and CTC-03 cultivars. On average, the PER values of the oat cultivars were 82% of casein; the net protein utilization was 88% of casein as determined in vivo and 49% by the estimation in vitro (PDCAAS). Key words: Cultivars, Nutritive value, Oat, Protein Introduction Oats represent an important alternative crop for the southern region of Brazil. It can be cultivated as pasture for cattle, as a green fertilizer for the soil and for grain production. Up until recently, oat production in Brazil had been low due mainly to lack of well adapted cultivars. Recently, breeding programs have been implemented to increase adaptability, yield of grain and protein content [1]. The use of oats as human and animal food has been justified by their taste and high nutritive value, when compared to other cereal grains, including a high protein content and protein value. The use of oats as human food has also been stimulated by the quality of its fiber components which act as hypoglycemic and/or hypocholesterolemic agents [2 4]. Protein nutritive value of oat grain was reported by Eggun & Gullord [5] and Eggun et al. [6], who found true protein digestibility in the range of 90.3 to 94.2%, biological value 74.5 to 79.6%, net protein utilization 69.1 to 72.4, and protein efficiency

2 298 ratio 2.25 to Proximate percent composition, amino acid profile, mineral and fatty acid composition of the Brazilian cultivars described in this paper have been reported elsewhere [7]. Materials and methods Oat cultivars. Four oat (Avena sativa L.) cultivars, UFP-15, UFP-16, CTC- 03 and UFRGS-14, were selected for this study. The oat kernels were initially cleaned (freed of impurities), dehulled in an Imack impact mill and held in a freezer ( 18 C) prior to grinding. Grinding was done immediately before use in a knife type Renard Mill, Adel MFC , to a particle size less than 500 µm. Analytical procedures. Amino acids were determined in an acid hydrolysate (6N HCl, 110 C, 22 h) using a Beckman 7300 autoanalyzer, according to the method of Spackman et al. [8]. The sulfur-containing amino acids (methionine and cysteine) were determined as methionine sulfone and cysteic acid, respectively, after complete oxidation of the sample protein with performic acid, prior to acid hydrolysis, according to the method of Moore [9]. Tryptophan was determined in an alkaline hydrolysate (4N LiOH) using the method of Lucas & Sotello [10]. Available lysine. Available lysine was determined by the method of Kakade & Liener [11] using a 0.1% water solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The samples were dissolved in an aqueous 4% NaHCO 3 solution for the reaction, and quantification of the lysine derivative was done using the Lambert Beer equation with a molar extinction coefficient of /M/cm. Essential amino acid scoring. The amino acid score was established by comparing the content of each essential amino acid in the oat protein with the corresponding FAO/WHO/UNU [12] standard reference value for the age group of 2 to 5 years. The essential amino acid in smallest quantity relative to the reference represented the chemical score for the protein under study. Protein digestibility corrected amino acid-scoring. PDCAAS was calculated by multiplying the most limiting essential amino acid score by the true protein digestibility. True protein digestibility was determined in vivo [13]. Biological assays with rat. The composition of diets used in the assays with rats is outlined in Table 1 and is in accordance with AOAC [14] methods

3 299 Table 1. Composition of diets utilized in the rat bioassays for determination protein and casein nutritive values of oat cultivars Components Diets a UFP-15 UFP-16 CTC-03 UFRGS-14 CAS Oat flour Casein Soy oil Mineral mix b Vitamin mix b Cellulose 20.0 Sucrose Corn starch a UFP-15, UFP-16, CTC-03, UFRGS-14, CAS cultivars of oats and casein utilized as sources of dietary protein (10% of the diet). b According to AIN-93. except for the mineral and vitamin mixtures which were based on AIN-93 [15]. Male rats of the Wistar strain, specific pathogen-free (SPF), days of age, and an average weight of 51.3 ± 4.1 g were furnished by the Experimental Animal Center of the State University of Campinas, São Paulo, Brazil, for use in these experiments. The rats (seven per treatment) were housed in individual stainless steel cages with screen bottom for 28 days, the duration of the experiment. Temperature of the laboratory was maintained at 21 ± 2 C with alternate periods of light and dark of 12 hours. The animals had free access to the diets and tap drinking water. Diet, protein consumption and body weight were recorded every other day for the duration of the experiment for growth evaluation and PER calculation. Feces and urine were collected during the second week of feeding for calculations of nitrogen balance and the protein quality indices derived from nitrogen balance. The procedures for obtaining the various indices of protein quality were those of Sgarbieri [16]. Statistical analysis. The statistical program SAS [17] was used for analysis of variance of the biological data, according to Cochran & Cox [18]. The t test of Tukey for statistical significant differences among means (p 0.05) was also used. Results and discussion Table 2 data show the essential amino acid contents of the four oat cultivars. Comparisons with the FAO/WHO/UNU reference standard [12] for require-

4 300 Table 2. Essential amino acid composition (g/16 g N) a of oat cultivars, compared to FAO/WHO/UNU reference pattern b Amino acid c Cultivars FAO/WHO/UNU UFP-15 UFP-16 CTC-03 UFRGS-14 Valine (95.1) Isoleucine Leucine (96.2) Threonine 2.68 (78.8) 2.83 (83.2) 2.88 (84.7) 2.41 (70.9) 3.4 Histidine (98.0) Lysine d 4.00 (69.0) 3.62 (62.4) 3.52 (60.7) 4.10 (70.7) 5.8 Tryptophane Phenylalanine Tyrosine Methionine Cysteine Phenylalanine Methionine a Average of duplicate determinations. b FAO/WHO/UNU (1985) reference profile. c Values in parenthesis indicate percentages of limiting amino acids with regard to FAO/WHO/UNU reference. d The most limiting essential amino acid. ments of children 2 to 5 years of age revealed that lysine was the most limiting amino acid, in the range of 60 70% of the reference. Threonine was the second limiting essential amino acids in all four cultivars. Sulfur-containing amino acids and tryptophan concentrations were above requirements, which makes oats a good complementary protein source for legume grains. The contents of essential amino acids encountered by other investigators [5, 19, 20] for a number of other oat cultivars are in the same range of values presented here. The available lysine data is shown in Table 3. Availability was in the 80 to 90% range. It was lowest (80% of total lysine) in the cultivar UFP-15, higher and similar (87 to 92%) for the other three cultivars. These values were lower than the 98% determined by Carpenter et al. [21] and in the same range of values reported for other cereal grains: wheat 90.8%, corn 91%, and barley 94% availability [22]. The limiting amino acid score and PDCAAS for the studied cultivars are also reported in Table 3. The highest limiting amino acid score and PDCAAS were found for the cultivar UFRGS-14.

5 301 Table 3. Available lysine, amino acid scores and protein digestibility corrected amino acid scoring (PDCAAS) of four oat cultivars Cultivars Available lysine Amino acid score a PDCAAS b (mg/16 g N) (%) (%) UFP (80) UFP (92) CTC (88) UFRGS (87) a Score based on available lysine (%) of the FAO/WHO/UNU reference pattern for children 2 to 5 years of age (FAO/WHO/UNU 1985). b PDCAAS = protein digestibility corrected amino acid-scoring, Henley & Kuster [13]. Figures between parentheses: Percent of total lysine. Table 4 data show the results of the nitrogen balance experiment, including nitrogen retention, apparent protein digestibility, apparent protein biological value, and net protein utilization for the oat cultivars compared with casein. Nitrogen retention in the UFRGS-14 fed rats did not differ statistically (p >0.01) from those of the casein fed animals. For the other three oat cultivars, nitrogen retention was significantly (p 0.05) lower than for casein. Apparent digestibility was significantly higher in the casein followed by UFRGS-14 and CTC-03 and lowest in UFP-15 and UFP-16. No stat- Table 4. Indices of protein nutritive value of four oat cultivars based on nitrogen balance experiment Cultivars Nitrogen Apparent Apparent Net protein retained digestibility biological value utilization (mg/24 h) (Da%) (BVa%) (NPUa%) UFP b c ns b UFP b c b CTC b bc b UFRGS ab b b Casein 78.0 a a a Mean CV (%) Mean value and coeficient of variation for the oat cultivars. a,b,c Different superscript letters within columns indicate statistically different values (p 0.05).

6 302 Figure 1. Protein consumption, body weight gain and PER determined for cultivars of oats and casein. Different superscript letters indicate significantly different PER values (p 0.05). istical difterences were found between casein and oat proteins and among oat cultivars for protein biological value. Casein was statistically (p 0.05) superior to the oat cultivars with respect to NPUa%. No statistical differences for NPUa% were detected among cultivars. Sarwar et al. [23] reported a protein digestibility in oats of 82%, similar to results reported in this paper. Digestibility for oat protein was superior to values encountered for wheat (77%), rice (67.6%) and corn (80.1%) [24]. The results for apparent biological value (Table 4) were superior to values reported by Kjaergaard & Bruzelius [25] and Eggum et al. [6], which were in the 73 to 75% range. Average NPUa% (Table 4) was slightly below the value of 70% reported by Eggum et al. [6] and Eggun & Gullord [5]. By comparing the average PDCAAS of 49% (Table 3) with the average NPUa of 68% (Table 4), one finds a very large discrepancy between the protein quality evaluation in vitro as suggested by Henly & Kuster [13] and the FDA [26] when compared with in vivo determination by the nitrogen balance technique or PER determination (Figure 1). Although in this research, apparent in vivo protein digestibility was used, rather than true digestibility, as recommended in the PDCAAS method [13], the criteria was the same for casein and for the oat cultivars. It seems that the casein amino acid-scoring of 1.0 recommended for calculation of PDCAAS [13, 25] for humans overestimates the casein nutritive value for rats. Data on protein consumption, body weight gain and protein efficiency ratio (PER) for casein and the oat cultivars are shown in Figure 1. The average PER for oat cultivars was 82% of the casein PER, and there were no statistical

7 303 differences (p >0.05) in PER among the oat cultivars. The oat cultivar PER values reported here are slightly higher than the range of 2.25 to 2.38 reported by Hischke et al. [27]. By examining the data of Tables 3 and 4 and Figure 1, a slight superiority of the cultivar UFRGS-14 can be noticed in terms of amino acid score, PD- CAAS, nitrogen retention, protein digestibility and body weight gain. UFRGS- 14 also had the highest protein content [7], although no statistically significant differences could be shown with regard to NPUa% and PER among the oat cultivars. If one takes the amino acid score of 1.0 for casein [23] and the digestibility of 94% (Table 4), the PDCAAS for casein would be 94%, essentially double the values calculated for the oat cultivars. This difference seems too large on the basis of other determined protein nutritional evaluation indices, as determined by biological assays. An overall examination of the data of Tables 3 and 4 and Figure 1 indicates that the in vitro (PDCAAS) method overestimates the protein nutritive value of casein and underestimates the protein nutritive value of the oat cultivar, for the rat. Acknowledgments The authors acknowledge the financial support to this work by FAPESP (Fundação de Aniparo à Pesquisa do Estado de São Paulo), Brazil. References 1. Carvalho FI, et al. (1987) Potencial genético da aveia como produtora de grãos no sul do Brasil. Pesq Agropec Brasil 22: Anderson JW, Chen WL (1986) Cholesterol-lowering properties of oat products. In Webster FH (ed), Oat chemistry and technology, American Association of Cereal Chemists, pp Ripsin CM, Keenan JM (1992) The effect of dietary oat products on blood cholesterol. Trends Food Sci Technol 3: Shinnich FL, Mathews R, Ink S (1991) Serum cholesterol reduction by oats and other fiber sources. Cereal Foods World 36: Eggum BO, Gullord M (1983) The nutritional quality of some oat varieties cultivated in Norway. Qual Plant-Plant Food Hum Nutr 32: Eggum BO, Hansen I, Larsen T (1989) Protein quality and digestible energy of selected food determined in balance trials with rats. Plant Foods Hum Nutr 39: Pedó I, Sgarbieri VC (1997) Caracterização química de cultivares de aveia (Avena sativa L.). Ciênc Tecnol Alim 17: Spackman DH, Stein WH, Moore S (1958) Automatic recording apparatus for use in chromatography of amino acids. Anal Chem 30: Moore S (1963) On determination of cystine as cysteic acid. J Biol Chem 238:

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