4.1. Kafirin
Kafirin is the dominant fraction of sorghum protein and Virupaksha and Sastry [
33] reported that kafirin constituted 54.1%, glutelin 33.4%, globulin 7.0% and albumin 5.6% of endosperm protein in sorghum. Kafirin is located in discrete protein bodies which are embedded in the glutelin protein matrix of sorghum endosperm where both kafirin and glutelin are intimately associated with starch granules. Subsequently, Taylor,
et al. [
34] reported average concentrations of 54.0 g/kg kafirin and 27.1 g/kg glutelin in 41 grain sorghum samples with an average crude protein content of 111 g/kg. Thus kafirin and glutelin comprised 48.0% and 27.7% of sorghum protein, respectively. However, kafirin, as a proportion of protein, was positively correlated (
r = 0.469;
p < 0.005) with sorghum protein concentrations; whereas, the proportion of glutelin was negatively correlated (
r = −0.401;
p < 0.01). This may suggest that screening sorghum quality by its protein concentration may result in sorghum grains with high kafirin concentrations.
Selle,
et al. [
35] proposed kafirin concentrations in Australian sorghums have increased in recent decades probably as an unintended consequence of breeding for increased yield and the use of high input nitrogen fertilizers. While this proposal is based on circumstantial evidence, the likely implications are important. In a quest to enhance grain weathering resistance, Henzell [
36] stated breeding programs commonly target sorghums with a relatively dense or corneous endosperm. As such, selecting sorghums with hard endosperms will result in higher kafirin contents. Importantly, the textures of Australian sorghums are relatively high by international standards. Our group has determined the Symes PSI texture [
37] of 38 sorghum varieties; the mean PSI value was 9.84 ± 1.603 which is in the more rigid end of the “very hard” scale and only two varieties or 5.3% of samples were “softer” in this scale. These findings are in contrast to an extensive global survey recorded by de Alencar Figueiredo,
et al. [
38]. In this survey the mean PSI texture of 117 sorghum samples was 12.7 but 44% of samples had softer textures than “very hard” as opposed to 5.3% in the local sorghum varieties. Moreover, the likelihood is that “soft” sorghums are superior in performance and nutrient utilisation to “hard” sorghums as the basis for broiler diets [
39].
It is instructive to compare the amino acid profiles of local sorghum varieties from related reports compiled by Ravindran,
et al. [
40] and subsequently by Bryden,
et al. [
5], where the same analytical methods were adopted. The mean protein content of sorghums in the two studies was very similar (98.7 g/kg
versus 103.6 g/kg). However, sorghums in the latter study contained 21.3% less lysine (1.89 g/kg
versus 2.40 g/kg;
p < 0.015) and 25.7% less arginine (3.32
versus 4.47 g/kg;
p < 0.005). Given that kafirin contains low levels of lysine and arginine, these reductions in concentrations of more than 20% are entirely consistent with the proposal that kafirin constitutes a greater proportion of total sorghum protein in the latter study.
This is a disadvantageous development because kafirin contains low levels of lysine but high levels of leucine. Moreover, kafirin is a poor source of digestible amino acids due to its inherent hydrophobicity and the structure of protein bodies. It is generally accepted disulphide cross-linking in the β- and γ-kafirin fractions, which are located in the periphery of the protein body, impedes the digestion of the central α-kafirin core and this is exacerbated by hydrothermal processes as they induce disulphide cross-linkages [
41].
Clearly, kafirin compromises sorghum protein quality [
2] but this may not be crucial as kafirin constitutes in the order of 15% of total protein in a sorghum-based broiler diet. However, if kafirin levels in local sorghums are indeed increasing then the real possibility that kafirin compromises starch/energy utilisation is pivotal. The mechanisms by which kafirin compromises starch utilisation have not been clarified but arguably their genesis stems from the close physical proximity of kafirin protein bodies, starch granules and glutelin protein matrices in sorghum endosperm. As discussed by Gidley,
et al. [
42] the quantity of starch in sorghum endosperm exceeds that of kafirin protein, which suggests that the magnitude of biophysical starch-protein interactions is capped. Intuitively, biochemical starch-protein interactions may hold more importance but despite the recognition of the importance [
43] they have yet to be precisely defined.
The proposition that kafirin compromises starch/energy utilisation, as enunciated by Taylor [
44], is generally accepted although reservations have been expressed by Gidley,
et al. [
42]. In sorghum endosperm the diameter of starch granules (~15 μm) is considerably larger than kafirin protein bodies (1–3 μm) and the endosperm contains considerably higher concentrations of starch than kafirin. Starch granules and kafirin protein bodies are both discrete structures in sorghum endosperm. The concept that hydrothermally induced disulphide cross-linkages in the cysteine-rich periphery of protein bodies impede the digestion of the central α-kafirin core is straightforward because α-kafirin would be “protected” by a layer of relatively insoluble protein. It is more difficult to conceptualise that these reactions are also a direct impediment to starch digestion, although they may physically hinder starch gelatinisation. Intuitively, it seems any interactions between starch and protein that might impede starch gelatinisation/digestion are more likely to occur between starch and the glutelin protein matrix because starch granules are embedded in the protein matrix. Another possible inherent impediment to starch digestion is the endosperm cell walls. Taylor [
44] considered it was probable that these cell walls constitute something of a barrier to digestion. The cell walls are rich in insoluble glucuronoarabinoxylan type pentosans and are intimately bound to the glutelin protein matrix, quite possibly via ferulic acid, a phenolic compound that is abundant in sorghum.
De Mesa-Stonestreet,
et al. [
45] discussed that kafirins are located primarily in spherical protein bodies, which are embedded in a glutelin protein matrix, and the integrity of which is dependent on disulphide cross-linkages. This is allied to studies using three-dimensional fluorescence microscopy completed by Ezeogu,
et al. [
46] and their conclusion was that low starch digestibility of cooked sorghum flour was the result of “the more disulfide-bonded protein matrix”. Another possibility, as considered by Truong,
et al. [
24], are interactions involving disulphide linkages between β- and γ-kafirin on the periphery of protein bodies and starch-granule associated proteins on the surface of starch granules may have the capacity to impede starch digestion. Collectively, it seems possible that disulphide cross-linkages involving kafirin protein bodies, glutelin protein matrices and starch-granule associated proteins in sorghum endosperm are a set of connections forming a web-like protein network that impedes starch utilisation.
Following the quantification of kafirin concentrations in sorghum, a series of feeding studies completed by Poultry Research Foundation indicated that kafirin has negative impact on energy utilisation in broiler chickens (unpublished data). The methodology involved to quantify kafirin has been fully documented by Truong,
et al. [
47]. In these studies, kafirin significantly depressed ME:GE ratios (
p < 0.04) and tended to decrease AMEn (
p = 0.074) in broiler chickens from meta-analyses with non-significant (
p > 0.10) experimental leverages (
Table 1). Thus our tentative conclusion is kafirin content and composition compromises starch/energy utilisation in sorghum-based diets and the real possibility that the chicken-meat industry is being confronted with “high-kafirin” sorghums is a predicament that needs to be considered by sorghum breeding programs. Put simply, the availability of relatively low protein sorghums with lesser kafirin proportions should be substantially beneficial to growth performance and energy utilisation in broiler chickens.
Table 1.
The relationship between dietary concentrations of kafirin and energy utilisation (ME:GE ratios and AMEn) in broiler chickens offered sorghum-based diets.
Table 1.
The relationship between dietary concentrations of kafirin and energy utilisation (ME:GE ratios and AMEn) in broiler chickens offered sorghum-based diets.
Response | Observations (n =) | Experiment Leverage (p =) | Dietary Kafirin Leverage (p =) | Whole Model |
---|
ME:GE | 10 | 0.109 | 0.034 | R2 = 0.71, p = 0.045 |
AMEn | 13 | 0.485 | 0.013 | R2 = 0.70, p = 0.074 |
There has been an increasing focus on the inclusion of exogenous proteases in pig and poultry diets. More specifically, Taylor [
44] suggested that proteases should be evaluated in sorghum-based broiler diets with the caveat that kafirin and glutelin are not readily hydrolysed by proteolytic enzymes. In this context it is relevant that most or all exogenous proteases are hydrolases and inherently lack the capacity to cleave disulphide cross-linkages which requires reductase activity.
Selle,
et al. [
48] reported that a
Bacillus lichenformis-derived protease improved N digestibility in conventional, sorghum-based broiler diets that were steam-pelleted at an 80 °C conditioning temperature. At the standard inclusion rate (300 units/gram), this protease significantly increased N digestibility coefficients by 16.5% in the distal jejunum (0.627
versus 0.538), by 5.91% in the proximal ileum (0.770
versus 0.727) and by 7.09% in the distal ileum (0.770
versus 0.719). Subsequently, Liu,
et al. [
49] reported that this protease significantly improved the digestibility of arginine (4.63%), histidine (8.23%), isoleucine (9.87%), leucine (12.7%), phenylalanine (10.5%), threonine (7.36%), valine (10.4%), alanine (12.8%), aspartic acid (9.60%), glutamic acid (9.38%), glycine (9.73%), proline (14.6%), serine (11.5%), and tyrosine (12.1%). Only the responses in lysine and methionine digestibility were not significant and this was probably because the diets contained quite high levels of these two amino acids in “free” or synthetic forms. In addition, protease supplementation significantly (
p < 0.05 - < 0.005) increased the rates of digestion of 12 of the 16 amino acids assessed. Moreover, as proposed by Liu and Selle [
22], it is probable digestion rates of amino acids were more indicative of broiler performance than static amino acid digestibility coefficients.
Instructively, this protease also significantly increased the digestibility of starch in the distal jejunum by 13.6% (0.770 versus 0.678; p < 0.005) and by 4.80% (0.851 versus 0.812; p < 0.05) in the proximal ileum and numerically enhanced starch digestibility in the proximal jejunum (15.8%) and the distal ileum (3.15%). Based on the presumption that this enzyme preparation did not contain any starch-degrading side-activities, these findings are consistent with the proposition that kafirin and/or glutelin are interfering with starch digestion when sorghum-based diets are offered to poultry.
4.2. “Non-tannin” Polyphenolic Compounds and Phenolic Acids
Phenolic compounds are a diverse group of phytochemicals ranging from highly-polymerised inert lignins to simple C
7-C
9 phenolic acids [
50] and their concentrations in sorghum (170–10,260 mg/100 g) are substantially more than maize (30.9 mg/100 g) and wheat (22–40 mg/100 g) [
51]. The polyphenolic compound condensed tannin is an anti-nutritive factor that depresses broiler performance [
52] and has been even described as a toxic factor [
53]. Sorghum cultivars may be divided into three categories depending on their genotypes and condensed tannin contents [
54]. Type I sorghums do not have a pigmented testa and are “tannin-free”, Type II sorghums have a pigmented testa layer that contains condensed tannin and Type III (“bird-proof”) sorghums contain condensed tannin in both the testa and the peripcarp. Dykes and Rooney [
55] reported condensed tannin concentrations of 0.28, 4.48 and 11.95 g/kg in Type I, Type II and Type III sorghums, respectively. As discussed, it is most likely Australian sorghums no longer contain condensed tannin; however, there is the very real possibility non-tannin phenolic compounds negatively influence digestion of starch and protein to compromise growth performance in broiler chickens.
Taylor [
44] concluded grain sorghum cultivars contain higher levels of phenolic compounds than other cereals and that red (non-tannin) sorghums are highly pigmented with polyphenols, anthocyanins and anthocyanidins and, importantly, these phenols bind strongly to starch. Also ferulic acid has been shown to influence starch pasting profiles which implies phenolic acids have the capacity to interact with starch [
17]. Thompson and Yoon [
56] found tannic acid depressed wheat starch digestion
in vitro and Thompson,
et al. [
57] reported intakes of phenolic compounds by humans were negatively correlated with blood glycaemic indices. Phenolic compounds and phytate are both located in the periphery of sorghum grain (pericarp and aleurone layer) rather than in the central endosperm where starch granules are located. Thus in native grain sorghum it seems unlikely any inter-reactions between phenolics and phytate with starch would take place; however, they may be initiated by processing (e.g., hammer-milling, steam-pelleting) and take place in the avian gut, perhaps particularly in the gizzard.
Phenolic compounds appear to be susceptible to processing and extrusion has been shown to reduce the molecular weight of phenolic polymers in sorghum [
58]. More recently, Dlamini,
et al. [
59] reported that extrusion reduced average total phenol contents by 51% (14.92
versus 7.37 mg catechin equivalents/g) in the whole grain of five sorghum varieties. The impact of decortication or dehulling was more profound with a reduction of 68% (14.92
versus 4.80 mg catechin equivalents/g) due to the location of phenolics in the outer layers of the grain. The effects of extrusion suggest steam-pelleting sorghum-based diets may reduce the size of phenolic polymers, which may influence any subsequent interactions between phenolics and starch in the avian gut.
Zhu [
60] reported non-covalent interactions involving starch and phenolic compounds influence physicochemical and nutritional properties of feedstuffs. Earlier, Tomasik and Schilling [
61] and Barros,
et al. [
62] opined that phenolics readily form starch complexes and are probably more likely to form starch-phenolic complexes with amylose than amylopectin. Kandil,
et al. [
63] reported that phenolic acids play an important role in the resistance of starch to hydrolysis in a study involving barley, maize, triticale and wheat but not sorghum. Welsch,
et al. [
64] found phenolic compounds were capable of inhibiting Na
+-K
+-ATPase or the “sodium pump” which suggested intestinal uptakes of nutrients including glucose via Na
+-dependent transporter systems could be compromised.
A wide range of polyphenolic compounds and phenolic acids have been quantified in a number of diverse sorghum samples by this institute. As a consequence, our contention is that the anti-nutritive properties of phenolic compounds are not the sole domain of CT and to some extent are shared by the balance of polyphenols and phenolic acids found in grain sorghum. Instructively, Elkin,
et al. [
65] concluded that condensed tannin is only partially responsible for variations in the nutrient quality. Also, phenolic compounds and phytate appear to share several anti-nutritive properties [
11]. Provisionally, there was a negative linear relationship (
r = −0.569;
p = 0.042) between dietary levels of total phenolic compounds and ME:GE ratios in broilers offered diets based on nine “tannin-free” sorghum varieties across five of feeding studies. This preliminary outcome supports the contention and prompts identification of which polyphenols or phenolic acids are responsible. Initial indications are that the flavan-4ols (polyphenolic compounds) and soluble conjugated and insoluble bound ferulic acid (a phenolic acid) might be involved.
The colour of red sorghums is attributable to polyphenolic pigments (anthocyanins) and axiomatically red sorghums contain more polyphenols than white sorghum varieties. Indeed, Beta and Corke [
66] reported that four red sorghums had a greater concentration of total polyphenols than six white sorghums by more than a three-fold factor. In Australia probably all white sorghums are of the Liberty variety. Interestingly, we have found that Liberty contains lower concentrations of phenolic acids than a number of red sorghum varieties albeit in a limited number of samples. Three categories of phenolic acid concentrations were quantified, free and conjugated phenolic acids which are soluble and the insoluble, bound phenolic acids. In two projects, nine red sorghums contained an average of 500 μg/g total phenolic acids and 392 μg/g total ferulic acid; whereas, in contrast, two white Liberty sorghums contained 328 μg/g phenolic acids and 235 μg/g ferulic acid. Thus red sorghums contained 28% more phenolic acids and 40% more ferulic acid than white sorghums in this comparison. Ferulic acid is the dominant phenolic acid in sorghum, representing 78% of total phenolic acids across the 11 varieties; however, ferulic acid is certainly not unique to sorghum amongst grains as it is present in maize and wheat.
Dykes,
et al. [
67] reported a positive correlation between total phenols and flavan-4ols (
r = 0.94;
p < 0.001) in non-tannin sorghums. A recent study at this institute involved offering sorghum-based diets (580 g/kg) based on six different varieties to broiler chickens. In this study, flavan-4ols in sorghum
per se was negatively correlated with ME:GE ratios (
r = −0.919;
p = 0.010) which supports the contention that “non-tannin” polyphenolic compounds in sorghum have the capacity to compromise energy utilisation in poultry.
4.3. Phytate
The inclusion of phytase in broiler diets is now a very common practice and the reciprocal impacts of dietary phytate and exogenous phytase on broiler performance have been reviewed extensively [
16,
20,
68,
69]. Briefly, to avoid repetition, sorghum invariably contains phytate at relative and absolute concentrations that are often somewhat higher than other cereal grains [
19,
70] and it is generally accepted that phytate negatively influences protein and energy utilisation in poultry [
20,
68,
71]. The polyanionic IP
6 phytate molecule may form binary or ternary complexes with proteins depending on the isoelectric point of protein and environmental pH [
70]. Phytic acid may affect starch digestion by directly binding starch through phosphate linkages or indirectly via starch granule-associated proteins [
72]. Also, phytate is a potent chelator of divalent cations including zinc and calcium. However, the fundamental role of phytase is to liberate phytate-bound P and this enhanced P utilisation is of astonishing environmental importance. Nevertheless, the suggestion is that the response of starch and protein digestion to phytase [
73] in sorghum-based broiler diets appears to be less robust in comparison to maize- and wheat-based diets [
74]. The likelihood is that the “extra-phosphoric” effects of phytase were largely a consequence of phytase attenuating protein-phytate complex formation.
As proposed by Cosgrove [
14], electrostatic attractions between positively-charged arginine, histidine, and lysine residues with polyanionic phytate molecules are pivotal to the initiation of binary protein-phytate complex formation. This takes place at pH levels less than the isoelectric point of proteins and the isoelectric point (iP) of kafirin is 5.9 [
75] which is considerably higher than pH levels usually found in the crop, proventriculus and gizzard. However, from first principles, it follows that phytate may not readily bind kafirin due to its paucity of basic amino acids. Also, the structure of the discrete, spherical kafirin protein body with its hydrophobic periphery may limit interactions with phytate. Both factors may in turn limit “extra-phosphoric” responses to exogenous phytases in sorghum-based diets simply because kafirin is not readily bound by phytate in binary complexes. It is also follows that if phenolic compounds are limiting performance in sorghum-based broiler diets, then phytase and other standard exogenous enzymes do not have the capacity to counteract this impact.
Both phytate and the various phenolic compounds are located in the outer layers of grain sorghum including the aleurone. Perhaps because of this we are consistently finding that concentrations of phytate and phenolics in sorghums are positively correlated. In this context it is relevant that Selle,
et al. [
11] gave consideration to the analogous anti-nutritive properties of phytate and phenolic compounds. As one related example, Kreydiyyeh,
et al. [
76] found that tea extracts reduced intestinal mucosal Na
+-K
+-ATPase or sodium pump activity and uptakes of glucose and sodium in rats. Subsequently, Kreydiyyeh [
77] reported that tannic acid, a constituent of tea extracts, inhibited intestinal sodium pump activity
in vitro and phenylalanine uptakes in rats. This was attributed to direct inhibition of Na
+-K
+-ATPase activity by tannic acid and dissipation of the Na gradient required for intestinal uptakes of phenylalanine. Alternatively, Liu,
et al. [
78] demonstrated the addition of 1000 FTU/kg phytase to corn-soy diets significantly increased Na
+-K
+-ATPase activity in the duodenum and jejunum of broilers by approximately 20%, and increased glucose concentrations in enterocytes. The implication is phytate depresses sodium pump activity and intestinal uptakes of glucose. Overall, the suggestion is that both phytate and phenolic compounds can impede sodium pump activity along the small intestine thereby compromising intestinal uptakes of glucose and amino acids via Na
+-dependent transport systems.