Antioxidant capacity of protein hydrolysates and phenolic compounds of flaxseed (Linum usitatissimum L.) and its modulatory effects on experimental colitis

Orientador: Flávia Maria NettoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: As proteínas da linhaça, assim como outras proteínas de origem vegetal, estão naturalmente associadas aos compostos fenólicos. Durante a hidrólise enzimática dessas proteínas, peptídeos e compostos fenólicos são liberados e podem exercer atividades antioxidante e anti-inflamatória. Este estudo avaliou a influência do complexo proteína-fenólico e da hidrólise com Alcalase e/ou enzimas digestivas sobre a capacidade antioxidante (CA) de produtos de linhaça. Investigou-se também a capacidade anti-inflamatória dos hidrolisados proteicos e compostos fenólicos da linhaça in vitro, pela inibição da produção de mediadores inflamatórios em macrófagos estimuladas por IFN-?/LPS e, in vivo, por meio da modulação da inflamação em animais com retocolite ulcerativa induzida por TNBS. O isolado proteico de linhaça (FPI) foi obtido a partir da farinha de linhaça desengordurada por extração alcalina (pH 9,0) e precipitação no ponto isoelétrico (pH 4,2) seguida de neutralização (pH 6,0). O hidrolisado de proteína de linhaça (FPH) foi produzido a partir da hidrólise do FPI com Alcalase. Para obter o isolado e hidrolisado proteico de linhaça com reduzido teor fenólico (phr-FPI e phr-FPH, respectivamente) a farinha de linhaça desengordurada foi lavada com etanol antes do processo de isolamento, para remoção da fração fenólica (Phi). A fração fenólica hidrolisada (Phh) foi obtida submetendo-se a Phi às mesmas condições de pH e temperatura utilizadas na hidrólise dos isolados proteicos. A análise de fluorescência intrínseca foi utilizada para avaliar a formação do complexo proteína: fenólico e a CA foi determinada pelos métodos ORAC e FRAP. As amostras Phi e Phh apresentam a maior CA seguida dos hidrolisados e isolados proteicos. A presença de compostos fenólicos nas amostras proteicas foi responsável pelo aumento de até 80% na CA do isolado proteico, medida pelos métodos ORAC e FRAP, enquanto que para o hidrolisado proteico ela foi responsável por um aumento de 15% da CA medida por ORAC, e 80%, quando medida por FRAP. Após a simulação da digestão, a CA dos isolados e hidrolisados proteicos aumentou e a influência dos compostos fenólicos na capacidade antioxidante persistiu, especialmente quando determinada por FRAP. Nossos resultados sugerem que CA de isolados deve-se principalmente à formação do complexo proteína:fenólico, enquanto que nos hidrolisados a CA deve-se possivelmente ao sinergismo entre fenólicos e peptídeos liberados a partir da hidrólise com Alcalase ou com as enzimas digestivas. Além disso, a transferência de H+ parece ser o principal mecanismo pelo qual as proteínas de linhaça atuam como antioxidantes. Em relação à capacidade antioxidante do phr-FPH, foram identificados quatro sequências de peptídeos usando LC-MS/MS e uma sequência foi identificada por degradação de Edman (GFPGRLDHWCASE) a qual apresentou CA maior do que a do antioxidante sintético BHA. Em relação ao efeito anti-inflamatório, as amostras Phi, Phh, FPH e phr-FPH foram capazes de inibir a produção de NO e TNF-? em macrófagos estimulados com IFN-?/LPS. No estudo in vivo, Phi e phr-FPH foram capazes de atenuar a perda de peso dos animais, as respostas inflamatórias das células TH1 e TH17, a proliferação de linfócitos TCD4+ e os níveis de citocinas inflamatórias em animais com retocolite induzida por TNBS. De forma geral os resultados demonstram que os hidrolisados proteicos e os compostos fenólicos da linhaça possuem atividade antioxidante e anti-inflamatória. Mais estudos são necessários para avaliar a interferência do complexo proteína:fenólico na biodisponibilidade de peptídeos e fenólicos, e a relação destes compostos com a microbiota intestinal para conhecer a real capacidade destes produtos em fornecer benefícios ao organismoAbstract: Flaxseed protein is a potential source of bioactive compounds. During the enzymatic hydrolysis of vegetable proteins, peptides and phenolic compounds, which are associated with proteins, are released and may act as antioxidants and anti-inflammatory. This study evaluated the influence of the hydrolysis with Alcalase and simulated gastrointestinal digestion on the antioxidant capacity of different flaxseed products. We also evaluated the inhibitory capacity of flaxseed protein hydrolysates and phenolic fractions on the production of inflammatory mediators by stimulated macrophages and the anti-inflammatory effects on TNBS-induced colitis. The Flaxseed protein hydrolysate (FPH) was produced with protein isolate obtained from defatted meal by alkaline extraction (pH 9.0) followed by precipitation (pH 4.2) and neutralization (pH 6.0). In order to obtain phenolic reduced flaxseed protein hydrolysate (phr-FPH), flaxseed was extracted from defatted meal with ethanol before isolating process. The hydrolysis was performed with Alcalase (pH 8.5; enzyme/substrate 1/90, w/w). Hydrolysed phenolics compounds (Phi) was obtained with the phenolics compounds extracted from the defatted flaxseed meal using the same conditions of protein hydrolysis. Four glycosylated phenolic compounds ¿ secoisolariciresinol and ferulic, p-coumaric, caffeic acids ¿ were identified using HPLC-MS/MS. Intrinsic fluorescence was used to analyze protein:polyphenol complex. A chromatographic characterization of the samples was performed and the AC was determined by FRAP and ORAC methods. A chromatographic separation of the phr-FPH by RP-HPLC was performed, and the AC of the six obtained fractions was determined. Flaxseed phenolic fractions exhibit the highest antioxidant capacity followed by flaxseed protein hydrolysates and isolates. The presence of phenolic compounds increased 80% of antioxidant capacity of the protein isolate, measured by ORAC and FRAP methods, while for the hydrolysates accounted for 15% measured by ORAC, and 80% when measured by FRAP. After simulated digestion, the antioxidant capacity of isolates and hydrolysates increased and the influence of phenolics on their antioxidant capacity persisted, especially when determined by FRAP. Our findings suggest that AC of isolates is mainly due to the protein: phenolic complex while in the hydrolysates is mainly by the synergism between phenolics and peptides released by Alcalase or simulated digestion. Also, the preferential mechanism that flaxseed protein may act as antioxidant is the H+ atom transfer. Four peptide sequences could be identified by using LC-MS/MS and one by Edman degradation. The peptide sequence GFPGRLDHWCASE was synthesized and showed a value higher than that of butylated hydroxyanisole. The identified sequences represent an advance in the molecular characterization of the flaxseed protein fraction. Regarding to the anti-inflammatory effect of flaxseed extracts, Phi, Phh, FPH and phr-FPH inhibit NO and TNF-? secretion in stimulated RAW cells and also downregulating weight loss, histological inflammation, TH1 and TH17 responses, T cell proliferation and inflammatory cytokine levels on TNBS-induced colitis in BALB/c mice. Together, the results demonstrate that flaxseed protein hydrolysates and phenolic isolated fractions had antioxidant activity and protective effects on a TNBS-induced colitis model. More studies are needed to evaluate the effect of the protein complex: phenol in the bioavailability of peptides and phenolics, and the relationship of these compounds with the intestinal microbiota to know the real ability of these products to provide health benefitsDoutoradoNutrição Experimental e Aplicada à Tecnologia de AlimentosDoutora em Alimentos e Nutrição2010/52680-7FAPES

Influence of protein-phenolic complex on the antioxidant capacity of flaxseed (Linum usitatissimum L.) products

The impact of the naturally present phenolic compounds and/or proteins on the antioxidant capacity of flaxseed products (phenolic fraction, protein concentrates, and hydrolysates) before and after simulated gastrointestinal digestion was studied. For that, whole and phenolic reduced products were assessed. Four glycosylated phenolic compounds (secoisolariciresinol and ferulic, p-coumaric, and caffeic acids) were identified in flaxseed products. Phenolic fraction exerts the highest antioxidant capacity that increased by alkaline hydrolysis and by simulated gastrointestinal digestion. The action of Alcalase and digestive enzymes resulted in an increase of the antioxidant capacity of whole and phenolic reduced products. Principal component analysis showed that proteinaceous samples act as antioxidant is by H transfer, while those samples containing phenolic compounds exert their effects by both electron donation and H transfer mechanisms. Protein/peptide-phenolic complexation, confirmed by fluorescence spectra, exerted a positive effect on the antioxidant capacity, mainly in protein concentrates.This work was supported by FAPESP (2010/52680-7), CNPq and UNICAMP (FAEPEX 607-11) grants, CSIC and Ministry of Economy and Competitiveness (Ramon y Cajal contract).Peer Reviewe

Antioxidante activity of flaxseed protein products (Linum usitatissimum L.)

Orientador: Flavia Maria NettoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Existem evidências numerosas sobre o papel dos radicais livres em uma série de condições patológicas, incluindo envelhecimento, câncer, esclerose múltipla, doenças cardiovasculares. Hidrolisados protéicos de diferentes fontes têm sido estudados por seu potencial antioxidante. A atuação antioxidante da proteína, na maioria das vezes, encontra-se limitada devido à conformação espacial, que concentra resíduos capazes de neutralizar radicais livres no interior da molécula, dificultando o acesso das espécies reativas aos centros nucleofílicos. A hidrólise da proteína contribui para aumentar a exposição desses resíduos de aminoácidos, aumentando sua atuação como antioxidante. Compostos fenólicos podem estar presentes em hidrolisados proteicos de origem vegetal, devido a sua associação com as proteínas. Métodos in vitro que simulam as condições do trato gastrointestinal permitem estudar como a digestão pode interferir na atividade antioxidante de peptídeos e compostos fenólicos. O presente trabalho tem como objetivos obter hidrolisados proteicos com capacidade antioxidante a partir da farinha de linhaça e avaliar o efeito da digestão in vitro pode interferir nessa atividade. A farinha de linhaça marrom foi desengordurada, obtendo-se a farinha de linhaça marrom desengordurada (FLMD). O concentrado proteico de linhaça (CPL) foi obtido a partir da FLMD por extração alcalina e precipitação no ponto isoelétrico seguida de neutralização. Para obtenção dos hidrolisados proteicos (HPL), a partir do CPL, com Alcalase, foi realizado um delineamento composto central rotacional (DCCR) 2². As variáveis independentes foram pH que variou entre 7,5 a 9,5 e relação enzima: substrato (E:S) que variou de 1:150 a 1:30. As variáveis dependentes foram grau de hidrólise (GH), teor de substâncias redutoras do reagente de Folin-Ciocalteau e atividade antioxidante, determinada por FRAP e ORAC. Teor de substâncias redutoras e atividade antioxidante foram avaliados a partir dos extratos aquosos e metanólico (metanol 70%). Os hidrolisados de maior atividade antioxidante, a FLMD e o CPL foram submetidos à digestão in vitro, simulando as condições da digestão gastrintestinal. As amostras antes e após a digestão in vitro foram caracterizadas por eletroforese em sistema SDS-PAGE Tricina e por cromatografia liquida de alta eficiência de fase reversa (HPLC- RP). O teor de substâncias redutoras e da atividade antioxidante das amostras FLMD, CPL e HPL foram avaliados antes e após a digestão in vitro. As condições ótimas para obtenção de HPL de maior GH (21,0%) são pH entre 7,5 e 8,0 e E:S entre 1:60 e 1:30, indicando que a faixa de pH ótimo da enzima e a alta E:S favorecem maior hidrólise do CPL. Para obtenção de HPL com maior teor de substâncias redutoras para os extratos aquoso (24 mg EAG/ g HPL) e metanólico (20 mg EAG/ g HPL) as condições ótimas são pH ~ 8,5 /E:S 1:30. Este resultado parece estar relacionado à liberação de compostos fenólicos ligados a proteína e também de peptídeos durante a hidrólise. Açúcares e aminoácidos aromáticos presentes no hidrolisado podem interferir na reação e superestimar o teor de fenóis dos HPL. A maior atividade antioxidante determinada pelo método de FRAP para o extrato aquoso (42 mg SF/ g HPL) se dá nas condições de pH ~ 9,5/E:S ~1:150 e para o extrato metanólico (40 mg SF/ g HPL) pH entre 8,5 e 9,0/E:S entre 1:90 a 1:150. Para o método de ORAC, as condições ótimas para maior atividade antioxidante no extrato aquoso (300 µmol TE/ g HPL) são pH entre 7,5 a 9,5/E:S ~ 1:30 ou ~1:150 e para o extrato metanólico (330 µmol TE/ g HPL) são pH ~ 8,5/E:S entre 1:150 e 1:30. Os hidrolisados de maior atividade antioxidante foram os obtidos em pH 8,5/E:S 1:90, e em pH 9,2/E:S 1:133 denominados HPL 0 e HPL 3, respectivamente. Para a FLMD, CPL e os hidrolisados, após a digestão in vitro, observou-se que o teor de substâncias redutoras totais aumentou (9 a 20 vezes) para todas as amostras. O teor de substâncias redutoras do CPL (~24 mg EAG/ g amostra), em ambos os extratos, após a digestão in vitro se igualou ao teor dos hidrolisados (~23 mg EAG/ g amostra). Este resultado sugere que tanto a hidrólise com Alcalase quanto o processo digestório liberam compostos redutores, dentre eles fenólicos da proteína de linhaça. A atividade antioxidante dos extratos de FLMD e CPL, determinada por FRAP, também aumentou (de 3 a 10 vezes) após a digestão, mas não se igualou à atividade antioxidante dos hidrolisados (48 mg SF/g amostra). No entanto, o CPL apresentou atividade antioxidante determinada por ORAC semelhante à dos hidrolisados no extrato aquoso (~420,24 µmol TE/ g amostra) e 10 % maior que o encontrado para os hidrolisados (~365 µmol TE/ g amostra) no extrato metanólico. Após a digestão in vitro, os hidrolisados apresentaram a maior atividade antioxidante medida por FRAP (50 mg SF/ g amostra), e o CPL, a maior atividade determinada pelo método de ORAC (~430 µmol TE/ g amostra). Estes resultados sugerem o processo digestório é tão ou mais eficiente que a Alcalase em liberar os compostos com atividade redutora no CPL. Uma vez que a metodologia de determinação da atividade antioxidante por ORAC tem maior proximidade com o mecanismo de oxirredução que ocorre in vivo, esses resultados sugerem o uso do CPL como melhor produto protéico da linhaça com maior potencial antioxidante para a formulação de nutracêuticos e alimentos funcionaisAbstract: There are several evidences which indicate the role of free radicals on a series of pathological conditions, including aging, cancer, multiple sclerosis and cardiovascular disease. Hydrolysates from different sources have been studied because of their antioxidant potential. The antioxidant activity of the protein, in most cases, is limited due to their conformation, which concentrates residues capable of neutralize free radicals in the molecule¿s core, hampering the access of the reactive species to nucleophilic sites. The protein hydrolysis contributes to increasing the exposure of these amino acid residues, increasing their role as antioxidants. Phenolic compounds may also be present in vegetable protein hydrolysates because of their association with proteins. In vitro methods that simulate the conditions of the gastrointestinal digestion are an important way to evaluate how the digestion affects the antioxidant activity of phenolic compounds and peptides. This study aims at obtaining hydrolysates with antioxidant capacity from defatted flaxseed flour and evaluate the effect of the in vitro digestion on this activity. The brown flaxseed flour was defatted, resulting in the brown defatted flaxseed meal (BDFM). The flaxseed protein concentrate (FPC) was obtained from the BDFM by alkaline extraction and precipitation at the isoelectric point followed by neutralization. To obtain the flaxseed protein hydrolysates (FPL), using FPC and Alcalase, a central composite rotational design (DCCR) was performed. The independent variables were pH ranging from 7.5 to 9.5 and enzyme: substrate ratio (E: S) that ranged from 1:150 to 1:30. The dependent variables were the degree of hydrolysis (DH), total phenolic content and antioxidant activity, determined by FRAP and ORAC. Phenolic and antioxidant activity were evaluated from the aqueous and methanol (70% methanol). The hydrolysates with the highest antioxidant activity, the CPL FLMD were submitted to the in vitro digestion. The samples obtained before and after the in vitro digestion were characterized by electrophoresis SDS-PAGE- tricine and HPLC. The total phenolic content and antioxidant activity of FLMD, CPL and HPL were evaluated before and after in vitro digestion. The optimum conditions to obtain HPL with the highest GDH (21.0%) are pH (7.5-8) and E:S ratio (1:60-1:30), which indicates that the Alcalase optimum pH and highest E:S ratio collaborates to highest hydrolysis of CPL. To obtain HPL with higher content of Folin-Ciocalteau reducing compounds content in aqueous (EAG 24 mg / g HPL) and methanol (20 mg EAG / g HPL) extracts, the optimum conditions were pH ~ 8.5 / E: S 1:30. This result seems to be related to the release of phenolic compounds bound to protein and also of peptides during hydrolysis. The highest antioxidant activity determined by the FRAP method in the aqueous extract (42 mg SF / g HPL) occurs under pH ~ 9.5 / E: S ~ 1:150 and the methanol extract (40 mg SF / g HPL) pH 8.5-9.0 / E: S 1:90-1:150. For the ORAC method, optimum conditions for increased antioxidant activity in aqueous extract (300 µmol TE / g HPL) are pH 7.5-9.5 / E: S ~ 1:30 or 1:150 and the methanol extract (330 µmol TE / g HPL) are pH ~ 8.5 / E: S 1:30-1:150. The hydrolysates with the highest antioxidant activities were obtained at pH 8.5 / E: S 1:90, and at pH 9.2 / E: S 1:133 denominated HPL ) and HPL 3, respectively. For FLMD, CPL and hydrolysates, after in vitro digestion, the content increased (9-20 times) for all samples. The Folin-Ciocalteau reducing capacity of the CPL (EAG ~ 24 mg / g sample) in both extracts after in vitro digestion equaled the content of hydrolysates (EAG ~ 23 mg / g sample). This result suggests that both hydrolysis with Alcalase and the digestion process are able to release phenolic compounds from the flaxseed products. The antioxidant activity of extracts of FLMD, CPL determined by FRAP, also increased (from 3 to 10 times) after digestion, but did not reached the antioxidant activity of hydrolysates (48 mg SF / g sample). However, when the activity was determined by ORAC, the FPC showed value similar to the hydrolysates, measured on the aqueous extract (~ 420.24 µmol TE / g sample) and 10% higher than on the methanol extract (~ 365 µmol TE / g sample). After in vitro digestion, hydrolysates showed the highest antioxidant activity measured by FRAP (SF 50 mg / g sample), and the FPC, the highest activity determined by ORAC method (~ 430 micromol TE / g sample). These results suggest that digestive process are equally or more effective than Alcalase in releasing peptides and phenolic compounds present in the FPC. Since the methodology for determining the antioxidant activity by ORAC utilizes a biologically relevant radical source, these results suggest the use of FPC as the best protein product of flaxseed with potential antioxidant in the formulation of nutraceuticals and functional foodsMestradoNutrição Experimental e Aplicada à Tecnologia de AlimentosMestre em Alimentos e Nutriçã

Identification of peptides released from flaxseed (Linum usitatissimum) protein by Alcalase® hydrolysis: Antioxidant activity

In this study, the hydrolysis of a flaxseed protein isolate with Alcalase was performed as a strategy to generate antioxidant peptides. A chromatographic separation of the hydrolysate was conducted by RP-HPLC. Both hydrolysate and six collected fractions were subjected to ORAC and FRAP assays to evaluate their antioxidant capacity. The higher antioxidant values were shown by fractions containing predominantly low molecular weight peptides, as it was demonstrated by MALDI analysis. Four peptides were identified by LC-MS/MS and one by Edman degradation. The peptide with sequence GFPGRLDHWCASE was synthesised showing a notable ORAC activity, 3.20 μmol Trolox equivalents/μmol of peptide. This value was higher than that reported for butylated hydroxyanisole. Therefore, the contribution of this peptide to the activity of the fraction where it had been found was 61%. The identified sequences represent an advance in the molecular characterization of the flaxseed protein fraction.This work has received financial support from projects AGL2015-66886-R and FAPESP (2010/52680-7). F.G.D. Silva acknowledges the CNPq for the scholarship granted, and B. Hernández-Ledesma thanks the Ministry of Economy and Competitiveness (MINECO) for her “Ramón y Cajal” post-doctoral contract.Peer Reviewe

Assessing the potential of flaxseed protein as an emulsifier combined with whey protein isolate

The potential use of flaxseed protein isolate (FPI) as an emulsifying agent was studied in combination with whey protein isolate (WPI) or alone. All the FPI and WPI–FPI emulsions were kinetically unstable. The increase of FPI concentration (0.7% w/v) led to a higher creaming stability of the FPI emulsions due partly to a reduction in interfacial tension between aqueous and oil phases, but mainly to the gel network formation. However at this same high FPI concentration, WPI–FPI emulsions showed a decrease in droplet size and creaming stability, which could be due to the presence of flaxseed gum in the protein isolate enhancing depletion effects. A protein excess was verified in the mixed systems (0.14 or 0.7% (w/v) FPI) and the increase of FPI concentration led to an even greater surface protein content. Increasing homogenization conditions (pressure and number of passes), the creaming stability of the FPI systems increased, mainly at higher concentration (0.7% w/v). Meanwhile, in the mixed systems, the creaming stability of the emulsions containing 0.7% (w/v) FPI decreased even more, but was improved for the emulsions with 0.14% (w/v) FPI. Thus, it was observed that systems containing only FPI at higher concentration were stabilized by gel formation, while in WPI–FPI systems there was a competition by interface between biopolymers with a consequent depletion process. As a result, more stable systems were obtained with WPI addition at lower FPI concentration (0.14% w/v) and using higher homogenization pressure and number of passes (60 MPa, two passes).588997CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140287/2009-4Sem informação2011/06083-0 ; 2009/54137-

Production of whey protein isolate – gellan microbeads for encapsulation and release of flaxseed bioactive compounds

Production of 1.5% (w/v) whey protein isolate (WPI) – 0.1, 0.3 or 0.5% (w/v) gellan gum microbeads from extrusion of the oil-in-water (O/W) emulsions into a 0.56% (w/v) calcium chloride (CaCl2) solution was evaluated to encapsulate flaxseed oil (15% v/v) and protein hydrolysate (FPH) (0, 0.25 or 0.5% w/v). Microgels resistance and controlled release of oil and FPH were also investigated. Microscopic images showed few free oil droplets and a prevailing presence of gellan on the external surface of the microbeads, indicating that oil and FPH were encapsulated. Microbeads produced at higher gellan concentrations (0.3 or 0.5% w/v) showed a more regular and spherical morphology. However a significant decrease in microbeads size (from ∼55 μm to ∼50 μm) and an increase in the polydispersity were observed with the FPH addition, which can be a consequence of the formation of a more dense biopolymers network. FPH presence (0.25% w/v) decreased the viscosity and shear thinning behavior of microbeads suspensions (10–90% w/v), which could be partly attributed to the smaller size of particles. The microbeads suspensions were stable at different salt concentrations (0.56, 1.11 or 2.22% w/v) regarding their shape, not releasing the encapsulated oil. 1.5% (w/v) WPI – 0.3% (w/v) gellan microbeads were resistant to simulated gastric conditions, but did not resist to intestinal conditions. Our results show that these microgels are adequate to encapsulate bioactive compounds to be released in the small intestine, passing intact in the stomach, which makes the process attractive in order to maintain the bioavailability and functionality of such compounds247104114CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140287/2009-4; 573913/2008-0Não temEMU 09/54137-1; 08/57906-3The authors are grateful to CNPq (140287/2009-4), CAPES and FAPESP (EMU 09/54137-1) for their financial support and to Professor Valdemiro Carlos Sgarbieri (DEPAN/UNICAMP) for the donation of whey protein isolate. The authors also thank the access to equipment and assistance provided by the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the University of Campinas; INFABIC is co-funded by FAPESP (08/57906-3) and CNPq (573913/2008-0

Identification of peptides released from flaxseed (linum usitatissimum) protein by alcalase(R) hydrolysis: antioxidant activity

In this study, the hydrolysis of a flaxseed protein isolate with Alcalase® was performed as a strategy to generate antioxidant peptides. A chromatographic separation of the hydrolysate was conducted by RP-HPLC. Both hydrolysate and six collected fractions were subjected to ORAC and FRAP assays to evaluate their antioxidant capacity. The higher antioxidant values were shown by fractions containing predominantly low molecular weight peptides, as it was demonstrated by MALDI analysis. Four peptides were identified by LC-MS/MS and one by Edman degradation. The peptide with sequence GFPGRLDHWCASE was synthesised showing a notable ORAC activity, 3.20 μmol Trolox equivalents/μmol of peptide. This value was higher than that reported for butylated hydroxyanisole. Therefore, the contribution of this peptide to the activity of the fraction where it had been found was 61%. The identified sequences represent an advance in the molecular characterization of the flaxseed protein fraction76A140146CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informação2010/52680-

Isosweetness concentrations of sucrose and high‐intensity sweeteners and antioxidant activity in white chocolate with functional properties

Healthier food products are an emerging trend in consumer demand. In this context, this study aimed at producing a prebiotic white chocolate with addition of an antioxidant source [goji berry (GB)] and replacement of sucrose by high‐intensity sweeteners (sucralose and rebaudioside A). The ideal sucrose level in white chocolate was determined as 40.46% (w/w). Different concentrations of dried GB (3%, 6% and 9%, w/w) in white chocolate did not affect consumers' preference. The isosweetness concentrations of the sweeteners were 0.05% for sucralose and from 0.10% to 0.16% (w/w) for rebaudioside A in prebiotic white chocolates. However, among the prebiotic chocolates containing GB, sucralose was the best sucrose substitute. The prebiotic white chocolates with GB showed antioxidant activity up to three times higher than the samples without the dried fruit, determined by ferric reducing antioxidant power and oxygen radical absorbance capacity methods. GB contributed to enhancing the nutritional value of white chocolate51921142122COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESnão te