Antioxidants are molecules capable of stabilizing and preventing oxidation. Certain peptides, protein hydrolysates, have shown antioxidant capacities, which are obtained once liberated from the native protein structure. Soy protein isolates (SPI) were enzymatically hydrolyzed by pepsin and pancreatin mixtures. The soy protein hydrolysates (SPH) were fractionated with sequential ultrafiltration (UF) and nanofiltration (NF) membrane steps. Heat pre-treatment of SPI at 95 degrees celsius (C) for 5 min prior to enzymatic hydrolysis was investigated for its effect on peptide distribution and antioxidant capacity. SPH were subjected to UF with a 10 kDa molecular weight cut off (MWCO) polysulfone membrane. UF permeate fractions (lower molecular weight than 10 kDa) were fractionated by NF with a thin film composite membrane (2.5 kDa MWCO) at pH 4 and 8. Similar peptide content and antioxidant capacity (α=0.05) were obtained in control and pre-heated SPH when comparing the respective UF and NF permeate and retentate fractions produced. FCR antioxidant capacities of the SPH fractions were significantly lower than their ORAC antioxidant capacities, and the distribution among the UF and NF fractions was generally different. Most UF and NF fractions displayed higher antioxidant capacities when compared to the crude SPI hydrolysates, showing the importance of molecular weight on antioxidant capacity of peptides. The permeate fractions produced by NF at pH 8 displayed the highest antioxidant capacity, expressed in terms of Trolox equivalents (TE) per total solids (TS): 5562 μmol TE/g TS for control SPH, and 5187 μmol TE/g TS for pre-heated SPH. Due to the improvement in antioxidant capacity of peptides by NF at pH 8, the potential for NF as a viable industrial fractionation process was demonstrated. Principal component analysis (PCA) of fluorescence excitation-emission matrix (EEM) data for UF and NF peptide fractions, followed by multi-linear regression analysis, was assessed for its potential to monitor and identify the contributions to ORAC and FCR, two in vitro antioxidant capacity assays, of SPH during membrane fractionation. Two statistically significant principal components (PCs) were obtained for UF and NF peptide fractions. Multi-linear regression models (MLRM) were developed to estimate their fluorescence and PCA-captured ORAC (ORAC-FPCA) and FCR (FCR-FPCA) antioxidant capacities. The ORAC-FPCA and FCR-FPCA antioxidant capacities for NF samples displayed strong, linear relationships at different pH conditions (R-squared>0.99). Such relationships are believed to reflect the individual and relative combined contributions of tryptophan and tyrosine residues present in the SPH fractions to ORAC and FCR antioxidant capacities. Therefore, the proposed method provides a tool for the assessment of fundamental parameters of antioxidant capacities captured by ORAC and FCR assays.

Antioxidants are molecules capable of stabilizing and preventing oxidation. Certain peptides, protein hydrolysates, have shown antioxidant capacities, which are obtained once liberated from the native protein structure. Soy protein isolates (SPI) were enzymatically hydrolyzed by pepsin and pancreatin mixtures. The soy protein hydrolysates (SPH) were fractionated with sequential ultrafiltration (UF) and nanofiltration (NF) membrane steps. Heat pre-treatment of SPI at 95 degrees celsius (C) for 5 min prior to enzymatic hydrolysis was investigated for its effect on peptide distribution and antioxidant capacity. SPH were subjected to UF with a 10 kDa molecular weight cut off (MWCO) polysulfone membrane. UF permeate fractions (lower molecular weight than 10 kDa) were fractionated by NF with a thin film composite membrane (2.5 kDa MWCO) at pH 4 and 8. Similar peptide content and antioxidant capacity (α=0.05) were obtained in control and pre-heated SPH when comparing the respective UF and NF permeate and retentate fractions produced. FCR antioxidant capacities of the SPH fractions were significantly lower than their ORAC antioxidant capacities, and the distribution among the UF and NF fractions was generally different. Most UF and NF fractions displayed higher antioxidant capacities when compared to the crude SPI hydrolysates, showing the importance of molecular weight on antioxidant capacity of peptides. The permeate fractions produced by NF at pH 8 displayed the highest antioxidant capacity, expressed in terms of Trolox equivalents (TE) per total solids (TS): 5562 μmol TE/g TS for control SPH, and 5187 μmol TE/g TS for pre-heated SPH. Due to the improvement in antioxidant capacity of peptides by NF at pH 8, the potential for NF as a viable industrial fractionation process was demonstrated. Principal component analysis (PCA) of fluorescence excitation-emission matrix (EEM) data for UF and NF peptide fractions, followed by multi-linear regression analysis, was assessed for its potential to monitor and identify the contributions to ORAC and FCR, two in vitro antioxidant capacity assays, of SPH during membrane fractionation. Two statistically significant principal components (PCs) were obtained for UF and NF peptide fractions. Multi-linear regression models (MLRM) were developed to estimate their fluorescence and PCA-captured ORAC (ORAC-FPCA) and FCR (FCR-FPCA) antioxidant capacities. The ORAC-FPCA and FCR-FPCA antioxidant capacities for NF samples displayed strong, linear relationships at different pH conditions (R-squared>0.99). Such relationships are believed to reflect the individual and relative combined contributions of tryptophan and tyrosine residues present in the SPH fractions to ORAC and FCR antioxidant capacities. Therefore, the proposed method provides a tool for the assessment of fundamental parameters of antioxidant capacities captured by ORAC and FCR assays.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-04T14:55:31Z No. of bitstreams: 1 arquivotal.pdf: 1914187 bytes, checksum: 451665c737c19a32a97906a57d305870 (MD5) / Made available in DSpace on 2017-09-04T14:55:31Z (GMT). No. of bitstreams: 1 arquivotal.pdf: 1914187 bytes, checksum: 451665c737c19a32a97906a57d305870 (MD5) Previous issue date: 2017-06-02 / The chicken’s comb and wattle are part of the by-products they are composed of a large number of collagenous proteins, they are considered a promising source for obtaining bioactive peptides. The objective of this thesis was to develop a technical procedure aimed at making better use of the chicken’s comb and wattle. For this, the authors carried out studies on the protein hydrolysis of a comb/wattle mixture (1:1 w/w), focusing on the production of peptides with bioactivities such as iron chelation and antioxidant capacity. At first, the following were evaluated: centesimal composition, mineral profile and protein quality (percentage of collagen, percentage of total amino acids and free amino acids, hydrophobicity profile and electrophoretic profile) of the comb, the wattle and a mixture of both (1:1 w/w). These by-products displayed a high protein concentration (61 to 83% dry base), especially proteins of collagenous origin (47 to 51% dry base). The amino acid profile presented a high level of glutamic acid (10.01 g/100g) aspartic acid (7,38 g/100g), glycine (9,05 g/100g). The minerals iron (8 mg/100g), phosphorus (337 mg/100g) and zinc (2,62 mg/100g) were present in high amounts, which emphasizes the byproducts as an excellent sources of these nutrients. Considering the protein quality of the chicken’s comb and wattle, and the absence of studies regarding their use in functional products, a study was then carried out to evaluate the obtaining of peptides from these by-products. Firstly, comparative analysis of proteolytic activity was carried out in order to select the enzyme (Alcalase, Flavourzyme, Protamex, Brauzyn and Colagenase) to be used in the hydrolysis of the comb and wattle mixture (1:1 w/w). The enzyme Alcalase displayed the highest degree of hydrolysis (DH = 19%) when compared to the other proteases (DH < 3%). After selecting the enzyme, the next step was to optimize the Alcalase hydrolysis process. The researchers selected the samples with the lowest degree of hydrolysis (< DG: enzyme substrate ratio of 1% and 60 min of hydrolysis) and the highest degree of hydrolysis (> DG: enzyme substrate ratio of 5% and 240 min of hydrolysis). These experimental samples were analyzed for their protein profile, (percentage of total amino acids and free amino acids, hydrophobicity profile and electrophoretic profile), mineral profile and were characterized according to their iron chelating and antioxidant (ABTS, DPPH e FRAP) capacities. The hydrolysate with the higher DH exhibited a higher quantity of peptides with low molecular weight and hydrophilic character, as well as a higher quantity of free amino acids. The minerals Fe, F and Zn were present in quantities that allow the samples to be characterized as sources. As for bioactivity, they showed excellent Fe2+ chelation capacity (> 94%) and antioxidant activity through the inhibition of ABTS● (> 46%), DPPH● (> 41%) and FRAP through the reduction of Fe3+ (> 95 mg/100g trolox equivalent). Comb and wattle have the potential to be used in the production of hydrolysates and are also sources of minerals and peptides with bioactivities antioxidant capacity and iron chelating. As a result of these studies, the process patent BR 10 2016 027430 3 was registered. / A crista e a barbela do frango fazem parte dos subprodutos que apresentam elevada quantidade de proteínas colagenosas, sendo consideradas matérias-primas promissoras para a obtenção de peptídeos bioativos. O objetivo desta pesquisa foi desenvolver procedimento tecnológico voltado a um melhor aproveitamento dos subprodutos cristas e barbelas de frango. Para tanto foram realizados estudos de hidrólise proteica da mistura de crista e barbelas (1:1 p/p) para produção de peptídeos com bioatividades de quelação ao mineral ferro e capacidade antioxidante. Inicialmente foi avaliada a composição centesimal, perfil de minerais e a qualidade proteica (% colágeno, % aminoácidos totais e livres, perfil de hidrofobicidade e perfil eletroforético) da crista, barbelas e sua mistura (1:1 p/p). Estes subprodutos apresentaram elevada concentração de proteínas (61 a 83% b.s.) com destaque para as proteínas de origem colagenosa (47 a 51% b.s.). O perfil de aminoácidos apresentou elevado teor de ácido glutâmico (10,01 g/100g), ácido aspártico (7,38 g/100g), glicina (9,05 g/100g). Os minerais ferro (8 mg/100g), fósforo (337 mg/100g) e zinco (2,62 mg/100g) também foram encontrados em grandes quantidades nos subprodutos, destacando-se como fontes destes nutrientes. Foi realizada uma analise comparativa de melhor atividade proteolítica para seleção da enzima (Alcalase, Flavourzyme, Protamex, Brauzyn e Colagenase), a ser utilizada na hidrolise da mistura crista e barbelas (1:1 p/p). A enzima Alcalase apresentou o maior grau de hidrólise (DH = 19%) em comparação com as demais proteases (DH < 3%). Definida a enzima foi realizada a otimização do processo de hidrólise com a Alcalase. Mediante a avaliação foram selecionados os experimentos que obtiveram o menor grau de hidrólise (< GH: relação enzima substrato de 1% e 60 min de hidrólise) e o maior grau de hidrólise (> GH: relação enzima substrato de 5% e 240 min de hidrólise). Os hidrolisados selecionados foram analisados em relação ao seu perfil proteico (% aminoácidos totais e livres, perfil de hidrofobicidade e perfil eletroforético), perfil de minerais e caracterizados quanto a sua capacidade quelante ao ferro e antioxidante (ABTS, DPPH e FRAP). O hidrolisado de >GH exibiu maiores quantidades de peptídeos com baixo peso molecular e caráter hidrofílicos, além de maiores quantidades de aminoácidos livres. Ambos os hidrolisados (> GH e < GH) apresentaram concentrações de minerais que os caracterizam como fontes Fe, F e Zn. Quanto a bioatividade os hidrolisados exibiram capacidade quelante ao Fe2+ (> 94%) e atividade antioxidante através da inibição dos radicais ABTS● (> 46%), DPPH● (> 41%) e FRAP 9 por redução do Fe3+ (> 95 mg / 100 g equivalente trolox). As cristas e barbelas apresentam potencial para elaboração de hidrolisados, fonte de minerais e peptídeos funcionais com bioatividades, como capacidade antioxidante e capacidade quelante ao mineral ferro. Com isto foi registrado a patente de processo de obtenção dos peptídeos bioativos da mistura de crista e barbela de frango - BR 10 2016 027430 3.