Orientador: Jaime Amaya-Farfan / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T13:16:03Z (GMT). No. of bitstreams: 1 Jackix_ElisadeAlmeida_M.pdf: 5040809 bytes, checksum: 9ccad63464c1464ceb7ded271d23cb5c (MD5) Previous issue date: 2008 / Resumo: A osteoporose constitui a enfermidade do esqueleto de maior incidência mundial e é caracterizada por redução da massa óssea levando ao aumento da susceptibilidade a fraturas. Nas mulheres, a osteoporose está associada à queda dos níveis de estrogênio, que acompanha a menopausa. O colágeno compreende cerca de 95% das proteínas dos ossos, e é parcialmente responsável pelas propriedades biomecânicas do osso. Os hidrolisados enzimáticos de colágeno (HC) são produtos obtidos a partir da hidrólise da gelatina e têm recebido atenção por suas propriedades no tratamento de doenças osteoarticulares. Objetivo: avaliar o efeito de um HC sobre características ósseas em modelo osteopênico com ratas. Métodos: 48 ratas adultas foram divididas em 6 grupos: 3 ovariectomizados, 1 sham-operated e 2 intactos, tratados com dieta AIN-93 M, suplementada com HC ou gelatina (controle), em dois níveis: (1) quantidade equivalente a 5X ao recomendado para humanos (10g/D), e outro (2) com nível 10X maior. Após 8 semanas, os ossos do fêmur e da coluna vertebral foram extirpados, e o sangue coletado; determinaram-se osteocalcina e fosfatase alcalina sérica, e no osso, determinou-se peso, teor protéico, conteúdo mineral (Ca, Na, Zn, Fe, K, P, Mg, Mn, Pb e Cu). Realizou-se também a análise de cortes histológicos dos diferentes grupos. Avaliou-se a resistência biomecânica por meio dos testes de flexão em três pontos nos fêmures e compressão nas vértebras. Resultados: As vértebras do grupo que recebeu a maior dosagem de HC suportaram uma carga 4X maior, e tiveram percentuais de proteína óssea e valores de osteocalcina maiores, do que aquelas que não tiveram suplementação ou receberam gelatina (p<0,05). A análise histológica mostrou maior espessura das trabéculas ósseas nos fêmures e vértebras das ratas que receberam HC. Conclusão: O HC contribuiu para uma maior conservação, composição e resistência óssea, quando comparado à gelatina / Abstract: Osteoporosis, the skeletal disease of highest incidence in the world is characterized by the reduction of bone mass and commonly leads to increased susceptibility of fractures. In women, osteoporosis is associated to the cessation of estrogen secretion and menopause. Collagen accounts for approximately 95% of the bone proteins and is to a large extent responsible for its biomechanical properties. Collagen hydrolyzates (CH) consist of mixtures of peptides obtained by a partial hydrolysis of gelatins, which have received considerable scientific attention as potential repositories of osteoraticular tissues. Objective: to evaluate the effect of supplementing the diets of ovariectomized rats with a commercial CH upon the compositional, biomechanical and histological characteristics of the bone. Methods: 48 adult female rats were divided into 6 groups: 3 of them ovariectomized, 1 sham-operated and 2 intact, fed the AIN 93-M diet, supplemented with either CH or gelatin (Control), in two levels: (1) a dose equivalent to 5x the amount suggested for humans (10g/D) and (2) a dose 10x greater. After eight weeks of treatment, the bones of femurs and vertebrae were excised the blood collected and serum osteocalcin and serum alkaline phosphatase determined. For the bone, gross weight, protein and mineral content (Ca, Na, Zn, Fe, K, P, Mg, Mn, Pb and Cu) were determined. Histological analysis of bone tissue sections was also performed to compare the different treatments. Biomechanical strength was assessed by means of 3-point flexion and compression tests of the femurs and vertebrae, respectively. Results: the vertebrae of the ovariectomized group that received the higher dosage of CH withstood a load 4x greater and exhibited higher levels of protein and osteocalcin content than those that received the gelatin or no supplementation at all. Histological analysis of the femurs and vertebrae of castrated rats that received the CH showed greater thickness than those receiving the gelatin supplementation. Conclusion: CH consumption as a diet supplement by the ovariectomized rat had an unequivocal contribution in the conservation or preservation of bone mass not seen when gelatin was used as a supplement / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

L'aquaculture est en pleine expansion et fournit aujourd'hui la moitié des produits aquatiques destinés à la consommationhumaine. Elle constitue ainsi un secteur clé pour le maintien et l'amélioration de la sécurité alimentaire dans lemonde. Le développement de l'aquaculture est étroitement lié à celui des formules alimentaires. Ces dernières années,la part des farines de poisson dans la formulation des aliments a particulièrement diminué au profit des farines d'originevégétale pour répondre aux nombreuses contraintes économiques et environnementales. Néanmoins, ces matièrespremières sont moins adaptées aux besoins nutritionnels des poissons carnivores et leur utilisation peut entraîner unebaisse des performances de croissance et d'efficacité alimentaire. L'ajout d'additifs et d'ingrédients fonctionnels devientalors indispensable. Les hydrolysats protéiques issus des co-produits de la pêche et de l'aquaculture sont des ingrédientsà fort potentiel appétence, nutritionnel et bioactif. Ces ingrédients sont des mélanges complexes riches en peptides hydrolytiqueset en acides aminés libres, mais dont la composition varie en fonction de l'origine de la matière première etdes paramètres d'hydrolyse appliqués lors de leur fabrication. Au cours de ces travaux de thèse, nous avons développéet mis en pratique des outils permettant d'approfondir la caractérisation structurale et les propriétés fonctionnellesdes hydrolysats de protéines. Dans un premier temps, nous avons développé une méthode analytique basée sur unenormalisation des échantillons suivie d'une détermination de l'abondance et de la richesse en peptides par chromatographied'exclusion stérique et chromatographie liquide couplée à la spectrométrie de masse de type électro-spray,respectivement. Les résultats présentés sous forme d'un diagramme 2D permettant de classer et comparer facilementles hydrolysats de protéines de forme galénique, d'origine et de process différents. Nous avons également développé unoutil in vitro sur l'intestin de bar européen, Dicentrarchus labrax, permettant de déterminer les activités myotropesdes hydrolysats. Nous avons pu notamment démontrer que l'hydrolysat de crevettes possède une plus forte activitémyotrope que les hydrolysats de poissons et que cette activité est portée par un pentapeptide KNPEQ clivé à partirde l'hémocyanine lors du process d'hydrolyse appliqué sur les co-produits de crevette. Enfin, dans un second temps,un conditionnement alimentaire de 65 jours a été conduit chez le bar européen nourri avec un aliment pauvre en farinede poisson supplémenté en hydrolysat de différentes origines et couplé à une analyse d'expression génique (approcheen RNA-Seq Illumina). Cette étude a permis de montrer que les hydrolysats de protéines appliqués sur un alimentfaible en farine de poisson (5%) sont capables de restaurer des performances de croissance équivalentes à celles d'unrégime contenant 20% de farine de poisson mais qu'ils portent par ailleurs des propriétés fonctionnelles spécifiques.Il a également été montré que le mélange des deux hydrolysats permet de moduler les transcriptomes intestinal ethépatique de façon plus profonde que lorsque que les hydrolysats sont utilisés séparément. Ces résultats confirmentl'intérêt des hydrolysats de protéines pour la formulation d'aliment à faible teneur en farines de poisson et apportentde nouveaux outils de caractérisation de ces ingrédients complexes, qui seront utiles pour leur optimisation et leurstandardisation ainsi que pour la compréhension de leurs mécanismes d'action chez les poissons. / Aquaculture is a key sector for supporting and improving the food security worldwide. The global production of farmedfish and shrimp has grown dramatically over the past decades and now contributes to half of the aquatic productsintended for human consumption. Aquaculture will require feeds to support its growth but availability of some raw materialssuch as fish meal are limited. The use of fish meal in aquafeed has particularly declined in favor of plant proteinsources to fit with economic and environmental constraints. But plant proteins do not meet perfectly the nutritionalrequirements of carnivorous fish and their utilization often results in lower growth and feed performances. Proteinhydrolysates manufactured from fishing and aquaculture co-products are ingredients with a high palatable, nutritionaland bioactive potential. They are rich in hydrolytic peptides and free amino acids, but they are complex mixtureswhose composition could vary according to raw material origin and hydrolysis parameters. During this PhD study, wedeveloped and implemented tools to further characterize the structure and functional properties hydrolysates. On afirst step, we developed a fast methodological tool based on sample standardization, followed by the determination ofthe abundance and richness of peptides using size exclusion chromatography and liquid chromatography coupled toelectro-spray mass spectrometry, respectively. We merged the results into a 2D diagram that made it easy to classifyand compare hydrolysates having different galenic, origin and process. We also developed a tool on isolated intestinefrom European seabass (Dicentrarchus labrax ) to rank protein hydrolysates according to their myotropic property.We demonstrated that shrimp hydrolysate showed a higher myotropic activity than fish hydrolysates and that thisactivity was carried by a unique pentapeptide KNPEQ produced by the enzymatic clivage of haemocyanin during thehydrolysis process applied on shrimp co-products. On a second step, a 65-day feeding trial was conducted in Europeanseabass fed a low fish meal diet supplemented with protein hydrolysates of different origin, and coupled to a studyof the intestine and liver transcriptomic response (Illumina RNA sequencing). It has been shown that protein hydrolysatesincluded in a low fish meal diet (5%) restored growth performances to the same level than a diet containing20% of fishmeal, and that they exhibited very specific functional properties. These results showed that a mixture oftwo hydrolysates impacted more deeply the intestine and liver transcriptomes than hydrolysate tested alone. ThisphD study confirmed that protein hydrolysates are very interesting candidates for formulating low fish meal feed andoffer new tools for characterizing such complex ingredients, which will be useful to optimize and standardize proteinhydrolysate while understanding their mechanisms of action in fish.

The production and use of protein hydrolysates, derived from animal and vegetable sources in specific formulations is an area of growing interest. Protein hydrolysates can be obtained mainly by three methods: alkaline hydrolysis, enzymatic hydrolysis and acid hydrolysis. However, enzymatic hydrolysis shows to be more beneficial, and with this purpose various enzymes from different sources are used in order to obtain protein rich hydrolysates. Three proteolytic enzymes called Papaína®, Flavourzyme® and Protamex® were used for hydrolysis of mechanically separated meat MSM and manually deboned chicken carcass MDC. The hydrolysis occurred in a thermostatic bath with controlled temperature, time and pH, specific to each of the three enzymes used. Proximal composition of raw materials and lyophilized hydrolysates obtained was performed. Also were performed control analysis of hydrolysis such as degree of hydrolysis, protein, total solids, ash and characterization of amino acid hydrolysates. The results were evaluated by analysis of variance and Tukey test. Protein hydrolysates obtained from MSM and MDC had high protein content characterizing them as a promising raw material in the formulation of special diets. The highest degree of hydrolysis with MCS was obtained with Papaína, and the most efficient MDC enzyme in the enzymatic hydrolysis was Papaína, followed by Protamex and Flavourzyme. The hydrolysates obtained from the MCS with Papaína obtained higher protein and soluble solids content and lower ash content when compared to other hydrolysates with Protamex and Flavourzyme. For the raw MDC, the highest levels of proteins were found in the hydrolyzate obtained with Papaína, soluble solids did not vary between Papaína and Protamex, but the enzyme Flavourzyme presented a smaller percentage of soluble solids (p <0.05) than the other two enzymes. The highest concentration of ash from the hydrolysates obtained from MDC with Protamex and Flavourzyme (6.75% to 7.97%) is the result of pH adjustment during hydrolysis. The amino acid composition of the hydrolysates demonstrates that both CMS and CMD derived from Papain obtained a composition closer to what is recommended by the control authorities. The functional properties of the hydrolysates are highly dependent on the degree of hydrolysis. The protein solubility increased with higher degree of hydrolysis and the hydrolyzate with highest solubility was MCS obtained from Papaína. The higher water retention capacity was presented by the enzyme hydrolyzate obtained with Flavourzyme on MSM and this property is inversely proportional to the degree of hydrolysis, such as the ability to retain oil which was higher with Flavourzyme on both raw materials. The emulsifying capacity was also higher with Flavourzyme on MSM, since this property also depends on the degree of hydrolysis. Therefore, based on the results, enzymatic hydrolysis becomes a useful tool for processing meat based raw materials. / A produção e a utilização de hidrolisados proteicos, oriundos de fontes animais e vegetais, em formulações específicas é uma área de crescente interesse. Os hidrolisados proteicos podem ser obtidos basicamente por três métodos, hidrólise alcalina, hidrólise enzimática e a hidrólise ácida. Porém, a hidrólise enzimática apresenta-se mais benéfica, e com este intuito várias enzimas das mais diferentes fontes são utilizadas, com a finalidade de obter hidrolisados proteicos, ricos em proteínas. Neste estudo foram utilizadas três enzimas proteolíticas denominadas de Papaína®, Flavourzyme® e Protamex® para a hidrólise de carne mecanicamente separada (CMS) e carcaça manualmente desossada (CMD) de frango. A hidrólise ocorreu em banho termostatizado com temperatura, tempo e pH controlados e específicos para cada uma das três enzimas utilizadas. Foi determinada a composição proximal das matérias-primas e dos hidrolisados liofilizados obtidos, também foram realizadas análises de controle da hidrólise como grau de hidrólise, teores de proteínas, sólidos totais, cinzas e caracterização de aminoácidos dos hidrolisados. Os resultados foram avaliados por análise de variância e teste de médias de Tukey. Os hidrolisados proteicos obtidos da CMS e da CMD de frango apresentaram alto conteúdo proteico caracterizando-se como uma matéria-prima promissora na formulação de dietas especiais. O maior grau de hidrólise com a CMS foi obtido com a Papaína, e para a CMD a enzima mais eficiente na hidrólise enzimática foi a Protamex seguida da Papaína e da Flavourzyme. Os hidrolisados obtidos a partir da CMS com a Papaína obtiveram maior teor proteico, de sólidos solúveis e menor teor de cinzas quando comparados com outros hidrolisados obtidos com a Flavourzyme e a Protamex. Para a matéria prima CMD, os maiores teores de proteínas foram encontrados no hidrolisado obtido com a Papaína, os sólidos solúveis não variaram entre a Papaína e Protamex, porém a enzima Flavourzyme apresentou um percentual de sólidos solúveis menores (p<0,05) que as outras duas enzimas. A maior concentração de cinzas dos hidrolisados provenientes da CMD obtidos com a Flavourzyme e com a Protamex (6,75% a 7,97%) é decorrente do ajustamento do pH durante a hidrólise. A composição de aminoácidos demonstra que os hidrolisados oriundos tanto da CMS como da CMD obtidos da Papaína possuem uma composição mais próxima da recomendada pelos órgãos de controle. As propriedades funcionais dos hidrolisados são altamente dependentes do grau de hidrólise. A solubilidade proteica aumentou com o aumento do grau de hidrólise e o hidrolisado que apresentou maior solubilidade foi com a CMS e com a papaína como enzima. A maior capacidade de retenção de água foi apresentada pelo hidrolisado obtido com a enzima Flavourzyme na CMS, e esta propriedade é inversamente proporcional ao grau de hidrólise. Da mesma forma a capacidade de retenção de óleo foi maior para a Flavourzyme, nas duas matérias-primas utilizadas. A capacidade emulsificante também foi maior para a Flavourzyme na CMS, já que esta propriedade também depende do grau de hidrólise. Assim, com base nos resultados obtidos conclui-se que a hidrólise enzimática torna-se uma ferramenta bastante útil na transformação das matérias-primas cárneas.