Conselho Nacional de Desenvolvimento Científico e Tecnológico / Amino acids are organic compounds abundant in animal tissues, especially in meat and meat products, in free form or polymers, participating in biochemical reactions and may be precursors of compounds of interest in the quality control of these foods. The aim of this study was to optimize and validate a method for determination of free amino acids by gas chromatography in Italian dry fermented sausage subjected to ultrasonic bath for 3, 6 and 9 min. Free amino acids were evaluated in salami pieces fermented/seasoned (initial time), at finished product (28 days) and 120 days after manufacture. The extraction was performed using 0.1 N HCl. An aliquot of samples were subjected to vacuum drying, followed by derivatization with MTBSTFA, which has been optimized with respect to time, temperature and reaction solvents (pyridine and acetonitrile). The instrumental and chromatographic conditions of injector temperature, constant pressure and column temperature program was optimized to ensure the method selectivity and efficiency. The use of a mixture of acetonitrile/MTBSTFA in the ratio 1:1 (v/v) using a temperature of 100 °C for 2 h promoted derivatizing all L-amino acids contained in the analytical standards.The injector temperature of 320 °C and 15 psi constant pressure gave the best efficiency in mass transfer of the analytes into the column, while also providing higher resolution and accuracy. The determination coefficients obtained for amino acids were higher than 0.99, except for glycine (0.98). Fourteen amino acids showed a linear range of from 1.0 to 160.0 mg L-1 with LOD and LOQ ranged between 0.3 and 10 g ml-1 and 1 and 20 mg L-1 and inter-day coefficients of variation accurately less than 15% for most analytes.The increase in the concentrations of most amino acids was observed over time processing/storage, indicating that there was an intense proteolytic activity with the release of free amino acids. In the finished product (28 days), treatment with ultrasound affected significantly (p <0.05) concentration of some amino acids. At this point, the amino acids alanine, glycine, valine, leucine, proline and methionine (non-polar) and tyrosine (polar uncharged) showed lower concentrations to control, on the other hand there was an increase of glutamic acid and arginine. The free amino acids found in salami are important compounds because through chemical reactions of decarboxylation or deamination can be precursors of volatile compounds responsible for characteristic processed meat product flavor/aroma. Thus, the employment of no conventional analytical techniques such as gas chromatography for determination of free amino acids allows obtaining information of great importance for the quality control and product development, correlating the technological and nutritional properties with the properties sensory involving amino acids as precursors. / Os aminoácidos são compostos orgânicos abundantes em tecidos animais, sobretudo em carne e produtos cárneos, na forma livre ou de polímeros, participando de reações bioquímicas, podendo ser precursores de compostos de interesse no controle de qualidade destes alimentos. O objetivo do presente trabalho foi otimizar e validar um método para determinação de aminoácidos livres por cromatografia em fase gasosa em salame tipo Italiano submetido a banho ultrassônico por 3, 6 e 9 min. Os aminoácidos livres foram avaliados na massa de salame não fermentada/maturada (tempo inicial), no produto acabado (28 dias) e após 120 dias da fabricação. A extração dos analitos foi realizada com a utilização de HCl 0,1 N. Uma alíquota das amostras foram submetidas à secagem a vácuo seguida de derivatização com MTBSTFA, a qual foi otimizada em relação ao tempo, temperatura e solventes de reação (piridina e acetonitrila). As condições instrumentais e cromatográficas de temperatura do injetor, pressão constante e programação de temperatura da coluna foram otimizadas para garantir a seletividade e eficiência do método. O emprego da mistura de acetonitrila/MTBSTFA na proporção de 1:1 (v/v), utilizando a temperatura de 100 °C por 2 h promoveu a derivatização de todos os L- aminoácidos contidos nos padrões analíticos. A temperatura do injetor a 320 °C e pressão constante de 15 psi proporcionou a melhor na eficiência na transferência de massa dos analitos para coluna, proporcionando também maior resolução e precisão. Os coeficientes de determinação obtidos para os aminoácidos foram maiores que 0,99, com exceção da glicina (0,98). Quatorze aminoácidos apresentaram faixa linear entre 1,0 160,0 μg mL-1, com LOD e LOQ variando entre 0,3 e 10 μg mL-1 e 1 e 20 μg mL-1 e precisão intra-dia com coeficientes de variação menores que 15% para a maioria dos analitos. O aumento nas concentrações da maioria dos aminoácidos foi observado com decorrer do tempo do processamento/armazenamento, indicando que houve uma intensa atividade proteolítica com liberação de aminoácidos livres. No produto pronto (28 dias), os tratamentos com ultrassom afetaram significativa (p<0,05) a concentração de alguns aminoácidos. Nesse ponto, os aminoácidos alanina, glicina, valina, leucina, prolina e metionina (apolares) e tirosina (polar sem carga) apresentaram concentrações inferiores ao controle, por outro lado foi observado um aumento dos de ácido glutâmico e arginina. Os aminoácidos livres encontrados nas amostras de salame são compostos importantes, pois através de reações químicas de descarboxilação ou desaminação podem ser precursores de compostos voláteis responsáveis pelo sabor/aroma característicos do produto cárneo processado. Dessa forma, o emprego de técnicas analíticas não convencionais, como a cromatografia em fase gasosa, para a determinação de aminoácidos livres, permite a obtenção de informações de grande importância no controle de qualidade e desenvolvimento de produtos, correlacionando as propriedades tecnológicas e nutricionais com as propriedades sensoriais que envolvem os aminoácidos como precursores.

The insertion of ω- amino acid residues as guests into host α-peptide sequences permits expansion of the range of polypeptide secondary structures. The term ω- amino acid is used to refer to the entire family of residues generated by homologation of the backbone of α - amino acid residues. This explores the consequences of insertion of substituted β-residues (β3) , unsubstituted β-residues , unsubstituted γ-residues (gamma aminobutyric acid) and unsubstituted δ-residues (delta aminovaleric acid) into host α -peptide sequences. Chapter 1 provides an introduction to the conformational properties of β-peptides and reviews current literature on the structural features of peptides containing ω-amino acid residues. The available crystallographic information is summarized. The conformational properties of β- residues may be described by three degrees of torsional freedom : φ (N – Cβ) , θ (Cβ -Cα) and ψ (Cα-CO). Similarly, the conformational properties of γ -residues is based on four torsional parameters ( φ , θ1 , θ2, ψ) and the conformational properties of δ - residues is based on five degrees of freedom ( φ , θ1 , θ2, θ3,ψ). The rational use of β -residues in peptide design requires an understanding of the nature of local conformations, which are readily accessible. The conformational space for β -residues can be represented in a three dimensional plot. The observed distribution of φ , θ and ψ values for β -residues in peptide crystal structures presented in this section permits a correlation - between the torsion angle θ and the secondary structure context. The gauche (g+ and g ) conformations induce helical folding and the trans conformation is generally observed in the strands of a hairpin. The most striking feature of hybrid sequences is the observation of novel hydrogen bonded rings in peptide structures. Chapter 2 describes the effects of insertion of β-residues into specific positions in the strand segments of designed peptide hairpins. Insertion of β -residues into the strands of a hairpin changes the orientation of peptide bonds, resulting in a “polar sheet” arrangement. The conformational analysis of three designed peptide hairpins composed of α/β - hybrid segments are described: Boc-Leu-βPhe-Val-DPro-Gly- Leu-βPhe-Val-OMe (BBH8) , Boc-βLeu- Phe-βVal-DPro-Gly-βLeu-Phe-βVal-OMe (BAB8) and CF3COO-H3N+-Leu-Val-Val-βPhe-DPro-Gly-βPhe-Leu-Val-Val-OMe (BHFF10). All the peptides have been characterized by 500 MHz 1H-NMR spectroscopy and several crucial long range NOEs confirm a predominant population of β-hairpin conformations in CD3OH. X-ray diffraction studies on single crystals of peptide BBH8 reveal a β-hairpin conformation, stabilized by three cross-strand hydrogen bonds and a Type II′β-turn at the DPro-Gly turn segment. Designed β-hairpin peptide scaffolds may be used to probe cross-strand sidechain interactions in β-sheet structures. A previously reported peptide β-hairpin, Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe exhibited an anomalous far UV CD spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (Zhao, C.; Polavarapu, P.L.; Das,C. and Balaram, P. J. Am. Chem. Soc., 2000, 122, 8228-8231). In BBH8 and BHFF10 the two cross-strand βPhe residues are at non-hydrogen bonding positions, with the benzyl sidechains pointing on opposite faces of the β- sheet. BBH8 yields a “hairpin –like” CD spectrum, with a minimum at 224 nm. The CD spectrum of BAB8 reveals a negative band at 234 nm and a positive band at 221 nm suggestive of an exciton split doublet. BHFF10 yields a “hairpine-like” CD spectrum, with a negative band at 220 nm. Chapter 3 describes the synthesis and conformational characterization of three hybrid decapeptides : Boc-Leu-Val-βGly-Val-DPro-Gly- Leu-βGly -Val-Val-OMe (BHB10), Boc-Leu-Val-γAbu-Val-DPro-Gly- Leu-γAbu -Val-Val-OMe (BHC10) and Boc- Leu-Val-δAva-Val-DPro-Gly- Leu-δAva -Val-Val-OMe (BHD10). These peptides were designed to systematically investigate the effect of insertion of additional methylene groups into the strands of a hairpin. The incorporation of additional carbon atoms changes the local polarity of the strands. 500 MHz NMR studies establish that BHB10 and BHD10 adopt predominantly β- hairpin conformations in methanol, with interstrand registry established by observation of long range NOEs. The observation of both DPro 4 (CαH) ↔ Gly 5 (NH) and Gly 5 (NH) ↔ Leu 6 (NH) NOEs provides evidence for a Type II ′β - turn for all the hairpins. In BHC10, no long range NOEs were observed. However, X-ray diffraction studies in single crystals reveal a β- hairpin conformation, nucleated by a DPro-Gly Type II′β-turn. Chapter 4 describes an attempt to incorporate one or two ω amino acid residues in the turn region of a potential hairpin, in order to assess the effect of expansion of the nucleating turn. The DPro-LPro segment has been shown to stabilize β-hairpin conformations in both cyclic (Shankaramma,S.C.; Moehle, K. ; James, S.; Vrijbloed, J.W.; Obrecht,D and Robinson, J.A. Chem Commun. 2003,1842-1843) and acyclic sequences ( Raj Kishore Rai ; S.Raghothama and P. Balaram , unpublished results) . In the present study the following turn segments have been considered: βDPro -αLPro , βLPro -αLPro and βLPro -αDPro. The synthesis and conformational analysis of three octapeptide sequences -Boc-Leu-Phe-Val-βDPro-αLPro-Leu-Phe-Val-OMe (βDPαLP8), Boc-Leu-Phe-Val-βLPro-αLPro-Leu-Phe-Val-OMe (βLPαLP8)and Boc-Leu-Phe-Val-βLPro-αDPro-Leu-Phe-Val-OMe (βLPαDP8) are described. In the βDPro-αLPro peptide, NMR evidence clearly supports a β-hairpin conformation, with a nucleating hybrid βα turn stabilized by a C11 (4 →1) hydrogen bond. In the other two octapeptides, no evidence for folded structures was obtained. These results suggest that nucleating turn formation is facilitated only in the heterochiral βD-αL case. Further expansion of the turn segment in potential hairpins has been investigated by inserting two contiguous β-residues into the center of a host α-peptide sequence. The conformational studies on two synthetic hexapeptides, Boc-Leu-Phe-βDPhe-βLPro-Phe-Leu-OMe (βDFβLP6) and Boc-Leu-Phe-βLPhe-βLPro-Phe-Leu-OMe (βLFβLP6) suggest that the βDPhe-βLPro segment is capable of forming a C12 turn in methanol. Two octapeptide sequences, Boc-Leu-Val-Leu-βDPhe-βLPro-Leu-Phe-Val-OMe (βDFβLP8N) and Boc-Leu-Val-Val-βDPhe-βLPro-Leu-Val-Val-OMe (βDFβLP8V) have also been investigated to probe the possible formation of hairpin structures. In these cases, spectroscopic analysis is hampered by the presence of multiple conformations, because of the tendency of the βDPhe-βLPro bond to exist in both cis and trans conformations. NMR studies on the conformational properties of a hexapeptide Boc-Leu-Val-βDPro-βLPro-Leu-Phe-OMe (βDPβLP6) in CDCl3 reveal that in the major conformer the Val 2(NH) ↔ Leu 5 (NH) NOE is observed, suggesting the presence of a 12-membered hydrogen bonded turn. A ββ - segment can give rise to two types of hydrogen bonded rings , 10 – membered (C10) and 12- membered (C12). In an attempt to generate C10 turns, an N-methylamino acid has been inserted next to a ββ - segment, preventing the formation of the 12 – membered turn. In such a situation formation of a 10-membered turn, with reverse hydrogen bond directionality, may be facilitated. The conformational properties of Boc-Leu-Val-βDPhe-βLPro-(N-Me) Leu- Phe-OMe (βDFβLPNMeL6) has been studied by 500 MHz NMR spectroscopy. The data suggests the formation of a C11 turn at the βLPro- (N-Me) Leu segment in CDCl3-DMSO mixtures, instead of formation of a C10 turn at the βDPhe -βLPro segment. Studies on the peptide Boc-Leu-Phe-Val-βLPro-(N-Me) Leu-Leu-Phe-Val-OMe (βLPNMeL8) also suggest the absence of turn formation and folded structures. In hybrid sequences, an important question to be addressed is whether ω amino acids can be accommodated into helical structures. Two contiguous β- residues have been inserted into a helical sequence. The conformational properties of a 11- residue peptide, Boc-Val-Ala-Phe-Aib-βVal-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA11) are described in Chapter 5. This sequence was based on the parent α- peptide Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, which adopted a complete helical conformation in crystals (Aravinda, S.; Shamala, N.; Das, C .; Sriranjini, A.; Karle, I.L. and Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308-5315). 500 MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution , as evidenced by diagnostic nuclear Overhauser effects. The molecular conformation in crystals reveals a continuous helical fold, stabilized by seven intramolecular hydrogen bonds. The characterization of two synthetic octapeptides Boc-Val-Ala-βPhe-Aib-Val-Ala-βPhe-Aib-OMe (VAβFU8) (βPhe residues have been incorporated at (i /i+4 positions) and Boc-Val-Ala-βPhe-Aib-βPhe-Ala-Val-Aib-OMe (βFUβF8) (βPhe residues have been incorporated at (i /i+2 positions) is also presented. NMR data suggests the retention of helical conformation in both the peptides. In order to delineate the conformations of hybrid peptides with three contiguous β-residues, two peptides have been synthesized Boc-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA10) and Boc-Val-Ala-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA12). NMR studies in chloroform support continuous helical conformation in the decapeptide.

PART I RESOLUTION AND DESYMMETRISATION Chapter I. ‘A Novel Racemate Resolution’. This describes a novel resolution strategy as applied to racemic α-amino acids in the solid state. The strategy is based on the possibility that second order asymmetric transformations (SOAT) would be more likely in the case of achiral molecules that form chiral crystals (i.e. a non- centrosymmetric space group).1 In such cases, a fundamental requirement of SOAT – that the molecules racemise in solution prior to crystallization – is obviated. Furthermore, the resulting enantiomerically-enriched crystals may be employed to effect a solid-state kinetic resolution of a different racemate (composed of chiral molecules). This strategy was explored with crystalline succinic anhydride (1, Scheme 1), which not only exists in a non-centrosymmetric space group (P212121) but also possesses reactive functionality to effect the resolution step.2 Thus, a finely-ground mixture of 1 (0.5 eqiv.) and a racemic α-amino acid (2, 1.0 eqiv.) was heated at ~ 70 oC over ~ 5 h without solvent. The resulting N-succinoyl derivative (3) was separated from the unreacted 2, which was found to possess significant levels of optical purity (typically ~ 70%). The strategy was applied to several common α-amino acids, the results being summarized in Table 1. These results, apart from establishing ‘proof-of-concept’ and the viability of the resolution strategy, indicate that crystalline succinic anhydride (1) is enantiomerically enriched as originally hypothesized. Chapter II. ‘Enantiospecific Alkylation and Desymmetrisations’. This deals with two enolate-mediated strategies of asymmetric synthesis: one describes approaches towards the alkylation of the stereogenic centre in benzoin without loss of stereogenicity (Section A), and the other the desymmetrisation of a meso tartarate derivative with a chiral base catalyst (Section B). Section A. This describes exploratory studies aimed at achieving the enantiospecific α-alkylation of optically-active benzoin (4, Scheme 2) via its enolate anion 5. The strategy depends on the possibility that 5 would exist in atropisomeric forms, because of steric interactions between the vicinal phenyl groups. (This is indicated in the crystal structure of the analogous enediol carbonate derived from racemic 4.)3 In such a case, remarkably, 5 would be chiral, despite its planar enediolate core! Thus, possibly, the configurational chirality in 4 (by virtue of the C2 stereogenic centre) would be transformed to the helical chirality in 5 (by virtue of the atropisomerism). Furthermore, enantioface-selective alkylation of 5 with achiral alkylating agents would, in principle, be possible. Preliminary studies were then directed towards establishing that controlled deprotonation of optically-active 4, followed by the protonation of the resulting enediolate 5, leads back to the original 4. (+)-Benzoin (4) was prepared via resolution,4 and deprotonated with KH in THF.5 The resulting enediolate (5) was neutralized with acetic acid at -70 oC/THF to recover 4, but with insignificant levels of optical activity (e.e. ~ 12%). The results possibly indicate that ortho-substituted benzoin analogs may show greater retention of chirality upon deprotonation, as the racemisation of the enediolate atropisomers would be suppressed by steric hindrance between the aryl moities. Section B. This describes studies directed towards the catalytic desymmetrisation of meso dimethyl tartarate (6, Scheme 3). The strategy involves the formation of the acetonide derivative 7 and its regioselective α-deprotonation with a chiral base catalyst. The enantioface-selective protonation of the resulting enolate (8) would lead to the chiral analog 9. The overall sequence offers a possible alternative to catalytic deracemisation, which is normally unviable for thermodynamic reasons.6 The above strategy hinges on the meso derivative 7 being thermodynamically less stable than the enantiomeric 9, which would thus be favoured at equilibrium. In fact, this is likely as the eclipsing interactions between the syn ester moieties in 7 would be relieved in 9, in which the ester moieties are anti. However, deprotonation of 7 at the other α position would compete to varying extents, depending on the selectivity induced by the chiral base. At total equilibrium, the sequence would occur via deprotonation at both α sites at equal rates, and no net optical induction would be observed. (This is a thermodynamic requirement via the principle of microscopic reversibility.) Thus, the success of the above strategy depends on stalling the deprotonation-protonation sequence at a quasi-equilibrium stage involving only one of the enantiomers (9).6 The other operational requirement was the compatibility of the pKa’s of 7 and the chiral base employed: too low a pKa of the base would result in inefficient deprotonation and slow overall rate, but a high pKa would generate a large quantity of the enolate 8 at equilibrium. After due consideration, the lithiated chiral fluorene derivative 11 (pKa ~ 22) was chosen as the chiral base catalyst [11 was prepared from fluorene (10) as indicated]. Treating 7 with 0.2 equivalent of 10 in THF at -65 oC over 2 h, led to the formation of a mixture of 7 and 9 in a 45:55 ratio (isolated in 85% total yield). Chromatographic separation of the mixture led to the isolation of pure (+)-9, which was identified spectrally; it was found to possess [α]D24 = +21.84 (c 1.0, CHCl3), corresponding to e.e. = 64%. (This implies the indicated (4S, 5S) configuration for 1, 3-dioxolane 9, as previously reported.)7 These results, despite the moderate e.e. levels obtained, indicate the viability of the above catalytic desymmetrisation strategy, bearing in mind the mechanistic ambiguities mentioned above. PART II SYNTHESES OF ALDEHYDES AND AMINO ACIDS Chapter III. ‘An Asymmetric Synthesis of Aldehydes’. This describes an oxazoline approach to the synthesis of chiral aldehydes. The oxazoline methodology for the synthesis of homochiral α-alkylated carboxylic acids is well known,8 and it was of interest to adapt this to the synthesis of the corresponding aldehydes. Essentially, it was envisaged that the reaction sequence could be diverted towards aldehydes via reduction of the alkylated oxazoline intermediate (Scheme 4). Thus, 2-ethyl-4(S)-methoxymethyl-5(R)-phenyl-1,3-oxazoline (12) was deprotonated with lithium diisopropylamide in THF, and the resulting anion treated with various alkyl halides, in the reported manner.8 The resulting alkylated product (13) was N-methylated with MeI in refluxing MeNO2 over 6 h, to obtain the quaternary salt 14. This was reduced with NaBH4 in MeOH to obtain the expected N- methyl oxazolidine 15, which was hydrolyzed in refluxing aqueous oxalic acid to the free aldehydes 16. These were isolated in moderate yields and e.e. values as shown. Chapter IV. ‘A Darzens Route to α-Amino Acids’. This describes a novel route to α-amino acids, based on the classical Darzens glycidic ester synthesis.9 In this approach (Scheme 5), the glycidic ester (19) was prepared from benzaldehyde (17) and t-butyl bromoformate (18), with KOH in THF as base, and tetrabutylammonium bromide (TBAB) as phase transfer catalyst.9b The oxirane ring in 19 was cleaved via nucleophilic attack with an amine (20), to furnish the two regio-isomeric hydroxy- amino acids (21) and (22). Generally, the β-hydroxy-α-amino acid product (21) predominated over the α-hydroxy-β-amino acid product (22), the two being separated chromatographically. The hydroxyl group in 21 was reductively cleaved via its xanthate derivative (23), by refluxing it in toluene with AIBN (10 mol %) over 4 h. The resulting α-amino acid derivatives (24) were obtained in moderate yields (< 60 %) upon chromatographic purification. (The β-amino analog 22, would lead to the corresponding β-amino acid, but this was not pursued further.) This strategy lends itself to creating structural diversity at the β-centre in the α- amino acid, drawing upon the wide scope of the well-established Darzens condensation reaction. Also, the introduction of the amino moiety is facilitated by the enhanced reactivity at the α-centre of the oxirane ring in the glycidic ester (19), presumably for both electronic and steric reasons.

<p>Diet is the number one leading modifiable cause of poor health globally, with poor diets accounting for 10.9 million (22%) of all deaths among adults in 2017. In addition, one of our generation’s forthcoming challenges is the rapid expansion of the population aged 60 years and older. Although people are living longer, there is an associated increase in the prevalence of aged-related chronic diseases and functional impairment, such as cardiometabolic diseases and sarcopenia. As such, dietary components can play a role in positively or negatively influencing the prevention and treatment of chronic cardiometabolic diseases and sarcopenia. One such dietary component is dietary protein, which is essential throughout the life course, from gestation through old age. Evidence supports dietary protein playing an important role in reducing the risk of developing age-related chronic diseases such as sarcopenia and cardiometabolic diseases. </p> <p><em><strong>     Study 1, Chapter 2:</strong></em> The Dietary Guidelines for Americans (DGA) recommends consuming a variety of “Protein Foods” based on “ounce equivalent” (oz-eq) portions. In addition, the 2020-2030 Strategic Plan for NIH Nutrition Research includes to “define the role of nutrition across the lifespan” with an objective to “assess the role of nutrition in older adults to promote healthy aging.” However, there is a paucity of primary research that directly compares EAAs bioavailability between young and older adults consuming the same oz-eq portions of varied Protein Foods. No study has assessed the same oz-eq portions of animal- versus plant-based Protein Foods on essential amino acids (EAAs) bioavailability for protein anabolism in young and older adults. Therefore, we conducted two sequential randomized, investigator-blinded, crossover, acute feeding trials with the same study design; first in a cohort of young adults and second in a cohort of older adults. The primary objective of this project was to assess the effect of consuming two oz-eq portions of animal-based (unprocessed lean pork or whole eggs) vs. plant-based (black beans or sliced almonds) Protein Foods as part of a mixed whole foods meal on plasma EAAs bioavailability for protein anabolism. Consistent with our hypotheses, participant age did not affect postprandial EAAs bioavailability, and consuming a meal with two oz-eq of unprocessed lean pork or whole eggs resulted in greater postprandial EAAs bioavailability compared to a meal with two oz-eq of black beans or raw sliced almonds in 1) young adults; 2) older adults; and 3) young and older adults combined. These findings show on the same oz-eq basis, consuming these animal- vs. plant-based Protein Foods more effectively provide bioavailable EAAs for protein anabolism. </p> <p><em><strong>     Study 2, Chapter 3:</strong></em> Poultry meat is the most consumed type of meat worldwide and in the US. Poultry is generally considered to be a “healthy” meat as it is a high-quality protein source and provides other essential nutrients. However, research assessing poultry and its effects on and relations with chronic diseases in humans is sparse, and the forms of poultry typically consumed in the US, are not necessarily in line with recommendations provided by the DGA. Therefore, we conducted a scoping review to systematically search and chronicle scientific literature pertinent to poultry intake and human health. Main findings from this project were 1) historically, little research, especially randomized diet-controlled feeding trials, has been conducted to understand associations between and effects of consuming poultry products on human health; 2) the majority of research is from observational studies assessing relationships between poultry intake and risks of morbidity and mortality from various types of cancer; 3) a paucity of research exists to support chicken as a health-promoting food in children; and 4) research taking into account poultry product processing and cooking methods is needed. Science and health professionals, the poultry industry, and the public will benefit from new observational and experimental research to address cutting-edge scientific, public policy, and consumer topics pertinent to poultry intake and human health. </p> <p><em><strong>     Study 3, Chapter 4:</strong></em> Emerging research on whey protein supplementation suggests it may be a potential modifier of type 2 diabetes mellitus (T2DM) risk factors, including glucose control. As systematic reviews and/or meta-analyses of randomized controlled trials are gaining importance in nutrition literature, we conducted an umbrella systematic review to search for and chronicle published systematic reviews and/or meta-analyses of randomized controlled trials pertinent to whey protein supplementation and T2DM modifiable risk factors (study 3, Chapter 4). Among the 13 systematic reviews, including 12 meta-analyses critically assessed for this umbrella review, no reviews reported any adverse effects of whey protein on any reported T2DM-related risk factor. Collectively, a preponderance of evidence indicates whey protein supplementation improves multiple clinical indicators of glucose control in apparently healthy adults and those at increased risk for type 2 diabetes mellitus. </p>

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease characterized by mutations in the dystrophin gene. Duchenne muscular dystrophy is the most common and most severe form of inherited muscle diseases, with an incidence of 1 in 3,500 male births1,2. Mutations in the dystrophin gene result in non-functional dystrophin or the complete absence of the protein dystrophin, resulting in necrosis and fibrosis in the muscle, loss of ambulation, cardiomyopathies, inadequate or failure of respiratory function, and decreased lifespan. Although there has been little research for effective nutritional strategies, dietary intervention may be effective as an adjuvant treatment. In this study, wild type (WT) and mdx animals were provided either a control or elevated branched chain amino acid (BCAA) diet nocturnally for 25 weeks to determine if the elevated BCAAs would attenuate muscle torque loss. Twenty-five weeks of chronic, elevated BCAA supplementation had no impact on muscle function measures. Interestingly, mdx and WT animals had the same torque responses in the low stimulation frequencies (1 Hz – 30 Hz) compared to higher stimulation frequencies. Tetanus was reached at a much lower stimulation frequency in mdx animals compared to WT animals (100 Hz vs +150 Hz). The mdx mouse consistently had more cage activity in the light cycle X- and Y-planes. Interestingly, animals on the BCAA diet increased X-, Y-, and Z-plane activity in the dark cycles at four weeks while animals on the control diet more Z-plane activity at 25 weeks, although not significant. All three BCAAs were elevated in the plasma at 25 weeks, although only Leu was significantly elevated. The BCAAs had no effect on. The diaphragm and skeletal muscle masses were larger in mdx animals, and WT animals had a significantly larger epididymal fat pad. The active state of BCKDC determined by phosphorylation of the E1α enzyme was greater in WT animals in white skeletal muscle, but not red skeletal muscle. Protein synthesis effectors of the mTORC1 signaling pathway and autophagy markers were similar among groups. Wild type animals had increased mTORC1 effectors and animals on the BCAA diet had decreased autophagy markers, although not significant. Although BCAAs did not affect muscle function, fibrosis, or protein synthesis effectors, this study illustrates the functionality of mdx muscles over time. It would be interesting to see how the different muscle fiber types are affected by DMD, noting the differences between the diaphragm, heart, red muscle, and white muscle fibrosis markers. Although there was no increase in mTORC1 effectors with an elevated BCAA diet, it would be interesting to determine muscle protein synthesis, myofibrillar protein synthesis, and total protein turnover in the mdx mouse with an elevated BCAA diet, although the dietary intervention started when mice arrived at 4 weeks of age, earlier intervention may be beneficial early in the disease process. / Doctor of Philosophy / Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease characterized by mutations in the dystrophin gene. Duchenne muscular dystrophy is the most common and most severe form of inherited muscle diseases, with an incidence of 1 in 3,500 male births1,2. Mutations in the dystrophin gene result in non-functional dystrophin or the complete absence of the protein dystrophin, resulting in necrosis and fibrosis in the muscle, loss of movement and walking ability, cardiomyopathies, inadequate or failure of respiratory function, and decreased lifespan. Although there has been little research for effective nutritional strategies, dietary intervention may be effective as an adjuvant treatment and palliative care. The branched chain amino acids (BCAAs) are known to directly stimulate muscle protein synthesis by direct activation of the mechanistic target of rapamycin complex 1 (mTORC1). This study aimed to illustrate the differences between diseased and healthy mice and determine if BCAAs can reduce muscle torque loss. Twenty-five weeks of chronic, elevated BCAA supplementation had no impact on muscle function measures. Interestingly, mdx and WT animals had the same torque responses in the low stimulation frequencies (1 Hz – 30 Hz) compared to higher stimulation frequencies. Tetanus was reached at a much lower stimulation frequency in mdx animals compared to WT animals (100 Hz vs +150 Hz). The mdx mouse consistently had more cage activity in the light cycle X- and Y-planes. Interestingly, animals on the BCAA diet increased X-, Y-, and Z-plane activity in the dark cycles at four weeks while animals on the control diet more Z-plane activity at 25 weeks, although not significant. All three BCAAs were elevated in the plasma at 25 weeks, although only Leu was significantly elevated. The BCAAs had no effect on. The diaphragm and skeletal muscle masses were larger in mdx animals, and WT animals had a significantly larger epididymal fat pad. The active state of BCKDC determined by phosphorylation of the E1α enzyme was greater in WT animals in white skeletal muscle, but not red skeletal muscle. Protein synthesis effectors of the mTORC1 signaling pathway and autophagy markers were similar among groups. Wild type animals had increased mTORC1 effectors and animals on the BCAA diet had decreased autophagy markers, although not significant. Although BCAAs did not affect muscle function, fibrosis, or protein synthesis effectors, this study illustrates the functionality of mdx muscles over time. It would be interesting to see how the different muscle fiber types are affected by DMD, noting the differences between the diaphragm, heart, red muscle, and white muscle fibrosis markers. Although there was no increase in mTORC1 effectors with an elevated BCAA diet, it would be interesting to determine muscle protein synthesis, myofibrillar protein synthesis, and total protein turnover in the mdx mouse with an elevated BCAA diet, although the dietary intervention started when mice arrived at 4 weeks of age, earlier intervention may be beneficial early in the disease process.

Soil carbon (C) and nitrogen (N) are predominantly in organic form. Proteins/ peptides, as an important organic form of N, constitute a substantial part of soil organic matter. On one hand, proteins/peptides are an important N source for plants and microorganisms, particularly in soils where inorganic N is limited. On the other hand, their stabilization in soils by forming organo-mineral associates or macromolecule complex reduces the C loss as CO2 into the atmosphere. Therefore, studies on the turnover, abundance, composition, and stability of proteins/peptides are of crucial importance to agricultural productivity and environmental sustainability. In the first part of this study, the bioavailability and distribution of amino acids, (building block of proteins/peptides), were investigated, in soils across the North-South and West-East transects of continental United States. The second part of this study aimed to understand the variations of organic C speciation in soils of continental United States. Previous investigations of the interactions between soil minerals and proteins/peptides were mostly limited to batch sorption experiments in labs, seldom of which gave the details at the molecular scales. Therefore, in the third part of this study, the molecular orientation of self-assembled oligopeptides on mineral surfaces was investigated by employing synchrotron based polarization-dependent Near Edge X-ray Adsorption Fine Structure Spectroscopy (NEXAFS) techniques. Specific aims of this study were: 1) to assess potentially bioavailable pool of proteinaceous compounds and the immediately bioavailable pool of free amino acids in surface and subsurface soils of various ecosystems; 2) to evaluate the relationship between environmental factors and levels/composition of the two pools; 3) to investigate the organic C speciation in soils of various land use; and 4) to understand molecular level surface organization of small peptides on mineral surfaces. The levels of free amino acids and hydrolysable amino acids which represent the potentially bioavailable pool of proteinaceous compounds in A-horizon soils were significantly high than in C-horizon soils due to the accumulation of organic matter in surface. On average, free amino acids accounted for less than 4 % of hydrolysable amino acids which represent the total proteinaceous compounds in soils. The composition of free amino acids was significantly different between surface soil and subsurface soil and was significantly influenced by mean annual temperature and precipitation. A relatively uniform composition of hydrolysable amino acids was observed irrespective of a wide range of land use. Significant variations were observed for the levels of free and hydrolysable amino acids along mean annual temperature and precipitation gradients, as well as among vegetation types of continental USA, suggesting levels of free and hydrolysable amino acids were associated with the above-ground biomass and root distribution. Organic C speciation investigation revealed the presence of carboxylic-C (38%), aliphatic-C (~ 22%), aromatic-C (~ 18%), O/N-alkyl-C (~ 16%), and phenolic-C (< 6%). Factors such as temperature and vegetation cover were revealed in this study to account for the fluctuations of the proportions of aromatic-C and phenolic-C, in particular. Phenolic-C may serve as a good indicator for the effect of temperature or vegetation on the composition of SOC. The average composition of soil organic C, over the continental scale, was relatively uniform over various soil ecosystems and between two soil horizons irrespective of surface organic C content. Polarization dependent NEXAFS analysis showed the oligopeptides tend to orient on mineral surface with an average tilt angle of 40 ° between the molecular chain and the mineral surface. / Ph. D.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The Dorper is numerically the second most abundent of all the sheep breeds in South Africa. It is the most numerous sheep breed in the dry arid areas of South Africa which make out a very big portion of the natural grazing in South Africa. In this study the feedlot performance was specifically investigated since so little research has been done in this regard. In the first study (Chapter 1), the production efficiency, body components and commercial yield of 13 Dorper and 12 Merino lambs were investigated. A pelleted diet was fed ad lib and the water intake per day was measured. Lambs were slaughtered at 30kg and at 40kg live weight and cut up according to the South African commercial cuts. The weights of the organs, head, feet, skin and carcass were also recorded. Results from the first study showed that Dorpers had a lower (P<0.05) water efficiency (285.97 mVkgO75/day) than the Merinos (240.07 mVkg075/day), but had a higher (P<0.05) nitrogen retention. The head, feet, skin, heart, lungs, kidneys and diaphragm of Merino lambs were heavier (P<0.05). Merinos had heavier (P<0.05) breast and shank and thick rib (only at 40kg live weight) cuts. But the Dorper had a significantly heavier hindquarter (buttock and loin) which is the high-priced cut of any carcass. It seems that the Dorper will do well under feedlot conditions, and would possibly make a better commercial meat producer than the Merino under all circumstances. In the second study (Chapter 2), the free-ranging Dorper on natural grazing were compared with 7 Dorper lambs in the feedlot study. The essential amino acids in the duodenal contents were compared. The free-ranging Dorpers were randomly slaughtered at 40 kg live weight and a duodenal sample was collected. The feedlot Dorpers were also slaughtered at 40 kg live weight and a duodenal sample was taken. The second study indicated no significant differences between the EAA composition of the duodenal samples, whether protein quantity was taken into account (gAA/100g crude protein (CP)) or not (expressed as % of lysine). The chemical scores indicated that the two most limiting amino acids in the duodenal digesta for whole empty body growth were: arginine and histidine. Excess levels of amino acids tend to be present in all three duodenal digesta samples (isoleucine, leucine, lusine, methionine, phenylalaline, threonine and valine). The whole empty body EAA composition (g AA/100 g crude protein) was as follows 7.10 arginine; 2.40 histidine; 3.31 isoleucine; 7.22 leucine; 6.61 lysine; 1.62 methionine; 3.91 phenylalanine; 3.77 threonine and 4.85 valine. The composition can serve as an example of the ideal EAA requirements for whole empty body growth between 30 and 40 kg live weight of Dorper ram lambs. The duodenal amino acid concentrations of the free-ranging Dorper compared well with those of the feedlot Dorpers which suggests that the microbial organisms have a big influence on the amino acid concentration which reaches the intestines. / AFRIKAANSE OPSOMMING: Die voerkraal prestasie en essentiele aminosuur behoeftes van groeiende Dorper lammers: The Dorper is getalgewys die tweede mees talryke skaapras in Suid-Afrika. Dit is die mees talryke skaapras wat in die drce woestynagtige gebiede van Suid-Afrika aangetref word. Die gebiede beslaan die grootste oppervlakte van Suid-Afrika se natuurlike weiveld. In die studie is voerkraal prestasie van die Dorper nagevors, spesifiek omdat daar so min navorsing op hierdie gebied gedoen is. In die eerste studie (Hoofstuk 1) is die produksie doeltreffendheid, liggaams komponente en kornrnersiele produksie van 13 Dorper- en 12 Merino lammers ondersoek. 'n Verpilde rantsoen is ad lib gevoer en die water inname per dag is ook gekontroleer. Die lammers is geslag op 30 en 40 kg lewende gewig en die karkasse is daarna opgesny volgens die Suid-Afrikaanse kornmersiele karkassnitte. Die resultate van die eerste studie het getoon dat Dorpers 'n laer water potentiaal (P<0.05) (285 97 mflkgO 75/dag) as die Merinos (240.07 mVkg075/dag) het. Dorper lammers het ook 'n hoer (P<0.05) stikstof retensie. Die kop, vel, pote, hart, longe, niere en diafragma van Merino lammers was swaarder (P<0.05). Merinos se brisket en dikrib snitte weeg ook swaarder (P<0.05). Die Dorper het 'n beduidende swaarder agterkwart (len de en boud) gehad, wat die duurste snit is van enige karkas. Die Dorper het baie goed gedoen in die voerkraal toestande en sal waarskynlik onder aile omstandighede 'n hcer vleisproduksie op die Merino lewer. In die tweede studie (Hoofstuk 2), is die vryweiende Dopers op natuurlike weiding vergelyk met die Dorpers in 'n voerkraal. Die twee groepe Dorpers se essensiele aminosuur samestelling in hul dunderms is bepaal en vergelyk om te bepaal of daar enige tekorte teenwoordig is. Die Dorpers in die veld en in die voerkraal is op 40 kg lewende gewig geslag en monsters van die dunderm inhoud is gekollekteer. Die monsters is ontleed om te bepaal of daar enige betekenisvolle verskille in die essensiele aminosure van die dunderm inhoud tussen die veld Dorpers en voerkraal Dorpers was. Die tweede studie het geen betekenisvolle verskille getoon tussen die dunderm monsters se essensiele aminosure, of die hoeveelheid proteien (gAA/100g) in aanmerking geneem is of nie (uitgedruk as % lisien). Die chemiese telling dui aan dat die twee mees beperkende aminosure in die dunderm inhoud vir lee liggaam groei arginien en histidien was. Oormaat vlakke van aminosure kom voor in al drie dunderm inhoud monsters (isoleusien, leusien, lisien, metionien, femielalanien, treonien en valien). Die totale lee liggaam essensiele aminosuursamestelling (g aminosuur/100g ruprotein) was soos volg: 7.10 arginien; 2.40 histidien; 3.31 isoleusien; 7.22 leusien; 6.61 lisien; 1.62 metionien; 3.91 fenielalanien; 3.77 treonien en 4.85 valien. Hierdie aminosuursamestelling kan as 'n aanduiding dien vir die ideale aminosuurbehoeftes van Dorper ram lammers vir totale lee liggaamsgroei vanaf 30 tot 40 kg lewende massa. Die dunderm aminosuur konsentrasie van die Dorper in die veld stem goed ooreen met die van die voerkraal Dorper. Dit toon waarskynlik dat die mikrobe organismes in die rumen In groot invloed het op die aminosuur samestelling wat die dunderm bereik.

Assignment (MPhil)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The study conducted was aiming on evaluating the influence of different levels of energy and amino acids, mainly lysine and methionine, on production performance of the layer bird. There were three treatments, namely the Control diet, a high energy, lysine and methionine diet (High spec.) and a low energy, lysine and methionine diet (Low spec.). The energy levels were 11.2 MJ/kg, 11.5 MJ/kg and 10.9 MJ/kg respectively. Lysine levels were 0.67%, 0.73% and 0.63% whereas methionine levels were 0.36%, 0.38% and 0.34% respectively. The experimental design was 3 x 4 factorial, which is 3 treatments with 4 replicates each. Results showed no significant difference (P>0.05) between treatments in egg production, egg mass, egg output, bodyweight and mortality. Feed intakes of the High spec. diet were significantly lower (P<0.05) than that of the control diet and the Low spec. diet. / AFRIKAANSE OPSOMMING: Hierdie studiestuk handeloor die evaluering van die invloed wat die verskillende vlakke van die energie en aminosure, veraiiisien en methionine op die produksie van 'n lê hoender het. Daar was gebruik gemaak van drie behandelings, naamlik die kontrole dieet, 'n hoë energie, lisien en methionien dieet (Hoë spesifikasie), en 'n lae energie, lisien en methionien dieet (Lae spesifikasie). Die energievlakke was 11.2 MJ/kg, 11.5 MJ/kg en 10.9 MJ/kg onderskeidelik. Lisienvlakke was 0.67%, 0.73% en 0.63% waarby methionienvlakke was 0.36%, 0.38% and 0.34% onderskeidelik. Die eksperimentele ontwerp was 3 x 4 fakulteitsfunksies: 3 behandelings met 4 replikas elk. Die resultate het geen noemenswaardige verskille (P>0.05) tussen die behandelinge in eierproduksie, eiergewig, eier-uitset, liggaamsgewig en mortaliteite nie. Die voerinnames van die hoë spesifikasie dieet was aansienlik laer (P<0.05) as die van die kontrole en lae spesifikasie dieet.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this study was to determine whether a lamb finishing ration that is balanced for essential amino acids (EAA), at a specific level of non-structural carbohydrates (NSC) would yield better feedlot performance when compared to a standard commercial finishing ration. Metionine (Met) and Lysine (Lys) were identified as the first limiting amino acids for growing lambs. These two amino acids, as well as Threonine (Thr), Arginine (Arg), Leucine (Leu), Isoleucine (Ile) and Phenylalanine (Phe) were included in an optimized protein (OP) feedlot ration at optimal levels. This ration was formulated to contain 157 g/kg crude protein (CP) and 477 g/kg NSC, with Met, Lys, Arg and Thr at 2.48, 7.45, 8.51 and 8.12 g/kg on dry matter (DM) basis respectively. The second treatment, FIN, was a standard commercial lamb finishing feed without optimized amino acids and contained similar total protein and NSC to OP of 152 g/kg CP and 468 g/kg NSC but with Met, Lys, Arg and Thr at 2.08, 5.49, 7.47 and 4.80 g/kg on DM basis respectively. A third treatment, a low protein (LP) diet served as a positive control and was formulated on lower specifications (139 g/kg CP, 455 g/kg NSC) and was also not optimized for amino acids. This treatment contained Met, Lys, Arg and Thr at 1.93, 4.99, 6.66 and 4.73 g/kg on DM basis respectively. Lambs grazing kikuyu pasture served as the negative control (CON) group. These lambs also received additional supplementary feed at 500 g/day as a production lick to be comparable to a scenario where lambs are finished on grazing. Forty cross-bred Merino x Döhne-Merino lambs with an average weaning weight of 24.35 ± 0.648 kg were finished in a feedlot for 57 days where after they were slaughtered at an average weight of 41.41 ± 1.259 kg. During the feedlot trial lamb performance was measured by monitoring daily growth rates and feed intake. Also, an in vivo digestibility study was carried out on the OP and FIN lambs. During the slaughter process the rumen was removed which was done to collect a sample of the rumen wall from next to the rumino-reticular fold. These rumen samples were mounted onto slides so that the development of the rumen could be examined. The M. longissimus dorsi from both sides of the carcass between the 2nd and 3rd last thoracic vertebra and the 4th and 5th lumbar vertebra were removed. There was no significant difference (P > 0.05) between any of the concentrate feed treatments with regards to the average daily gain (ADG), feed conversion ratio (FCR) or dressing percentage (DP). The CON lambs, as expected, had lower growth rates (P < 0.05) than the concentrate fed lamb and thus showed significant differences (P < 0.05) in terms of ADG. The DP of 45.96 % ± 0.711 for the CON lambs differed significantly (P < 0.05) from the OP (51.44 % ± 0.358), FIN (52.72 % ± 0.653) and LP (51.74 % ± 0.611) treatments. As expected the concentrate feeds were much more effective in maintaining higher growth rates when compared to the CON lambs while the optimizing of EAA in the OP diet did not lead to improved feedlot performance as the FIN and LP treatments were able to achieve similar (P > 0.05) growth rates. Within the feedlot treatments there was no significant difference (P > 0.05) with regard to the papillae length and rumen wall thickness. There was however a numerical increase in the papillae length as the NSC levels in the feed increased. The CON lambs differed from the OP lambs (P < 0.05) in terms of papillae length. This illustrated the importance of having increased levels of NSC in a feedlot diet as it is this fraction that is responsible for the initialisation and maintenance of rumen morphological development. The in vivo digestibility study therefore confirmed that the commercial finishing feed was just as effective as the optimized feed in terms of nitrogen retention as well as in maintaining suitable energy balance. Although the in vivo digestibility for Met and Lys in the OP feed was higher (P < 0.05) than the FIN feed, this did not lead to improved feedlot performance of the OP lambs. The increased digestibility of these amino acids is due to the fact that the OP diet was higher in levels of bypass amino acids than the FIN feed. The in vitro true digestibility (IVTD) of the OP feed was higher (P < 0.05) than that of the FIN feed. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om te bepaal of ‘n lamafrond rantsoen wat gebalanseerd is vir beperkende essiensiële aminosure (EAA) teen ‘n spesifieke vlak van nie-strukturele koolhidrate (NSK) beter voerkraal prestasie teweeg sou bring wanneer gemeet word teen ‘n standaard kommersiële afrond rantsoen. Metionien (Met) en Lisien (Lis) is geïdentifiseer as die eerste beperkende aminosure vir groeiende lammers. Hierdie twee aminosure, asook Treonien (Tre), Leusien (Leu), Isoleusien (Ile) en Fenielalanien (Fen) is teen optimale vlakke ingesluit in ‘n geoptimeerde voerkraal rantsoen, OP. Hierdie rantsoen is geformuleer om 157 g/kg RP en 477 g/kg NSK te bevat asook Met, Lis, Arg en Tre teen 2.48, 7.45, 8.51 en 8.12 g/kg onderskeidelik. Die tweede behandeling, FIN, was ‘n kommersiële lamafrond voer waarin die aminosure nie geoptimeer is nie en het soortgelyke vlakke van proteïene en NSK bevat teen 152 g/kg RP en 468 g/kg NSK met Met, Lis, Arg en Tre teen 2.08, 5.49, 7.47, 4.80 g/kg onderskeidelik. ‘n Derde voer, LP, het gedien as ‘n positiewe kontrole en was ‘n lae proteïen voer met laer spesifikasies (139 g/kg RP, 455 g/kg NSK) waarin die aminosure ook nie geoptimeer is nie. Die LP voer het Met, Lis, Arg en Tre bevat teen 1.93, 4.99, 6.66 en 4.73 g/kg onderskeidelik. Die negatiewe kontrole behandeling, CON, is verteenwoordig deur lammers wat op kikuyu gewei het terwyl addisionele supplementêre voeding teen 500g/lam/dag voorsien is. Hierdie supplementêre voeding het gedien as ‘n produksie lek om sodoende vergelykbaar te wees met scenario waar lammers op weiding afgerond word. Veertig kruisgeteelde Merino x Döhne-Merino lammers met ‘n gemiddelde gewig van 24.35 ± 0.648 kg is vir 57 dae in ‘n voerkraal afgerond waarna hulle, teen ‘n gemiddelde gewig van 41.41 ± 1.259 kg, geslag is. Tydens die voerkraal proef is prestasie gemonitor deur die meet van daaglikse groei en voerinname. Hiertydens is daar ook ‘n in vivo verteringsproef op die OP en FIN lammers gedoen. Tydens die slagproses is die rumen verwyder waarna ‘n monster van die rumenwand langs die rumino retikulêre vou geneem is. Hierdie rumenmonsters is op skyfies geplaas sodat die ontwikkeling van die rumen ondersoek kan word. Die M. longissimus dorsi was aan beide kante van die karkas tussen die 2de en 3de laaste torakale werwels en die 4de en 5de lumbale werwels verwyder. Daar was geen betekenisvolle verskille (P > 0.05) tussen enige van die konsentraat behandelings ten opsigte van gemiddelde daaglikse toename (GDT), voeromset verhouding (VOV) of uitslag persentasie nie. Die CON lammers het egter, soos verwag, beduidend (P < 0.05) stadiger gegroei en het dus verskille getoon ten opsigte van GDT. Die uitslag persentasie van 45.96 % ± 0.711 vir die CON lammers het ook betekenisvol verskil (P < 0.05) van die OP (51.44 % ± 0.358), FIN (52.72 % ± 0.653) en LP (51.74 % ± 0.611) behandelings. Binne die voerkraal behandelings was daar geen betekenisvolle verskille (P > 0.05) ten opsigte van die papillae lengte en rumenwand dikte nie, alhoewel daar ‘n numeriese toename in papillae lengte was soos die NSK vlakke in die voer gestyg het. Die CON lammers het wel van die OP lammers verskil (P < 0.05) ten opsigte van papillae lengte. Hierdie bevinding het bevestig hoe belangrik NSK is in die inisiasie en instandhouding van die morfologiese ontwikkeling van die rumen. Die in vivo verterings studie het daarop gedui dat die kommersiële afrond voer net so effektief soos die geoptimeerde voer was in terme van stikstof retensie asook die handhawing van ‘n geskikte energie balans. Alhoewel die in vivo verteerbaarheid van Met en Lis in die OP hoër was (P < 0.05) as in die FIN voer, het hierdie verskille nie gelei tot beter groei in die OP lammers nie. Hierdie verskil in verteerbaarheid is toegeskryf aan die feit dat die aminosure in die OP voer meer rumen-beskermd was as dié in die FIN voer. Die in vitro verteringstudie het daarop gedui dat die OP voer beduidend beter (P < 0.05) verteer is as die FIN voer.

Thesis (PhD (Chemistry and Polymer Science)--University of Stellenbosch, 2006. / Synthetic peptides derived from the active core of a natural antimicrobial peptide were used as a template for the design of novel bolaamphiphilic peptides and hybrid molecules. The amphiphilic character of the original compounds was modified by using non-natural amino acids (AAs) – such as ω-AA – and varying the hydrophobic content. The outcomes of these modifications were studied focusing on structural and biological properties. Because of the bolaamphiphilic character, the alternation of polar and non-polar AAs and the use of hydrophobic AAs such as tyrosine and leucine, these novel molecules were designed to undergo self-assembly in response to certain stimuli (e.g. a pH increase). This significant property was investigated by means of different tools, such as fluorescence measurements, electron microscopy (EM), Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). By using fluorescence it was possible to determine the critical aggregation concentration (CAC) of the new compounds. Differences in amino acid composition, which were reflected into diverse secondary structures and hydrophobicity (H), resulted in different CAC values and aggregation profiles. The data were consistent with the literature and showed that (i) the aggregation of these basic compounds was triggered by a pH increase, (ii) the use of hydrophobic AA highly augmented the self-assembly tendency while (iii) the presence of proline strongly reduced it. EM revealed the morphology of the peptide assemblies: microtubes and microvesicles were identified and characterised by dimensions of 500 nm to 2 μm. The presence of 3-way junctions and vesicles budding out of the microtubes demonstrated that the self-assembly is a dynamic process. The aggregation was confirmed by FT-IR spectroscopy, by studying the dried peptide assemblies and the significant spectral signs the process left, especially in the amide II envelope. The relationship between hydrophobicity and self-assembly was expanded by experimentally and theoretically determining the hydrophobic content of the novel bolaamphiphiles. Data from liquid chromatography and computational calculations (two common ways used to determine the hydrophobicity of a given molecule) correlated well with the tendency to self-assemble, as expressed by CAC values. Importantly, some structural parameters (such as the presence of β-turn induced by proline) also showed significant influence on the aggregation, highly limiting the role of the peptides’ hydrophobicity. These novel peptide bolaamphiphiles displayed a very low haemolytic action and retained some antimicrobial activity at high concentrations against both Gram-positive and -negative bacteria. Unfortunately, the activity was greatly reduced at low concentrations, as clearly demonstrated by the use of two antimicrobial tests. The inability to provoke cell lysis was also evident when using liposomes mimicking a negative bacterial membrane. The loss of activity is possibly related to the modifications of the three-dimensional structure caused by the use of ω-AA and proline, which strongly alter the secondary structure. The results of this study were valuable in terms of understanding the relationships between self-assembly and structural parameters, such as AA compositions, hydrophobicity and secondary structure. Possible applications of the synthesised compounds were however limited as a result of the loss of the biological activity at low concentrations.