Biostimulants offer an innovative approach to potentially improve crop yield and quality under abiotic stresses. Particularly, plant-derived protein hydrolysates (PH), a mixture of amino acids and soluble peptides from enzymatic or chemical hydrolysis of agricultural waste, are gaining global interest due to their sustainability and positive effects on crops. However, a functional role of the PH in crop yield and quality remains uncertain and is proposed to be associated with its phytohormone-like activities or serve as an additional nitrogen (N) source. Besides, the effects of PH on crop yield and quality are limited in intensive production systems such as greenhouse facilities. The purposes of this research are to examine the effects and mechanisms of PH on crops and to assess the potential of PH application to reduce fertilizer use in crop production. The specific objectives were to; 1) elucidate the hormone-like activities of PH in the adventitious rooting formation of cuttings, 2) evaluate the effects of different PH application methods on greenhouse crop yield and quality under different N levels when plants are grown with a commercial growing medium, and 3) examine the effects of PH application methods on yield and quality of hydroponically grown lettuce under different N levels and forms. Three conclusions were that 1) <a>the hormonal effects of PH are attributed to brassinosteroid-mediated processes, and PH has overlapping functions with auxin during adventitious rooting of cuttings in a plant species-specific manner</a>, 2) root application of PH (PH-R) effectively improves nutrient uptake compared to foliar spray of PH (PH-F), subsequently, increases the lettuce and tomato yield and quality regardless of N levels while PH-R did not change the chemical properties of growing media, and 3) PH-R effectively increases root growth, and subsequently, improving shoot yield and quality with significant PH × N levels and PH × NO₃:NH₄ratios interactions. Also, PH-R counteracted the negative effects of low NO₃:NH₄ratios on lettuce yield. The outcomes provide the optimization of PH and N fertilization in modern sustainable greenhouse production and the development of a new strategy for producing high-quality greenhouse crops with improved nutrient use efficiency.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24678号 / 農博第2561号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5459(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 教授 舟場 正幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The heavy reliance on live feeds is currently restricting the growth and sustainability of the aquaculture industry, therefore, the overall goal of this research was to improve the utilization of formulated dry diets at first feeding of larval fish. This was done with a specific focus on the production and provision of the optimal dietary protein form and composition. Chapter 2 aimed to provide an efficient protein source for larval fish by using same-species muscle and endogenous enzymes to produce hydrolysates and by providing a series of diets with increasing molecular weight protein fragments through larval development. Largemouth Bass (Micropterus salmoides) (LMB) muscle was mixed with the digestive enzymes from adult LMB and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, an intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. The molecular weight profile of those diets were formulated to vary based on the inclusion level of each hydrolysate. To account for gut development, one group of larval LMB was fed a weekly series of diets with an increasing molecular weight profile. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The results from this study suggest that the inclusion of same-species hydrolysates can improve the initial growth of first-feeding LMB, but further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds to improve the overall growth performance during the larval stage. Chapter 3 compared the effect of dietary inclusion of a fish muscle hydrolysate produced from species-specific muscle and enzymes to hydrolysates produced from those of a different species, in diets for larval Walleye (Sander vitreus). Four intact and hydrolyzed protein products were produced from each combination of Walleye muscle and endogenous enzymes, and muscle and endogenous enzymes from Nile Tilapia (Oreochromis niloticus). The hydrolyzed products were continuously mixed for 3 h during the hydrolysis, (at 22oC and 28oC for Walleye and Tilapia enzymes, respectively), and the pH was adjusted throughout the process to mimic gastric and intestinal digestion conditions. Four diets were produced with the dietary protein supplied as a 50/50 ratio of the intact and hydrolyzed muscle from the respective muscle/enzyme combination. There was a significant interaction effect between muscle and enzyme source on the growth of larval Walleye. At the conclusion of the study, the larval Walleye that received the diet with muscle hydrolysate produced with Walleye muscle and Walleye endogenous enzymes had a significantly higher average weight than all other groups, and significantly higher postprandial levels of total free amino acids and indispensable amino acids in the muscle. Each hydrolysate-based diet led to a significant reduction in skeletal deformities and survival, compared to a group fed with a commercial diet. The results from this study suggest that species-specific muscle and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products, but further research should focus on improving the physical properties of the formulated diets to improve survival of fish larvae. Chapter 4 proposed a practical controlled hydrolysis method to utilize the endogenous enzymes within the fish body for the breakdown of tissues proteins, and to produce a species-specific meal that is tailored to the nutritional requirements and absorptive capacity of fish larvae. Four Zebrafish (Danio rerio) meals were produced from whole-body adult Zebrafish, three hydrolysates that were hydrolyzed for 1, 2, and 3 h, respectively, and an unhydrolyzed meal. From these meals, three diets were produced, each defined by their supply of dietary protein. The Unhydro diet was solely based on the unhydrolyzed Zebrafish meal. The 50% Hydro diet was based on 50% Zebrafish hydrolysate mix and 50% unhydrolyzed Zebrafish meal. The 100% Hydro diet was 100% based on the Zebrafish meal hydrolysate. The hydrolysate mix contained equal parts of the 1, 2, and 3 h hydrolysates. Proteomic analysis showed that the proposed hydrolysis method was able to efficiently hydrolyze the protein within Zebrafish body. The feeding trial found no significant differences in the final weight, total length, or survival between the Unhydro, 50% Hydro, and 100% Hydro groups, but the 50% Hydro group did express a significant upregulation of PepT1 at 24 h after feeding, compared to the Unhydro group. The growth results paired with PepT1 gene expression potentially indicate Zebrafish larvae to be adapted to dry feeds at first feeding and able to utilize dietary protein in different molecular forms efficiently for growth. Overall, the proposed hydrolysis method provides a practical and cost-effective approach to producing species-specific fishmeal hydrolysates. Further research is necessary to determine whether the produced hydrolysates can improve the growth of larval fish in other fish models. Further insight into behavioral and physiological responses in fish to imbalanced dietary amino acid profiles was provided in Chapter 5. The objective of this study was to determine how stomachless fish respond to diets deficient in the main limiting IDAA (lysine, methionine, and threonine), using Zebrafish as a model species. Six semi-purified diets were formulated for this study. The CG diet contained casein and gelatin as its only protein sources, while FAA50 diet had 50% of is dietary protein supplied with crystalline amino acids. Both were formulated to contain identical, balanced amino acid profiles. The remaining diets were supplied with the same amino acid mix as the FAA50 diet, but with minor adjustments to create deficiencies of the selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the free amino acid (FAA) mix, respectively, and the Def diet was deficient in all three. The fish were fed to apparent satiation three times a day, and each feeding was carefully observed to ensure all feed added to the tanks was consumed. The results showed that although the singular deficiency of the three main limiting amino acids did not induce significant changes in feed intake, the combined deficiency of the three IDAA significantly increased the feed intake of juvenile Zebrafish. This increased feed intake prevented the IDAA deficiencies from significantly reducing growth, however, the feeding efficiency was also reduced. There was also an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group, compared to the FAA50 group. The outcomes of this study provide insight into the behavioral and physiological response to dietary amino acid imbalances of stomachless fish and suggests stomachless fish increase their feed intake when challenged with IDAA-deficient diets, and that the regulation of NPY might play a role in this response. Chapter 6 assessed the postprandial FAA dynamics in the plasma, liver, and muscle of three species; 1) Largemouth Bass – warm-water, stomach-possessing carnivorous species; 2) Walleye – cool-water, stomach-possessing carnivorous species; and 3) Zebrafish– tropical, stomachless omnivorous species. Two diets were formulated for this study, a diet based on intact casein and gelatin (CG), and a diet with 50% of its protein supplied in FAA form (FAA50). Forty-two fish from each species were utilized, with one group of 21 receiving the CG diet, and the other 21 receiving the FAA50 diet. All fish were starved for 24 hours prior to the final feeding before sampling. Three fish were sampled at each time point, with three samples (plasma, liver, and muscle) taken from each fish. Samples were taken prior to feeding (0 h) and then at 0.5, 1, 2, 3, 6, and 12 h after feeding, for all species. A significant three-way interaction was observed between the diet, species, and postprandial time on the total FAA, IDAA, and DAA levels in the plasma, liver, and muscle, indicating that the postprandial FAA patterns were significantly different between species and in response to the different diets. In stomach-possessing species, dietary amino acids from the FAA50 diet were absorbed more rapidly than those from the CG diet, resulting in fewer correlations with the dietary IDAA profiles. The absorption of FAA in cool-water Walleye was more gradual and prolonged than the warm-water LMB, leading to more significant correlations with the dietary IDAA and more sustained peaks. The postprandial peaks of FAA typically occurred at the same time in the stomachless Zebrafish fed with the CG or FAA50 diet. The levels of FAA were noticeably lower after feeding with the FAA50 diet in Zebrafish, compared to the CG diet. These results provide a reference for differences in the FAA dynamic patterns of three species with differing physiological characteristics, when fed diets with intact protein or supplemented with FAA. The findings presented in this dissertation provide support and novel methods for the production and inclusion of species-specific protein hydrolysates as an ideal protein source in formulated diets for first-feeding larval fish. This research contributes to the development of larval diets that can release the limitations of growth placed on the aquaculture industry by the reliance on live feeds, particularly within the hatchery sector. This research also provides further understanding of dietary protein utilization and delivers new fish nutrition knowledge that will benefit the aquaculture industry as a whole.

Les hydrolysats protéiques ont une haute valeur ajoutée pour des secteurs industriels variés, de par leurs propriétés nutritives, fonctionnelles et / ou nutraceutiques. Pour améliorer les propriétés des hydrolysats, l’ultrafiltration est utilisée. Cependant, le manque d’outils de modélisation lié à la complexité des mélanges est un verrou pour une mise en œuvre rationnelle du procédé. Ces travaux ont permis de valider une stratégie de prédiction basée sur des caractéristiques classiques des hydrolysats et un étalonnage expérimental de la membrane d’ultrafiltration. Cette méthode permet de prédire les rendements et enrichissements en fraction(s) ou peptide(s) cible(s), ainsi que la productivité du procédé. Le modèle global de prédiction de l’ultrafiltration obtenu est alors utilisé afin d’optimiser la mise en œuvre de ce procédé. La démarche d’optimisation consiste à maximiser l’enrichissement de fractions ou de peptides cibles en minimisant la consommation d’eau et la durée du procédé / Protein hydrolysates are high added value mixtures for various industrial areas, thanks to their nutritive, functional or nutraceutical properties. To enhance hydrolysates performances, fractionation processes such as ultrafiltration are used. However, the lack of tools to predict ultrafiltration performances is a major bottleneck for a rational implementation of the process. This research thesis work enables to validate a prediction strategy based on classical characteristics of hydrolysates and an experimental calibration of the membrane. Yields and enrichment factors in targeted peptides or fractions during ultrafiltration as well as the productivity of the process can be predicted. This global methodology of performances prediction is then used to optimize the implementation modes of ultrafiltration. The multiobjective optimization approach consists in maximizing the enrichment in targeted peptides or fractions while water consumption and / or process duration is minimized

Hemp seed proteins (HSP) were investigated for physicochemical and functional properties in model food systems. In addition, the HSP were enzymatically digested and the released peptides investigated as potential therapeutic agents. Membrane isolated HSP (mHPC) were the most soluble with >60% solubility at pH 3-9 when compared to a maximum of 27% for isoelectric pH-precipitated proteins (iHPI). However, iHPI formed emulsions with smaller oil droplet sizes (<1 µm) while mHPI formed bigger oil droplets. The iHPI was subjected to enzymatic hydrolysis using different concentrations (1-4%) of six proteases (pepsin, pancreatin, flavourzyme, thermoase, papain and alcalase) to produce various HSP hydrolysates (HPHs). HPHs had strong in vitro inhibitions of angiotensin converting enzyme (ACE) and renin activities, the two main enzyme systems involved in hypertension. Oral administration of the HPHs to spontaneously hypertensive rats led to fast and persistent reductions in systolic blood pressure. The HPHs also inhibited in vitro activities of acetylcholinesterase (AChE), a serine hydrolase whose excessive activities lead to inadequate level of the cholinergic neurotransmitter, acetylcholine (ACh). Inadequate ACh level in the brain has been linked to neurodegenerative diseases such as dementia and Alzheimer’s disease (AD); therefore, AChE inhibition is a therapeutic target. The 1% pepsin HPH was the most active with up to 54% AChE inhibition at 10 µg/mL peptide concentration. The 1% pepsin HPH (dominated by <1 kDa) was subjected to reverse-phase HPLC peptide purification coupled with tandem mass spectrometry, which led to identification of several peptide sequences. Some of the peptides inhibited activities of both animal and human AChE forms with LYV being the most potent against human AChE (IC50 = 7 µg/ml). Thus the LYV peptide may serve as a useful template for the development of future potent AChE-inhibitory peptidomimetics. In conclusion, several novel AChE-inhibitory peptides were discovered and their amino acid sequences elucidated for the first time. Results from this work identified HSP products that could serve as functional ingredients in the food industry. The work also produced and confirmed the in vitro AChE-inhibitory activities of several new peptide sequences that may serve as therapeutic agents for AD management. / October 2015

Orientador: Jaime Amaya-Farfan / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-09T15:53:11Z (GMT). No. of bitstreams: 1 Carvalho_IaraRibeiro_M.pdf: 1875369 bytes, checksum: 01034f576b4ca76425fe296fed74054e (MD5) Previous issue date: 2008 / Resumo: Estudos recentes sugerem que o consumo das proteínas do soro de leite, quando parcialmente hidrolisadas, resulta em efeitos fisiológicos diferentes daqueles produzidos pelas proteínas intactas. Desse modo, o objetivo do presente trabalho foi verificar as alterações metabólicas causadas nos tecidos do estômago e coração, e na resistência à exaustão, utilizando ratos Wistar alimentados com isolado do soro de leite, ou seu proteolisado enzimático, e submetidos a exercício físico. Foram investigadas possíveis alterações na atividade enzimática da pepsina, glicogênio sintase, glicogênio fosforilase, creatina quinase e glicogênio do miocárdio e a perfusão de peptídeos através do estômago. O ensaio biológico teve duração de 42 dias, com 120 ratos divididos em 12 grupos (n=10), sendo três fontes protéicas: caseína (C), proteína hidrolisada (H), proteína intacta do soro de leite (I) e quatro tipos de atividade física: treinado (T), treinado-exausto (TX), sedentário (S), sedentário-exausto (SX), em esteira rolante, por quatro semanas. Foram observados valores mais altos na atividade da enzima creatina quinase (sem diferença significativa) nos grupos T e TX, quando esses consumiram dieta H. Em relação ao treinamento, foram encontradas maiores quantidades de glicogênio miocárdico nos animais submetidos a menor atividade física. Em relação à dieta, as concentrações de glicogênio variaram aleatoriamente. Entretanto, os animais que consumiram a dieta H exibiram menor atividade da glicogênio sintase do que aqueles que receberam dieta I, e esses também menores que a dieta C. Na enzima glicogênio fosforilase, notou-se valores maiores de atividade enzimática nos grupos de animais do grupo T em relação aos outros grupos, sendo que a dieta H mostrou resultados menores em comparação com as outras dietas. Quanto à exaustão, foram comparados os grupos H e I, sendo que os grupos que foram submetidos a treinamento prévio (TX) mostraram-se mais resistentes à exaustão do que aqueles que foram mantidos sedentários (SX). Na atividade enzimática da pepsina, os resultados entre grupos foram semelhantes, notando-se aumento no grupo sedentário que consumiu o hidrolisado, mas desaparecendo com a exaustão. Estudo da possibilidade de perfusão de peptídeos através da parede estomacal encontrou evidências de que, ao menos, um peptídeo rico em valina é detectado no perfusado externo ao cabo de duas horas. Foi observada maior quantidade de picos eletroforéticos no conteúdo do estômago dos animais dos grupos SX e TX que receberam infusão do hidrolisado e teores de aminoácidos livres mais elevados no grupo S do que nos demais. Associando os valores encontrados para aminoácidos livres com os valores de aminoácidos totais, no interior e exterior do estômago, pode se afirmar que o conteúdo de peptídeos formados no estômago, no grupo que recebeu a infusão de hidrolisado, foi superior à que recebeu o isolado. Conclui-se que ambos o tipo de atividade física e a fonte protéica da dieta podem influenciar aspectos fisiológicos, tais como a atividade da pepsina, a facilidade com que peptídeos são gerados e acumulados no órgão, sendo que alguns peptídeos podem atravessar a parede estomacal. As implicações decorrentes destes fenômenos ainda aguardam maior investigação / Abstract: Recent studies suggest that the consumption of milk whey proteins, if partially hydrolyzed, result in different physiological effect from those produced by the ingestion of the unbroken proteins. Therefore, the objective of the present work was to verify eventual metabolic alterations caused in the stomach and heart tissues of exercising Wistar rats fed an enzymatic milk whey hydrolyzate, as compared to cohorts receiving the unhydrolyzed proteins. The enzymatic activity of pepsin and those of glycogen synthase, glycogen phosphorylase, creatine kinase, as well as glycogen stores of the myocardium were thus investigated. Additionally, the perfusion of peptides through the stomach wall using capillary electrophoresis and liquid chromatography was also verified. The biological assay was conducted with 120 rats, during 42 days. The animals were divided into 12 groups (n=10), as follows: three protein sources: casein (C), protein hydrolyzate (H) and the unbroken whey protein (I), in addition to four types of physical activity: trained (T), trained-exhausted (TX), sedentary (S), sedentary-exhausted (SX) for four weeks. For creatine kinase, higher activities were observed (without significant difference) in groups T and TX, when these consumed diet H. With regard to myocardial glycogen, higher stores were found in the animals with lesser physical activity, while glycogen concentrations varied randomly in response to the diet. However, the animals that consumed diet H exhibited lower glycogen synthase activity in comparison to those that received diet I, which in turn were lower than those that received diet C. As for glycogen phosphorylase, higher values were noticed in the groups subjected to training (T) in relation to the other groups. Similarly, the animals on diet H also responded with lower activities. With regard to exhaustion time, only those groups that underwent training (TX) appeared to be more resistant to exhaustion than those that remained sedentary (SX). The enzymatic activity of pepsin did not show significant differences among groups, except for the increase of the sedentary that consumed diet H. The increase, however, disappeared when the animals were brought to exhaustion. Study of the possibility of perfusion of whey protein peptides through the stomach wall suggested that at least one peptide rich in valine promptly perfused to the external fluid. A higher number of capillary electrophoretic peaks were also observed in the stomach contents of the animals of groups SX and TX that received the hydrolyzate infusion and, by liquid chromatography, it was possible to notice that the group S stomachs had greater levels of free amino acids than the other groups. Comparing the values found for free amino acids with the total amino acids, in both the inside and outside of the stomach, it could be stated that the content of peptides formed in the stomach was considerably greater in the group infused with the hydrolyzate than with the isolate. It is concluded that chronic consumption of the whey protein hydrolyzate as the only source of protein results in enzymatic changes consistent with a more efficient physiologic state, favorable to higher physical performance, such as higher myocardial creatine kinase and lower glycogen kinase and phosphorylase activities. No significant changes in pepsin activity in the rat stomach were observed, but the readiness with which the hydrolyzate peptides accumulate and traverse the organ wall was evident. Further data to provide a better understanding of the implications of consuming prehydrolyzed proteins await investigation / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Orientadores: Flávia Maria Netto, Maria Teresa Bertoldo Pacheco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T00:05:18Z (GMT). No. of bitstreams: 1 Caetano-Silva_MariaElisa_M.pdf: 1696296 bytes, checksum: 13e371ea02446fff6b254e550f086832 (MD5) Previous issue date: 2013 / Resumo: A deficiência de ferro é um dos principais problemas nutricionais no mundo, sendo a suplementação de alimentos com sais de ferro uma importante estratégia para combater essa deficiência. Porém, nessa forma, o mineral apresenta baixa biodisponibilidade e pode causar dor de estômago, diarreia, alterações de sabor e aparência dos produtos. Quelatos ferro-peptídeos têm sido apontados como uma promissora fonte de ferro mais biodisponível e com redução desses efeitos adversos. O presente estudo teve por objetivo avaliar o potencial de quelação de ferro dos peptídeos obtidos da hidrólise enzimática de isolado proteico de soro de leite (IPS) com as enzimas alcalase (HA), pancreatina (HP) ou flavourzyme (HF). Os hidrolisados foram ultrafiltrados (membrana de corte de 5 kDa) e as frações permeada (< 5 kDa) e retida (> 5 kDa) foram liofilizadas. Os hidrolisados e suas frações foram caracterizados quanto ao perfil aminoacídico, perfil de hidrofilicidade por cromatografia líquida de alta eficiência em fase reversa (CLAE-FR), perfil de massa molecular (MM) por cromatografia líquida de alta eficiência de exclusão molecular (CLAE-EM) e eletroforese SDS-PAGE Tricina. As frações foram avaliadas quanto à sua capacidade quelante de ferro, utilizando-se FeCl2 na proporção 40:1 proteína:Fe, pH 7,0, 25±2 °C/1h sob agitação, seguida de centrifugação. Para avaliação do ferro livre e/ou fracamente ligado aos peptídeos, o pH dos sobrenadantes da reação de quelação foi ajustado para 3,5 e o Fe2+ solúvel foi determinado. Foi selecionado o hidrolisado com maior capacidade quelante (frações HP > 5 kDa e HP < 5 kDa) para o prosseguimento do trabalho. Para avaliação da estabilidade do quelato, essas frações foram submetidas à digestão gástrica in vitro e posterior neutralização (pH 7,0), seguida de centrifugação. Os peptídeos das amostras selecionadas foram isolados por cromatografia de afinidade por íons metálicos imobilizados (IMAC-Fe3+). Os peptídeos com afinidade pelo ferro foram sequenciados por espectrometria de massas (MS/MS). O grau de hidrólise (GH) dos hidrolisados HA, HP e HF foi 16,8%, 16,4% e 9,1%, respectivamente. O perfil eletroforético das frações < 5 kDa não apresentou bandas, enquanto as frações > 5 kDa apresentaram bandas de MM inferior; porém, por CLAE-EM, foi verificado que todas as frações apresentaram peptídeos de MM aparente superior a 5 kDa, sugerindo que nas condições usadas houve formação de agregados. Na reação de quelação, HP > 5 kDa reteve 70,6% do ferro em solução, enquanto as demais frações variaram entre 37,4% e 66,1%. A fração HP > 5 kDa apresentou maior teor de ferro precipitado em pH 3,5 (65,3%), sugerindo maior interação peptídeos-ferro. A mesma amostra, após digestão gástrica, apresentou solubilidade do ferro inicialmente presente entre 57,8 e 59,0% em pH 7,0, sugerindo que a digestão com ou sem pepsina não desfez totalmente o complexo formado. Esse teor foi superior ao obtido com HP < 5 kDa (40,1 a 43,0%), bem como ao ensaio controle com FeCl2 (9,9%). No isolamento de peptídeos por IMAC-Fe3+, verificou-se maior teor de peptídeos com capacidade quelante de ferro na fração HP > 5 kDa (70%) do que na fração HP < 5 kDa (50%). O sequenciamento por MS/MS mostrou, em todos os fragmentos, presença de Glu e/ou Asp, cujos grupos carboxílicos estão entre os principais sítios de ligação com o ferro. Os resultados sugerem que a hidrólise do IPS com pancreatina origina peptídeos com alta capacidade quelante de ferro. Esses peptídeos podem ser usados para obtenção de quelatos Fe2+-peptídeos que, futuramente, sejam aplicados para fortificação de alimentos no intuito de elevar a biodisponibilidade do ferro, além de potencialmente reduzir seus efeitos pró-oxidantes / Abstract: Iron deficiency is one of the major nutritional problems in the world, being the food supplementation with iron salts an important strategy to combat this deficiency. However, in salt form, this mineral has low bioavailability and may lead to stomachache, diarrhea and even cause changes in flavor and appearance of food products. Iron-peptides chelates have been suggested as a promising source of more bioavailable iron, reducing these side effects. This study aimed at evaluating the iron-binding ability of peptides obtained from enzymatic hydrolysis of whey protein isolate (WPI) with alcalase (AH), pancreatin (PH) or flavourzyme (FH). Hydrolysates were ultrafiltered in 5 kDa membrane and permeate (< 5 kDa) and retentate (> 5 KDa) fractions were lyophilized. Hydrolysates and their fractions were characterized by aminoacidic profile, hydrophilicity profile by reversed-phase high performance liquid chromatography (RP-HPLC), molecular weight (MW) profile by size-exclusion high performance liquid chromatography (SE-HPLC) and SDS-PAGE Tricine). Fractions were evaluated by iron-binding ability using FeCl2 (40:1 protein:Fe ratio) at pH 7.0 and 25±2 °C for 1h under stirring, followed by centrifugation. For evaluation of free and/or weakly bound iron, the pH of the supernatant from the chelation reaction was adjusted to 3.5 and soluble Fe2+ was determined. The hydrolysate with higher iron-binding ability was selected (fractions PH > 5 kDa and PH < 5 kDa) for further proceeds. To evaluate the chelate stability, these fractions were subjected to in vitro gastric digestion and further neutralization, followed by centrifugation. The peptides of selected samples were isolated by immobilized metal affinity chromatography (IMAC-Fe3+). The peptides with iron-binding affinity were sequenced by mass spectrometry (MS/MS). The degree of hydrolysis (DH) of hydrolysates AH, PH and FH was 16.8%, 16.4% and 9.1%, respectively. Electrophoretic profile of fractions < 5 kDa did not present any band, while fractions > 5 kDa presented peptides with lower MW. However, by SE-HPLC, it was verified peptides with apparent MW above 5 kDa for all samples, suggesting that, under the conditions studied, there was aggregates formation. In the chelation reaction, PH > 5 kDa retained 70.6% of iron in solution, while other samples ranged from 37.4% to 66.1%. PH > 5kDa showed higher content of precipitated iron in pH 3.5 (65.3%), suggesting greater peptide-iron interaction. After gastric digestion, the same sample showed initial iron solubility ranging from 57.8 and 59.0% in pH 7.0, suggesting that digestion with or without pepsin was not able to completely break the complex formed. This content was higher than that obtained in both PH < 5 kDa (40.1 to 43.0%) and the control assay with FeCl2 (9.9%). IMAC-Fe3+ isolation showed higher content of iron-binding peptides in PH > 5 kDa (70%) than in PH < 5 kDa (50%). The MS/MS sequencing showed Glu and/or Asp in all fragments, which carboxylic groups are among the main iron-binding sites. The results suggest that WPI hydrolysis with pancreatin yields peptides with high iron-binding ability. These peptides may be used for obtaining iron-peptide chelates, which, in future, may be applied in food fortification in order to increase iron bioavailability and potentially reduce its pro-oxidant effects / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T13:38:26Z (GMT). No. of bitstreams: 1 Castro_RuannJanserSoaresde_D.pdf: 3791426 bytes, checksum: 08b166335935f4a40427f1ddea405399 (MD5) Previous issue date: 2015 / Resumo: O presente trabalho teve como objetivo utilizar a técnica de delineamento experimental de misturas como estratégia para a produção de proteases por Aspergillus niger LBA02 em fermentação semissólida utilizando formulações contendo diferentes resíduos agroindustriais e produção de hidrolisados proteicos com atividades biológicas e funcionais utilizando a hidrólise enzimática simultânea de proteínas de diferentes fontes. Efeitos sinérgicos e significativos entre as misturas quaternárias de farelo de trigo, farelo de soja, farelo de algodão e casca de laranja foram observados durante a fermentação de A. niger LBA02, atingindo aumentos de 33,7, 7,6, 30,8 e 581,7%, respectivamente, na produção de proteases em comparação com os substratos utilizados de forma isolada. O estudo das características bioquímicas das preparações enzimáticas mostrou que a linhagem de A. niger LBA02 foi capaz de secretar diferentes tipos de proteases em resposta a cada substrato. De um modo geral, as proteases apresentaram atividade ótima a 50 °C e na faixa de pH de 3 a 4. As maiores diferenças entre as preparações de proteases foram observadas para os parâmetros cinéticos e termodinâmicos de ativação e inativação térmica. Na hidrólise enzimática de misturas contendo proteína isolada de soja, proteínas do soro de leite e da clara de ovo utilizando a preparação comercial Flavourzyme® 500L foram observados efeitos sinérgicos entre as formulações contendo misturas binárias ou ternárias, para vários parâmetros. Para atividade antioxidante determinada pelo método DPPH, a mistura contendo proteínas do soro de leite e proteínas da clara de ovo apresentaram aumentos de 45,1 e 37,3% na atividade, quando comparada aos hidrolisados obtidos com as duas fontes de forma isolada, respectivamente. Entre as propriedades funcionais, a capacidade emulsificante foi a que apresentou maior efeito sinérgico, onde os hidrolisados contendo a mistura ternária de proteína isolada de soja, proteínas do soro de leite e da clara de ovo, alcançaram valores 2 a 12 vezes superiores, em relação aos hidrolisados obtidos de forma isolada. A determinação da atividade anti-adipogênica dos hidrolisados revelou que o tratamento de células pré-adipócitas 3T3-L1 com a mistura binária de proteínas do soro de leite e da clara de ovo na concentração de 1.200 ppm reduziu o acúmulo relativo de lipídeos nas células em até 47,9%. Em relação à atividade antimicrobiana, a linhagem de Staphylococcus aureus ATCC 6538 foi a única que apresentou inibição do crescimento quando cultivada em meio suplementado com uma mistura binária de proteína isolada de soja e proteínas da clara de ovo não hidrolisadas, resultando em inibição de 16,82%. Os hidrolisados obtidos com misturas binárias de proteínas do soro de leite e da clara de ovo ou proteína isolada de soja e soro de leite estimularam o crescimento de bactérias lácticas e probióticas, resultando em aumentos de 29,4 a 100% comparados aos meios não suplementados. A utilização de composições contendo diferentes preparações comerciais de proteases para hidrólise de proteína isolada de soja e estudo da atividade antioxidante mostrou diferentes resultados para cada método utilizado. Para inibição dos radicais DPPH, os hidrolisados obtidos com Flavourzyme® 500L combinada com Alcalase® 2.4L mostraram o maior efeito sinérgico, com aumentos de 10,9 e 13,2% da atividade antioxidante, em comparação aos hidrolisados produzidos com as enzimas isoladas. Os hidrolisados obtidos utilizando a mistura ternária de Flavourzyme® 500L, Alcalase® 2.4L e YeastMax® A apresentaram o maior poder de inibição da auto-oxidação do ácido linoleico / Abstract: This study aimed to use the mixture experimental design technique as a strategy to produce proteases by Aspergillus niger LBA02 under solid state fermentation, using formulations containing different agroindustrial wastes. It also aimed to produce protein hydrolysates with biological and functional activities using the simultaneous enzymatic hydrolysis of proteins from the different sources. Synergistic and significant effects between the quaternary mixtures of wheat bran, soybean meal, cottonseed meal and orange peel were observed during fermentation by A. niger LBA02, reaching increases of 33.7, 7.6, 30.8 and 581.7%, respectively, for the production of proteases as compared to the isolated substrates. The study of the biochemical properties of the enzyme preparations showed that the strain of A. niger LBA02 was able to secrete different types of proteases in response to each substrate. In general, the proteases showed optimal activity at 50 °C in the pH range from 3 to 4. The major differences between the protease preparations were observed for the kinetic and thermodynamic parameters of thermal activation and inactivation. In the enzymatic hydrolysis of mixtures containing soy protein isolate, bovine whey protein and egg white protein using the commercial preparation FlavourzymeTM 500L, synergistic effects were observed for various parameters between formulations containing binary or ternary mixtures,. For antioxidant activity as determined by the DPPH assay, the mixture containing bovine whey protein and egg white protein showed increases of 45.1 and 37.3% in their activities as compared to hydrolysates obtained with the isolated proteins, respectively. Of the functional properties, the emulsifying capacity showed the greatest synergistic effect, the hydrolysates containing the ternary mixture of soy protein isolate, bovine whey protein and egg white protein, showing increases ranging from 2 to 12-fold as compared to the hydrolysates obtained using isolated substrates. The determination of the anti-adipogenic activity of the hydrolysates indicated that the treatment of 3T3-L1 preadipocyte cells with 1200 ppm of the mixture containing bovine whey protein and egg white protein reduced the relative lipid accumulation to 47.9%. With respect to antimicrobial activity, the strain of Staphylococcus aureus ATCC 6538 was the only one which showed growth inhibition when cultivated in a medium supplemented with a non-hydrolyzed binary mixture of soy protein isolate and egg white protein, resulting in inhibition of 16.82%. The hydrolysates obtained with binary mixtures of bovine whey protein and egg white protein or soy protein isolate and bovine whey protein stimulated the growth of probiotic and lactic acid bacteria, reaching increases from 29.4 to 100% when compared to non-supplemented media. The use of formulations containing various commercial preparations of proteases for the hydrolysis of soy protein isolate and the study of antioxidant activities showed different results for each method. For DPPH radical scavenging, the hydrolysates obtained with FlavourzymeTM 500L combined with AlcalaseTM 2.4L showed greater synergistic effects, with increases of 10.9 and 13.2% in antioxidant activity as compared to the hydrolysates produced with individual enzymes. The hydrolysates obtained from ternary mixtures of FlavourzymeTM 500L, AlcalaseTM 2.4L and YeastMaxTM A showed the greatest power of inhibition of linoleic acid autoxidation / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

As proteínas têm sido objeto de extenso estudo e um dos passos mais importantes que se obteve industrialmente nas últimas décadas foi a produção de proteínas hidrolisadas, viabilizando sua aplicação em uma gama de produtos cosméticos e alimentícios. Os hidrolisados de proteína são incorporados em formulações destinadas aos cuidados de cabelo e pele, possuindo facilidade de penetração na cutícula dos cabelos proporcionando brilho, hidratação e condicionamento. Também são incluídos em cremes e loções para o corpo, possuindo propriedades amaciantes, hidratantes e de proteção à pele. Neste trabalho, a técnica de eletroforese capilar foi utilizada para a obtenção do perfil eletroforético de diversos hidrolisados, a saber, de soja, leite, seda, queratina, pele de animal bovino, incluindo proteína de origem marinha, variando-se condições analíticas e instrumentais. Os hidrolisados apresentaram um perfil pouco resolvido e uma investigação com extração em fase sólida foi realizada, proporcionando um fracionamento de peptídeos e, posteriormente um perfil eletroforético com maior resolução. O método de Kjeldhal foi empregado para a determinação de proteínas totais dos diversos hidrolisados de proteína estudados. Duas metodologias para a obtenção das massas molares dos hidrolisados de proteína foram implementadas, utilizando a eletroforese capilar em gel. Um dos métodos empregou o polímero linear de acrilamida para a separação de padrões de proteína na faixa de 14 a 94 kDa. Outro método desenvolvido utilizou dextran (com massa molar de 2.000.000), como rede polimérica, para a separação de padrões de proteína entre 2.512 a 16.949 Da e de 14 a 67 kDa. Os hidrolisados de proteína estudados apresentaram massa molar na faixa de 10-70 kDa. O método com detecção indireta foi desenvolvido para a separação de uma mistura de 20 aminoácidos, sendo que dezesseis aminoácidos foram efetivamente separados em dois métodos distintos. A metodologia estudada foi aplicada para a identificação dos aminoácidos dos hidrolisados de proteína (QUERATAN, SILKION, LACTOSOL, PROSOJA, PROMARIN e PROTEINAN). A quantificação dos aminoácidos foi realizada para o hidrolisado de queratina (QUERATAN), apresentando concentrações na faixa de mgL-1. / Proteins have been object of extensive investigation and in the last years, an important step has been reached from an industrial point of view, which was the production of protein hydrolysates, employed in cosmetic and nourishing products. Protein hydrolysates are employed in shampoo formulations and skin care products because they confer to the products characteristics associated to protection, conditioning and repair of the hair fiber whereas to the body they contributes to moisturization. In this work, the profile of protein hydrolysates from a variety of sources was determined by capillary electrophoresis optimizing analytical and instrument conditions. The protein hydrolysates presented a poorly resolved peak profile. However, when the hydrolisates were submitted to solid phase extraction procedures, a rich peptide mapping was obtained. The Method of Kjeldhal was employed to determine the total protein of several protein hydrolysates. Two methodologies to determine molar masses of protein hydrolysates were implemented using capillary gel electrophoresis. Acrilamide was used to separate protein standards from 14,4 to 94 kDa and dextran, a physical gel, was used to separated protein of 2,5- 16 kd and 14 - 67 kDa. Additionally, an indirect UV/vis absorbance detection methodology was developed to identify and quantify free amino acids in the protein hydrolysates.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-11-29T17:12:51Z No. of bitstreams: 1 2013 - Renata Silva Cabral de Souza.pdf: 1753830 bytes, checksum: 2dff8752eb3f7974e34f8809d059f126 (MD5) / Made available in DSpace on 2018-11-29T17:12:51Z (GMT). No. of bitstreams: 1 2013 - Renata Silva Cabral de Souza.pdf: 1753830 bytes, checksum: 2dff8752eb3f7974e34f8809d059f126 (MD5) Previous issue date: 2013-05-23 / CAPES / The aim of this study was to evaluate the process of protein concentration in bovine whey proteins by ultrafiltration process and subsequently the protein hydrolysate obtained by enzymatic hydrolysis to produce bioactive peptides with potential antimicrobial and antioxidant activities. For concentration process was used a ceramic ultrafiltration membrane with a molecular range cut-off of 10-20 kDa, transmembrane pressure of 5 bar and, temperature of 30 ?C, 40 ?C and 50 ?C . The optimum temperature condition was at 40 ?C. The Volume Concentrate Factor (VCF) parameter was used as a end-point of the ultrafiltration process and fixed at 2, corresponding on concentrating the initial volume twice, in volume. At the temperature of 40 ?C, VCF had a correspondence on final protein concentration on the concentrated fraction by ultrafiltration and confirmed by Bradford method. Two commercial enzymes were tested Alcalase, Flavourzyme and an equivalent mixture of both 50:50 (w/w) in the hydrolysis reaction. The hydrolysis conditions were determined according to manufacturer instructions and confirmed by other studies: 60 ?C and pH 8 for Alcalase; 50 ?C and pH 7 for Flavourzyme; 50 ?C and pH 8 for enzyme mixture with enzyme / substrate ratio (w / w) 5/100 for all enzymes. The reaction was monitored by pH Stat method. The final Degree of Hydrolysis (DH) achieved was 15%, 52% and 63% for Flavourzyme, Alcalase and enzyme mixture, respectively. Five aliquots were collected along the hydrolysis for each enzyme reaction corresponding to differents DH in order to evaluatethe antioxidant activity by ORAC and ABTS assays with final values between 597- 1092 m? TE (ABTS) and from 1615 to 2920 ?M TE (ORAC) for Flavourzyme; 998-6290 ?M TE (ABTS) and 3092-7567 ?M TE ( ORAC) for Alcalase and finally 913-2678 ?M TE (ABTS) and 2547-5903 ?M TE (ORAC) for the enzyme mixture. The samples from all hydrolysates showed no antimicrobial activity against strains of Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) and Listeria monocytogenes (ATCC 9117). / A proposta do presente trabalho foi avaliar a concentra??o das prote?nas do soro de leite bovino por ultrafiltra??o e posterior obten??o de hidrolisados proteicos deste concentrado via hidr?lise enzim?tica visando obter pept?deos bioativos com potencial atividade antimicrobiana e antioxidante. Para concentra??o das prote?nas do soro foi utilizada membrana cer?mica de ultrafiltra??o com massa molar de corte de 10-20 kDa, press?o aplicada ? membrana de 5 bar, temperaturas testadas (30 ?C, 40 ?C e 50 ?C) . A temperatura ?tima selecionada foi de 40 ?C. O Fator de Concentra??o Volum?trica foi o par?metro utilizado para indicar o final do processo de ultrafiltra??o sendo fixado em duas vezes o volume inicial da alimenta??o. Na temperatura de 40 ?C foi obtida correspond?ncia entre a concentra??o volum?trica e a concentra??o proteica final na fra??o retida pela UF, que tamb?m foi o dobro da encontrada na fra??o alimenta??o, avaliada pelo m?todo de Bradford. Foram testadas duas enzimas comerciais: Alcalase, Flavourzyme e uma mistura equivalente de ambas, na propor??o 50:50 (m/m) na rea??o de hidr?lise. As condi??es de rea??o enzim?tica foram determinadas de acordo com instru??es do fabricante e corroboradas por outros estudos em: 60 ?C, pH 8 para Alcalase; 50 ?C, pH 7 para Flavourzyme; 50 ?C, pH 8 para mistura enzim?tica e rela??o enzima/substrato (g/g) foi de 5/100 para todas as enzimas. A rea??o de hidr?lise foi monitorada pelo m?todo pH Stat. Os Graus de Hidr?lise (GH) finais alcan?ados foram de 15 %, 52 % e 63 % para Flavourzyme, mistura enzim?tica e Alcalase, respectivamente. Foram coletadas cinco al?quotas correspondentes a diferentes GH ao longo da rea??o para cada condi??o enzim?tica utilizada e avaliadas quanto a atividade antioxidante pelos m?todos ABTS e ORAC tendo valores entre 597 a 1092 ?M TE (ABTS) e 1615 a 2920 ?M TE (ORAC) para Flavourzyme, 998 a 6290 ?M TE (ABTS) e 3092 a 7567 ?M TE (ORAC) para Alcalase e por fim, 913 a 2678 ?M TE (ABTS) e 2547 a 5903 ?M TE (ORAC) para a mistura enzim?tica. Nenhuma das amostras de hidrolisado com diferentes GH apresentou atividade antimicrobiana contra cepas de Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) e Listeria monocytogenes (ATCC 9117).