Introdução As doenças cardiovasculares estão entre as principais causas de morte no Brasil e no mundo. Evidências epidemiológicas e clínicas estabelecem associação entre dieta, dislipidemia e aumento do risco de morte. O consumo de proteína isolada de amaranto tem efeito hipocolesterolemizante e por isso pode reduzir, de modo significativo, os fatores de risco das doenças cardiovasculares. Objetivo Avaliar o efeito da ingestão do isolado protéico de amaranto, no perfil de lipoproteínas plasmáticas e na expressão de proteínas relacionadas à modulação da síntese do colesterol hepático. Métodos Vinte e oito ratos Wistar (Ratus novergicus) foram distribuidos em quatro grupos e receberam dietas diferenciadas pela fonte protéica. Os grupos experimentais (I e Icol) receberam dieta com 20por cento de proteína de amaranto e os grupos controle (C e Ccol) receberam dieta com 20por cento de caseína. As dietas col apresentavam 1por cento de colesterol. Ao grupo controle foi fornecida a média da quantidade de ração ingerida pelos grupos experimentais I e Icol (controle pair feeding). Para determinar o efeito da ingestão das dietas no metabolismo do colesterol, foram avaliadas as concentrações plasmáticas de triacilgliceróis, colesterol total e HDL-c, e as concentrações hepáticas de colesterol e lipídios totais. O efeito da ingestão da proteína de amaranto na regulação das vias de síntese do colesterol hepático foi investigado pela avaliação da expressão das proteínas nucleares: receptor X hepático alfa (LXR alfa), receptor ativado por proliferadores de peroxissoma alfa (PPAR alfa) e proteína ligadora do elemento regulado por esterol 2 (SREBP-2). Resultados A dieta Icol promoveu menor concentração plasmática de colesterol total e triacilgliceróis (36por cento e 47por cento , respectivamente) em comparação ao grupo Ccol. Observou-se, no fígado dos animais alimentados com dieta contendo proteína isolada de amaranto (I e Icol), menor concentração de lipídios totais e de fração colesterol. A digestibilidade entre as dietas Icol e Ccol não apresentou diferença significativa, enquanto a da dieta I foi menor que a da dieta C. Não foi observada alteração na expressão das proteínas PPAR alfa e LXR alfa em nenhum dos grupos. Uma redução significativa na expressão da proteína SREBP-2 foi verificada no fígado dos ratos que receberam dieta Icol em relação aos do grupo Ccol. Conclusão A ingestão de dieta Icol reduz de forma significativa a expressão do SREBP-2 no fígado de ratos. Essa redução sugere que o efeito hipocolesterolemizante promovido pela proteína de amaranto pode estar relacionado ao metabolismo endógeno do colesterol. Esse efeito independe da ação dos fatores de transcrição PPAR alfa e LXR alfa e pode estar associado à formação de peptídeos bioativos, muito embora os mecanismos não estejam claros. O isolado protéico apresenta efeito hepatoprotetor por diminuir o acúmulo de lipídios hepáticos mesmo quando o colesterol está presente na dieta / Introduction - Cardiovascular diseases are among the most important causes of death in Brazil and around the world. Epidemiologic and clinical evidences associate diet, dyslipidemia, and increased risk of death. Consumption of amaranth protein isolate has a hypocholesterolemic effect that may reduce, significantly, cardiovascular disease risk factors. Objective To assess the effect of amaranth protein isolate intake on plasma lipoprotein profile and on expression of proteins that modulate hepatic cholesterol synthesis. Methods Twenty eight Wistar rats were distributed in four groups and fed on different protein diets. The experimental groups (I e Icol) diets contained 20per cent amaranth protein and the control groups (C e Ccol) diets contained 20per cent casein. The col diets also contained 1per cent cholesterol. It was offered to the control group the mean of the amount of food consumed by the experimental groups (pair feeding control). In order to determine the effects of dietary intake on cholesterol metabolism, plasma total cholesterol, triglycerides, and HDL-c levels were assessed, as well as hepatic total lipids and cholesterol levels. The effect of amaranth protein on pathways of cholesterol synthesis was investigated by liver X receptors alpha (LXR alpha), peroxisome proliferator activated receptors alpha (PPAR alpha) and sterol regulatory element binding protein 2 (SREBP-2) expressions. Results Rats fed on Icol diet showed lower concentrations of plasma total cholesterol and triglycerides (36per cent and 47per cent, respectively) than those observed in Ccol diet group. A lower cholesterol and hepatic lipid concentration was observed in rats fed on amaranth protein isolate (I e Icol). There was no significant difference shown between the digestibility of the Icol and Ccol diets, although the digestibility of the I diet was lower than the digestibility of the C diet. No change was noticed in PPAR alpha and LXR alpha expression in any of the studied groups. There was a significantly down-regulation in SREBP-2 expression in the liver of rats fed on Icol diet when compared to those fed on Ccol diet. Conclusions The consumption of Icol diet reduces significantly SREBP-2 expression in the liver of rats. This decrease in SREBP-2 expression suggests that the hypocholesterolemic effect of the amaranth protein may be related to the endogenous metabolism of cholesterol. This effect does not depend on the transcription factors PPAR alpha and LXR alpha, and may be associated with bioactive peptides formation, although the mechanisms involved are not yet clear. The protein isolate has a hepatic-protective effect because it lowers hepatic lipid accumulation even when cholesterol was present in the diet

The purpose of this study was to evaluate the effectiveness of a whey protein isolate (WPI), a reported glutathione (GSH) booster, on exercise performance, immune function, and antioxidant status during weight maintenance and energy restriction in humans. Twenty well-trained, college age, male cyclists performed a cycling exercise test for 45 min, the first 7 min at 70% of VO2peak and the remaining 38 min at 55% VO₂peak immediately followed by a performance test set at 90% VO2peak until exhaustion. Blood samples were collected prior to the exercise test, after 45 min of exercise, within 5 min of exhaustion, and 1 h after exercise. Blood samples were analyzed for GSH, GSH/GSSG ratio, glutathione peroxidase (GPx), lipid hydroperoxides (LPO), phagocytosis, oxidative burst, peripheral blood mononuclear cell (PBMC) proliferation, and PBMC phenotyping. Subjects consumed 40g/day of WPI or casein placebo (P) along with their normal diet for 2 wk, repeated the exercise test, and then began a low energy period continuing the same supplementation for 4 d before the final exercise test. WPI was not associated with superior exercise performance or antioxidant status following exercise or weight loss. WPI supplementation did result in 33% greater lymphocyte proliferation capacity following exercise. Following exhaustive exercise for all trials, tGSH and GPx increased 7% and 11%, respectively, while WBCGSH decreased 13%. For WPI, GPx activity was 10% lower than P following exhaustive exercise for all trials combined. Weight loss (2.67 ± 0.26 kg) resulted in increases in phagocytosis (65%), white blood cell (WBC) GSH (40%), and GPx (35%) while decreasing the GSH/GSSG ratio (55%) and LPO (16%). Exhaustive exercise caused a 28% increase in CD8+ PBMCs and decreased CD4+ (34%), CD3+ (15%), the CD4+/8+ ratio (45%), and phagocytosis (8%) with all values returning to baseline after 1 h recovery. Supplementation with WPI did not enhance GSH status or exercise performance in trained cyclists, during weight maintenance or energy restriction. Following exercise, WPI is associated with greater lymphocyte proliferation of PBMCs which may help maintain an athlete's health during heavy training or competition. / Ph. D.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

<p dir="ltr">Pea protein is one of the most used plant proteins in food products, acting as an alternative to conventional animal protein sources due to its abundant, nutritious, and ease in supply chain characteristics. The objective of this study was to investigate the impact of homogenization and UHT processing parameters on the properties of protein emulsion. Protein emulsions (8% w/w pea protein isolate and 1% w/w sunflower oil) were freshly prepared prior to processing, and the untreated sample was considered as the control (NT). The pilot-scale aseptic processing system (APS) used in this study consisted of two coil-in-shell heaters and two coolers. Samples flowed through each section of the APS system following this order: balance tank, pre-heater, final heater, hold tube, pre-cooler, and final cooler. The homogenizer was located either after the pre-cooler (AC) or the pre-heater (AH) with a controlled temperature of 165F. A third setup was utilized by bypassing the homogenizer in the UHT system. An additional 8-hour continuous run was conducted to mimic a commercial manufacturing operation by recirculating the protein emulsion in the UHT system, and fouling detections were made using a non-intrusive sensor (NICS). 5% w/w soy protein, 1% w/w sunflower oil oil-in-water emulsion was also used for fouling tests. Protein concentration, pH and zeta potential, Cryo-SEM microscopic image, particle size distribution, flocculation index (FI), coalescence index (CI), viscosity and color data were collected and analyzed. The protein concentration had a 23.20 ± 4.00 %, 28.35 ± 5.02 %, 27.98 ± 5.05% and 21.38 ± 5.75% reduction for AC, AH, UHT and NT samples, respectively, when compared with the initial concentration in the formula. AC, AH, UHT and NT samples had pH values of 7.24 ± 0.01, 7.27 ± 0.01, 7.28 ± 0.02, 7.41 ± 0.01, and zeta potential values of -42.91 ± 0.89, -47.30 ± 0.91, -46.91 ± 1.40 and -50.11 ± 1.47 mV. AC sample had a smaller and NT sample had a bigger, respectively, mean weighted size D 4,3 value than AH and UHT samples, which could also be seen in Cryo-SEM images where only AC images contained more visually observable smaller particles. FI and CI for AC, AH and UHT indicated the formation of flocs but no irreversible aggregations were found. Shear-thinning AC, AH, UHT and NT samples had viscosity decreases from 4.00 to 3.56, 3.88 to 3.75, 4.02 to 3.79 and 10.42 to 9.56 mPa*s in 1 1/s to 100 1/s shear rate range. NT sample had a very noticeable color difference from the other three treated samples. Overall, AC samples had similar or better emulsion stability in all aspects than AH and UHT samples, suggesting that AC processing could potentially be used in the protein beverage industry for manufacturing products with improved shelf stability. Severe foulants buildups were neither observed nor detected by a non-intrusive continuous sensor (NICS) in the UHT system within 8 hours of process for both pea protein and soy protein emulsion, indicating that this UHT-homogenization processing can potentially be adapted to current industrial practices for higher-quality protein beverages.</p>

Introdução: Dentre os distúrbios relacionados à alimentação, o aumento do colesterol e, conseqüentemente, a incidência de doenças cardiovasculares, representa um importante problema de Saúde Pública. A proteína do amaranto reduz o colesterol plasmático, possivelmente pela presença de peptídeos bioativos, liberados durante sua digestão parcial. Objetivo: Verificar a ocorrência de peptídeos hipocolesterolemizantes após digestão in vitro do isolado protéico de amaranto. Métodos: Proteína isolada do amaranto foi submetida à digestão enzimática in vitro por duas metodologias distintas. Os peptídeos menores que 3000 Da foram injetados em espectrômetro de massa para sua identificação. Resultados: Foi obtido isolado protéico com grau de pureza acima de 90%. O isolado protéico e a farinha integral apresentaram a mesma quantidade de aminoácidos essenciais e de aminoácidos presentes nas seqüências dos peptídeos de interesse. O isolamento protéico também não promoveu alterações nas principais frações moleculares. As estruturas terciária e quaternária provavelmente foram alteradas, pois houve redução da solubilidade protéica. O grupo dissulfeto é um dos responsáveis pela ocorrência de agregados, entretanto outras ligações também podem estar envolvidas. Essas forças podem interferir no acesso aos sítios de clivagem das ligações peptídicas e dificultar a ação enzimática. Devido ao aumento da força iônica do meio obteve-se alto grau de hidrólise em ambos os métodos enzimáticos. A digestão protéica resultou em fragmentos que, em sua maioria, apresentaram pesos moleculares inferiores a 30 kDa. O perfil peptídico, para a maior parte das amostras, mostrou-se complexo, com difícil separação de picos. A amostra hidrolisada que apresentou menor grau de hidrólise e menor quantidade de picos no cromatograma continha um dos peptídeos hipocolesterolemizantes procurados, o fragmento IAEK. Conclusões: O isolado protéico de amaranto apresenta pelo menos um peptídeo hipocolesterolemizante quando submetido às digestões enzimáticas in vitro estudadas, similares à digestão in vivo. / Introduction: Among the problems associated to food habits, the increase of cholesterol levels, and thus the incidence of cardiovascular diseases represents an important problem for Public Health. The amaranth protein reduces the blood cholesterol levels, possibly due to the presence of peptides released during its incomplete digestion. Objective: To verify the occurrence of hypocholesterolemic peptides after in vitro digestion of amaranth protein isolate. Methods: Amaranth protein isolate was submitted to in vitro enzymatic digestion by two distinct methodologies. Peptides smaller than 3000 Da were injected in mass spectrometer for identification. results: Protein isolate presented a high purity degree, above 90%. The fat extraction and the protein isolation were efficient and did not modify significantly amaranth chemical composition, preserving the quantities of essential amino acids present in the sequence of the investigated peptides. Protein isolation did not promote changes in the main molecular fractions. The tertiary and quaternary structures were probably altered, given that protein solubility decreased. Disulfide bonds are responsible for aggregate arrangement; however, other bonds probably occurred and were also responsible for the decrease in solubility. These bonds may interfere in enzymatic hydrolysis, impeding the enzymes to cleave the peptide bonds. It was obtained a great hydrolysis degree in both enzymatic methods because ionic strength of the solution was high. Most of the protein digestion fragments presented molecular weight lower than 30kDa, demonstrating the efficiently of both digestion methods. Peptide mixture, for most samples, presented a complex profile and difficulties in peaks separation. The hydrolyzed sample that presented the lowest hydrolysis degree and the lowest quantity of peaks in chromatogram presented one of the hypocholesterolemic peptides, the sequence IAEK. Conclusions: The amaranth protein isolate presents at least one hypocholesterolemic peptide when submitted to the studied in vitro enzymatic digestion that is similar to in vivo digestion

Introdução: Doenças cardiovasculares constituem importante causa de morte em todo mundo e a hipercolesterolemia está diretamente relacionada a elas. A dieta desempenha papel importante neste processo e alguns alimentos como o feijão caupi (Vigna unguiculata L. Walp), especialmente sua proteína, tem sido apontado com potencial capacidade de redução do colesterol plasmático. Os efeitos hipocolesterolêmicos já observados indicaram o uso da proteína do feijão caupi, ou dos seus peptídeos, como ingrediente funcional de alimentos para a promoção da saúde e a redução do risco de doenças. Entretanto, as consequências da digestão gastrointestinal na absorção destes peptídeos são claramente complexas tornando essenciais estudos in vitro e in vivo para avaliar a sua bioacessibilidade e sua resistência à degradação gastrointestinal, além da disponibilidade e real eficácia destes peptídeos. Objetivo: Analisar a biodisponibilidade de peptídeos e avaliar parâmetros ligados ao metabolismo do colesterol em modelos animais após ingestão de isolado proteico de feijão caupi. Métodos: A farinha de feijão caupi foi desengordurada e sua proteína isolada. O isolado proteico foi submetido a métodos de hidrólise in vitro, para verificação das frações peptídicas formadas e inferência sobre a capacidade de ligação à albumina. Dois experimentos in vivo foram conduzidos. No primeiro, o isolado proteico do feijão caupi foi administrado a ratos e a concentração dos peptídeos monitorada no sangue, por 2 horas. O experimento in vivo 2 consistiu na alimentação de hamsters com dietas normo (N) - e hipercolesterolêmicas por 21 dias, contendo a proteína do feijão caupi como única proteína da ração (I), comparada ao controle de caseína (H). Neste experimento foram analisados no plasma: colesterol total (CT) e frações (LDLc, VLDLc e HDLc), triglicerídeos (TG) e peptídeos; nas fezes: colesterol total (CF) e ácidos biliares (AB); no fígado: colesterol (CH) e lipídeos totais (LH), HMGCR (atividade enzimática e expressão) e expressão de SREBP2, LDLR, ABCA1, ABCG1, ABCG5, ABCG8, LXRa e AMPK. Resultados: Os peptídeos identificados a partir da hidrólise proteica do feijão caupi, ou a partir do plasma dos animais estudados não evidenciaram similaridades entre os experimentos ou corresponderam a sequências previamente identificadas para o feijão caupi a partir de banco de dados. CT, VLDLc, HDLc, TG, CH dos hamsters foram maiores nos grupos H e I quando comparado ao N; LDLc foi maior para I comparado aos demais; LH foi maior em H comparado a N, sendo que I não diferiu dos demais; CF foi maior para I comparado a N, sendo que H não diferiu dos demais. A expressão de ABCA1 foi maior para I em relação aos demais; LXRa foi maior para I em relação a H, mas N não diferiu dos demais; SREBP2 foi menor em H em comparação aos demais; HMGCR foi mais expressa em N em comparação aos demais, ao passo que a atividade desta enzima foi maior em I quando comparado a N, sendo que H não diferiu dos demais. Não houve diferença entre os grupos quanto a AB ou expressão de ABCG8 ou AMPK. Não foram obtidos resultados de expressão para LDLR, ABCG1 e ABCG5. Conclusão: Apesar de pesquisas anteriores a este trabalho terem evidenciado a capacidade do isolado proteico do feijão caupi em inibir a atividade da HMGCR, inibir a solubilização micelar ou melhorar o perfil de lipídeos plasmáticos, no trabalho atual esta matéria prima não mostrou atuação positiva quanto ao metabolismo do colesterol de hamsters nas condições experimentais utilizadas. Os fragmentos indicados como peptídeos obtidos a partir da hidrólise proteica do feijão caupi, ou do plasma dos animais estudados não corresponderam a peptídeos com comprovada, ou até mesmo, com indicação de bioatividade / Introduction: Cardiovascular diseases are important cause of death worldwide and hypercholesterolemia is directly related to them. Diet plays an important role in this process and some foods such as cowpea (Vigna unguiculata L. Walp), especially its protein, have been shown to have a potential for reducing plasma cholesterol. The hypocholesterolemic effects already observed indicated the use of cowpea protein, or its peptides, as a functional food ingredient for health promotion and reduction of disease risk. However, the consequences of gastrointestinal digestion on the absorption of these peptides are clearly complex, making in vitro and in vivo studies essential to assess their bioaccessibility and resistance to gastrointestinal degradation, as well as the availability and actual efficacy of these peptides. Objectives: To analyze the bioavailability of peptides and evaluate parameters related to cholesterol metabolism in animal models after ingestion of protein isolate of cowpea. Methods: Cowpea flour was defatted and its protein isolated. The protein isolate was subjected to in vitro hydrolysis methods to verify the formed peptide fractions and inference about albumin binding ability. Two in vivo experiments were conducted. In the first, the cowpea protein isolate was administered to rats and the concentration of the peptides monitored in the blood for 2 hours. The in vivo experiment 2 consisted of feeding hamsters with normal (N) - and hypercholesterolemic diets for 21 days, containing the cowpea protein as the sole dietary protein (I), compared to casein control (H). In this experiment were analyzed in the plasma: total cholesterol (TC) and fractions (LDLc, VLDLc and HDLc), triglycerides (TG) and peptides; In feces: total cholesterol (CF) and bile acids (AB); In the liver: cholesterol (CH) and total lipids (LH), HMGCR (enzymatic activity and expression) and expression of SREBP2, LDLR, ABCA1, ABCG1, ABCG5, ABCG8, LXRa and AMPK. XX. Results: The peptides identified from the protein hydrolysis of cowpea or from the plasma of the animals studied did not show similarities among the experiments or correspond to sequences previously identified for the cowpea from the database. CT, VLDLc, HDLc, TG, CH of hamsters were higher in groups H and I when compared to N; LDLc was higher for I compared to the others; LH was higher in H compared to N, and I did not differ from the others; CF was higher for I compared to N, and H did not differ from the others. The expression of ABCA1 was higher for I than the others; LXRa was higher for I than H, but N did not differ from the others; SREBP2 was lower in H compared to the others; HMGCR was more expressed in N compared to the others, whereas the activity of this enzyme was higher in I when compared to N, and H did not differ from the others. There was no difference between groups regarding AB or expression of ABCG8 or AMPK. No expression results were obtained for LDLR, ABCG1 and ABCG5. Conclusion: Although previous research to this work evidenced the ability of the cowpea protein isolate to inhibit HMGCR activity, inhibit micellar solubilization or improve the plasma lipid profile, in the current work this raw material did not show a positive cholesterol metabolism of hamsters under the experimental conditions used. The fragments indicated as peptides obtained from the protein hydrolysis of cowpea beans, or from the plasma of the animals studied did not correspond to peptides with proven, or even, with indication of bioactivity

Orientador: José Paschoal Batistuti / Banca: Maria Helena Martini / Banca: Valdir Augusto Neves / Banca: José Alfredo Gomes Arêas / Banca: João Bosco Faria / Resumo: A partir da farinha da semente de goiaba (Psidium guajava), cuja composição centesimal é de aproximadamente 6,17 l 0,04% de umidade, 8,43 l 0,12% de proteína e alto teor de fibras, 60,88 l 0,9%, obteve-se isolado protéico (IP) através da precipitação isoelétrica (pI 4,5), cuja fração majoritária pertence à classe das glutelinas. As condições para o preparo do IP foram definidas a partir da curva de solubilidade em água x pH e temperatura de 25 l 3 ºC. Tais condições permitiram obter isolados protéicos com rendimento de extração de 45,2 l 0,5% (pH10,0) e 66,2 l 0,5% (pH11,5) e elevado conteúdo protéico 96,4 l 0,5% e 93,5 l 0,4% respectivamente. A capacidade de absorção de água e óleo foram baixas, apresentando 1,05 l 0,07 e 2,3 l 0,01 mL/g proteína respectivamente para IP 10,0 e 1,65 l 0,07 e 1,70 l 0,07 mL/g pr oteína respectivamente para IP 11,5. A maior capacidade de emulsificação, foi observado para o IP 11,5, 140 l 8 g óleo/g prot., comparado com o IP 10,0, 37 l 2 g óleo/g prot. A formação de gel foi observada em pH neutro e ausência de sal, apresentando as concentrações de 8% para IP 10,0 e 10% para IP 11,5. A cromatografia revelou a presença de dois picos para ambos isolados com sete frações de proteínas de diferentes pesos moleculares. As curvas TG-DTG / DSC revelaram maior quantidade de água para o IP 10,0 e elevada temperatura de estabilidade térmica 200 oC para ambos isolados. / Abstract: From the guava seed flour (Psidium guajava), whose centesimal composition belongs to about 6,17 ??0,04% of moisture, 8,43 ??0,12% of protein and high content of fibers, 60,88 ??0,9%, the protein isolate (PI) was obtained through the isoelectric precipitation (Ip 4,5) whose majority belongs to glutelins class proteins. The conditions for the preparation of the PI was defined from the solubility curve in water x pH and temperature of 25 ??3ºC. Such conditions allowed to obtain protein isolated with extraction yield of 45,2 ??0,5% (pH10,0) and 66,2 ??0,5% (pH11,5) and high protein content of 96,4 ??0,5% and 93,5 ??0,4% respectively. The absorption capacity for water and oil were low, showing 1,05 l 0,07 and 2,3 l 0,01 mL/g protein, respectively, for PI 10,0 and 1,65 l 0,07 and 1,70 l 0,07 mL/g protein, respectively, for PI 11,5. The most emulsification capacity was observed for PI 11,5 (140 l 8 g oil/g prot.), compared to PI 10,0 (37 l 2 g oil/g prot.). The gel formation was observed in neutral pH and salt absence, showing the concentrations of 8% to PI 10,0 and 10% to PI 11,5. The chromatography shows the presence of two peaks for both protein isolated with seven fractions of proteins with different molecular weights. The curves of TG-DTG / DSC revealed high water quantity for PI 10,0 and high temperature for thermal stability 200 °C for both isolates. / Mestre

Introdução: Dentre os distúrbios relacionados à alimentação, o aumento do colesterol e, conseqüentemente, a incidência de doenças cardiovasculares, representa um importante problema de Saúde Pública. A proteína do amaranto reduz o colesterol plasmático, possivelmente pela presença de peptídeos bioativos, liberados durante sua digestão parcial. Objetivo: Verificar a ocorrência de peptídeos hipocolesterolemizantes após digestão in vitro do isolado protéico de amaranto. Métodos: Proteína isolada do amaranto foi submetida à digestão enzimática in vitro por duas metodologias distintas. Os peptídeos menores que 3000 Da foram injetados em espectrômetro de massa para sua identificação. Resultados: Foi obtido isolado protéico com grau de pureza acima de 90%. O isolado protéico e a farinha integral apresentaram a mesma quantidade de aminoácidos essenciais e de aminoácidos presentes nas seqüências dos peptídeos de interesse. O isolamento protéico também não promoveu alterações nas principais frações moleculares. As estruturas terciária e quaternária provavelmente foram alteradas, pois houve redução da solubilidade protéica. O grupo dissulfeto é um dos responsáveis pela ocorrência de agregados, entretanto outras ligações também podem estar envolvidas. Essas forças podem interferir no acesso aos sítios de clivagem das ligações peptídicas e dificultar a ação enzimática. Devido ao aumento da força iônica do meio obteve-se alto grau de hidrólise em ambos os métodos enzimáticos. A digestão protéica resultou em fragmentos que, em sua maioria, apresentaram pesos moleculares inferiores a 30 kDa. O perfil peptídico, para a maior parte das amostras, mostrou-se complexo, com difícil separação de picos. A amostra hidrolisada que apresentou menor grau de hidrólise e menor quantidade de picos no cromatograma continha um dos peptídeos hipocolesterolemizantes procurados, o fragmento IAEK. Conclusões: O isolado protéico de amaranto apresenta pelo menos um peptídeo hipocolesterolemizante quando submetido às digestões enzimáticas in vitro estudadas, similares à digestão in vivo. / Introduction: Among the problems associated to food habits, the increase of cholesterol levels, and thus the incidence of cardiovascular diseases represents an important problem for Public Health. The amaranth protein reduces the blood cholesterol levels, possibly due to the presence of peptides released during its incomplete digestion. Objective: To verify the occurrence of hypocholesterolemic peptides after in vitro digestion of amaranth protein isolate. Methods: Amaranth protein isolate was submitted to in vitro enzymatic digestion by two distinct methodologies. Peptides smaller than 3000 Da were injected in mass spectrometer for identification. results: Protein isolate presented a high purity degree, above 90%. The fat extraction and the protein isolation were efficient and did not modify significantly amaranth chemical composition, preserving the quantities of essential amino acids present in the sequence of the investigated peptides. Protein isolation did not promote changes in the main molecular fractions. The tertiary and quaternary structures were probably altered, given that protein solubility decreased. Disulfide bonds are responsible for aggregate arrangement; however, other bonds probably occurred and were also responsible for the decrease in solubility. These bonds may interfere in enzymatic hydrolysis, impeding the enzymes to cleave the peptide bonds. It was obtained a great hydrolysis degree in both enzymatic methods because ionic strength of the solution was high. Most of the protein digestion fragments presented molecular weight lower than 30kDa, demonstrating the efficiently of both digestion methods. Peptide mixture, for most samples, presented a complex profile and difficulties in peaks separation. The hydrolyzed sample that presented the lowest hydrolysis degree and the lowest quantity of peaks in chromatogram presented one of the hypocholesterolemic peptides, the sequence IAEK. Conclusions: The amaranth protein isolate presents at least one hypocholesterolemic peptide when submitted to the studied in vitro enzymatic digestion that is similar to in vivo digestion

Introdução: Doenças cardiovasculares constituem importante causa de morte em todo mundo e a hipercolesterolemia está diretamente relacionada a este problema de saúde pública. A dieta desempenha papel importante neste processo e alguns alimentos, como o amaranto (Amaranthus cruentus L. BRSAlegria), têm mostrado capacidade de redução do colesterol plasmático. Estudos sugerem que este efeito está relacionado a peptídeos liberados durante a digestão das proteínas, os quais atuam na modulação do metabolismo lipídico. Considerando-se que os efeitos da digestão gastrointestinal e da absorção destes peptídeos são claramente complexos torna-se importante a realização de estudos visando avaliar bioacessibilidade e mecanismos de ação destes peptídeos nos locais alvo do organismo. Objetivo: Analisar a biodisponibilidade de peptídeos em modelos animais após ingestão de isolado proteico de amaranto e relacioná-la com parâmetros ligados ao metabolismo do colesterol. Métodos: O amaranto teve sua proteína isolada. Os peptídeos da proteína do amaranto foram analisados após digestão in vitro. Dois experimentos in vivo foram conduzidos: um de fase aguda e outro de média duração. No primeiro, o isolado proteico de amaranto foi administrado a ratos e os peptídeos no sangue foram monitorados por 2 horas para verificação de fragmentos que resistissem à digestão gastrointestinal. O experimento in vivo 2 consistiu na alimentação de 3 grupos de hamster, um com dieta recomendada pela AIN93 (grupo N) e dois com dietas hipercolesterolêmicas por 21 dias, contendo a proteína de amaranto como única proteína da ração (grupo I), comparada ao controle de caseína (grupo H). Neste experimento foram analisados no plasma: peptídeos, colesterol total e frações; nas fezes: colesterol total e ácidos biliares; no fígado: colesterol, lipídeos totais, ácidos graxos, atividade enzimática da Hmgcr, expressão de Hmgcr, Srebf2, Lxr, Abca1, Abcg8 e Ampk. Resultados e discussão: Foram identificados fragmentos peptídicos provenientes da digestão in vitro do isolado proteico de amaranto, e outras dezenas de sequencias peptídicas em ratos após administração aguda de amaranto foram analisadas. Destaca-se a identificação do peptídeo ALGV, presente em proteína do amaranto de acordo com banco de dados, e similar a fragmentos com ação hipocolesterolemizante. No sangue de hamsters foram encontrados seis peptídeos com 100 por cento de cobertura e similaridade a base de dados de proteínas de amaranto, merecendo investigação sobre seus efeitos. Verificou-se que o isolado proteico de amaranto foi capaz de suprimir a hipercolesterolemia quando a dieta hipercolesterolemizante foi introduzida em paralelo a este ingrediente, com valores inferiores em 72 por cento (triglicerídeos), 64 por cento (colesterol total), 80 por cento (LDL-c) do grupo I em relação ao grupo H. Foi observada ainda menor concentração de colesterol e lipídeos totais no fígado dos animais do grupo I em relação ao grupo H (177 x 464 mg de colesterol/100 g de tecido; 2,06 x 2,86 g de lipídeos/100 g de tecido, respectivamente). Parâmetros lipídicos do sangue, das fezes e do fígado foram similares aos do grupo N, cuja dieta seguiu a preconização para roedores. Foi observada maior excreção de colesterol total no grupo I em relação ao grupo H, mas não houve maior excreção de ácidos biliares nas fezes. Não houve mudança na expressão dos genes analisados neste estudo, mas o amaranto reduziu a atividade da enzima Hmgcr. Postulase que parâmetros como expressão de Ldlr e atividade da Acat sejam alterados pela ingestão de amaranto. O perfil de ácidos graxos também foi modificado de forma a se assimilar ao grupo N, porém deve-se verificar parâmetros inflamatórios devido à maior proporção de ácido araquidônico em relação aos demais grupos estudados. Conclusão: Verifica-se biodisponibilidade dos peptídeos do amaranto e ação hipocolesterolemizante e hipolipemiante em diversas vias metabólicas, promovendo proteção cardiovascular. / Introduction: Cardiovascular diseases are important causes of death worldwide, and hypercholesterolemia is directly related to this public health problem. Diet plays an important role in this process and some foods such as amaranth (Amaranthus cruentus L. BRS-Alegria) have been shown to reduce plasma cholesterol. Studies suggest that this effect is related to peptides released during the digestion of proteins, which would play an important role in the modulation of lipid metabolism. Considering that the effects of gastrointestinal digestion and the absorption of these peptides are clearly complex, it is important to carry out studies aiming to evaluate their bioaccessibility and evaluation of the mechanisms of action of these peptides in the target sites of the organism. Objective: To analyze the bioavailability of peptides in animal models after ingestion of amaranth protein isolate and to relate it to parameters associated to cholesterol metabolism. Methods: The amaranth was crushed, the flour was defatted and its protein isolated. Amaranth peptides were analysed after in vitro digestion. Two in vivo experiments were conducted: one of acute phase and one of medium duration. In the first, the amaranth protein isolate was administered to rats and the peptides in the blood were monitored for 2 hours to check for fragments that resisted gastrointestinal digestion. The in vivo experiment 2 consisted of feeding three groups of hamsters, one with a diet recommended by AIN93 (group N) and two with hypercholesterolemic diets for 21 days, containing amaranth protein as the only dietary protein (group I), compared to casein control (group H). In this experiment were analyzed in the plasma: peptides, total cholesterol and fractions; In feces: total cholesterol and bile acids; In the liver: cholesterol, total lipids, fatty acids, Hmgcr enzymatic activity, Hmgcr expression, Srebf-2, Lxr, Abca1, Abcg8 and Ampk. Results and discussion: Peptide fragments from the in vitro digestion of amaranth protein isolate were identified and other dozens of peptide sequences were found in rats after acute amaranth administration. A higher number of peptides were found in the serum in relation to the plasma of the animals. Remarkably, ALGV peptide was found in serum of rats. This peptide is present in amaranth protein, according to databases, and is similar to fragments that present hypocholesterolemic action. In the blood of hamsters it could be found six peptides with 100 per cent coverage and similarity to the database of amaranth proteins, deserving investigation about their effects. Amaranth protein was able to suppress hypercholesterolemia when the hypercholesterolemic diet was introduced in parallel with this ingredient, with values lower for group I in 72 per cent (triglycerides), 64 per cent (total cholesterol), 80 per cent (LDL-c) in relation to the H group. A lower concentration of cholesterol and total lipids were observed in the liver of the group I compared to the H group (177 x 464 mg cholesterol / 100 g of tissue, 2.06 x 2,86 g lipids / 100 g of tissue, respectively). Lipid parameters of blood, faeces and liver were similar to those of group N, whose diet followed the recommendation for rodents. There was greater excretion of total cholesterol in group I in relation to group H, but there was no greater excretion of bile acids in feces, indicating that the effect of amaranth protein may be due to increased transintestinal cholesterol excretion, decreased micellar solubilization of cholesterol and / or modification in the expression of cholesterol transport related proteins in the intestine. There was no change in the expression of the genes analyzed in this study, but amaranth reduced the activity of the Hmgcr enzyme. It is postulated that parameters such as Ldlr expression and Acat activity are altered by amaranth intake. The fatty acid profile was also modified in order to assimilate to the N group, but inflammatory parameters related to amaranth intake should be verified due to the higher proportion of arachidonic acid in relation to the higher proportion of arachidonic acid in relation to the other groups studied. Conclusion: The bioavailability of amaranth peptides and hypocholesterolemic and hypolipidemic activity in several metabolic pathways is verified, therefore promoting cardiovascular protection.