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Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy
ARTICLE IN PRESS
Journal of Cereal Science 44 (2006) 342–346 www.elsevier.com/locate/yjcrs
Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy Israel Salinasa,, Arturo Pro´a, Yolanda Salinasb, Eliseo Sosac, Carlos Miguel Becerrila, Manuel Cucaa, Miguel Cervantesd, Jaime Gallegosa a
Ganaderı´a, Campus Montecillo, Colegio de Postgraduados, 56230 Montecillo, Estado de Me´xico, Me´xico b Laboratorio de maı´z INIFAP, Estado de Me´xico, Me´xico c Universidad Auto´noma Chapingo, 56230 Chapingo, Estado de Me´xico, Me´xico d Instituto de Ciencias Agrı´colas, Universidad Auto´noma de Baja California, Mexicali, Baja California, Me´xico Received 20 March 2006; received in revised form 24 August 2006; accepted 29 August 2006
Abstract Kafirins are stored proteins that negatively affect the nutritional quality of sorghum grain. Kafirin concentration and other chemical characteristics were determined in 12 sorghum hybrids and varied significantly, from 58% (HB1) to 42% (HB12) as percent total protein. Kafirin concentration correlated negatively with crude protein (CP) (0.49), with acid detergent fiber (0.40), apparent metabolizable energy (0.61), and true metabolizable energy corrected for N (0.63). HB12 was the hybrid with the lowest content of kafirins, amylose and tannins, and the highest content of apparent metabolizable energy. No differences were observed in the concentration of starch, but differences were found in apparent metabolizable energy (3325–2944 kcal kg1) probably due to a greater availability of starch, related to differences in kafirin concentration. r 2006 Elsevier Ltd. All rights reserved. Keywords: Sorghum hybrids; Kafirin; Metabolizable energy
1. Introduction Sorghum is the world’s fifth most important cereal; it is high-yielding and resistant to drought stress. Its protein content is higher than that of corn although its nutritional protein quality is lower (Dowling et al., 2002; Gualtieri and Rapaccini, 1990). Sorghum grain quality is affected by factors such as genotype, climate, soil type and fertilization, among others, which affect the chemical composition and nutrient value (Ebadi et al., 2005). The crude protein (CP) content of sorghum grain is highly variable (5.44–12.9%).
Abbreviations: ADF, acid detergent fiber; AME, apparent metabolizable energy; AAOAC, American Association of Official Analytical Chemists; CF, crude fiber; CP, crude protein; NDF, neutral detergent fiber; TMEn, true metabolizable energy corrected for nitrogen Corresponding author. Tel.: +55 595 95 2 02 0 0 ext. 1707; fax: +55 55 58 04 59 79. E-mail address: [email protected] (I. Salinas). 0733-5210/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.08.008
The storage proteins in sorghum grain are mostly kafirins, which are prolamins that are soluble in aqueous alcohol in the presence of a reducing agent. According to Watterson et al. (1993), kafirins are the most abundant storage proteins in sorghum grain. They are of low nutritional quality, very heterogeneous (Sastry et al., 1986), deficient in lysine, threonine and tryptophan, and rich in leucine, proline and glutamic acid (Duodu et al., 2003; Shewry et al., 1995). The kafirin content of sorghum grain depends on agronomic and genetic factors (Hicks et al., 2001) and accounts for up to 70% of the total protein content of the grain (Hamaker et al., 1995). Kafirins are deposited primarily in the endosperm during grain development (Shewry et al., 1995) forming protein bodies that surround starch granules and prevent access of amylases during digestion (Chandrashekar and Kirleis, 1988). For this reason, the quantity and nutritional quality of sorghum grain protein depends mainly on kafirin concentration. Recent studies have focussed on increasing the digestibility of proteins such as kafirins (Dowling et al.,
ARTICLE IN PRESS I. Salinas et al. / Journal of Cereal Science 44 (2006) 342–346
2002; Elkin et al., 2002). In broiler chickens, the quantity and nutritional quality of the proteins is important, especially in cereal-based diets, since proteins in the cereals contribute more than 30% of the CP in the diet (Dowling et al., 2002). Genetic studies on sorghum improvement have considered mainly agronomic traits but their effects on nutritional quality are unknown. The objective of this study was to determine the chemical composition and variability of metabolizable energy of 12 commercial sorghum hybrids and their relationship to kafirin concentration. 2. Materials and methods 2.1. Materials The sorghum hybrids studied were: HB1 through HB12, and were cultivated under similar commercial conditions during the fall-winter crop cycle, 2003–2004, in the state of Morelos, Mexico, located at latitude 181300 N and longitude 981450 W and at an altitude of 1030 m. Samples of grain (30 kg) from each hybrid, were collected directly from the field during harvest. From each sample, subsamples were taken for chemical analyses. 2.2. Chemical analyses The chemical characteristics of the sorghum grains studied were estimated: CP and crude fiber (CF) as described by AOAC (1990). Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were quantified by the methods proposed by Van Soest (1963) and Van Soest and Wine (1967). Starch was determined according to Herrera and Huber (1989). Amylose concentration was estimated
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by the method described by Juliano (1971). Kafirins were extracted with aqueous ethanol (70%) containing 0.6% 2mercaptoethanol as described by Landry and Moureaux (1970). Tannin content was determined by the method of Maxson and Rooney (1972). Apparent (AME) and true metabolizable energy corrected for nitrogen (TMEn) were estimated as described by Sibbald (1986), using adult cecectomized roosters. Analysis of tannins and amylose were conducted in duplicate for each hybrid studied and, for the other characteristics, in triplicate. For the determination of AME and TMEn, each sample of sorghum was fed to six Leghorn roosters housed in individual cages. Excreta from each rooster fed each hybrid were collected for 48 h and freeze dried. From the mixed of excreta from two roosters, a compound replication was formed and AME and TMEn were determined.
2.3. Statistical analysis A completely random experimental design was used; least square means were compared and correlations were obtained using the SAS program. The characteristics measured in percentage were transformed to the arc-sine function (Steel and Torrie, 1988). The statistical model used was Y ij ¼ m þ H i þ E ij , where Yij is the response variable in the jth observation of the ith hybrid, m is the constant that characterizes the population, Hi is the fixed effect of the ith hybrid i ¼ 1,2y12; Eij is the effect of the random error of the jth observation of the ith hybrid j ¼ 1,2,3; EijIDN (0, s2).
Table 1 Concentration of kafirins, protein, apparent metabolizable energy (AME), true metabolizable energy corrected by N (TMEn), starch, amylose and tannins in twelve sorghum hybrids (dry matter basis) Hybrid
Kafirin (%)*a
Crude protein (%)*
AME (kcal kg1)*
TMEn (kcal kg1)
Starch (%)
Amylose (%)*b
Tannins (%)*
HB1 HB2 HB3 HB4 HB5 HB6 HB7 HB8 HB9 HB10 HB11 HB12 Mean Standart error of mean7
57.59 57.52 53.59 50.93 48.86 47.61 44.56 44.29 44.25 43.83 43.12 42.35 48.29 1.09
8.72 6.61 9.64 10.05 8.59 11.42 10.27 9.88 9.71 9.51 9.89 11.19 9.62 0.37
3000 2944 3196 2996 3268 3089 3159 3091 2999 3070 3325 3195 3119 40
3301 3325 3605 3397 3591 3593 3574 3510 3562 3424 3459 3540 3497 71
71.24 73.71 68.62 67.73 72.81 69.53 65.17 70.68 71.38 71.21 68.23 67.58 69.82 2.44
25.28 26.38 26.27 26.83 27.92 26.67 26.46 27.33 28.26 27.11 27.01 25.36 26.74 0.32
0.98 1.01 0.55 1.02 0.65 0.62 0.65 0.75 0.68 0.55 1.05 0.68 0.77 0.08
*pp0.05. Extreme values are given in bold type. a As protein percentage. b As starch percentage.
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Table 2 Crude fiber and neutral and acid detergent fiber in twelve sorghum hybrids, expressed on dry matter basis Hybrid
HB1 HB2 HB3 HB4 HB5 HB6 HB7 HB8 HB9 HB10 HB11 HB12 Mean Standart error of mean 7
Crude fiber (%)
Neutral detergent fiber (%)
Acid detergent fiber (%)
1.96 2.67 4.17 2.23 2.10 1.94 1.71 1.84 2.24 3.06 2.85 2.73 2.45 0.07
33.14 22.22 28.62 24.24 25.47 26.01 24.75 19.47 22.57 24.87 26.35 35.18 26.07 3.18
7.94 5.86 9.51 9.02 6.80 9.47 7.47 7.51 8.02 9.07 7.13 9.52 8.07 0.47
Table 3 Correlations of the analyzed characteristics
TMEn AME ADF CP Starch Amylose
Kafirins
AME
0.63 ** 0.61 ** 0.4 * 0.49 ** 0.23 NS 0.28 NS
0.91 ** 1 0.01 NS 0.19 NS 0.08 NS 0.11 NS
CP
0.6 ** 1 0.5 * 0.13 NS
NS ¼ not significant; TMEn ¼ true metabolisable energy nitrogen corrected by N; AME ¼ apparent metabolisable energy; ADF ¼ acid detergent fiber; CP ¼ crude protein. *pp0.05. **pp0.01.
Pp0.05. Extreme values are given in bold type.
3. Results The results are presented on a dry matter basis and in descending order of kafirin concentration (Tables 1 and 2). The characteristics analyzed were statistically different (pp0.05), except for starch and TMEn. Kafirin content varied by 15 percentage units between HB12 and HB1 (Table 1). The highest concentration of protein was observed in HB6 and the lowest in HB2. The extreme concentrations of amylose were observed between HB9 and HB1 with a maximum variation of three percentage units. Tannin content varied widely among HB11, HB10 and HB3. CF, NDF and ADF values for the sorghum hybrids are presented in Table 2. HB7 had the lowest content of CF (pp0.05), while HB3 had the highest concentration; the difference between the two hybrids was 2.5%. NDF and ADF were highest in HB12 contrasting with HB8 and HB2 which had the lowest concentrations, differences in percentage units were 16 and 4, respectively. The correlations observed are summarized in Table 3. Kafirins correlated negatively with TMEn, AME, ADF, and CP, while CP correlated negatively with starch. Positive correlations were observed between CP and ADF, and between AME and TMEn. 4. Discussion The nutrient composition and variability among sorghum hybrids available on the market were analyzed with special emphasis on the kafirins present in the grain (Tables 1 and 2). Kafirin concentrations in the sorghum samples were similar to those reported by Hamaker et al. (1995). Evers and Millar (2002) point out that the protein matrix and the protein bodies found in the starchy endosperm of
the mature grain have a high kafirin content and that these prolamin polymers are intermolecularly associated through disulfide linkages with the protein matrix composed of glutelins (Wall, 1971). Duodu et al. (2002) report that even after fine milling of sorghum grain, wedges of protein bodies are held together by matrix proteins with attached starch and/or cell walls. In addition, the matrix is less resistant to digestion than protein bodies, which surround the starch granules and form a covering that impedes starch gelatinisation and access by enzymes (Rom et al., 1992). In treated, cooked sorghum flour a reducing agent such as sodium bisulfite or 2-mercaptoethanol increases starch gelatinisation by reducing intermolecular disulfide linkages between the matrix and protein bodies (Rom et al., 1992; Zhang and Hamaker, 1998). In this study, the hybrids that had lower kafirin content probably have higher gelatinisation capacity, i.e. the starch granules become less stable and more likely to lose their original structure. This implies higher values for AME, as was found for the hybrids HB12 and HB11, in contrast to HB1, HB2 and HB4 (Table 1), which had higher concentrations of kafirins. Kafirin concentration correlated negatively with TMEn and AME (Table 3), indicating that with higher kafirin concentrations, the availability of energy decreases. Also, when kafirin concentration increases, grain hardness increases due to the rigidity of protein bodies (Marzhar and Chandrashekar, 1995), which are difficult to separate from starch even with milling (Rooney and Pflugfelder, 1986). Endosperm hardness is thus most likely related to the quantity of kafirins present. Watterson et al. (1993) reported that vitreous (hard) endosperms contain up to twice as much protein rich in b-g kafirins as opaque (soft) endosperms. This suggests that hybrids with higher nutritional value are lower in kafirin content and softer (although the latter trait was not evaluated in our study).
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The negative correlation observed between CP and kafirins is similar to that reported by Hicks et al. (2001), who analyzed different fractions of kafirins (a, b and g), while in our study the total concentration of kafirins was considered. Although no studies were found in which CP, kafirins and metabolizable energy were analyzed simultaneously, it is known that CP deposition depends on genetic variation, availability of water, soil fertility and environmental temperature (da Silva and Taylor, 2004), and kafirins are particularly sensitive to availability of nitrogen (Banda-Nyirenda et al., 1987). In the hybrids analyzed, no statistical differences were observed in the content of starch, but there were differences in AME, and therefore the energy content of sorghum seems to depend more on the availability than on the content of starch. Starch availability in the sorghum grain is influenced by interactions between protein and cell walls (Chandrashekar and Kirleis, 1988), non-starch polysaccharides (Kavitha and Chandrashekar, 1997) and tannins (Duodu et al., 2003). When sorghum starch is gelatinised, more disulfide bridges are formed in the protein matrix which further delays the digestion of starch and protein itself (Zhang and Hamaker, 1998). In this study kafirin concentration was the only fraction of the sorghum grain that correlated negatively with TMEn and AME (Table 3); these results suggest that the energy value of sorghum grain depends on the kafirin concentration, which reduces availability of starch. Zhang and Hamaker (1998) point out that the lower the digestibility of kafirins, the lower the digestibility of gelatinized sorghum starch. In contrast, Elkin et al. (2002), comparing mutant sorghum P851171 (with highly digestible protein) and normal sorghums P712N and 611Y observed lower values of TMEn and poor feed conversion in mutant sorghum, because the endosperm had less starch and more non-starch polysaccharides than normal sorghums. Thus, it may be important that future studies in sorghum breeding to focus on increasing protein digestibility and perhaps on reducing kafirin concentrations without affecting the agronomic characteristics. A negative correlation between kafirins and ADF (Table 3) was observed. Although this had not previously been reported, Bach-Knudsen et al. (1988) point out that the content of dietary fiber in sorghum flour reduces digestible energy. Evers and Millar (2002) and Duodu et al. (2002) state that cell walls surrounding protein bodies embedded in a protein matrix and starch granules are found in cereal endosperm, and therefore it is possible that these structures make access difficult for digestive enzymes. In this study no correlation was observed between ADF and the types of energy evaluated; thus, it is likely that when kafirins increase, the negative effect on energy is greater than the effect of the reduction of ADF associated with an increase in kafirins. The positive correlation of ADF with CP is similar to that reported by Bach-Knudsen and Munck (1985), who mention that significant amounts of protein are associated with ADF. However, in agreement with the
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correlations observed in this study (Table 3), when CP and ADF increase, kafirin concentration decreases. The tannin content (Table 1) of the hybrids studied is lower than that previously reported for bird-resistant sorghum, but it is considered medium-high by Lucbert and Castaing (1986). These authors state that when tannin concentration is above 0.23% (low content), metabolizable energy decreases by 40 kcal for every additional 0.1%, although sorghums with less than 1% tannin can be used for broiler chicken with no negative effect on feed conversion. The HB11 hybrid had the highest content of tannins as well as AME, possibly because of the lower kafirin content with which indigestible complexes could be formed. It is important to take into account the capacity of tannins to associate with protein (they can precipitate up to 12 times their own weight of protein; Butler et al., 1984) and their great affinity for proteins such as kafirins which are rich in proline. In Table 3, four negative correlations stand out: starch with CP, kafirins with CP, kafirins with TMEn and kafirins with AME. When starch concentration decreased, CP increased. When this occurs, kafirin concentration also decreases. It is possible that when the kafirin concentration decreases, starch availability is higher, and this resulted in higher levels of TMEn and AME. High kafirin concentrations negatively affect metabolizable energy values in sorghum. HB12 and HB11 were the hybrids with the lowest concentrations of kafirin, and they had high content of apparent metabolizable energy (AME). Starch content was similar in all samples analyzed. Differences, however, were observed in the levels of AME of the hybrids when kafirin concentration changed, suggesting that other factors such as the association of kafirins with ADF can affect the energy content of sorghum. Because starch availability depends on many factors, more studies are needed to determine, from agronomic and nutritional perspectives, the impact of a reduced kafirin concentration.
Acknowledgement We thank D.V.M. Juan Carlos Gordoa Garcı´ a for assistance in performing the surgery on the roosters.
References Association of Official Analytical Chemists (AOAC), 1990. Official Methods of Analysis. Washington, DC. Bach-Knudsen, B.K.E., Munck, L., 1985. Dietary fibe contents and compositions of sorghum and sorghum-based foods. Journal of Cereal Science 3, 153–164. Bach-Knudsen, B.K.E., Kirleis, A.W., Eggum, B.O., Munck, L., 1988. Carbohydrate composition and nutritional quality for rats of sorghum toˆ prepared from decorticated white and whole grain red flour. Journal of Nutrition 118, 588–597. Banda-Nyirenda, B.G.D., Vohra, P., Ingebretson, K.H., 1987. Nutritional evaluation of some varieties of sorghums (Sorghum bicolor (L.) Moench). Cereal Chemistry 64, 413–417.
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Butler, L.G., Riedl, D.J., Lebryk, D.G., Blytt, H.J., 1984. Interaction of proteins with sorghum tannin: mechanism, specificity and significance. Journal of the American Oil Chemists Society 61, 916–920. Chandrashekar, A., Kirleis, A.W., 1988. Influence of protein on starch gelatinization in sorghum. Cereal Chemistry 65, 457–462. da Silva, L.S., Taylor, J.R.N., 2004. Sorghum bran as a potential source of kafirin. Cereal Chemistry 81, 322–327. Dowling, L.F., Arndt, C., Hamaker, B.R., 2002. Economic viability of high digestibility sorghum as feed for market broilers. Agronomy Journal 94, 1050–1058. Duodu, K.G., Nunes, A., Delgadillo, I., Parker, L.M., Mills, E.N.C., Belton, P.S., Taylor, R.N., 2002. Effect of grain structure and cooking on sorghum and maize in vitro protein digestibility. Journal of Cereal Science 35, 161–174. Duodu, K.G., Taylor, J.R.N., Belton, P.S., Hamaker, B.R., 2003. Factors affecting sorghum protein digestibility. Journal of Cereal Science 38, 117–131. Ebadi, M.R., Pourreza, J., Jamalian, J., Edriss, M.A., Samie, A.H., Mirhadi, S.A., 2005. Amino acid content and availability in low medium and high tannin sorghum grain for poultry. International Journal of Poultry Science 1, 27–31. Elkin, G.R., Elaine, A., Hamaker, B.R., Axtell, J.D., Douglas, W.M., Parsons, C.M., 2002. Nutritional value of a highly digestible sorghum cultivar for meat-type chickens. Journal of Agricultural and Food Chemistry 50, 4146–4150. Evers, T., Millar, S., 2002. Cereal grain structure and development: some implications for quality. Journal of Cereal Science 36, 261–284. Gualtieri, M., Rapaccini, S., 1990. Sorghum grain in poultry feeding. World’s Poultry Science Journal 46, 246–254. Hamaker, B.R., Mohamed, A.A., Habben, J.E., Huang, C.P., Larkins, B.A., 1995. Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chemistry 72, 583–588. Herrera, S.R., Huber, J.T., 1989. Influence of varying protein and starch degradabilities on performance of lactating cows. Journal of Dairy Science 72, 1477–1483. Hicks, C., Bean, S.R., Lookhart, G.L., Pedersen, J.F., Kofoid, K.D., Tuinstra, M.R., 2001. Genetic analysis of kafirins and their phenotypic correlations with feed quality traits, in vitro digestibility, and seed weight in grain sorghum. Cereal Chemistry 78, 412–416. Juliano, B.O., 1971. A simplified assay for milled rice amylose. Cereal Foods World 16, 334–340. Kavitha, R., Chandrashekar, A., 1997. Content and composition of nonstarchy polysaccharides in endosperms of sorghum varying in hardness during four developmental stages. Cereal Chemistry 74, 22–24.
Landry, J., Moureaux, T., 1970. He´te´roge´ne´ite´ des glute´lines du grain de mais: extraction se´lective et composition en acides amines des trois fractions isole´es. Bulletin de la Socie´te´ de chimie Biologique 52, 1021–1037. Lucbert, J., Castaing, J., 1986. Utilisation de sorghos diferentes teneurs en tannins pour l’alimentation des poulets de chair. In: Proceedings of the 7th European Poultry Conference, vol. 1, pp. 472–476. Marzhar, H., Chandrashekar, A., 1995. Quantification and distribution of kafirins in the kernels of sorghum cultivars varying in endosperm hardness. Journal of Cereal Science 21, 155–162. Maxson, E.D., Rooney, L.W., 1972. Two methods of tannin analysis for Sorghum bicolor (L.) Moench grain. Crop Science 12, 253–254. Rom, D.L., Shull, J.M., Chandrashekar, A., Kirleis, A.W., 1992. Effects of cooking and treatment with sodium bisulfite on in vitro protein digestibility and microstructure of sorghum flour. Cereal Chemistry 69, 178–181. Rooney, L.W., Pflugfelder, R.L., 1986. Factors affecting starch digestibility with special emphasis on sorghum and corn. Journal of Animal Science 63, 1607–1623. Sastry, L.V.S., Paulis, J.W., Bietz, J.A., Wall, J.S., 1986. Genetic variation of storage proteins in sorghum grain: Studies by isoelectric focusing and high-performance liquid chromatography. Cereal Chemistry 63, 420–427. Shewry, R.P., Naiper, A.J., Tatham, S.A., 1995. Seed storage proteins: structures and biosynthesis. The Plant Cell 7, 945–956. Sibbald, I.R., 1986. The T.M.E. system of feed evaluation: methodology, feed composition data and bibliography. Technical Bulletin. 1986-4E. Animal Research Centre, Ottawa, Ont., Canada. Steel, R.G.D., Torrie, J.H., 1988. Bioestadı´ stica: Principios y Procedimientos, 2a edicio´n. McGraw-Hill, Me´xico. Van Soest, P.J., 1963. Use of detergents in analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. Journal of the Association of Official Analytical Chemists 46, 829–835. Van Soest, P.J., Wine, R.H., 1967. Use of detergents in analysis of fibrous feeds. IV. Determination of plant cell-wall constituents. Journal of the Association of Official Analytical Chemists 50, 50–55. Wall, J.S., 1971. Disulfide bonds: determination, location and influence on molecular properties of proteins. Journal of Agricultural and Food Chemistry 19, 619–625. Watterson, J.J., Shull, J.M., Kirleis, A.W., 1993. Quantitation of a-, b-, and g-kafirins in vitreous and opaque endosperm of Sorghum bicolor. Cereal Chemistry 70, 452–457. Zhang, G., Hamaker, R.B., 1998. Low a-amylase starch digestibility of cooked sorghum flours and the effect of protein. Cereal Chemistry 75, 710–713.
Journal of Cereal Science 44 (2006) 342–346 www.elsevier.com/locate/yjcrs
Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy Israel Salinasa,, Arturo Pro´a, Yolanda Salinasb, Eliseo Sosac, Carlos Miguel Becerrila, Manuel Cucaa, Miguel Cervantesd, Jaime Gallegosa a
Ganaderı´a, Campus Montecillo, Colegio de Postgraduados, 56230 Montecillo, Estado de Me´xico, Me´xico b Laboratorio de maı´z INIFAP, Estado de Me´xico, Me´xico c Universidad Auto´noma Chapingo, 56230 Chapingo, Estado de Me´xico, Me´xico d Instituto de Ciencias Agrı´colas, Universidad Auto´noma de Baja California, Mexicali, Baja California, Me´xico Received 20 March 2006; received in revised form 24 August 2006; accepted 29 August 2006
Abstract Kafirins are stored proteins that negatively affect the nutritional quality of sorghum grain. Kafirin concentration and other chemical characteristics were determined in 12 sorghum hybrids and varied significantly, from 58% (HB1) to 42% (HB12) as percent total protein. Kafirin concentration correlated negatively with crude protein (CP) (0.49), with acid detergent fiber (0.40), apparent metabolizable energy (0.61), and true metabolizable energy corrected for N (0.63). HB12 was the hybrid with the lowest content of kafirins, amylose and tannins, and the highest content of apparent metabolizable energy. No differences were observed in the concentration of starch, but differences were found in apparent metabolizable energy (3325–2944 kcal kg1) probably due to a greater availability of starch, related to differences in kafirin concentration. r 2006 Elsevier Ltd. All rights reserved. Keywords: Sorghum hybrids; Kafirin; Metabolizable energy
1. Introduction Sorghum is the world’s fifth most important cereal; it is high-yielding and resistant to drought stress. Its protein content is higher than that of corn although its nutritional protein quality is lower (Dowling et al., 2002; Gualtieri and Rapaccini, 1990). Sorghum grain quality is affected by factors such as genotype, climate, soil type and fertilization, among others, which affect the chemical composition and nutrient value (Ebadi et al., 2005). The crude protein (CP) content of sorghum grain is highly variable (5.44–12.9%).
Abbreviations: ADF, acid detergent fiber; AME, apparent metabolizable energy; AAOAC, American Association of Official Analytical Chemists; CF, crude fiber; CP, crude protein; NDF, neutral detergent fiber; TMEn, true metabolizable energy corrected for nitrogen Corresponding author. Tel.: +55 595 95 2 02 0 0 ext. 1707; fax: +55 55 58 04 59 79. E-mail address: [email protected] (I. Salinas). 0733-5210/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.08.008
The storage proteins in sorghum grain are mostly kafirins, which are prolamins that are soluble in aqueous alcohol in the presence of a reducing agent. According to Watterson et al. (1993), kafirins are the most abundant storage proteins in sorghum grain. They are of low nutritional quality, very heterogeneous (Sastry et al., 1986), deficient in lysine, threonine and tryptophan, and rich in leucine, proline and glutamic acid (Duodu et al., 2003; Shewry et al., 1995). The kafirin content of sorghum grain depends on agronomic and genetic factors (Hicks et al., 2001) and accounts for up to 70% of the total protein content of the grain (Hamaker et al., 1995). Kafirins are deposited primarily in the endosperm during grain development (Shewry et al., 1995) forming protein bodies that surround starch granules and prevent access of amylases during digestion (Chandrashekar and Kirleis, 1988). For this reason, the quantity and nutritional quality of sorghum grain protein depends mainly on kafirin concentration. Recent studies have focussed on increasing the digestibility of proteins such as kafirins (Dowling et al.,
ARTICLE IN PRESS I. Salinas et al. / Journal of Cereal Science 44 (2006) 342–346
2002; Elkin et al., 2002). In broiler chickens, the quantity and nutritional quality of the proteins is important, especially in cereal-based diets, since proteins in the cereals contribute more than 30% of the CP in the diet (Dowling et al., 2002). Genetic studies on sorghum improvement have considered mainly agronomic traits but their effects on nutritional quality are unknown. The objective of this study was to determine the chemical composition and variability of metabolizable energy of 12 commercial sorghum hybrids and their relationship to kafirin concentration. 2. Materials and methods 2.1. Materials The sorghum hybrids studied were: HB1 through HB12, and were cultivated under similar commercial conditions during the fall-winter crop cycle, 2003–2004, in the state of Morelos, Mexico, located at latitude 181300 N and longitude 981450 W and at an altitude of 1030 m. Samples of grain (30 kg) from each hybrid, were collected directly from the field during harvest. From each sample, subsamples were taken for chemical analyses. 2.2. Chemical analyses The chemical characteristics of the sorghum grains studied were estimated: CP and crude fiber (CF) as described by AOAC (1990). Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were quantified by the methods proposed by Van Soest (1963) and Van Soest and Wine (1967). Starch was determined according to Herrera and Huber (1989). Amylose concentration was estimated
343
by the method described by Juliano (1971). Kafirins were extracted with aqueous ethanol (70%) containing 0.6% 2mercaptoethanol as described by Landry and Moureaux (1970). Tannin content was determined by the method of Maxson and Rooney (1972). Apparent (AME) and true metabolizable energy corrected for nitrogen (TMEn) were estimated as described by Sibbald (1986), using adult cecectomized roosters. Analysis of tannins and amylose were conducted in duplicate for each hybrid studied and, for the other characteristics, in triplicate. For the determination of AME and TMEn, each sample of sorghum was fed to six Leghorn roosters housed in individual cages. Excreta from each rooster fed each hybrid were collected for 48 h and freeze dried. From the mixed of excreta from two roosters, a compound replication was formed and AME and TMEn were determined.
2.3. Statistical analysis A completely random experimental design was used; least square means were compared and correlations were obtained using the SAS program. The characteristics measured in percentage were transformed to the arc-sine function (Steel and Torrie, 1988). The statistical model used was Y ij ¼ m þ H i þ E ij , where Yij is the response variable in the jth observation of the ith hybrid, m is the constant that characterizes the population, Hi is the fixed effect of the ith hybrid i ¼ 1,2y12; Eij is the effect of the random error of the jth observation of the ith hybrid j ¼ 1,2,3; EijIDN (0, s2).
Table 1 Concentration of kafirins, protein, apparent metabolizable energy (AME), true metabolizable energy corrected by N (TMEn), starch, amylose and tannins in twelve sorghum hybrids (dry matter basis) Hybrid
Kafirin (%)*a
Crude protein (%)*
AME (kcal kg1)*
TMEn (kcal kg1)
Starch (%)
Amylose (%)*b
Tannins (%)*
HB1 HB2 HB3 HB4 HB5 HB6 HB7 HB8 HB9 HB10 HB11 HB12 Mean Standart error of mean7
57.59 57.52 53.59 50.93 48.86 47.61 44.56 44.29 44.25 43.83 43.12 42.35 48.29 1.09
8.72 6.61 9.64 10.05 8.59 11.42 10.27 9.88 9.71 9.51 9.89 11.19 9.62 0.37
3000 2944 3196 2996 3268 3089 3159 3091 2999 3070 3325 3195 3119 40
3301 3325 3605 3397 3591 3593 3574 3510 3562 3424 3459 3540 3497 71
71.24 73.71 68.62 67.73 72.81 69.53 65.17 70.68 71.38 71.21 68.23 67.58 69.82 2.44
25.28 26.38 26.27 26.83 27.92 26.67 26.46 27.33 28.26 27.11 27.01 25.36 26.74 0.32
0.98 1.01 0.55 1.02 0.65 0.62 0.65 0.75 0.68 0.55 1.05 0.68 0.77 0.08
*pp0.05. Extreme values are given in bold type. a As protein percentage. b As starch percentage.
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Table 2 Crude fiber and neutral and acid detergent fiber in twelve sorghum hybrids, expressed on dry matter basis Hybrid
HB1 HB2 HB3 HB4 HB5 HB6 HB7 HB8 HB9 HB10 HB11 HB12 Mean Standart error of mean 7
Crude fiber (%)
Neutral detergent fiber (%)
Acid detergent fiber (%)
1.96 2.67 4.17 2.23 2.10 1.94 1.71 1.84 2.24 3.06 2.85 2.73 2.45 0.07
33.14 22.22 28.62 24.24 25.47 26.01 24.75 19.47 22.57 24.87 26.35 35.18 26.07 3.18
7.94 5.86 9.51 9.02 6.80 9.47 7.47 7.51 8.02 9.07 7.13 9.52 8.07 0.47
Table 3 Correlations of the analyzed characteristics
TMEn AME ADF CP Starch Amylose
Kafirins
AME
0.63 ** 0.61 ** 0.4 * 0.49 ** 0.23 NS 0.28 NS
0.91 ** 1 0.01 NS 0.19 NS 0.08 NS 0.11 NS
CP
0.6 ** 1 0.5 * 0.13 NS
NS ¼ not significant; TMEn ¼ true metabolisable energy nitrogen corrected by N; AME ¼ apparent metabolisable energy; ADF ¼ acid detergent fiber; CP ¼ crude protein. *pp0.05. **pp0.01.
Pp0.05. Extreme values are given in bold type.
3. Results The results are presented on a dry matter basis and in descending order of kafirin concentration (Tables 1 and 2). The characteristics analyzed were statistically different (pp0.05), except for starch and TMEn. Kafirin content varied by 15 percentage units between HB12 and HB1 (Table 1). The highest concentration of protein was observed in HB6 and the lowest in HB2. The extreme concentrations of amylose were observed between HB9 and HB1 with a maximum variation of three percentage units. Tannin content varied widely among HB11, HB10 and HB3. CF, NDF and ADF values for the sorghum hybrids are presented in Table 2. HB7 had the lowest content of CF (pp0.05), while HB3 had the highest concentration; the difference between the two hybrids was 2.5%. NDF and ADF were highest in HB12 contrasting with HB8 and HB2 which had the lowest concentrations, differences in percentage units were 16 and 4, respectively. The correlations observed are summarized in Table 3. Kafirins correlated negatively with TMEn, AME, ADF, and CP, while CP correlated negatively with starch. Positive correlations were observed between CP and ADF, and between AME and TMEn. 4. Discussion The nutrient composition and variability among sorghum hybrids available on the market were analyzed with special emphasis on the kafirins present in the grain (Tables 1 and 2). Kafirin concentrations in the sorghum samples were similar to those reported by Hamaker et al. (1995). Evers and Millar (2002) point out that the protein matrix and the protein bodies found in the starchy endosperm of
the mature grain have a high kafirin content and that these prolamin polymers are intermolecularly associated through disulfide linkages with the protein matrix composed of glutelins (Wall, 1971). Duodu et al. (2002) report that even after fine milling of sorghum grain, wedges of protein bodies are held together by matrix proteins with attached starch and/or cell walls. In addition, the matrix is less resistant to digestion than protein bodies, which surround the starch granules and form a covering that impedes starch gelatinisation and access by enzymes (Rom et al., 1992). In treated, cooked sorghum flour a reducing agent such as sodium bisulfite or 2-mercaptoethanol increases starch gelatinisation by reducing intermolecular disulfide linkages between the matrix and protein bodies (Rom et al., 1992; Zhang and Hamaker, 1998). In this study, the hybrids that had lower kafirin content probably have higher gelatinisation capacity, i.e. the starch granules become less stable and more likely to lose their original structure. This implies higher values for AME, as was found for the hybrids HB12 and HB11, in contrast to HB1, HB2 and HB4 (Table 1), which had higher concentrations of kafirins. Kafirin concentration correlated negatively with TMEn and AME (Table 3), indicating that with higher kafirin concentrations, the availability of energy decreases. Also, when kafirin concentration increases, grain hardness increases due to the rigidity of protein bodies (Marzhar and Chandrashekar, 1995), which are difficult to separate from starch even with milling (Rooney and Pflugfelder, 1986). Endosperm hardness is thus most likely related to the quantity of kafirins present. Watterson et al. (1993) reported that vitreous (hard) endosperms contain up to twice as much protein rich in b-g kafirins as opaque (soft) endosperms. This suggests that hybrids with higher nutritional value are lower in kafirin content and softer (although the latter trait was not evaluated in our study).
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The negative correlation observed between CP and kafirins is similar to that reported by Hicks et al. (2001), who analyzed different fractions of kafirins (a, b and g), while in our study the total concentration of kafirins was considered. Although no studies were found in which CP, kafirins and metabolizable energy were analyzed simultaneously, it is known that CP deposition depends on genetic variation, availability of water, soil fertility and environmental temperature (da Silva and Taylor, 2004), and kafirins are particularly sensitive to availability of nitrogen (Banda-Nyirenda et al., 1987). In the hybrids analyzed, no statistical differences were observed in the content of starch, but there were differences in AME, and therefore the energy content of sorghum seems to depend more on the availability than on the content of starch. Starch availability in the sorghum grain is influenced by interactions between protein and cell walls (Chandrashekar and Kirleis, 1988), non-starch polysaccharides (Kavitha and Chandrashekar, 1997) and tannins (Duodu et al., 2003). When sorghum starch is gelatinised, more disulfide bridges are formed in the protein matrix which further delays the digestion of starch and protein itself (Zhang and Hamaker, 1998). In this study kafirin concentration was the only fraction of the sorghum grain that correlated negatively with TMEn and AME (Table 3); these results suggest that the energy value of sorghum grain depends on the kafirin concentration, which reduces availability of starch. Zhang and Hamaker (1998) point out that the lower the digestibility of kafirins, the lower the digestibility of gelatinized sorghum starch. In contrast, Elkin et al. (2002), comparing mutant sorghum P851171 (with highly digestible protein) and normal sorghums P712N and 611Y observed lower values of TMEn and poor feed conversion in mutant sorghum, because the endosperm had less starch and more non-starch polysaccharides than normal sorghums. Thus, it may be important that future studies in sorghum breeding to focus on increasing protein digestibility and perhaps on reducing kafirin concentrations without affecting the agronomic characteristics. A negative correlation between kafirins and ADF (Table 3) was observed. Although this had not previously been reported, Bach-Knudsen et al. (1988) point out that the content of dietary fiber in sorghum flour reduces digestible energy. Evers and Millar (2002) and Duodu et al. (2002) state that cell walls surrounding protein bodies embedded in a protein matrix and starch granules are found in cereal endosperm, and therefore it is possible that these structures make access difficult for digestive enzymes. In this study no correlation was observed between ADF and the types of energy evaluated; thus, it is likely that when kafirins increase, the negative effect on energy is greater than the effect of the reduction of ADF associated with an increase in kafirins. The positive correlation of ADF with CP is similar to that reported by Bach-Knudsen and Munck (1985), who mention that significant amounts of protein are associated with ADF. However, in agreement with the
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correlations observed in this study (Table 3), when CP and ADF increase, kafirin concentration decreases. The tannin content (Table 1) of the hybrids studied is lower than that previously reported for bird-resistant sorghum, but it is considered medium-high by Lucbert and Castaing (1986). These authors state that when tannin concentration is above 0.23% (low content), metabolizable energy decreases by 40 kcal for every additional 0.1%, although sorghums with less than 1% tannin can be used for broiler chicken with no negative effect on feed conversion. The HB11 hybrid had the highest content of tannins as well as AME, possibly because of the lower kafirin content with which indigestible complexes could be formed. It is important to take into account the capacity of tannins to associate with protein (they can precipitate up to 12 times their own weight of protein; Butler et al., 1984) and their great affinity for proteins such as kafirins which are rich in proline. In Table 3, four negative correlations stand out: starch with CP, kafirins with CP, kafirins with TMEn and kafirins with AME. When starch concentration decreased, CP increased. When this occurs, kafirin concentration also decreases. It is possible that when the kafirin concentration decreases, starch availability is higher, and this resulted in higher levels of TMEn and AME. High kafirin concentrations negatively affect metabolizable energy values in sorghum. HB12 and HB11 were the hybrids with the lowest concentrations of kafirin, and they had high content of apparent metabolizable energy (AME). Starch content was similar in all samples analyzed. Differences, however, were observed in the levels of AME of the hybrids when kafirin concentration changed, suggesting that other factors such as the association of kafirins with ADF can affect the energy content of sorghum. Because starch availability depends on many factors, more studies are needed to determine, from agronomic and nutritional perspectives, the impact of a reduced kafirin concentration.
Acknowledgement We thank D.V.M. Juan Carlos Gordoa Garcı´ a for assistance in performing the surgery on the roosters.
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