Peptides represent a source of novel therapeutics for recalcitrant human diseases, but screening for bioactivity from natural or synthetic sources can be uneconomic. In contrast, in vivo expression of peptides from DNA libraries in a heterologous host such as Escherichia coli may combine production with screening. This dissertation aimed to use such an approach to discover novel bioactive peptides in a high throughput and cost-effective manner, with a focus on antimicrobials and antiaggregants as proof-of-principle. Antimicrobial peptides (AMPs) are innate defence effectors that may combat antibiotic-resistant pathogens. An inducible, autocleaving fusion tag was utilised to produce the model murine cathelicidin K2C18, along with a number of variants, which exhibited varying degrees of antimicrobial activity against a panel of microbes. Importantly, K2C18 also exhibited a bacteriostatic effect in vivo when secreted to the periplasm. This allowed for the implementation of an in vivo whole cell screen for novel AMPs, using genomic DNA libraries as an input. One putative hit, the peptide S-H4, showed similar in vivo behaviour to K2C18 and was active when added exogenously to microbial cultures. A second in vivo screen was constructed to search for inhibitors of Aβ42 aggregation, a process implicated in Alzheimer’s disease. The aggregation state of Aβ42 was coupled to the fluorescence of a chromophore fusion partner, and used to screen co-expressed peptides from a random DNA library for putative antiaggregants. Additionally, the system incorporated an internal fluorescent reference to allow ratiometric comparison between samples. Several hits were identified and further validated using flow cytometry, with work ongoing to assess their activity in vitro. Proof-of-principle of these two screens was achieved, indicating that such in vivo approaches to bioactive peptide discovery could lead to the development of new and useful therapeutics.

Master of Science / Department of Grain Science and Industry / Yonghui Li / Antioxidants are widely used in food industries to delay lipid oxidation and prevent oxidative deterioration. In recent years, growing interests in developing safe and efficient antioxidants from natural sources due to the health-related risks associated with synthetic antioxidants. Recently, peptide antioxidants have drawn growing interests as since proteins are a macronutrient with various functionalities and high consumer acceptability. A lot of dietary proteins have been validated for their antioxidant potentials especially those obtained from animal proteins, nuts and pulses. Relatively less information is available on characterizing the antioxidant profile of cereal protein, and even less for sorghum protein. Sorghum is the fifth largest crop worldwide and is the third in United States. U.S. is leading in global sorghum production and distribution, and the state of Kansas is producing nearly half of U.S. sorghum. Currently, about one third of the U.S. sorghum is being used for ethanol production, resulting in more than 450 kilotons of by-products (e.g., DDGS) annually, which were often discarded or underutilized. DDGS is a premium protein source (~ 30% protein) that could be potentially modified into value-added products such as peptide antioxidants. In this study, relevant literatures detailing the extraction of cereal proteins, enzymatic hydrolysis of proteins, purification and characterization of hydrolysates, and evaluation of antioxidant profiles were extensively reviewed in Chapter 1. As preliminary experiments, sorghum kafirin protein was extracted from defatted sorghum white flour and hydrolyzed by 10 different types of enzymes from microbial, plant and animal sources. Hydrolysates prepared with Neutrase, Alcalase, and Papain displayed the most promising antioxidant activities as well as total protein recovery were primarily selected and investigated in depth described in Chapter 2, Chapter 3, and Chapter 4. The reaction conditions including substrate content, enzyme-to-substrate ratio, and hydrolysis time are critical parameters in producing peptides with desired activity and consistency, were therefore examined and optimized for each case of kafirin hydrolysates. The antioxidant capacity of the resulting hydrolysates was measured for antioxidant capacity through in vitro assays (DPPH, ABTS, ORAC, reducing power, and metal chelating) and then demonstrated in model systems (oil-in-water emulsion and ground meat). The fractions of hydrolysates possessing strongest activities were further fractionated by gel filtration and HPLC. Peaks representing the largest areas from HPLC were identified for major sequences by MALDI-TOF-MS. The experiment results indicated that all the three selected fractions of kafirin hydrolysates revealed excellent inhibition effects against oil and fat oxidations, which could be employed as tools to predict their performances in real food products. In addition, the structure studies showed that medium-sized hydrolysates of Neutrase (3 – 10 kDa) and Alcalase (5 – 10 kDa), and small-sized hydrolysates of Papain (1 – 3 kDa) exhibited relatively stronger activities. This study provided a workable processing method and critical reaction parameters for the production of peptide antioxidants from sorghum protein. The experiment results revealed that the sorghum peptide antioxidant could act through multiple mechanisms including free radical scavenging, metal ion chelation, hydrogen donating, and forming physical barriers to minimize the contact of oxidative agents to targets. These antioxidative peptides are a promising ingredient that can be potentially incorporated to food and feed products as alternatives to synthetic antioxidants or synergetic elements to nonpeptic antioxidants for protection of susceptible food ingredients. This study also made a positive impact to sorghum ethanol industry by guiding the conversion of sorghum protein-rich by-products into value-added antioxidant products as an additional revenue stream.

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

Bioactive peptides constitute a major class of signaling molecules in animals and have been shown to play a role in diverse physiological processes, including hypertension, appetite and sleep. As a result, knowing the identity of these molecules and understanding the mechanisms by which they are regulated has basic and medical significance. In this dissertation, I describe the development and application of novel methods for discovering bioactive peptides and the molecular pathways that control their activity. Recent analyses of mammalian RNAs have revealed the translation of numerous short open reading frames (sORFs). However, it is unknown whether these translation events produce stable polypeptide products that persist in the cell at functionally relevant concentrations. In Chapter 1, I describe a study in which we used a novel mass spectrometry-based strategy to directly detect sORF-encoded polypeptides (SEPs) in human cells. This analysis identified 115 novel SEPs, which is the largest number of mammalian SEPs discovered in a single study by more than a factor of 25. We observed widespread translation of SEPs from non-canonical RNA contexts, including polycistronic mRNAs and sORFs defined by non-AUG start codons. We also found that SEPs possess properties characteristic of functional proteins, such as stable expression, high cellular copy numbers, post-translational modifications, sub-cellular localization, the ability to participate in specific protein-protein interactions and the ability to influence gene expression. Taken together, these findings provide the strongest evidence to date that coding sORFs constitute a significant human gene class. In chapter 3, I describe a study in which we combine quantitative in vivo peptidomics, classical biochemical experiments and pharmacological studies in animal models to elucidate the metabolism of the neuropeptide substance P in the spinal cord. We identified two physiological substance P metabolites: the N- terminal fragments SP(1-9) and SP(1-7). Focusing our efforts on the SP(1-9)- producing pathway, we determined that an activity sensitive to the inhibitor GM6001 is the dominant SP(1-9)-generating activity in the spinal cord. We also show that GM6001 treatment causes a nearly three-fold increase in endogenous substance P levels in the spinal cords of mice, highlighting the functional relevance of the pathway blocked by this inhibitor.

Entre as diferentes propriedades de uma superfície capazes de influenciar a deposição óssea ao redor de implantes, suas composições químicas e bioquímicas podem interferir no processo de reconhecimento a partir do tecido ósseo circundante. O presente trabalho se propôs a investigar se a funcionalização de superfícies de implante poderia influenciar a deposição óssea ao redor de implantes em um modelo animal. Para tanto, quatro grupos experimentais com mesma topografia microtexturizada, porém variando quanto à adição ou não de uma concentração de peptídeo bioativo foram testados. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos e após 12 semanas, foram instalados 6 implantes em cada cão, constituindo uma amostra de 48 implantes. Durante o período cicatricial de 2 meses, uma marcação policromática fluorescente foi realizada com o intuito de investigar a dinâmica de remodelação óssea. Estes marcadores ósseos foram administrados no terceiro dia após a instalação dos implantes, bem como após 1, 2, 4 e 6 semanas. A análise histomorfométrica revelou que a superfície microtexturizada modificada pela adição de uma baixa concentração peptídica obteve maior densidade óssea adjacente (54,6 ± 16,6%) quando comparada aos outros grupos (microtexturizada + veículo de hidroxiapatita = 46,0 ± 21,0%, somente microtexturizada = 45,3 ± 11,3% e microtexturizada com adição de alta concentração peptídica = 40,7 ± 15,3%), no entanto estas diferenças numéricas não foram estatisticamente significantes (p>0,05). Adicionalmente, em relação à análise de fluorescência, a comparação entre grupos demonstrou uma diferença estatisticamente significante em favor da superfície composta pela baixa concentração do peptídeo bioativo na área adjacente aos implantes no período de 4 semanas (p<0,001). Pode-se concluir que a funcionalização da superfície de implantes pode interferir na aposição óssea, em particular na densidade óssea, ressaltando que diferentes concentrações peptídicas podem conduzir a diferentes resultados. Dentro do padrão de remodelação óssea observado entre superfícies microtexturizadas, sendo estas funcionalizadas ou não, aquelas com baixa concentração do peptídeo bioativo estudado favoreceram a formação óssea adjacente aos implantes quando comparadas às demais no período avaliado. / Among the different surface properties that influence the bone apposition around implants, the chemical or biochemical composition may interfere in its acceptance by the surrounding bone. The aim of this study was to investigate if a biofunctionalization of implant surface influences the bone apposition in a dog model and to compare it with other surfaces, such as a microstructured created by the grit-blasting/acid-etching process. The mandibular bilateral premolars of 8 dogs were extracted and after 12 weeks each dog received 6 implants, totaling 48 implants in the experiment. All the 4 experimental groups had the same microrough topography with or without some biofunctionalization treatment. After histomorphometric analysis it was observed that the modified microstructured surface with a low concentration of the bioactive peptide provided a higher adjacent bone density (54.6%) when compared to the other groups (microstructured + HA coating = 46.0%, microstructured only = 45.3% and microstructured + high concentration of the bioactive peptide = 40.7%), but this difference was only numeric and not statistically significant. The fluorescence analysis showed that bone remodeling is an active process resulting from the alternation of resorptive and formative activities. A similar pattern of bone remodeling was observed among the microstructured surfaces, biofunctionalized or not; however the addition of an adhesive peptide in low concentration favored the bone formation adjacent to the implants when compared to the other surfaces during the period evaluated. In conclusion the biofunctionalization of the implant surface could interfere in the bone apposition around implants in particular in terms of bone density and different concentrations of bioactive peptide lead to different results.

Entre as diferentes propriedades de uma superfície capazes de influenciar a deposição óssea ao redor de implantes, suas composições químicas e bioquímicas podem interferir no processo de reconhecimento a partir do tecido ósseo circundante. O presente trabalho se propôs a investigar se a funcionalização de superfícies de implante poderia influenciar a deposição óssea ao redor de implantes em um modelo animal. Para tanto, quatro grupos experimentais com mesma topografia microtexturizada, porém variando quanto à adição ou não de uma concentração de peptídeo bioativo foram testados. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos e após 12 semanas, foram instalados 6 implantes em cada cão, constituindo uma amostra de 48 implantes. Durante o período cicatricial de 2 meses, uma marcação policromática fluorescente foi realizada com o intuito de investigar a dinâmica de remodelação óssea. Estes marcadores ósseos foram administrados no terceiro dia após a instalação dos implantes, bem como após 1, 2, 4 e 6 semanas. A análise histomorfométrica revelou que a superfície microtexturizada modificada pela adição de uma baixa concentração peptídica obteve maior densidade óssea adjacente (54,6 ± 16,6%) quando comparada aos outros grupos (microtexturizada + veículo de hidroxiapatita = 46,0 ± 21,0%, somente microtexturizada = 45,3 ± 11,3% e microtexturizada com adição de alta concentração peptídica = 40,7 ± 15,3%), no entanto estas diferenças numéricas não foram estatisticamente significantes (p>0,05). Adicionalmente, em relação à análise de fluorescência, a comparação entre grupos demonstrou uma diferença estatisticamente significante em favor da superfície composta pela baixa concentração do peptídeo bioativo na área adjacente aos implantes no período de 4 semanas (p<0,001). Pode-se concluir que a funcionalização da superfície de implantes pode interferir na aposição óssea, em particular na densidade óssea, ressaltando que diferentes concentrações peptídicas podem conduzir a diferentes resultados. Dentro do padrão de remodelação óssea observado entre superfícies microtexturizadas, sendo estas funcionalizadas ou não, aquelas com baixa concentração do peptídeo bioativo estudado favoreceram a formação óssea adjacente aos implantes quando comparadas às demais no período avaliado. / Among the different surface properties that influence the bone apposition around implants, the chemical or biochemical composition may interfere in its acceptance by the surrounding bone. The aim of this study was to investigate if a biofunctionalization of implant surface influences the bone apposition in a dog model and to compare it with other surfaces, such as a microstructured created by the grit-blasting/acid-etching process. The mandibular bilateral premolars of 8 dogs were extracted and after 12 weeks each dog received 6 implants, totaling 48 implants in the experiment. All the 4 experimental groups had the same microrough topography with or without some biofunctionalization treatment. After histomorphometric analysis it was observed that the modified microstructured surface with a low concentration of the bioactive peptide provided a higher adjacent bone density (54.6%) when compared to the other groups (microstructured + HA coating = 46.0%, microstructured only = 45.3% and microstructured + high concentration of the bioactive peptide = 40.7%), but this difference was only numeric and not statistically significant. The fluorescence analysis showed that bone remodeling is an active process resulting from the alternation of resorptive and formative activities. A similar pattern of bone remodeling was observed among the microstructured surfaces, biofunctionalized or not; however the addition of an adhesive peptide in low concentration favored the bone formation adjacent to the implants when compared to the other surfaces during the period evaluated. In conclusion the biofunctionalization of the implant surface could interfere in the bone apposition around implants in particular in terms of bone density and different concentrations of bioactive peptide lead to different results.

L’α-casozépine (α-CZP) est un décapeptide porteur des propriétés anxiolytiques de l’hydrolysat trypsique de caséine αs1 bovine. Différentes propriétés ont pu rapprocher ce peptide de la famille des benzodiazépines, les anxiolytiques les plus prescrits. Cependant, certaines différences, dont notamment une absence d’effets secondaires, permettent aussi de distinguer l’α-CZP des benzodiazépines. Bien que de nombreux éléments laissent penser qu’une action centrale reste l’hypothèse principale du mécanisme d’action de l’α-CZP, aucune régulation de l’activité de zones cérébrales n’avait été montrée jusqu’à présent. Ce travail de thèse aura donc pu montrer que les propriétés anxiolytiques de l’α-CZP sont associées à une modification de l’activité cérébrale chez la souris, après une unique injection intrapéritonéale, dans différentes régions impliquées dans la régulation de l’anxiété, notamment l’amygdale, la formation hippocampale, le noyau accumbens et certains noyaux de l’hypothalamus et du raphé. De plus, ces modifications de l’activité cérébrale ne sont pas exactement les mêmes que celles observées avec le diazépam, une benzodiazépine de référence, ni de celles obtenues avec YLGYL, un peptide dérivé de l’α-CZP, bien qu’il existe des similitudes dans le comportement de l’animal suite aux différents traitements effectués. Enfin, il a été démontré qu’une situation anxiogène est indispensable pour révéler cet effet central. L’ensemble de ce travail aura permis d’avancer dans la compréhension du mode d’action d’un peptide alimentaire ayant des effets positifs sur le comportement et les émotions de son consommateur / Α-casozepine (α-CZP) is a decapeptide that mediates the anxiolytic-like properties of the tryptic hydrolysate of bovine αs1-casein. Different properties of α-CZP leads to consider this peptide close to the benzodiazepine family, the most commonly used anxiolytic molecules. In contrast, other results suggest a distinct mode of action between α-CZP and benzodiazepines, especially the fact that the peptide does not have side effects. Although a central action remains the main hypothesis of the mode of action of α-CZP, no regulation of the brain activity has been shown before. The work achieved in this thesis displayed the fact that the anxiolytic-like properties of α-CZP, after a single intraperitoneal injection of the peptide, are associated with a modulation of cerebral activity in several regions linked to anxiety regulation in mice brains, such as the amygdala, the hippocampal formation, the accumbens nucleus and some nuclei of the hypothalamus or raphe. Besides, these modulations of neural activity are not exactly the same as those obtained after an injection of diazepam, a reference benzodiazepine, or YLGYL, a derivative of α-CZP, even though observed behaviours are similar. Eventually, it has been demonstrated that an anxiety-inducing situation is needed to trigger the central effects of α-CZP. This work allowed a better understanding of the mode of action of a bioactive peptide from alimentary origin that has a positive action on its consumer’s mood and behaviour

<p>  </p> <p>The ever-growing interest in novel food ingredients and their dietary influence on human health and wellbeing has driven the study of bioactive peptides (BAP). BAP are protein-derived fragments composed of (2-20 amino acids) that could positively affect bodily function and chronic diseases. This dissertation explores the health-promoting properties of a novel source of BAPs, namely canary seed, by encompassing three specific aims: 1) evaluate the <em>in vitro </em>potential of hairless canary seed peptides (CSP) as a nutraceutical ingredient, 2) develop an understanding of CSP's bioavailability and molecular interactions with its biological targets, and 3) evaluate CSP's antioxidant and antiobesity activity at the organism level using a nematode (<em>C. elegans</em>) and murine (C57BL/6J mice) model, respectively. First, CSP were generated by implementing mechanical oil extraction followed by commercial enzymatic hydrolysis with Alcalase™. In addition, CSP were also subject to simulated gastrointestinal digestion (SGD) to assess their gastric stability and <em>in vitro</em> bioavailability. The results showed that canary seed proteins were mainly composed of prolamins fractions followed by glutelins, globulins, and albumins. CSP extracts with low molecular weight (< 3 kDa and 3–10 kDa) showed the highest bioactivity. Furthermore, after SGD, CSP inhibitory activity remained stable toward angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPP-IV), and pancreatic lipase but unstable for α-glucosidase. The digested peptides were transported efficiently (>10%) through the Caco-2 monolayer, indicating a potential high absorption capacity through the intestinal epithelium. During kinetic analysis by Lineweaver-Burk plots, it was observed that CSP-SGD interacted by mixed-type inhibition for DPP-IV and α-glucosidase, non-competitive inhibition for ACE, and uncompetitive inhibitor for pancreatic lipase. Furthermore, CSP-SGD were especially potent as antihypertensive (ACE inhibitors) and antiobesity (pancreatic lipase) agents. Consequently, molecular docking and <em>in silico</em> analyses were targeted to understand CSP-SGD interactions with ACE and pancreatic lipase. CSP-SGD with ACE inhibitory activity were found to be rich in proline, glutamine, and cationic residues and could have inhibited ACE by destabilizing the tetrahedral transition state and zinc ions interaction leading to conformational changes in the enzyme structure. For peptides with antiobesity properties, it was also found that arginine, glycine, and hydrophobic amino acids from CSP-SGD hold critical interactions with the lid domain (CYS238-CYS262) of pancreatic lipase, disrupting its proper function and preventing fat hydrolysis.</p> <p>In the second part of this dissertation, the relevant health-promoting properties of CSP were further investigated by testing the effects of peptide supplementation on obesity and oxidative stress animal models. The studies showed that exposure to CSP significantly mitigated the acute and chronic oxidative damage in <em>C. elegans</em>, extending the lifespan of the nematodes by 88 and 61%, respectively. Furthermore, it was established that the CSP prevented oxidative stress by scavenging free radicals and antioxidant gene upregulation. Concerning this, CSP caused a drop in reactive oxygen species (ROS) to safe levels and induce the upregulation of the GST-4 gene encoding antioxidant enzyme Glutathione S-transferase. Concerning antiobesity properties, the daily supplementation with CSP successfully prevented metabolic implications of western diet-induced obesity in C57BL/6J mice, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Likewise, CSP promoted a drop in fatty acid uptake gene, LPL, and fatty acid biosynthesis genes FAS and ACC while unaffecting lipid oxidation genes PPAR-α and ACO in the liver. While both moderate and high CSP supplementation levels exhibit hypolipidemic effects, only moderate levels induce satiety and significantly prevent weight gain. Together, these results suggest that CSP's weight gain prevention depended on a dual mechanism involving lipid metabolism retardation to modulate satiety. Overall, the results presented in this dissertation establish the effectiveness of canary seed peptides as nutraceutical ingredients for antioxidant and antiobesity functional food applications.</p>

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-06T14:55:01Z No. of bitstreams: 1 Vilma Sobral Bezerra.pdf: 3241045 bytes, checksum: 1dbe7e33df34dacca3124c036d6370bd (MD5) / Made available in DSpace on 2016-06-06T14:55:01Z (GMT). No. of bitstreams: 1 Vilma Sobral Bezerra.pdf: 3241045 bytes, checksum: 1dbe7e33df34dacca3124c036d6370bd (MD5) Previous issue date: 2011-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dairy proteins have bioactive peptides production great potential, however, their bioactivity is achieved only after enzymatic hydrolysis, which produces substances beneficial to health when incorporated into food or pharmaceuticals. Among them, casein has been used for this purpose. This study aims to determine peptide profile and amino acid sequence of bioactive peptides derived from Moxotó goat milk casein hydrolysis, using proteolytic enzymes such as trypsin, pepsin, papain and a protease extracted from Penicillium aurantiogriseum URM 4622. Enzymatic hydrolysis was performed using a statistical experimental design, which independent variables were pH, enzyme substrate (E: S), temperature and reaction time in Moxotó goat milk casein hydrolysis. Hydrolysis products were visualized in SDS-PAGE electrophoresis. Hydrolysates subjected to ultrafiltration (cut off 3000Da) were used to determine biological properties. Antioxidant activity was evaluated by ABTS + [2,2 '-Azin-bis (3-ethylbenzothiazoline) 6-sulfonic acid] method, using the pool of peptides (permeate <3000Da; retentate >3000Da). Antimicrobial activity was determined by the Clinical and Laboratory Standards Institute. Binding through the zinc solubility of zinc in the sample by Mass Spectrometry Inductively Coupled Plasma. Peptide profile and amino acid sequences were determined by mass spectrometry MALDI-TOF-MS/MS. The best hydrolysis degree (38.27%) was obtained with pepsin enzyme pH 3.0, 1:100 E: S, temperature of 40°C and 5 hours time reaction. However, a high hydrolysis degree precluded the use of these hydrolysates for bioactive peptides obtention. Casein tryptic hydrolysates demonstrated antioxidant activity up to 3242.3 μmol.L-1TROLOX/mg peptide in retentate (> 3000Da). By papain use, we obtained an activity up to 2329.6 μmol.L-1TROLOX /mg of peptide in permeate (<3000Da). The peptides produced by P. auratiogriseum protease action showed activity from 843.17 to 2587.30 μmol.L-1of Trolox / mg of 10 and 29% peptides hydrolysates, respectively, which were compatible with natural antioxidants, such as C vitamin and α-tocopherol. Goat casein tryptic hydrolysates demonstrated antimicrobial activity against Enterococcus faecalis ATCC 6057, Escherichia coli ATCC 2508, Klebisiela pneumoniae ATCC 29665, Bacillus subtilis ATCC 6633. Casein hydrolysates showed IC50 of 4.46 mg/g for zinc binding. Mass spectrometry MALDI TOF MS\MS allowed the visualization of caprine casein peptides in permeate (<3000Da), ranging from 568 to 2923 Da. It also showed LLYQEPVLGPV and HPINHQGLSPEVPNENLLR amino acids sequences for αs1 and β-casein, respectively, from casein tryptic hydrolysates; LLYQEPVLGPV sequences of β-casein, the NPWDQVK αs2 NENLL-casein and-casein in the αS1 casein hydrolysates by papain use; LLYQEPVLGPVRGPFPI β-casein sequence from casein hydrolysates obtained by the use of P. aurantiogriseum URM 4622 protease. The casein hydrolysates bioactive properties are referent to the prevalence of hydrophobic amino acids, which possibility of their use as bioactive peptides. / Proteínas lácteas apresentam grande potencial na produção de peptídeos bioativos. A caseína se destaca na produção de bioativos. Entretanto, a sua bioatividade só é conseguida após hidrólise enzimática, ao qual se produz substâncias benéficas à saúde quando incorporados em alimentos ou produtos farmacêuticos. O objetivo deste trabalho foi o de caracterizar e a avaliar atividades biológicas e determinando o perfil peptídico e a sequência de aminoácidos dos peptídeos bioativos obtidos a partir de hidrólise da caseína do leite de cabra Moxotó, usando enzimas proteolíticas tripsina, pepsina, papaína e protease extraída de Penicillium aurantiogriseum URM 4622. A hidrólise enzimática foi realizada através de planejamento experimental estatístico, os quais as variáveis independentes foram pH, relação enzima substrato (E:S), temperatura e tempo de reação na hidrólise da caseína do leite de cabra Moxotó. Os produtos de hidrólise foram visualizados em eletroforese SDS‑PAGE. Os hidrolisados submetidos à ultrafiltração (Cut off 3000Da) foram utilizados para determição das propriedades biológicas. A atividade antioxidante foi avaliada no pool de peptídeos, permeado (<3000Da) e retentado (>3000Da) usando-se o método ABTS+ [2,2’-azino-bis (3-etilbenzotiazolin) 6-ácido sulfônico]. A atividade antimicrobiana foi determinada pelo método do Clinical and Laboratory Standards Institute. O carreamento de zinco através da solubilidade do zinco na amostra através da Espectrometria de Massa em Plasma Indutivamente Acoplado. O perfil peptídico e as sequências de aminoácidos foram determinados por espectrometria de massa MALDI-TOF-MS/MS. O melhor grau de hidrólise (38,27%) foi obtido com a enzima pepsina usando pH 3,0, E:S de 1:100, 40ºC e 5 horas de reação. Entretanto, um alto grau de hidrólise impossibilitou o uso desses hidrolisados para obtenção de peptídeos bioativos. Os hidrolisados trípicos da caseína mostraram atividade antioxidante de até 3.242,3μmol.L-1TROLOX/mg de peptídeo no retentado (>3000Da). Com o uso da papaína, obteve-se uma atividade de até 2.329,6μmol.L-1TROLOX/mg de peptídeo no permeado (<3000Da). Os peptídeos produzidos pela ação da protease produzida por P. auratiogriseum apresentaram atividade de 843,17 a 2.587,30μmol.L-1de TROLOX/mg de peptídeos para os hidrolisados com 10 e 29% respectivamente, os quais foram compatíveis a antioxidantes naturais, como a vitamina C e α-tocoferol. Os hidrolisados trípticos da caseína caprina apresentaram atividade antimicrobiana frente a Enterococcus faecalis ATCC 6057, Escherichia coli ATCC 2508, Klebisiela pneumoniae ATCC 29665, Bacillus subtilis ATCC 6633. Os hidrolisados de caseína mostraram IC50 de 4,46mg/g para carreamento de zinco. A espectrometria de massa MALDI TOF MS permitiu visualizar os peptídeos no permeado (<3000Da) da caseína caprina, na faixa de 568 a 2.923 Da. Mostraram as sequências LLYQEPVLGPV e HPINHQGLSPEVPNENLLR e da β- e αs1-caseina para hidrolisados trípticos da caseína; as sequências LLYQEPVLGPV da β-caseína, NPWDQVK da αs2-caseina e NENLL da αS1-caseína nos hidrolisados da caseína com o uso da papaína e a sequência LLYQEPVLGPVRGPFPI da β-caseina no hidrolisado da caseína com o uso da protease produzida por P. aurantiogriseum URM 4622. As propriedades bioativas dos hidrolisados da caseína referem-se à prevalência de aminoácidos hidrofóbicos, o que possibilita o uso destes como peptídeos bioativos.