Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Key research areas in modern enology are related to the production of wine of consistent quality, as well as to the improvement of existing wine quality through the enhancement of the sensory properties of wine. The formation of glycerol during alcoholic fermentation is highly relevant to both these issues. Since the early years of the zo" century, glycerol has been positively associated with the quality of wine and it is thought to impart important mouth-feel sensations such as "viscosity", "smoothness" and "body". In general, it is considered that glycerol concentrations higher than those normally found in wine, can contribute towards the improvement of wine quality. It has also been suggested that increased concentrations of glycerol can enhance the aroma of wine. On the basis of these perceptions, several strategies have been developed to favour the production of glycerol during the fermentation process and over a period of years, a large volume of data has been collected that relates to various aspects regarding glycerol production during alcoholic fermentation. To date, however, several aspects regarding the relationship between glycerol and wine quality remain unclear. The reasons for this situation can mainly be ascribed to the lack of reliable analytical data to serve as a basis for investigating the relationship between glycerol and wine quality, as well as the preponderance of empirical and anecdotal evidence. Despite numerous opinions regarding optimal glycerol concentrations in wine, glycerol is indeed seldom assayed on a routine basis and targets with respect to specific wine grape cultivars and glycerol concentrations have largely remained unspecified. To date, very little information regarding glycerol concentrations in South African wines has been published. The analytical techniques that are most frequently used for the quantification of glycerol in grape juice, fermenting must and wine are not easily automated and this aspect placed severe limitations on the generation of large volumes of analytical data on glycerol concentrations in these matrices. This project was undertaken with the aim to holistically address some of the unresolved issues relating to the relationship between glycerol and wine quality. This also implied the development and optimisation of analytical techniques suitable for the rapid and accurate determination of glycerol in fermentation media, as well as in finished wine. In the first stage of this project a quantitative database was established that contained the analytical data on the glycerol concentrations of a statistically significant number of wines of adjudged quality, as well as additional information for each wine regarding the geographic origin, vintage, routine chemical analyses and the yeast strain(s) used for the production of the wine. The relevance of glycerol in wine for the modern South African winemaker was evaluated through the establishment of a quantitative database that contained the opinions of an expert panel of 15 South African winemakers, enologists and wine chemists on topics relating to glycerol in wine. In the second stage of the project the data captured in the databases were used to investigate aspects regarding the relationship between glycerol and wine quality. From the data captured in the qualitative database, it was clear that the topic regarding glycerol in wine was important to the South African winemakers and it was also evident that there was a need for the development and optimisation of methods suitable for the routine analysis of the glycerol concentrations in grape juice, fermenting must and wine. The opinions of the panel members also highlighted the issue that the mouth-feel property of wine was considered to be an area where the quality of some wines could be further improved. The quantitative database contained the information on the glycerol concentrations of 450 commercial South African table wines of adjudged quality. The premium cultivars Chardonnay, Chenin blanc, Sauvignon blanc, Cabernet Sauvignon, Merlot, Shiraz and Pinotage were used for the purpose of investigating the relationship between glycerol concentration and wine quality. The wines represented a wide variety of wine styles, including dry white, off-dry white, dry red and late harvest wines. The average glycerol concentration was significantly associated with the wine style. In white wines the average glycerol concentration was much lower than in the red wines (6.82 gIL versus 10.49 gIL, respectively). No significant relationship between the final glycerol concentration and the geographic origin, vintage and the yeast strain used for the fermentation was found. Wine quality could not be significantly associated with glycerol concentration in the red wines. In the white wines, the relationship between glycerol concentration and wine quality was significant, but due to the very small differences in the average glycerol concentrations of the wines of different quality ratings, the statistical significance is probably of little practical value. The effect of glycerol on the volatility of a selection of esters and higher alcohols was also investigated. Solid-phase microextraction, followed by gas chromatography, was used to analyse the composition of the headspace at equilibrium between the liquid phase and the gas phase of a model wine, and a dry white wine that contained a basal concentration of 5.4 gIL glycerol. Results showed that incremental increases in the glycerol concentrations over a range of 1 -10 gIL in the model wine and in a Chenin blanc wine, were not accompanied by a proportional increase or decrease in the abundance of the aroma components in the headspace. The volatile components tested were isoamyl acetate, ethyl butyrate, ethyl valerate, ethyl lactate, ethyl hexanoate, hexyl acetate, isoamyl alcohol and isobutanol. For all the volatile components tested, the difference between the headspace composition of samples containing the lowest glycerol concentration, and those containing the highest glycerol concentration, was not significant. However, sufficient experimental evidence was obtained to indicate that increasing glycerol concentrations had an effect on the volatility of aroma components, and that the effect is of a complex and non-linear nature. In the third phase of the work Fourier transform infrared spectroscopy (FT-IR) was used to establish and optimise methods for the accurate and rapid quantification of glycerol in wine. For this purpose calibrations were developed for the quantification of glycerol in dry wine and late harvest wines. The accuracy of prediction was evaluated by means of the standard error of prediction that was 0.38 gIL for the dry wines and 0.65 gIL for the sweet wines. Large variations are introduced in the FT-IR spectra of wine by factors such as process technology, cultivar and geographic origin, and this variation can have an effect on the accuracy of the analytical data generated when employing FT-IR spectroscopy. Using glycerol prediction in wine as a model system, principal component analysis of the FT-IR spectra was done in order to establish quality control measures for the detection of poorly predicted, or outlier samples. A classification model, based on principal component analysis, was established that enabled the interpretation and classification of the outlier samples in the data set in 100% of the cases tested. This work forms the basis for expanding the quality control measures for the detection of wines of which the FT-IR spectra are highly unnatural, as well as for establishing quality control measures to ensure that accurate analytical data are generated when FT-IR is used. FT-IR spectroscopy was also used to develop a rapid screen for the evaluation of the fermentation profiles of wine yeasts. For this purpose, a selection of wine yeasts, which included commercial wine yeasts frequently used in winemaking in South Africa, as well as a selection of hybrid Saccharomyces cerevisiae yeasts, that were obtained through a selective breeding strategy aimed at increasing glycerol concentrations were used. Calibrations necessary for the accurate quantification of glycerol, volatile acidity, ethanol, reducing sugar and glucose, in Chenin blanc must and a synthetic must were developed and optimised. This work forms the basis upon which the scope of the analysis, both in terms of the number of components that can be measured, as well as the medium in which the yeasts are being evaluated, can be enlarged. This would be valuable for future applications in both the research as well as the industrial environment. The method that was developed serves to illustrate how this application can play a supportive role in yeast development programmes, through the speeding up of the initial stages of yeast strain evaluation. / AFRIKAANSE OPSOMMING: Navorsing in moderne wynkunde is sterk gefokus op die produksie van wyn waarvan die kwaliteit van In volhoubare goeie peil is, sowel as die verdere verbetering van bestaande wynkwaliteit, deur In verhoging van die sensoriese eienskappe van wyn. Die vorming van gliserol tydens alkoholiese fermentasie het betrekking op beide hierdie aspekte. In Opvatting wat wyd gehuldig word en wat reeds sedert die vroeë jare van die 20e eeu geld, impliseer dat gliserol In positiewe bydrae lewer tot wynkwaliteit. Oor die algemeen word dit beskou dat gliserolvlakke hoër as wat normaalweg in wyn aangetref word, kan bydra om die kwaliteit van die wyn nog verder te vehoog. Daar is ook spekulasie dat verhoogde gliserolvlakke in wyn die intensiteit van die aroma van wyn kan verhoog. Hierdie opvattings het tot gevolg gehad dat veskeie strategieë ontwikkel is om die gliserol vlakke wat tydens die fermentasieproses gevorm word, te verhoog. Baie min inligting oor die gliserolinhoud van Suid-Afrikaanse wyn is tot onlangs gepubliseer. Ten spyte van die opvattings oor gliserol en wynkwaliteit wat reeds oor In lang tydperk gehuldig word, bly sekere aspekte van die verwantskap tussen gliserol en wynkwaliteit nog steeds onduidelik. Redes vir hierdie situasie kan hoofsaaklik toegeskryf word aan die totale afwesigheid van betroubare en substansiële eksperimentele data wat as basis kan dien vir die evaluering van die algemene opvattings aangaande die verwantskap tussen gliserol en wynkwaliteit. Die inligting wat wel beskikbaar is, is verder ook oorwegend van In empiriese aard. Huidig word gliserol selde op In roetine basis in die analitiese laboratorium bepaal, hoofsaaklik omdat die bestaande metodes tydrowend is en nie maklik geoutomatiseer kan word nie. In hierdie studie is In kwantitatiewe databasis opgestel waarin die inligting ten opsigte van die gliserolvlakke van 450 kommersiële Suid-Afrikaanse tafelwyne waarvan die kwaliteit beoordeel is, vervat is. Die kultivars Chardonnay, Chenin blanc, Sauvignon blanc, Cabernet Sauvignon, Merlot, Shiraz en Pinotage is gebruik vir hierdie doel en die seleksie van wyne was verteenwoordigend van In wye verskeidenheid wynstyle, insluitende droë wit-, halfdroë wit-, droë rooi- en laatoeswyne. Die gemiddelde gliserolvlakke in die witwyne was heelwat laer as die gemidelde gliserolvlakke in die droë rooiwyne (6.82 gIL teenoor 10.49 gIL, onderskeidelik). Geen beduidende verwantskap kon aangetoon word tussen die gliserolkonsentrasie in die wyn en die geografiese oorsprong, oesjaar, en die gisras wat gebruik is in die produksie van die wyn nie. Die kwaliteit van rooiwyn kon nie beduidend met die gliserolkonsentrasie geassosieer word nie. In die geval van wit wyn was die verwantskap statisties beduidend, maar die verskille was klein en moontlik nie van veel praktiese waarde nie. Die aktualiteit van gliserol vir die moderne Suid-Afrikaanse wynmaker is geëvalueer op grond van die opinies van In paneel van 15 Suid-Afrikaanse kundiges, rakende aspekte wat verband hou met die algemene opvattings oor gliserol en wyn. Die paneel het bestaan uit wynmakers, wynkundiges en chemici. Die opinie van die panel is deur middel van "n vraelys bekom en is vervat in "n kwalitatiewe databasis. Die resultate van hierdie menigspeiling het getoon dat gliserol wel vir die Suid- Afrikaanse wynmakers belangrik is en dit het ook die behoefte uitgewys vir die ontwikkeling en optimisering van metodes wat geskik is vir die roetine analyses van gliserol in wyn. Die invloed van gliserol op die vlugtigheid van "n seleksie van hoëralkohole en esters is ondersoek in "n model wyn sowel as "n wit wyn, waarvan die basiese gliserolkonsentrasie 5.4 gIL was. Soliede-fase mikroekstraksie van die gasfase van wyn is opgevolg met "n gaschromatografiese analise. Resultate het getoon dat "n stapsgewyse toename in die gliserol konsentrasie, oor "n konsentrasie reeks van 0 - 10 gIL in die model wyn, en 0 - 15 gIL in die wit wyn, nie gepaard gegaan het met "n reglynige toename of afname in die konsentrasie van die aromakomponente nie. Vir al die komponente wat ondersoek is, was die samestelling van die gasfase in monsters wat die laagste gliserol konsentrasie gehad het, teenoor dié wat die hoogste gliserol konsentrasie gehad het, nie beduidend nie. Nietemin het die resultate getoon dat gliserol wel "n effek het op die vlugtigheid van die aroma komponente wat in hierdie ondersoek gebruik is, maar dat die aard van die effek kompleks en nie-liniê is. Fourier-transformasie-infrarooispektroskopie (FT-IR) is gebruik om die metodes vir die analise van gliserol in wyn sodanig te optimiser, dat vinnige en akkurate bepalings op "n roetine basis in wyn gedoen kan word. Kalibrasies is ontwikkel vir die kwantifisering van gliserol in droëwyn en laatoeswyn. Die standaard voorspellingsfout van die kalibrasies wat ontwikkel is, was 0.38 gIL in droëwyn en 0.65 gIL in die geval van laatoeswyn. Inherente variasie in die FT-IR-spektra word deur eksterne faktore soos die proses tegnologie wat gebruik is om die wyn te berei, die kultivar en geografiese oorsprong van die wyn veroorsaak en hierdie variasie kan "n effek hê op die voorspelingsakkuraatheid van FT-IR spektroskopie. Om die effek van hierdie variasie op die akkuraarheid van die analitiese data te evalueer, is die voorspelling van gliserol as modelsisteem gebruik. Hoofkomponentanalise van die FT-IR spektra is gedoen om dié wyne met onnatuurlike FT-IR-spektra te identifiseer, ten einde beheer oor die akkuraatheid van die analitiese data uit te oefen. "n Verdere model wat ook gebaseer is op hoofkomponentanalise van die FT-IR-wynspektra is ontwikkel vir die interpretasie en klassifikasie van wynmonsters met onnatuurlike FT-IRspektra. Met die model wat ontwikkel is kon onnatuurlike wynmonsters met "n akkuraatheid van 100% gëidentifiseer word. Hierdie werk vorm die basis waarop kwaliteitsbeheer verder uitgebrei kan word sodat wyne met onnatuurlike spektra gëidentifiseer kan word, sowel as om te verseker dat akkurate data gegenereer word as FT-IR as analitiese metode gebruik word vir die kwantifisering van gliserol in wyn. FT-IR spektroskopie is ook gebruik om "n vinnige skandeerproses te ontwikkel waarmee die fermentasieprofiele van wyngiste geëvalueer kan word. Die seleksie giste wat vir hierdie doel gebruik is, sluit kommersiële Suid-Afrikaanse wyngiste in, sowel as hibriede Saccharomyces cerevisia-giste wat normaalweg hoër vlakke van gliserol tydens die fermentasieproses produseer. Kalibrasies is ontwikkel vir die akkurate kwantifisering van gliserol, vlugtige suur, alkohol, reduserende suiker en glukose, in Chenin blanc-mos sowel as in 'n sintetiese medium. Hierdie werk vorm die basis waarop verdere uitbreidings gedoen kan word, sodat meer metaboliete gemeet kan word en 'n groter verskeidenheid van fermentasiemedia gebruik kan word. Hierdie ontwikkeling is waardevol vir toekomstige toepassings in die navorsings- sowel as die industriële omgewing. Die metode wat ontwikkel is illustreer ook hoe hierdie toepassing 'n ondersteunende rol kan speel in wyngisontwikkelingsprogramme deur die aanvanklike evalueringsproses van die giste te versnel.

Orientador: Jane Maria Bertocco Ezequiel / Banca: Mauro Dal Secco de Oliveira / Banca: Cyntia Ludovico Martins / Resumo: A utilização de resíduos agroindustriais e aditivos alimentares na dieta de bovinos de corte tem crescido nos últimos anos em decorrência da ação positiva destes na fermentação ruminal, principalmente com o intuito de melhorar a eficiência alimentar e reduzir os custos da produção. Neste sentido, este trabalho foi realizado com o objetivo de avaliar a substituição parcial do milho (50% da MS na dieta total) e da casca de soja (13,27% da MS na dieta total) pelo coproduto do processamento do biodiesel (glicerina bruta) e sua associação aos aditivos óleos essenciais (Essential®) ou a monensina sódica (Rumenpac®) na dieta de bovinos de corte. Foram utilizados cinco bovinos machos da raça Nelore, castrados, com 36 meses de idade, peso médio de 500 kg e providos de cânulas permanentes no rúmen. Dietas experimentais semelhantes nos teores de PB e EM foram fornecidas para os animais na relação volumoso:concentrado de 30:70, e denominadas: CONT - Tratamento controle (sem glicerina bruta ou aditivos); OE - Tratamento óleos essenciais (adição do produto Essential®, sem glicerina bruta); MON - Tratamento monensina sódica (adição do produto Rumenpac®, sem glicerina bruta); OEG - Tratamento óleos essenciais com introdução de glicerina bruta; MONG - Tratamento monensina sódica com introdução de glicerina bruta. A introdução de glicerina bruta nos tratamentos OEG e MONG foi de 200g/kg MS, e as inclusões de monensina sódica e óleos essenciais foram respectivamente de 0,03g/kg e 0,5g/kg de MS da dieta total. Avaliou-se o consumo e digestibilidade in vitro da MS e nutrientes das dietas, os parâmetros ruminais (pH e nitrogênio amoniacal), a produção e qualidade da massa microbiana e a produção de gases metano e carbônico das dietas. Os animais foram distribuídos em delineamento quadrado latino (5 x 5), em esquema fatorial 2 × 2 + 1 (monensina sódica ou óleos essenciais × ausência ou presença de ... / Abstract: The use of agroindustrial residues and food additives in the diet of beef cattle has increased in recent years due to positive action of these on ruminal fermentation, mainly with the aim of improve feed efficiency and reduction of production costs. Thus, this work was carried to evaluate the partial substitution of corn (50% DM in the total diet) and soybean hulls (13.27% DM in the total diet) by biodiesel coproduct (crude glycerin) and their association with essential oils (Essential ®) or sodium monensin (Rumenpac ®) in the diet of beef cattle. Were utilized five Nellore steers with 36 months of age, average weight of 500 kg and provided with permanent rumen cannulas. Similar experimental diets on the CP and EM were given for the animals in forage to concentrate ratio of 30:70, and denominated: CONT - Treatment control (no additives or crude glycerin), OE - Treatment Essential Oils (Addition of Essential® without crude glycerin); MON - Treatment sodium monensin (addition of Rumenpac® without crude glycerin); OEG - Treatment essential oils with introduction of crude glycerin; MONG - Treatment sodium monensin with introduction of crude glycerin. The introduction of the crude glycerine on the treatments OEG and MONG was 200g/kg MS, and the inclusions of sodium monensin and essential oils were respectively 0.03 g / kg and 0.5 g / kg DM in the diet. It was evaluated the intake and digestibility in vitro DM and nutrients in the diets, ruminal parameters (pH and ammonia nitrogen), and the production and quality of microbial mass, production of methane and carbon of the diets. The animals were distributed in Latin square design (5 x 5) in a factorial 2 × 2 + 1 (sodium monensin or essential oil vs absence or presence of crude glycerin + control). The data were analyzed using PROC MIXED of SAS version 9.2, a 5% probability. The inclusion of crude glycerin treatments (OEG and MONG) resulted in reduced intake of DM, OM, NDF, EE and STARCH ... / Mestre

Orientador: Telma Teresinha Berchielli / Coorientador: Giovani Fiorentini / Banca: Saulo da Luz e Silva / Banca: Otavio Rodrigues Machado Neto / Banca: Paulo Henrique Moura Dian / Banca: Juliana Duarte Messana / Resumo: O glicerol é um substrato utilizado por bactérias que metabolizam o lactato ruminal e a virginiamicina é um antibiótico não ionóforo que inibe o crescimento de bactérias gram-positivas produtoras de lactato. Foram realizados dois experimentos para avaliar os efeitos da glicerina bruta (GB) e da virginiamicina (VM) na ingestão, digestibilidade, fermentação ruminal, população microbiana, desempenho, características de carcaça e perfil de ácidos graxos da carne de bovinos Nelore. Os tratamentos experimentais foram organizados em um arranjo fatorial 2 × 2: dietas sem virginiamicina (VM-) ou 25 mg de virginiamicina/kg de matéria seca (VM+) combinadas com dietas sem glicerina bruta (GB-) ou 100 g de glicerina bruta/kg de matéria seca (GB+). O bagaço de cana-de-açúcar foi usado como forragem na proporção de 20% na matéria seca (MS) da dieta e a GB substituiu o milho na formulação da dieta. No primeiro experimento, foram utilizados oito bovinos Nelore fistulados no rúmen (Peso corporal = 600 ± 34 kg, 26 ± 3 meses) em um quadrado latino 4×4 replicado (período= 21 dias) para se avaliar a digestibilidade dos nutrientes, fermentação ruminal e população microbiana. A ingestão de MS teve uma tendência a aumentar em dietas com GB (P = 0,07). As digestibilidades aparentes totais dos nutrientes foram semelhantes entre as dietas (P ≥ 0,10). As dietas com GB ou VM apresentaram valores de pH similares (média = 6,15; P ≥ 0,10). A proporção de propionato aumentou 27,5% nas dietas com GB+, independ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glycerol is a substrate used for bacteria that metabolize ruminal lactate and virginiamicyn is a non-ionophore antibiotic that inhibits the growth of gram-positive lactate-producing bacteria. Two experiments were conducted to evaluate the effects of crude glycerin (CG) combined with virginiamycin (VM) on intake, digestibility, ruminal fermentation, microbial population, performance, carcass traits and fatty acid profile of meat from feedlot Nellore cattle. Treatments were arranged in 2 × 2 factorial design: diets without virginiamycin (VM-) or virginiamycin at 25 mg/kg DM (VM+) combined with diets without crude glycerin (CG-) or CG (80% glycerol) at 100 g/kg DM (CG+). The sugar cane bagasse was used as the exclusive roughage in the proportion of 20% in the dry matter (DM) of diet and crude glycerin replaced corn in the diet formulation. In the first experiment, eight rumen fistulated bulls (BW= 600 ± 34 kg; 26 ± 3 months) were used in a replicated 4 × 4 Latin square (21-d periods) to evaluate the digestibility, ruminal fermentation and microbial population. The intake of DM had a tendency to be greater in CG+ than CG- diets (P = 0.07). Apparent total tract digestibilities of nutrients were similar among diets (P ≥ 0.10). Diets with CG or VM had similar values of pH (mean=6.15; P ≥ 0.10). The proportion of propionate increased 27.5% in CG+ diets, regardless of VM inclusion (P = 0.01). In the second experiment, forty-eight bulls with initial BW (408.4 ± 22.2 kg; 21 ± 2 months) ... (Complete abstract click electronic access below) / Doutor

Orientador: Jane Maria Bertocco Ezequiel / Banca: Mauro Dal Secco de Oliveira / Banca: Márcia Helena Machado da Rocha Fernandes / Banca: Viviane Correa Santos / Banca: Rosemary Lais Galati / Resumo: O objetivo deste trabalho foi avaliar os efeitos do uso da glicerina bruta (GB) em até 30% de inclusão na MS em dietas de alto concentrado e baixo amido, substituindo o milho total da dieta de cordeiros em terminação sobre o consumo, digestibilidade, comportamento ingestivo, parâmetros sanguíneos, desempenho, características da carcaça, qualidade e perfil de ácidos graxos da carne. Utilizaramse 40 animais com peso inicial de 23,5 ± 1,35 kg que foram distribuídos em delineamento em blocos casualizados com cinco tratamentos. Os tratamentos foram: sem inclusão da GB; inclusão de 7,5% de GB; inclusão de 15% de GB; inclusão de 22,5% de GB e inclusão de 30% de GB na MS. O volumoso utilizado foi o feno de Tifton 85 na proporção de 18,19% e o concentrado (81,81%) foi composto por milho, GB, glutenose, óleo de milho, ureia, farelo de girassol, casca de soja e sal mineral. O experimento teve duração média de 72 ± 7 dias e os animais foram abatidos com aproximadamente 38 kg. Os dados foram analisados como modelos mistos, utilizando-se o procedimento MIXED do pacote estatístico SAS. Os efeitos dos tratamentos foram decompostos em contrastes linear, quadrático, bem como o efeito da dieta com 0% de GB versus a média de todos os tratamentos que continham GB. Os contrastes foram considerados significativos quando P ≤ 0,05 e tendência quando P < 0,10. O consumo da MS e dos nutrientes não foi afetado pela GB (P > 0,10), exceto o consumo de FDN que diminui linearmente no ensaio de comportamento e desempenho com o aumento da GB (P = 0,01). A digestibilidade da MS, MO e PB apresentou comportamento linear crescente a medida que a glicerina foi adicionada e a digestibilidade do EE apresentou comportamento quadrático (P = 0,01). Na avaliação de comportamento, o tempo despendido para ruminação e ingestão+ruminação em min/g de FDN aumentou linearmente com inclusão da GB (P ≤ 0,02). A glicose, insulina e o desempenho dos animais ... / Abstract: This study aimed to evaluate the effects of crude glycerin (GB) inclusion until 30% of DM in diets with high level of concentrated and low starch, replacing total maize of lambs feedlot diet on intake, digestibility, feeding behavior, blood parameters, performance, carcass characteristics, quality and fatty acid profile of meat. Forty animals with 23.5 ± 1.35 kg initial body weight were distributed in a randomized block design with five treatments. The treatments were: no inclusion of GB; inclusion of 7.5% of GB; inclusion of 15% GB; inclusion of 22.5% and inclusion of 30% GB on DM basis. The roughage used was Tifton 85 hay at 18.19% and the concentrate (81.81%) was composed of corn, GB, corn gluten meal, corn oil, urea, sunflower meal, soybean hulls and mineral salt. Duration of experiment was on average 72 ± 7 days, and the animals were slaughtered around 38 kg of body weight. The procedure used for data analysis was the SAS program and the treatment effects were decomposed into linear, quadratic contrasts, as well as the effect of diet with 0% vs. GB (average of all treatments containing GB). The contrasts were considered significant when P ≤ 0.05 and trend when P> 0.10. The DM and nutrientes intake were not affected by GB (P> 0.10) except for NDF intake that decreases linearly in feeding behavior and performance with increasing GB (P = 0.01). The DM, OM and CP increased linearly with inclusion of GB and EE digestibility showed a quadratic behavior (P = 0.01). In the feeding behavior trail, time spent for rumination and intake+rumination on min/g NDF increased linearly with inclusion of GB (P ≤ 0.02). Blood parameters and animals performance had no significant differences (P ≥ 0.11). The weight and yield of hot and cold carcass linearly decreased with the inclusion of crude glycerin (P ≤ 0.02). The AOL, EGS, weight and yield of omental, mesenteric and renal fat had no differences with the inclusion of GB (P ≥ 0.14). The ... / Doutor

Thesis (Ph. D.)--Institute of Paper Chemistry, 1948. / Bibliography: leaves 84-86.

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (M. Sc.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references (leaves 76-78). Also available via the World Wide Web.

Techn. Hochsch., Diss., 2003--Aachen.

Orientador: Telma Teresinha Berchielli / Banca: Ana Claudia Ruggieri / Banca: Juliana Duarte Messana / Banca: André Soares de Oliveira / Banca: Mateus Pies Gionbelli / Resumo: Foram realizados quatro experimentos distribuídos durante a época de seca e águas, com o objetivo de avaliar a inclusão de glicerina bruta (80% de glicerol) no suplemento de bovinos Nelore criados em pastagem tropical, sobre o consumo, digestibilidade, fermentação ruminal, perfil de microrganismo no rumem, desempenho, emissões de metano, qualidade de carne e características de carcaça. Os tratamentos consistiram de suplementos com níveis crescentes (0, 70, 140, 210, e 280 g/kg MS do suplemento) de glicerina bruta, fornecidos aos animais em uma relação de 700 g/100kg de peso corporal da época da seca, e 300 g/100kg de peso corporal durante a época das águas. Na estação da seca, cinquenta tourinhos Nelore (279.52 ± 16.31 kg peso corporal inicial) foram usados para avaliação de performance animal, e dez novilhos Nelore canulados (408.8 ± 38.5 kg) foram usados para investigar a digestibilidade, fermentação ruminal e perfil de microrganismos do rumem. Na estação das águas, os experimentos foram repetidos com os mesmos animais utilizados na fase anterior. Inclusão de glicerina bruta no suplemento de bovinos Nelore castrados, criados a pasto na época da seca, não afeta o consumo e digestibilidade aparente. No entanto, altera a fermentação ruminal, aumentando a proporção de butirato e valerato enquanto reduz acetato e ácidos graxos voláteis totais, sem apresentar efeito negativo sobre a população relativa de bactéria celulolíticas e protozoários no rúmen. Contudo, a inclusão de glicerina até o nível de 28% da matéria seca do suplemento de tourinhos Nelore em crescimento criados em pastagens tropicais na época da seca, melhora o ganho de peso e a eficiência alimentar. A inclusão de glicerina bruta até a concentração de 28% da matéria seca do suplemento não altera consumo, as características de carcaça e qualidade de carne, e emissão de metano. No entanto, promove ... / Abstract: Four experiments were conducted during the dry and rainy season, in order to assess the increasing concentrations of crude glycerin (80% glycerol) in the supplement of young Nellore grazing tropical grass, on intake, digestibility, ruminal fermentation, rumen microorganism profile, performance, methane emission, and carcass and meat quality traits. The treatment consist of supplements with increasing concentrations (0, 70, 140, 210, and 280 g/kg DM basis of supplement) of crude glycerin, fed to the animals in a ratio of 700 g/ 100kg of body weight in the dry season and 300 g/100kg of body weight in the rainy season. In the dry season, fifty young Nellore bulls (279.52 ± 16.31 kg initial body weight) were used for animal performance evaluation, and ten ruminal cannulated Nellore steers (408.8 ± 38.5 kg) were used to investigate the digestibility, ruminal fermentation and rumen microorganism profile, in the two experimental phases. In the rainy season, the experiments were replicated with the same animals used in the previous phase. Inclusion of crude glycerin in the supplement of young Nellore steers grazing tropical grass in the dry season, does not affect intake and apparent total tract digestibility. However, alters rumen fermentation whereas increases butyrate and valerate while reducing acetate and total VFA, showed no negative effect on relative proportion of cellulolytic bacteria and protozoa population. Nevertheless, inclusion concentration of glycerin at up to 28% DM in the supplement of growing Nellore bulls raising tropical grass in the dry season, improved BW gain and feed efficiency. Inclusion of crude glycerin up to the level of 28% of dry matter in the supplement does not alter the carcass characteristics, the meat quality and methane emission. However, glycerin supplementation promotes additional daily gain with potential ... / Doutor