Protein is an important fraction within bovine milk. This milk protein is not only vital for calf growth and development, but also includes bioactive proteins and peptides that have been shown to enhance the health of animals and humans. Research efforts are focusing on factors, such as nutrition, that can influence the quantity and profile of proteins within the bovine milk proteome. The research outlined herein investigated the impact of diet on the bovine milk proteome. The first experiment examined whether dietary inclusion of grape marc (GM), a condensed tannin (CT) containing by-product from the viticulture industry, could alter the bovine milk proteome through altered nitrogen (N) metabolism. In this experiment, 10 lactating Holstein cows were fed either 2.0 kg dry matter (DM)/ cow/ day of beet pulp: soy hulls in a 50% mixture (control), or 1.5 kg DM/ cow/ day of GM as part of a balanced dairy cow ration for a 28-d trial. Milk samples were obtained for analysis of the high- and low-abundance protein fractions. Skimmed milk samples collected for high-abundance protein analysis were measured using high performance liquid chromatography (HPLC), and liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to identify proteins in the low-abundance protein enriched fraction. Skimmed milk samples collected for low-abundance milk protein analysis were fractionated and enriched to remove higher abundance proteins. Enriched milk samples were then digested and labeled with isobaric tandem mass tags (TMT) prior to protein identification using LC-MS/MS analysis. There were no changes in the high-abundance protein fraction in response to diet; however, 16 of 127 low-abundance proteins were identified at different relative-abundances due to diet (P ≤ 0.05). While there were no alterations in the metabolic or N status of animals due to GM supplementation, the 12% change in the low-abundance milk protein fraction highlighted the potential for dietary alteration of the bovine milk proteome. A second experiment evaluated the inclusion of alternative forage crops (AFC) as a means to alter the bovine milk proteome. In this experiment, both the skimmed milk and milk fat globule membrane (MFGM) protein fractions were included in analysis. Milk samples were collected from 16 lactating Jersey cattle included in a 21-d grazing experiment, where cows were offered one of two diets. The control group (CON, n=8) grazed a grass-legume pasture mixture containing orchardgrass (Dactylis glomerata), timothy (Phleum pratense), Kentucky bluegrass (Poa pratensis), and white clover (Trifolium repens). The treatment group (AFC, n=8) grazed a similar base pasture that was strip-tilled with oat (Avena sativa), buckwheat (Fagopyrum esculentum), and chickling vetch (Lathyrus sativus) so that the AFC species comprised 10% of the AFC group’s pasture DM intake (DMI). Milk samples were collected for HPLC analysis of the high abundance milk proteins, and LC-MS/MS analysis of the low abundance protein enriched skim milk fraction and MFGM-associated protein fraction. Cows that grazed pastures containing AFC had higher αs1-CAS content (P = 0.005), and higher relative-abundances of 7 low-abundance proteins within the skim milk and MFGM fractions (P ≤ 0.05). While it is plausible that the inclusion of AFC in pasture increased nutrient availability to the mammary gland, the specific mechanisms that could have caused the shifts observed remain unclear. Further investigation is necessary to fully understand the role of diet and the milk protein profile.

The topic of my dissertation is Comparison of the quality of eggs depending on the method of feeding. The aim of this study was to investigate whether the quality of hen eggs changes after adding crushed grape seeds in the compound feed of the hens. Grape seeds are formed as a side product during wine making processes and contain large amounts of biologically active substances and suitable acids, which can improve the feed conversion and subsequently also improve the technological properties of eggs. With processing of grape seeds the effective utilization of crop residues in vineyards comes about. The experiment was conducted in two experimental periods and in each period 100 laying hens were surveyed. They were separated into two groups an experimental and a control one. In the control group the hens were fed with a standard mixture of N2 feed and in the experimental group there was a supplement of 5 weight percent of crushed grape seeds in the compound feed. Eggs were collected daily and once every two weeks an analysis of technological properties of eggs was conducted. The results of the experiment showed that the addition of crushed grape seeds containing a biologically active substance did not have a statistically significant effect on the quality and selected technological parameters of eggs.

Questa tesi di dottorato ha riguardato il recupero di composti ad alto valore aggiunto dai sottoprodotti agro-alimentari e il loro utilizzo in campo alimentare. In particolare, si è studiato il frazionamento lignocellulosico di raspi d’uva e residui di potatura di melo per ottenere emicellulosa, cellulosa, lignina e antiossidanti attraverso processi di auto-idrolisi e organosolv, investigando successivamente l’influenza di sei cultivar sul frazionamento lignocellulosico dei raspi. Un processo composto da quattro fasi (trattamento di lavaggio seguito da un’idrolisi acida, un’idrolisi basica e, infine, una decolorazione basica) è stato applicato su raspi di uva rossa (Barbera, Pinot Noir, Nebbiolo) e bianca (Müller, Chardonnay and Moscato). I raspi sono stati confrontati in termini di composizione chimica della materia prima (umidità, ceneri, minerali, lipidi, fibre, proteine zuccheri liberi) e di frazioni recuperate ( lavaggio, liquors acidi e basici e residui di cellulosa finali). Si è inoltre studiato l’influenza varietale sull’estrazione di antiossidanti da vinacce di Barbera, Pinot Noir and Nebbiolo, i diversi estratti sono stati confrontati per il loro profilo fenolico e per la loro capacità antiossidante. Infine, un estratto liofilizzato di vinacce di Barbera è stato utilizzato per migliorare la shelf-life di una pasta di nocciole valutando l’ossidazione cinetica. / This thesis has focused on the recovery of high value compounds from agri-food by-products and their use in food. In particular, we studied the lignocellulosic fractionation of grape stalks and apple tree pruning for the recovery of hemicelluloses, cellulose, lignin and antioxidants through processes of auto-hydrolysis and organosolv, investigating subsequently the influence of six grape cultivar,on the lignocellulosic fractionation of grape-stalks. A four-step process (a washing treatment followed by an acid hydrolysis, a basic hydrolysis and, finally, a basic bleaching) was applied to stalks obtained from six different red (Barbera, Pinot Noir, Nebbiolo) and white (Müller, Chardonnay and Moscato) grape cultivars. The different stalks were compared in terms of chemical composition of raw material (moisture, ash, minerals, lipids, fiber, proteins, free sugars) and of the recovered fractions (washing, acid and basic liquors and final cellulose residues). We also studied the influence of variety on the antioxidants extraction from Barbera, Pinot Noir and Nebbiolo grape marc. The different extracts were compared in terms of phenolic profile and antioxidant capacity. Finally, a freeze-dried extract from Barbera grape marc was used to improve the shelf-life of hazelnut paste evaluating the oxidation kinetics.

Dans un contexte de réduction des intrants chimiques, l’utilisation des phytosanitaires naturels stimulant l’immunité végétale ouvre la porte vers une nouvelle approche de protection des plantes. Ces composés éliciteurs regroupés sous le terme de «Stimulateurs des Défenses naturelles des Plantes» (SDP) activent le système défensif de la plante la rendant plus résistante aux bio-agresseurs. Les SDP, de nature diverse, se présentent sous forme de composés uniques ou en mélange dans les extraits végétaux. Au cours de ma thèse, nous avons démontré l’activité SDP des extraits de marc de raisin. Les extraits issus de sous-produits de la vigne, marc de raisin rouge, marc de raisin blanc et pépins de raisin induisent diverses réactions de défense au sein de plantes modèles. Nous avons focalisé notre étude sur l’extrait de marc de raisin rouge (EMR) stimulant l’immunité chez le tabac. Infiltré sur feuilles, l’EMR induit la réponse de type HR caractérisée par l’apparition de lésions chlorotiques et accumulation de composés autofluorescents dans les tissus infiltrés. Ces réactions de défense locales ont été observées également chez l’arabette et la tomate. L’EMR déclenche les réponses LAR et SAR avec l’accumulation des transcrits des gènes de défense dans les feuilles de tabac et ce quelque soit son mode d’application (infiltration ou pulvérisation). Le mode d’action de l’EMR a été abordé sur cultures cellulaires de tabac BY-2. L’EMR induit une forte alcalinisation du milieu extracellulaire avec une mobilisation du calcium (Ca2+), l’expression des gènes de défense et la mort cellulaire. Une étude pharmacologique de la mort cellulaire suggère la mise en place de mort cellulaire programmée (PCD) dans les cellules de tabac. La caractérisation de la voie de signalisation activée par l’EMR a été étudiée avec le mutant NahG de tabac incapable d’accumuler l’acide salicylique (SA). Les réponses de défense (HR, LAR et SAR) sont faiblement induites par l’EMR chez le mutant nahG. L’EMR provoque une réponse de type HR fortement réduite avec une faible accumulation des composés autofluorescents et une diminution drastique de l’accumulation des transcrits des gènes PR suggérant l’intervention du SA dans l’induction des réactions de défense. Le degré de protection induit par l’EMR a été déterminé sur le pathosystème tabac/Phytophthora parasitica. Pulvérisé sur feuilles, l’EMR réduit de 45% les zones infectées par l’oomycète. Ce degré de protection semble être le résultat de l’activité antimicrobienne de l’EMR combinée à l’activité SDP. Aucune protection n’a été observée chez le mutant nahG confirmant l’implication de SA dans la résistance induite par l’EMR. Le fractionnement de l’EMR a permis de simplifier la formule active des extraits de raisin et d’identifier un mélange de molécules potentiellement capables d’induire l’activité SDP. Les composés actifs sont de nature polyphénolique et contiennent de la procyanidine B2 capable à elle seule d’induire la réponse de type HR et l’expression de l’antimicrobien PR1. Cependant, il semble que cette molécule agisse en association avec d’autres composés polyphénoliques pour stimuler le système défensif de la plante. / In order to reduce chemical inputs, the use of natural phytosanitary products stimulating plant immunity are emerging approaches in phytoprotection. These elicitor compounds known as "Plant Defense Inducers" (PDI) activate the plant defense system and improve their resistance to pests attack. PDI are single molecule or mixture of compounds extracted from plant. In my thesis, we demonstrated the PDI activity of different grape marc extracts. The winery byproducts, red grape marc extract, white grape marc extract and grape seed extract all induced various defense reactions in several plant models. We focused our study on the red grape marc extract (GME) which stimulates the immunity system in tobacco plants. When infiltrated into tobacco leaves, GME induced HR-like response characterized by the appearance of chlorotic lesions and accumulation of autofluorescent compounds in infiltrated tissues. Similar local defense reactions have been observed in Arabidopsis thaliana and tomato. GME also triggered LAR and SAR responses and induced defense gene transcript accumulation in tobacco leaves after infiltration or spraying. The GME mode of action was studied using the suspension-cultured cells of tobacco BY-2. GME induced rapid alkalinization of extracellular medium with calcium mobilization, expression of defense genes and cell death. A pharmacological approach of this defensive phenomenon suggests the establishment of programmed cell death (PCD) in tobacco cells. The characterization of the signaling pathway activated by GME was studied using tobacco nahG mutant unable to accumulate salicylic acid (SA). Defense responses (HR, LAR and SAR) induced by GME were impaired in the nahG mutant. GME drastically reduced HR-like response symptoms and PR transcript accumulation. These data suggest the implication of SA in the GME-induced plant defense reactions. The GME-induced protection was evaluated in the model pathosystem of compatible interaction between Nicotiana tabacum and Phytophthora parasitica var. nicotianae (Ppn). GME could reduce by 45% the infected areas induced by the oomycete on tobacco leaves. This level of protection was the result of the combined antimicrobial and PDI actions of GME. GME had no protecting effect against Ppn on NahG leaves evidencing the involvement of SA in the GME-induced resistance. GME fractionation led to identification of a bioactive molecule mixture capable of inducing the PDI activity. The active compounds are polyphenolics and involve procyanidin B2 which is by itself able to induce the HR-like response and PR1 transcript accumulation. This compound should act in combination with other polyphenolic molecules to stimulate the full plant defense reactions.

Ce travail de thèse s’inscrit dans le contexte identifié de la nécessité de développer de nouveaux polymères plus respectueux de l’environnement. Nous sommes convaincus que l’essor de ces matériaux « verts » exige d’utiliser des additifs « verts » lors de la formulation. Dans le but d’élaborer des additifs respectueux de l’environnement capables d’assurer la stabilisation du polymère au regard du cycle de vie, et en attachant une attention particulière à la valorisation de la biomasse, trois approches ont été étudiées. La première est une voie innovante qui consiste à utiliser un mélange de polyphénols issus des sous-produits de l’industrie vinicole, et de l’industrie du thé. La seconde voie est la valorisation d’une molécule issue du sous-produit majeur de l’industrie du blé. La troisième est une voie d’innovation récente, qui propose l’utilisation de nanocomposites hydroxydes doubles lamellaires (HDL) fonctionnalisés avec des stabilisants biosourcés. L’évaluation de leurs propriétés stabilisantes, est réalisée au regard des processus de thermo- et photo-oxydation, dans les polyoléfines. Les résultats très prometteurs de ce projet mettent en évidence le fort potentiel des molécules et composites d’origine biosourcée à maintenir les propriétés des polymères en conditions d’usages. / This thesis work falls within the identified context of the need to develop new polymers that are more environmentally friendly. We are convinced that the development of these "green" materials requires the use of "green" additives during formulation. To develop environmentally friendly additives capable of stabilizing the polymer, with respect to the life cycle, and paying special attention to biomass valorization, three approaches have been studied. The first is an innovative way of using a mixture of polyphenols from winery by-products and tea industry. The second way is the valorisation of a molecule from the major by-product of the wheat industry. The third is a recent route of innovation, which proposes the use of layered double hydroxides nanocomposites (LDH) functionalized with bio based stabilizers. The evaluation of their stabilizing properties is carried out in polyolefins, against thermo- and photo-oxidation processes. The very promising results of the project highlight the strong potential of bio based molecules and composites to maintain polymers properties under conditions of use.