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Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Grape and wine phenols consist of flavanols which is the building blocks for tannins. These building blocks are called monomers which consist of catechins, epicatechins, epigallocatechins and epicatechin-gallate. Tannin is important in wine as it contributes to bitterness, mouth feel (astringency) and maturation potential of the wine. Futhermore it has a health benefit as an antioxidant. Anthocyanins are responsible for the colour of red wine. The anthocyanins combine with tannins to form stable polymeric pigments. Due to the importance of tannins and anthocyanins in wine, it is imperitative that different winemaking techniques are used to extract as much of these components as possible and that the analysis is done quickly and accurately. The aim of this study was to evaluate different winemaking techniques and their extraction of tannins and anthocyanins into the wine. Too much tannin extraction can have a negative effect on the sensory quality of the wine. Therefore a second aim was to evaluate the mouth feel properties of a Shiraz wine. A third aim was to compare the two tannin precipitation methods in terms of time efficiency, repeatability and the ease of practice. To investigate the amount of tannin concentration extracted by different winemaking techniques, two cultivars (Cabernet Sauvignon and Shiraz) were used. These treatments included the addition of an enzyme during fermentation [E], cold maceration [CM], post maceration [PM] and the combination of cold and post maceration [CM+PM]. The grapes were harvested in two different climatic areas during the 2008 and 2009 vintages. The two climatic areas were classified according to the Winkler scale as a III (Morgenster) and a IV (Plaisir de Merle). The grapes were harvested at two different ripeness levels in order to evaluate the effect of the different winemaking processes on the extraction of tannins and anthocyanins. One harvest was before (LB) and the other after (HB) the commercial harvest. The results of this study showed significant differences in the phenolic composition of the wines. It was found that the warmer area showed higher tannin concentrations than the cooler area for both cultivars. In the 2008 Cabernet Sauvignon the CM extracted higher concentrations of tannin from the cooler area at both ripeness levels. In the warmer area, CM extracted the highest tannin concentration HB, but the CM+PM extracted the highest tannin concentration from Cabernet Sauvignon at the LB and CM at the HB of the warmer area. In 2009 the PM extracted the highest concentration of tannin at the lower ripeness level, while the E treatment extracted the highest concentration from the warmer area. In the cooler area the CM+PM extracted the highest concentration of tannin at a lower ripeness level, while there were no siginicant differences between the different treatments at the higher ripeness level. The highest anthocyanin concentration was found in the cooler area. The CM treatment was found to have no effect on anthocyanin extraction. Different methods are available to quantify the tannin concentration in wine. Two of the most popular tannin analytical methods are the bovine serum albumin (BSA) and the methyl cellulose precipitable tannin (MCP) methods. The BSA method is a very complex method which uses at least 3 times more reagents than the MCP method. The MCP method only analyzes tannins, while the BSA method analyzes tannins, monomeric pigments (MP), small polymeric pigments (SPP) and large polymeric pigments (LPP). In this study a good correlation was found between the two tannin precipitation methods (R2 – 0.88). There is controversy regarding the variability of these methods. Some scientists found that the two methods show a good correlation with HPLC, while others found that there was no such correlation between the precipitation methods and the HPLC. The MCP method had a practical advantage as it could be performed in half the time required for the BSA method. This has a significant impact in scenarios where a high sample throughput is required although it only measures total tannin. The phenolic composition and mouth feel of the wine was strongly influenced by the climatic area. In the warmer area the effect of tannin concentration on mouth feel was much less than in the cooler area. The wine made of riper grapes, was more grippy, bitter and numbing than the wines made from greener grapes. The E treatment was especially associated with a dry, grippy sensation. / AFRIKAANSE OPSOMMING: Druif en wyn fenole bestaan uit flavanole wat weer die boublokke is van tanniene. Hierdie boublokke, wat bekend staan as monomere, betsaan uit katesjiene, epikatesjiene, epigallokatesjiene an epikatesjien-gallaat. Tanniene is belangrik in wyn aangesien dit bydra tot bitterheid, mondgevoel (vrankheid) asook die verouderingspotensiaal van wyn. As antioksidante hou dit ook gesondheidsvoordele in. Antosianiene dra by tot die kleur van rooiwyn. Antosianiene kombineer met tanniene om meer stabiele polimeriese pigmente te vorm. As gevolg van die belangrikheid van tanniene en antosianiene is dit van uiterse belang dat verskillende wynmaak tegnieke gebruik word om ekstraksie in die wyn te bevoordeel en dat die analitiese metode so vinnig en akkuraat as moontlik gedoen word. Die eerste doel van hierdie studie was om die ekstraksie van tanniene en antosianiene deur middel van verskillende wynmaak tegnieke te evalueer. Te veel tanniene in die wyn kan negatiewe sensoriese kwaliteit tot gevolg het. Daarom is die tweede doel om die sensoriese kwaliteit van Shiraz wyn te evalueer. Die derde doel van hierdie studie was die twee tannien presipitasie metodes met mekaar te vergelyk in terme van die moeilikheidsgraad van die metode, tyd doeltreffendheid en herhaalbaarheid. Verskillende wynmaak tegnieke (ensiem byvoegings [E], koue maserasie [CM], verlengde dopkontak [PM] en ‘n kombinasie van koue maserasie en verlengde dopkontak [CM+PM]) is vergelyk ten opsigte van tannien en antiosianien ekstraksie. In 2008 en 2009 is twee kultivars (Cabernet Sauvignon en Shiraz) in twee verskillende klimatologiese areas gepars. Hierdie areas is geklassifiseer in die Winklerskaal as ‘n IV (Plaisir de Merle) en ‘n III (Morgenster). Om die effek van die verskillende wynmaak tegnieke op die ekstraksie van antosianiene en tanniene te vergelyk, is hierdie twee kultivars by twee verskillende rypheidsgrade geoes. Die eerste oes was net voor kommersiële oes (LB) en die tweede oes het net na kommersiële oes (HB) plaasgevind. Die 2009 Shiraz wyn is organolepties beoordeel om die effek van die verskillende wynmaak tegnieke op die wyn se mondgevoel te vergelyk. Die resultate van hierdie studie toon beduidende verskille in die fenoliese samestelling van die wyne. Dit is gevind dat die warmer area hoër tannien konsentrasies het as die koeler area. In 2008 het die CM+PM die meeste tanniene uit die Cabernet Sauvignon geëkstraheer by LB en die CM by HB in die warmer area. Die CM het in die koeler area meer tanniene geëkstraheer by beide die LB en HB rypheidsgrade. In 2009 het PM die meeste tanniene geëkstraheer by LB terwyl E die meeste tanniene geëkstraheer in die warmer area. In die koeler area het CM+PM die meeste tanniene geëkstraheer, terwyl geen van die behandelings ‘n effek gehad het by HB. Die meeste antosianien konsentrasie was in die koeler area gevind as in die warmer area. In beide 2008 (LB en HB) en 2009 (LB) het CM die meeste antosianiene geëkstraheer, terwyl geen behandeling ‘n effek gehad het by HB. Twee van die mees populêre tannien analitiese metodes is die BSA (bovine serum albumien) en die MCP (metielsellulose presipitasie) metodes. Die BSA metode is ‘n baie meer ingewikkelde metode waarvoor drie keer meer reagense gebruik word as vir die MCP metode. Maar waar die MCP net tanniene ontleed, ontleed die BSA metode tanniene, monomere (MP), klein polimeriese pigmente (SPP) en groot polimeriese pigmente (LPP). Dit help indien daar gekyk wil word na die evolusie van polimeriese pigmente. In hierdie studie is bevind dat daar ‘n redelike korrelasie (R2 – 0.88) tussen die BSA en MCP metode bestaan. Die herhaalbaarheid van die metodes het redelike kontroversie veroorsaak, waar sommige navorsers bevind het dat die BSA metode nie so herhaalbaar is soos eers bevind is nie. Die MCP metode het ’n praktiese voordeel aangesien dit in die helfde van die tyd van die BSA metode uitgevoer kan word. Dit het ‘n groot impak indien ‘n groot hoeveelheid monsters ontleed moet word. Die fenoliese samestelling en mondgevoel word sterk beïnvloed deur die klimatologiese area. In die warmer area was die effek van tannien konsentrasie op mondgevoel kleiner as in die koeler area. Die wyn van ryper druiwe het meer harder, verdowingseffek en bitter nasmaak gehad as by die wyn van groener druiwe. Die ensiem behandeling was meer geassossieerd met droë mond gevoel.

L'astringence résulte de l'interaction des tanins, polyphénols abondants dans les végétaux, avec les protéines salivaires et plus particuliÈrement les protéines salivaires riches en proline (PRP), appartenant à la famille des protéines peu structurées. Les tanins participent aux mécanismes de défense des végétaux et présentent des effets antinutritionnels dus à leur capacitè à inhiber les enzymes digestives. La synthÈse de PRP salivaires constitue un mècanisme d'adaptation à la consommation d'aliments riches en tanins. Ce travail vise à caractèriser les complexes ètablis en solution entre les PRP et les tanins, par une approche basèe sur la spectromètrie de masse (MS). Pour cela, les protèines salivaires humaines IB5, PRP basique, et II-1, PRP glycosylèe, ont ètè produites par voie hètèrologue. AprÈs purification, les deux protèines ont ètè caractèrisèes par MS avec une source d'electronèbullisation (ESI-MS) et avec une source MALDI (Matrix-Assisted Laser Desorption/Ionisation). L'ètude des interactions par ESI-MS a confirmè la prèsence en solution de complexes non-covalents IB5tanin et permis de prèciser leurs stchiomètries. Des expèriences de compètition entre diffèrents tanins et de dissociation des complexes IB5tanin ont mis en èvidence l'influence des principales caractèristiques structurales des tanins sur cette interaction. L'ètude structurale des èdifices IB5tanin, par diffèrentes techniques de MS/MS (Collision Induced Dissociation, Electron Capture Dissociation et photodissociation) et par mobilitè ionique couplèe à la MS, a mis en èvidence la prèsence de plusieurs sites d'interaction sur IB5 ainsi que des changements conformationnels liès à l'interaction / Astringency is an important organoleptic property of plant-based food. It is attributed to interactions of tannins, which are polyphenolic compounds, with salivary proteins and especially proline rich proteins (PRPs), which belong to the group of intrinsically unstructured protein (IUP). Tannins play an important part in plant defence mechanisms. Indeed, they have an antinutritional effect as they inhibit digestive enzymes. Production of salivary PRP is thus an adaptation process to tannin-rich diets. The purpose of this work is to provide a closer look at PRPtannin supramolecular edifices in solution, using a mass spectrometry (MS) approach. The human salivary proteins IB5, a basic PRP, and II-1, a glycosylated PRP, have been produced by heterologous expression. After purification, both proteins have been characterized by MS using electrospray (ESI) and Matrix-Assisted Laser Desorption/Ionisation (MALDI) sources. The study of the interaction between IB5 and model tannins by ESI-MS confirmed the presence of IB5tannin non covalent complexes in solution and provided new information on their stoichiometries. Competitive interaction experiments between IB5 and two tannins, along with IB5tannin complexes dissociation studies revealed the impact of the main tannin chemical features on this interaction. Structural studies performed on IB5tanin edifices by Collision induced dissociation (CID), Electron Capture Dissociation (ECD) and photodissociation MS/MS experiments and by ion mobility coupled with MS showed the presence of several interaction sites on IB5 and conformational changes arising from the interaction.

Les nématodes gastro-intestinaux (NGI) représentent un problème sanitaire majeur dans le monde entier en systèmes de production à l’herbe des élevages de moutons et de chèvres. La maîtrise de ces parasites est désormais compliquée par l'apparition de résistances aux anthelminthiques (AH) chez les nématodes. L'utilisation de plantes riches en tanins (PRT) comme AH non conventionnels est une des alternatives possibles au contrôle chimique de ces maladies parasitaires. Cependant, le mécanisme d'action de ces plantes sur les vers adultes demeure inconnu. Cet objectif a constitué le premier but de cette étude en particulier en essayant de déterminer quelle part jouent les effets directs et indirects dans l’action des PRT sur les populations de Nématodes adultes dans le tractus digestif des petits ruminants. Un effet direct correspond à une action de type pharmacologique sur la biologie, la structure et l’ultrastructure des nématodes liés à des composés biochimiques présents dans les PRT. L'hypothèse d’un effet indirect correspondrait une modification de la réponse cellulaire dans les muqueuses gastro-intestinales de l'hôte, affectant la biologie des NGI. Cette thèse est divisée en deux parties: 1) Deux expériences in vivo réalisées respectivement, au Mexique et en France, visant à déterminer les effets directs ou indirects sur la biologie des NGI liés à la consommation de PRT tel le tzalam (Lysiloma latisiliquum), une Légumineuse arbustive du Yucatan, le sainfoin (Onobrychis viciifolia) une Légumineuse fourragère tempérée, et ainsi que le quebracho(Schinopsis spp.), un extrait riche en tannins condensés Les agneaux ont été infestés par Haemonchus contortus (au Mexique) ou H. contortus et Trichostrongylus colubriformis (en France). La consommation de tzalam a provoqué une perturbation de croissance des vers ou de fertilité des femelles d’H. contortus. Selon la durée de consommation, le quebracho a réduit la population de H. contortus et T. colubriformis, et affecte également la fertilité de T. colubriformis. Le sainfoin a affecté la fécondité de H. contortus sans affecter celle de T. colubriformis. Le comptage de cellules effectrices dans les muqueuses pour évaluer l’implication d’un effet indirect n'a pas montré de différences significatives entre les lots recevant ou non des PRT. La seconde partie s’est fondée sur un test in vitro et deux essais in vivo chez des chèvres infestées par H. contortus pour mieux comprendre l'effet de la consommation de PRT (tzalam, au Mexique ou sainfoin, en France) sur la structure et l’ultrastructure d’H. contortus. Les principales altérations ont été trouvées dans la cuticule et la région céphalique des vers. Des agrégats de matériel végétal autour de la vulve n’ont été trouvé qu’in vitro. L’examen de l’ultrastructure des H. contortus a montré des signes de vacuolisation, surtout visible dans les tissus intestinaux et musculaires. Ces lésions suggèrent que des composés secondaires présents dans le feuillage des PRT peuvent être impliqués dans les fonctions vitales d’ H. contortus, telles que la mobilité, la nutrition et éventuellement la reproduction. / Gastrointestinal nematodes (GIN) represent a major health problem worldwide in grazing sheep and goat production systems. The control of these parasites has been complicated by the emergence of nematodes which are resistant to the commercially available anthelmintics (AHs). The use of tannin rich plants (TRP), as non-conventional Ahs, represents an alternative for the control of these parasites. However, the mechanism of action of such plants against adult populations of GIN has not been determined. The objective of the study was to determine the direct and indirect effect of TRP against adult populations of GIN in small ruminants. A direct effect is considered to be any action against the biology, structure and/or ultrastructure of the nematodes which is similar to a chemical AH. The indirect effect is a modification of the immune response of the host at the level of the gastrointestinal mucosa which affects the biology of the GIN. This study is divided into two stages: First stage) Two in vivo experiments conducted in Mexico and France respectively, determined the direct and indirect effects on the biology of GIN due to the consumption of the TRP forage of tzalam (Lysiloma latisiliquum), sainfoin (Onobrychis viciifolia) as well as a tannin-rich quebracho extract (Schinopsis spp). Lambs were artificially infected with Haemonchus contortus (Mexico) or H. contortus and Trichostrongylus colubriformis (France). The consumption of tzalam affected the length and fertility of H. contortus females. Meanwhile, the quebracho extract reduced the population of H. contortus and T. colubriformis also affecting the fecundity of T. colubriformis. The sainfoin affected the fecundity of H. contortus without affecting T. colubriformis. The indirect effect was not evident. Second stage) An in vitro assay and two in vivo experiments with goats infected with H. contortus allowed to identify the effect of the consumption of TRP (tzalam or sainfoin) on the structure and ultrastructure of H. contortus. Alterations in cuticle and cephalic region were found. The aggregates in the vulva were only found in vitro. The ultrastructure of H. contortus showed a vacuolization process in the intestinal and muscular tissues of these nematodes. The lesions suggest that the compounds contained in the foliage of TRP may be involved in vital functions of H. contortus such as mobility, nutrition and possibly reproduction. This study is a contribution towards the understanding the mechanisms of AH action of the TRP against GIN.

A indústria alimentícia tem oferecido ao consumidor muitas opções para aquisição de pães, dentre essas estão os pães enriquecidos e os integrais. A linhaça é um alimento com alegação funcional que vem se destacando pela presença do Omega-3, fibra solúvel e proteína, porém seu consumo é baixo devido à falta de hábito e também por escassez de informação dos consumidores. O objetivo do estudo foi formular pães com adição de grãos de linhaça nas concentrações de 3%, 6% e 9% e compará-los ao controle, sem adição de linhaça. Foram realizadas análises de determinação do perfil de ácidos graxos, composição centesimal, quantificação do teor de minerais, determinação de antinutricionais (ácido fítico, taninos, compostos fenólicos totais), digestibilidade de proteína in vitro, análise da qualidade dos pães através do peso da massa crua, peso da massa assada, perdas durante a cocção e índice de conversão. As propriedades físicas dos pães também foram avaliadas através da textura, volume e cor. As características sensoriais foram analisadas através de teste de aceitabilidade realizado em padaria com 116 provadores e também pela Análise Descritiva Quantitativa (ADQ) com 13 provadores treinados. Com os dados obtidos das análises químicas, elaborou-se um rótulo nutricional para cada formulação. Dentre os ácidos graxos quantificados, o palmítico, o oléico e o linoléico foram os maiores representantes nas 4 amostras de pães o que contribuiu para a frágil estabilidade dos produtos e para a formação de compostos indesejáveis, porém a presença de antioxidantes naturais nas sementes de linhaça controlou a oxidação lípidica. Nenhuma das amostras pode ser considerada fonte de fibra alimentar pelo fato de não conter 3g/100g de alimento pronto para consumo. O teor de fitatos presente nas amostras aumentou de acordo com a adição de sementes de linhaça. Nas 4 formulações de pães o teor de fitato diminuiu significativamente quando comparado ao da semente de linhaça. As amostras controle e com 6% de linhaça foram classificadas como volume específico Muito bom e as amostras com 3% e 9% de linhaça foram classificadas como volume específico Bom. Entre as amostras que receberam adição de linhaça, a amostra com 9% foi a melhor aceita pelos provadores e na ADQ, essa amostra apresentou médias superiores às demais em relação a maioria dos atributos levantados pelo provadores. Não foram identificados atributos indesejáveis para nenhuma das amostras através da ADQ. / Food industries offer to consumer several types of breads options which vary from whole wheat to enrich with multigrain such with flax seed. Flax seed is considered a functional food that has important content of omega 3 fat acids, soluble fiber and protein, however it intakes is very low in our diet. The aim of the research was to prepare a wheat bread enrich with flax seed at 3% or 6% or 9% concentration and to compare with a wheat bread control with no flax seed added. Analysis of fat acids composition, mineral composition, antinutritionals content (phytic acid, tannin, total phenolic compounds) and in vitro protein digestibility were carried out in the bread samples. Bread quality was evaluated analyzing the weight of the raw dough, the baked bread, and the lost during baking process and its conversion index. Physical properties of breads were evaluated for texture, volume and color. The sensory characteristics were evaluated using acceptability test with 116 panelists and by quantitative descriptive analysis (QDA) with 13 trained panelists. A nutritional label was formulated to each one of the bread preparations. The quantified fat acids profile showed that palmitic, oleic and linolenic acids were the most representative fat acids in the 4 bread sample analyzed. The presence of those fat acids possibly contributes to poor stability of product and for the development of undesirable compound. However, the presence of natural antioxidants in flax seed may help to control fat acid oxidation. None of the samples were considered source of dietary fiber because they have less then 3g/100 g of bread. The phytate content present in the samples increased according to the increase of flax seed concentration in the bread. Compared to raw flax seed, the phytate content in the bread samples were lower. The control and the sample with 6% of flax seed had the ranking for specific volume described as Very good and the samples with 3% and 9% of flax seed as Good. Between the bread samples, the best score in the acceptability test was obtained in the sample with 9% of flax seed. The same was observed for QDA which showed that the panel had high attributes for this sample compared to other ones. No undesirable attributes were identified in any other sample analyzed in QDA.

As leguminosas constituem uma importante fonte de alimentos para os ruminantes e podem ser exploradas para pastejo direto ou, se conservadas, para fornecimento na forma de feno ou silagem. Algumas leguminosas possuem compostos fenólicos em sua composição. A caracterização química dessas as plantas possibilita melhor uso das mesmas na alimentação animal assim como um melhor entendimento dos efeitos positivos e negativos dos compostos fenólicos na nutrição dos animais. Objetivou-se com o presente estudo: (i) avaliar a composição química de cinco leguminosas de interesse para a alimentação de ruminantes; (ii) estudar os efeitos dos taninos de diferentes leguminosas na produção de gases in vitro; e (iii) estudar os efeitos de dietas constituídas com estas leguminosas no consumo voluntário e digestibilidade aparente dos nutrientes em ovinos. O primeiro trabalho (Capítulo 3) refere-se à caracterização in vitro das leguminosas forrageiras (Leucaena leucocephala (Leucena), Arachis pintoi (Amendoim forrageiro), Stylosanthes guianenses cv mineirão (Estilosantes mineirão), Stylosanthes guianenses cv Campo Grande (Estilosantes Campo Grande e Calopogonio sp. (Calopogônio). Foram avaliadas a composição química, a quantificação de taninos, a fermentabilidade ruminal e a síntese microbiana e os resultados obtidos demonstraram valores de proteína bruta compatíveis com a literatura, exceto para o Calopogônio e Estilosantes Campo Grande (< 60 g kg-1 MS). Os teores de taninos variaram significativamente dentre as plantas estudadas, entretanto o teor de tanino condensado pode ser considerado seguro para os animais (entre 30 a 40 eq-g leucocianidina kg-1 MS). As cinco leguminosas apresentaram boa fermentabilidade in vitro, com baixo tempo de colonização (~ 4 h) e T ½ (tempo gasto para atingir metade do valor da Produção Potencial de Gases) inferior a 25 h. A técnica in vitro de incorporação de radiofósforo mostrou efeito significativo da adição de polietileno glicol (PEG) na avaliação da síntese microbiana, demonstrando o efeito dos taninos presentes nas leguminosas estudadas sobra a síntese de proteína no rúmen. O segundo trabalho (Capítulo 4) refere-se à caracterização e avaliação nutricional in vitro de Medicago sativa (Alfafa), Cajanus cajan (Feijão guandu), Mucuna aterrina (Mucuna preta) e Mucuna pluriens (Mucuna cinza). A composição química, a quantificação de taninos e a cinética de fermentação destas leguminosas e dietas experimentais constituídas de fenos de Tifton-85 (Cynodon sp), milho triturado, sal mineralizado na proporção de 30:18:2 com a adição (50%) de cada uma das leguminosas foram estudadas (tratamento ALF, GND, MCZ, MPT respectivamente para Alfafa, Feijão guandu, Mucuna cinza e Mucuna preta). Alfafa apresentou o maior conteúdo de proteína bruta (185 g kg-1 MS) e os teores de taninos variaram significativamente entre as plantas estudadas. Mucuna cinza apresentou maior e Alfafa menor valores de tanino condensado (50 e 0,2 eg. G leucocianidina kg-1 MS respectivamente). Com exceção da Alfafa, todas as outras leguminosas apresentaram incremento de gases quando incubadas in vitro na presença de PEG; o que reflete a atividade biológica dos taninos presente nestas plantas. Em relação às dietas experimentais, apenas as dietas GND e MCZ apresentaram incrementos de gases na presença de PEG. O terceiro trabalho (Capítulo 5) descreve a avaliação nutricional in vivo das dietas experimentais utilizadas no Capítulo 4 (ALF, GND, MCZ, e MPT). Foram utilizados ovinos da raça Santa Inês, machos, castrados com peso vivo médio de 53 ± 5,1 kg. As dietas foram oferecidas de acordo com o peso vivo de cada animal (3%) durante o ensaio de consumo voluntário, enquanto que durante o ensaio de digestibilidade, foi oferecida em cerca de 90% do consumo voluntário determinado anteriormente. Nenhuma dieta apresentou quantidade de taninos condensados considerado prejudicial aos animais. Todas as dietas estudadas apresentaram consumo voluntário e consumo de nutrientes semelhantes. Apenas ALF apresentou maior digestibilidade de PB, enquanto que a MCZ apresentou menor digestibilidade da mesma fração. É concluído com esses trabalhos que as leguminosas estudadas constituem uma importante fonte de nutrientes para os ruminantes, contudo, os teores de taninos condensados devem ser monitorados a fim de que sejam preparadas dietas que não alterem o consumo voluntário nem a fermentação ruminal, e consequentemente disponibilização dos nutrientes aos animais / Leguminous plants constitute an important source of food for the ruminants and they can be explored as direct grazing or, if conserved, for supply in the form of hay or silage. Some of those plants possess phenolic compounds in its composition. The chemical characterization of such plants make possible better use of them in the animal feeding as well as a better understanding of the positive and negative effects of the phenolic compounds in the nutrition of the animals. It was aimed with the present study: (i) to evaluate the chemical composition of five leguminous plants of interest for the feeding of ruminant; (ii) to study the effects of the tannins of different leguminous plants in the in vitro gas production assay; and (iii) to study the effects of diets constituted with these leguminous plants in the voluntary intake and apparent digestibility of the nutrients in sheep. The first work (Chapter 3) refers to the characterization in vitro of the leguminous foragers (Leucaena leucocephala (Leucena), Arachis pintoi (Amendoim forrageiro), Stylosanthes guianenses cv mineirão (Estilosantes mineirão), Stylosanthes guianenses cv Campo Grande (Estilosantes Campo Grande) and Calopogonio sp. (Calopogônio). They were appraised for the chemical composition, the quantification of tannins, the ruminal fermentability and the microbial synthesis. The obtained results demonstrated compatible values for crude protein content, except for Calopogônio and Estilosantes Campo Grande (< 60 g kg-1 DM). The tannin content varied significantly among the studied plants, however the condensed tannin content can be considered safe for the animals (between 30 to 40 eq-g leucocianidin kg-1 DM). The five leguminous plants presented good in vitro fermentability, with low time of colonization (~ 4 h) and T ½ (time spent to reach half of the value of the potential gas production) less than 25 h. The in vitro technique of radio labeled phosphorus incorporation showed significant effect of the addition of polyethylene glycol (PEG) in the evaluation of the microbial synthesis, demonstrating the effect of the presence of tannins in the studied leguminous plants. The second work (Chapter 4) refers to the characterization and in vitro nutritional evaluation of Medicago sativa (Alfalfa), Cajanus cajan (Feijão guandu), Mucuna aterrina (Mucuna preta) and Mucuna pluriens (Mucuna cinza). The chemical composition, the quantification of tannins and the kinetics of fermentation of these leguminous plants and four constituted experimental diets with Tifton-85 hay (Cynodon sp), corn grain, mineralized salt (30:18:2) with the addition (50%) of each one of the leguminous plants were studied (treatment ALF, GND, MCZ, MPT respectively for Alfalfa, Feijão guandu, Mucuna cinza and Mucuna preta). Alfalfa presented the highest content of crude protein (185 g kg-1 DM) and the tannin content varied significantly among the studied plants. Mucuna cinza presented greater and Alfalfa lower values of condensed tannin (50 and 0.2 eg g leucocianidin kg-1 DM respectively). Except for the Alfalfa, all the other leguminous plants presented increment of gases when incubated in vitro in the presence of PEG; what reflects the biological activity of the tannins present in these plants. In relation to the experimental diets, just the diets GND and MCZ presented increments of gases in the presence of PEG. The third work (Chapter 5) describes the in vivo nutritional evaluation of the experimental diets used in the Chapter 4 (ALF, GND, MCZ, and MPT). Santa Inês males, castrated with live weight of 53 ± 5.1 kg sheep were used. The diets were offered in agreement with the liveweight of each animal (3%) during the voluntary intake assay, while during the digestibility trial, it was offered in about 90% of the voluntary intake determined previously. No diet presented amount of condensed tannins considered harmful to the animals. All of the studied diets presented similar voluntary intake and nutrients consumption. Just ALF presented higher crude protein digestibility, while MCZ presented lower digestibility of the same fraction. It is concluded with those works that the studied leguminous plants constitutes an important source of nutrients for the ruminants, however, the condensed tannins content should be monitored so that it can prepare diets to neither alter the voluntary intake nor the ruminal fermentation, and consequently guaranteeing the supply of nutrients to the animals

O presente trabalho visou analisar comparativamente as alterações fisiológicas e estruturais de caquis adstringentes (PVA) e não adstringentes (PCNA) desde o início do desenvolvimento até a maturação avançada. Além disso, na pós-colheita, foram realizados tratamentos de destanização e testadas diferentes condições de armazenamento para \"Giombo\". Este trabalho está sendo apresentado em seis capítulos, sendo que os últimos cinco capítulos referem-se aos experimentos realizados entre 2010 a 2013. No capítulo 2 é apresentado o desenvolvimento de caquis \"Giombo\" (PVA) e \"Fuyu\"(PCNA). Foi possível observar que a diferenciação das células taníferas e o processo de acúmulo de taninos no vacúolo não variou entre as cultivares, porém houve diferença significativa em relação à densidade de células taníferas e aos teores de taninos solúveis. O capítulo 3 apresenta o estudo comparativo de sete estádios de maturação de caquis \"Giombo\" e \"Rojo Brillante\"(PVA) e \"Fuyu\" e \"Hana Fuyu\" (PCNA). A redução da firmeza do mesocarpo de caquis de ambos os grupos está relacionada ao avanço da maturação e não à perda natural da adstringência. A capacidade antioxidante e o teor de sólidos solúveis em caquis dos dois grupos indicam que pode haver relação com a perda natural da adstringência, pois houve decréscimo somente nas cultivares PVA durante a maturação. Os taninos delimitados às células taníferas no teste com vanilina clorídrica, e o conteúdo do vacúolo com aspecto alveolar observado ao Cryo-SEM podem estar relacionados à polimerização dos taninos durante o amadurecimento dos frutos. No experimento descrito no capítulo 4, caquis \"Giombo\" de fim e início de safra foram submetidos a diferentes tempos de exposição ao etanol (1,70 mL Kg-1). No fim da safra os frutos obtiveram índice de adstringência e teores de taninos solúveis adequados para consumo em 24 horas e, no início da safra, em 36 horas. O capítulo 5 discute a influência das pectinas na remoção da adstringência de caquis \"Giombo\" sob o armazenamento refrigerado a 5?C. Os resultados indicaram que não houve interação das pectinas com os taninos solúveis na remoção da adstringência, pois mesmo com a redução da firmeza de polpa e aumento da solubilização das pectinas, o índice de adstringência e o teor de taninos solúveis permaneceram elevados nos frutos não destanizados. No capítulo 6 é apresentada a influência das temperaturas (1, 5 e 10?C) de armazenamento na qualidade de caquis \"Giombo\" destanizados tratados ou não com 1-MCP. A qualidade foi melhor preservada quando os frutos foram destanizados e tratados com 1-MCP e armazenados a 1ºC por até 35 dias. Os frutos que foram apenas destanizados tiveram a estrutura celular do mesocarpo e a firmeza mais alteradas em todas as temperaturas analisadas. Provavelmente, a temperatura de 5ºC torna os frutos mais susceptíveis a danos por frio com redução drástica da firmeza e alterações estruturais. / This study purpose was the comparison between the physiological and structural changes of astringent persimmons (PVA) and non-astringent (PCNA) from the start of development to advanced maturation. Furthermore, postharvest treatments of deastringency were made and tested for different storage conditions at \"Giombo\". This work is presented in six Chapters, and the last five chapters refer to experiments conducted between 2010 and 2013. Chapter 2 describes the development of persimmons \"Giombo\" (PVA) and \"Fuyu\" (PCNA). It was observed that the differentiation of tannin cells and the accumulation process of tannin in vacuole did not vary among cultivars, but there was a significant difference in the density of tannin cells and soluble tannins. Chapter 3 presents the comparative study of seven stages of ripening persimmons \"Giombo\" and \"Rojo Brillante\" (PVA), and \"Fuyu\" and \"Hana Fuyu\" (PCNA). Reduction of mesocarp firmness of both groups of persimmons is related to maturation advance and not due to natural loss of astringency. Antioxidant capacity and total soluble solids of persimmons from both groups indicate a possible relation with the natural loss of astringency, once only PVA cultivars showed astringency decrease during ripening. The tannins delimited in tannin cells for the test with vanillin hydrochloric, and the contents of the vacuole with alveolus aspect observed by Cryo-SEM may be related to tannin polymerization during fruit ripening. In the experiment described in Chapter 4, persimmons \"Giombo\" from late and early harvest were submitted to different periods of exposure to ethanol (1.70 mL kg-1). Fruits obtained at late harvest presented index astringency and soluble tannin content suitable for consumption within 24 hours, while early harvest in 36 hours. Chapter 5 regards to the influence of pectins in astringency removal of persimmons \"Giombo\" under cold storage at 5?C. Results indicated no interaction of pectins with soluble tannins astringency removal, because even with the reduction of flesh firmness and increased solubilization of pectins, the index of astringency and soluble tannins remained high in fruits not deastringenced. The influence of temperature (1, 5 and 10?C) storage on the quality of persimmons \"Giombo\" treated or not with 1-MCP is presented in Chapter 6. Better quality was observed when the fruits were deastringenced, treated with 1-MCP and stored at 1°C for up to 35 days. Fruits that had been only deastringenced had bigger changes on their mesocarp cellular structure and firmness at all temperatures experimented. The temperature of 5°C may be responsible for making fruitsmore susceptible to chilling injury with structural changes and a drastic reduction of the firmness.

Les travaux présentés dans ce manuscrit sont consacrés à l’étude et à l’élaboration de résines de collage bio-sourcées pour remplacer les résines Urée-Formol (UF) d’origine pétrochimique, sources de formaldéhyde, utilisées actuellement dans l’industrie panneautière. Les résines étudiées dans le cadre de cette thèse sont formulées à partir de ressources locales (dextrines issues d’amidon de maïs et tanins d’écorces de pin maritime) et doivent respecter certaines contraintes industrielles. Pour répondre à ces contraintes, les travaux de recherche présentés dans ce mémoire vont de la compréhension structurale des mélanges des produits de base jusqu’à la fabrication de panneaux à l’échelle du laboratoire, en passant par la formulation et la réticulation des différentes résines thermodurcissables. Ces travaux montrent qu’il est nécessaire de travailler en milieu alcalin pour solubiliser les tanins. Dans cette situation, les mélanges tanins/dextrines donnent des suspensions colloïdales. Les fractions massiques en tanins et en dextrines permettant d’obtenir des paramètres respectant le cahier des charges varient de 0 à 40%. La réticulation de ces mélanges par l’épichlorhydrine a permis l’obtention de colles thermodurcissables insolubles dans l’eau ayant une bonne stabilité dimensionnelle à sec, mais un pouvoir gonflant plus ou moins important selon la formulation. Les propriétés adhésives, mécaniques et thermiques de ces matériaux ont été caractérisées et ont permis de sélectionner des formulations de colles ayant les meilleures performances thermomécaniques. Enfin, tout en respectant un protocole industriel, des composites ont été fabriqués à partir de ces colles et des particules de bois. Il a été montré que les panneaux de particules obtenus ont des performances mécaniques équivalentes à ceux fabriqués avec des colles UF. / This work aims to substitute resins based on formaldehyde as UF by “green” thermosetting adhesives based on natural and renewable local products, in the wood composite industry. The resins studied in this thesis are made from local resources (dextrins from corn starch and tannins from maritime pine barks) and must respect certain industrial requirements. To meet these industrial constraints, the research work of this manuscript deals with studies ranging from the structural understanding of basic product mixtures, up to the characterization of the properties of panels manufactured in the lab, including the formulation and reticulation of resins. These studies show that it is necessary to work in an alkaline medium to solubilize the tannins. In this situation, the tannins / dextrin mixtures give colloidal suspensions. The mass fractions in tannins and dextrins allowing to obtain parameters respecting the specifications vary from 0 to 40%. The epichlorohydrin has been used as hardener to cross-link these resins. Thermosetting materials with good dimensional stability have been obtained. However the resin swelling depends on the composition of the formulation. Adhesive, mechanical and thermal properties of these materials were characterized. Thus, adhesive formulations with the best thermomechanical performances have been selected. Finally, composites have been made from these adhesives and wood particles, respecting an industrial procedure. It has been shown that these panels and those obtained with UF adhesives have equivalent mechanical performances.

Les tanins jouent un rôle clé dans les qualités organoleptiques du vin rouge. Ils sont à l’origine de l'Astringence, une sensation de sècheresse perçue en bouche lors de la dégustation. Cette sensation est la conséquence d'une interaction spécifique entre des tanins et des protéines salivaire, principalement les Protéines Riches en Prolines (PRPs). Une première partie de ce travail a consisté à étudier l'influence de divers sucres sur le processus d'auto-association des tanins ainsi que sur les interactions tanins-protéines. Le comportement colloïdal d’un tanin (l’Epigallocatechine Gallate – EGCG), ainsi que son interaction avec un peptide modèle IB9-14 représentatif des PRPs, ont été étudiés en présence de différents sucres simples et polysaccharides. Les paramètres de l’interaction ont été déterminé pour l’ensemble des systèmes, mettant en évidence l’existence d’une interaction entre EGCG et sucres dont l’affinité semble dépendre du degré de polymérisation du sucre. Cette interaction ne perturbe pas, dans les conditions expérimentales testées, l’association entre les tanins et le peptide. Une deuxième partie de ce travail correspond à la synthèse complète de la protéine IB9 incluant la séquence peptidique d’IB9-14, et à l’étude de son interaction avec deux procyanidines : l’EGCG et le dimère B3. Cette étude a permis d’observer et de confirmer une influence de la longueur de la chaîne peptidique sur les interactions avec les tanins. / Tannins play a key role in the organoleptic qualities of red wine. They are responsible for wine astringency, a dry, rought and puker sensation perceived in the mouth while tasting. This sensation is the consequence of a specific interaction between tannins and saliva proteins, mainly Proline Rich Protein (PRPs). The first part of this work was to study the influence of various sugars on the self-association of tannins process as well as tannins - proteins interactions. The colloidal behavior of a tannin (the epigallocatechin gallate - EGCG), as well as its interaction with a representative peptide IB9-14 of PRPs was studied in the presence of various simple sugars and polysaccharides. The parameters of the interaction were determined for all systems, highlighting the existence of an interaction between EGCG and sugars whose affinity seems to depend on the sugar polymerization degree. This interaction does not interfere, under the experimental conditions tested, on the association between tannins and peptide. The second part of this work was to realize the full synthesis of the protein IB9 including the peptidic sequence of IB9-14, and to study its interaction with two procyanidins: EGCG and dimer B3. The results show and confirm the influence of the length of the peptide chain interactions with tannins.

The broad objective of the current study was to determine the response of pigs fed on incremental levels of Acacia tortilis leaf meal-based diet. Eight trees of each of the following five dominant leguminous leaf meals; A. tortilis, A. robusta, A. nilotica, A. nigrescens and A. xanthophloea, were individually hand harvested from the same grazing camp at Makhathini Research Station, Jozini, South Africa. The leaf meals were harvested between April and May 2013 after the rainy season at advance stage of maturity. Following the nutritive evaluation of Acacia species, A. tortilis was selected for the feeding experiment. Thirty finishing male F1 hybrid (Landrace × Large White) pigs with an initial weight of 60.6 (s.d. = 0.94) kg were randomly allotted to six dietary treatments containing 0, 50, 100, 150, 200, 250 g/kg DM inclusion levels of A. tortilis leaf meal. Each treatment diet was offered ad libitum to five pigs in individual pens for 21 days. Average daily feed intake (ADFI), body weight, average daily gain (ADG) and gain: feed ratio was measured every week. Blood was collected at the end of the experimental period for the determination of nutritionally-related blood metabolites and activity of Aspartate aminotransferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP). Following a feed withdrawal period of 12 hours, pigs with a mean body weight of 80 ± 15 kg were slaughtered, eviscerated for the collection of livers, kidneys, lungs and heart from each pig and weighed individually using a digital scale. Acacia tortilis and A. xanthophloea leaf meals had the highest CP and fat content (P < 0.01) among all the Acacia species. The NDF and ADF varied significantly across Acacia species (P < 0.05). Acacia robusta had significantly the highest levels of non-structural carbohydrates, whilst A. tortilis had the lowest. The concentration of proanthocynidins varied across the species. Acacia tortilis had significantly high levels (23 g/kg DM) of phosphorus compared to the other species. Acacia nilotica had the highest (P < 0.001) concentration of iron, but it had the lowest (P < 0.01) level of manganese. There was a quadratic decrease in both ADFI and ADG (P < 0.001) with as A. tortilis leaf meal increased. The gain: feed ratio was linearly reduced (P < 0.001) with incremental levels of A. tortilis leaf meal in the diets. Serum concentrations of Fe, AST and ALP increased quadratically (P < 0.01) as A. tortilis leaf meal increased. There was a linear increase in ALT activity with increased leaf meal inclusion. Hepatosomatic index (HSI: liver weight/ body weight x 100), scaled kidney weight and scaled heart weight increased linearly (P < 0.001) as A. tortilis leaf meal increased. There was, however, a quadratic increase in the relative weight of lungs (P < 0.001) as leaf meal increased. In conclusion, the piecewise regression (brocken-stick) NLIN procedure (SAS, 2008; SASA institute Inc.) showed that A. tortilis leaf meal can be included in finisher pig diet up to 150 g/kg DM of feed without negatively affecting growth performance, nutritionally-related blood metabolites, liver enzymes and internal organs of pigs. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.