Digestion-resistant whey peptides promote antioxidant effect on Caco-2 cells.

Enteric endothelial cells are the first structure to come in contact with digested food and may suffer oxidative damage by innumerous exogenous factors. Although peptides derived from whey digestion have presented antioxidant potential, little is known regarding antioxidant pathways activation in Caco-2 cell line model. Hence, we evaluated the ability to form whey peptides resistant to simulated gastrointestinal digestive processes, with potential antioxidant activity on gastrointestinal cells and associated with sequence structure and activity. Using the INFOGEST method of simulated static digestion, we achieved 35.2% proteolysis, with formation of peptides of low molecular mass (<600 Da) evaluated by FPLC. The digestion-resistant peptides showed a high proportion of hydrophobic and acidic amino acids, but with average surface hydrophobicity. We identified 24 peptide sequences, mainly originated from ß-lactoglobulin, that exhibit various bioactivities. Structurally, the sequenced peptides predominantly contained the amino acids lysine and valine in the N-terminal region, and tyrosine in the C-terminal region, which are known to exhibit antioxidant properties. The antioxidant activity of the peptide digests was on average twice as potent as that of the protein isolates for the same concentration, as evaluated by ABTS, DPPH and ORAC. Evaluation of biological activity in Caco-2 intestinal cells, stimulated with hydrogen peroxide, showed that they attenuated the production of reactive oxygen species and prevented GSH reduction and SOD activity increase. Caco-2 cells were not responsive to nitric oxide secretion. This study suggests that whey peptides formed during gastric digestion exhibit biological antioxidant activity, without the need for previously hydrolysis with exogenous enzymes for supplement application. The study's primary contribution was demonstrating the antioxidant activity of whey peptides in maintaining the gastrointestinal epithelial cells, potentially preventing oxidative stress that affects the digestive system.

In vitro inhibition of glucose gastro-intestinal enzymes and antioxidant activity of hydrolyzed collagen peptides from different species.

The growing value of industrial collagen by-products has given rise to interest in extracting them from different species of animals. Intrinsic protein structure variation of collagen sources and its hydrolysis can bring about different bioactivities. This study aimed to characterize and evaluate the differences in vitro biological potential of commercial bovine (BH), fish (FH), and porcine hydrolysates (PH) regarding their antioxidant and hypoglycemic activities. All samples showed percentages above 90% of protein content, with high levels of amino acids (glycine, proline, and hydroxyproline), responsible for the specific structure of collagen. The BH sample showed a higher degree of hydrolysis (DH) (8.7%) and a higher percentage of smaller than 2 kDa peptides (74.1%). All collagens analyzed in vitro showed inhibition of pancreatic enzymes (α-amylase and α-glucosidase), with the potential to prevent diabetes mellitus. The PH sample showed higher antioxidant activities measured by ORAC (67.08 ± 4.23 µmol Trolox Eq./g) and ABTS radical scavenging (65.69 ± 3.53 µmol Trolox Eq./g) methods. For the first time, DNA protection was analyzed to hydrolyzed collagen peptides, and the FH sample showed a protective antioxidant action to supercoiled DNA both in the presence (39.51%) and in the absence (96.36%) of AAPH (reagent 2,2'-azobis(2-amidinopropane)). The results confirmed that the source of native collagen reflects on the bioactivity of hydrolyzed collagen peptides, probably due to its amino acid composition. PRACTICAL APPLICATIONS: Our data provide new application for collagen hydrolysates with hypoglycemiant and antioxidant activity. These data open discussion for future studies on the additional benefits arising from collagen peptide consumption for the prevention of aging complications or hyperglycemic conditions as observed in chronic diseases such as diabetes mellitus type II (DM 2). The confirmation of these results can open new market areas for the use of collagen with pharmacological applications or to produce new supplements. Furthermore, provides a solution for waste collagen from meat industries and adds value to the product.

Impact of Gluten-Free Sorghum Bread Genotypes on Glycemic and Antioxidant Responses in Healthy Adults.

Sorghum is used to provide good quality gluten-free products due to phytochemicals and low glycemic index (GI). This study aimed to determine the chemical composition, the antioxidant activity and capacity, and the glycemic and insulinemic responses of gluten-free (GF) sorghum bread. GF bread samples were produced with three different sorghum genotypes. The samples were analyzed for chemical composition, resistant starch and dietary fiber content; antioxidant activity by ORAC; antioxidant capacity by FRAP; GI; and insulinemic responses. This double-blind, crossover, randomized clinical trial was conducted with 10 healthy men aged 28.0 ± 4.9 years (77.6 ± 11.7 kg and 24.2 ± 2.3 kg/m2). All sorghum bread showed significantly more fiber than rice bread (control). Brown sorghum bread was classified as low GI, bronze and white as medium GI, and control as high GI. Brown sorghum bread presented a low carbohydrate content, a significant amount of fiber, and a significantly lower 3 h AUC glucose response than those of the control, aside from the highest antioxidant activity value (p ≤ 0.001). Therefore, brown sorghum was superior to other genotypes analyzed in this study, and its production should be encouraged to provide gluten-free products with a better nutritional profile. More research is required to explore the effects of different sorghum genotypes in food products on human health.

Copper-Binding Peptides Attenuate Microglia Inflammation through Suppression of NF-kB Pathway.

SCOPE: Activation of microglia, the resident immune cells of the central nervous system, has been related to the etiology and progression of neurodegenerative diseases; thus, finding novel approaches to suppress the neuroinflammatory process is of utmost relevance. METHODS AND RESULTS: The anti-inflammatory activity of whey Cu-, Fe-, and Zn-binding peptides and their possible underlying mechanism of action were evaluated in microglia. Whey metal-binding peptides decreased nitric oxide production and tumor necrosis factor α (TNF-α) at mRNA and protein levels by stimulated BV-2 microglia in comparison to the control with no peptide treatment. The hydrophobicity, specific sequences, and possible synergistic effects seem to play a role. Cu-binding peptides (Cu-bp) presented anti-inflammatory activity both in BV-2 and primary microglia cultures. These peptides exert their action by suppressing nuclear factor kappa B (NF-kB) pathway since nuclear translocation of NF-kB p65 is decreased by roughly 30% upon Cu-bp treatment. Specific sequences identified in Cu-bp showed high affinity to bind NF-kB p65 by molecular docking (up to -8.8 kcal mol-1 ), corroborating the immunofluorescence studies. CONCLUSION: Cu-bp represent food-derived peptides that may be useful for neuroprotective purposes. Chelation of copper excess in the CNS and the bioavailability of such peptides, as well as their behavior in in vivo models, deserve further research for future applications.

Isolation and Sequencing of Cu-, Fe-, and Zn-Binding Whey Peptides for Potential Neuroprotective Applications as Multitargeted Compounds.

This study aims to isolate metal-binding peptides and synthesize promising amino acid sequences to potentially act as neuroprotective compounds in the future, targeting different mechanisms. Fractions of whey metal-binding peptides (Cu, Fe, and Zn) isolated by immobilized metal affinity chromatography showed different amino acid profiles according to the metal. The Cu-binding peptides presented roughly twofold increase in the in vitro antioxidant, as assessed by oxygen radical absorbance capacity and anticholinesterase activities over the hydrolysate. This is probably because of the higher concentration of aromatic and basic residues, the latter being crucial for binding to the anionic sites of acetylcholinesterase. Six peptide sequences were synthesized based on the metal-binding sites, molecular mass, hydrophobicity, and bioactivity probability. Among the synthetic peptides, the VF dipeptide stood out both for its in vitro antioxidant and anticholinesterase activities. This peptide, as well as the fraction of Cu-binding peptides, should be further studied because it may act through different mechanisms related to neurodegenerative diseases, in addition to the chelation of the excess of metals in the central nervous system.

Identification of Angiotensin I-Converting Enzyme-Inhibitory and Anticoagulant Peptides from Enzymatic Hydrolysates of Chicken Combs and Wattles.

Peptides from protein hydrolysate of a mixture of chicken combs and wattles (CCWs) were obtained through enzymatic hydrolysis, and their anticoagulant and inhibitory effects on angiotensin I-converting enzyme (ACE) were investigated. The protein hydrolysate exhibited anticoagulant capacity by the intrinsic pathway (activated partial thromboplastin time) and potent ACE-inhibitory activity. The peptides were sequenced by LC-MS to identify those with higher inhibitory potential. From the pool of sequenced peptides, the following three peptides were selected and synthesized based on their low molecular weight and the presence of amino acids with ACE-inhibitory potential at the C-terminus: peptide I (APGLPGPR), peptide II (Piro-GPPGPT), and peptide III (FPGPPGP). Peptide III (FPGPPGP) showed the highest ACE-inhibitory capacity among the peptides selected. In conclusion, a peptide (FPGPPGP) of unknown sequence was identified as having potent ACE-inhibitory capacity. This peptide originated from unconventional hydrolysates from poultry slaughter waste, including combs and wattles.

Microencapsulation performance of Fe-peptide complexes and stability monitoring.

Iron supplementation presents several challenges, such as low bioavailability, high reactivity and a metallic taste. Iron absorption is enhanced by complexing with organic compounds such as peptides, while microencapsulation is an alternative to protect the mineral and mask undesirable flavors. Fe-peptide complexes were obtained by reacting small whey peptides (< 5 kDa) with iron (from ferrous sulfate) under controlled conditions. Maltodextrin (MD) and polydextrose (PD) were used as the wall materials and spray dried to form particles containing the active Fe-peptide. The conditions of enzymatic hydrolysis with the bacterial endopeptidase produced from Bacillus licheniformis were optimized to achieve a high degree of cleavage (~20% degree of hydrolysis). The physicochemical and structural properties of the microparticles were evaluated during storage (365 days). The encapsulation process showed high efficiency (84%) and process yield (≥90%). The iron dialyzability and uptake by Caco-2 cells from microparticles were at least 3-fold higher than the ferrous sulfate. The water content and water activity varied from 3.0 to 5.7% and from 0.29 to 0.44, respectively, after 365 days. SEM revealed morphological stability during storage and EDX showed the presence of iron ions at the surface of the microparticles, which could be free or complexed. The microparticles can be an alternative of higher bioavailable iron besides the further protection and iron stability which the microparticles may present when compared with the Fe-peptide complexes. Future studies could demonstrate the feasibility of applying these microparticles in formulation for food supplementation, concerning bioavailability and sensory aspects.

Roasting soybeans in a microwave for manufacturing chocolate dragées / Torração de soja em micro-ondas para desenvolvimento de drágeas de chocolate

ABSTRACT: This study aimed to evaluate the effect of different microwave-roasting timeson different sizes of soybean grains with black tegument to achieve the best crunchiness, flavor, and hardness. Black soybean dragées- containing a soybean core coated with layers of chocolate (dragée) - were manufactured, and their nutritional value, microbiological risk, acceptability, and consumer-purchase intentions were verified. Flavor and crunchiness of the roasted black soybeans wereonly affected by the roasting time in the microwave, whereas the hardness was only affected by grain size. The best core of the dragéewas obtainedusing grains ofsieve size 15 (large grains) roastedin the microwave for 11 min; this core presented higher frequencies for extremely crunchy notes (56%), toasted flavor (61%), and an instrumental hardness close to that of the existing commercial product (33.17 N). The manufactured dragées did not present resistant starch, anthocyanins, and microbiological risk, and they were sensorially accepted by the tasters (mean scores of 7.77 for texture and 8.36 for appearance on a scale of 1 to 9). Thus, roasted black soybean dragéeshave a high marketing potential from technological, nutritional, and sensorial points of view.

RESUMO: Este estudo objetivou avaliar o efeito do tempo de torra no micro-ondas para diferentes tamanhos de grãos de soja tipo alimento de tegumento preto, de forma a atingir a melhor crocância, sabor e dureza, e em seguida caracterizar o seu confeito, estruturado na forma de núcleo e camadas de cobertura de chocolate (drageados), em relação ao valor nutricional, risco microbiológico, aceitabilidade e intenção de compra pelos consumidores. O sabor e a crocância da soja preta torrada foram afetados somente pelo tempo de torra em micro-ondas, enquanto a dureza somente foi afetada pela dimensão dos grãos. O melhor núcleo (selecionado) foi obtido com os grãos de peneira 15 (grãos grandes), no tempo de 11 min de torra em micro-ondas, pois apresentou maior frequência para as notas de extremamente crocante (56%), sabor de torrado (61%), e uma dureza instrumental próxima ao produto comercial (33,17 N). As drágeas não apresentaram amido resistente, antocianinas e risco microbiológico, e foram sensorialmente aceitas pelos provadores (scores médios de 7,77 para textura e 8,36 para aparência, em uma escala de 1 a 9). Portanto, o drageado de soja preta torrada com chocolate é viável do ponto de vista tecnológico, nutricional e sensorial, com alto potencial de comercialização.

Short communication: Identification of iron-binding peptides from whey protein hydrolysates using iron (III)-immobilized metal ion affinity chromatography and reversed phase-HPLC-tandem mass spectrometry.

Peptides with iron-binding capacity obtained by hydrolysis of whey protein with Alcalase (Novozymes, Araucaria, PR, Brazil), pancreatin, and Flavourzyme (Novozymes) were identified. Hydrolysates were subjected to iron (III)-immobilized metal ion affinity chromatography, and the bound peptides were sequenced by mass spectrometry. Regardless of the enzyme used, the domains f(42-59) and f(125-137) from ß-lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case of peptides derived from α-lactalbumin, with sequences deriving from diverse regions. Iron-bound peptides exhibited common structural characteristics, such as an abundance of Asp, Glu, and Pro, as revealed by mass spectrometry and AA analysis. In conclusion, this characterization of iron-binding peptides helps clarify the relationship between peptide structure and iron-chelating activity and supports the promising role of whey protein hydrolysates as functional ingredients in iron supplementation treatments.

Efeito de um hidrolisado de proteínas de soro de leite e de seus peptídeos na proteção de lesões ulcerativas da mucosa gástrica de ratos / Effects of a whey protein concentrate and it's peptides in the protection of ulcerative lesions at rat gastric mucosa

OBJETIVO: Avaliar a atividade do hidrolisado das proteínas de soro de leite bovino e uma fração de peptídeos de baixo peso molecular (peso molecular <1kDa), na proteção do epitélio da mucosa do estômago de ratos Wistar adultos contra o processo ulcerativo, induzidos por três diferentes agentes. MÉTODOS: Nesse estudo foram utilizados os modelos de indução de úlcera pelo antiinflamatório indometacina (30mg/kg de peso), por etanol absoluto (1ml/animal) e por estresse causado por imobilização e frio (4ºC/2h) em ratos Wistar adultos. RESULTADO: O hidrolisado protéico de soro de leite foi obtido por tratamento com pancreatina, ao grau de hidrólise de 20 por cento, e fracionado em membrana de fluxo tangencial com faixa de corte de 1kDa, para obtenção de uma fração contendo peptídeos de baixo peso molecular denominada peptídeos do hidrolisado protéico de soro (<1kDa). A administração aguda do hidrolisado protéico de soro, de acordo com o modelo etanol, resultou em 65,5 por cento de redução dos índices de lesões ulcerativas, sendo obtida 77,4 por cento de inibição em dose dupla. CONCLUSAO: O efeito citoprotetor dos peptídeos de baixo peso molecular foi mais elevado para o modelo de indução por antiinflamatório, em relação ao hidrolisado integral, tanto em dose única como em dupla (53,1 por cento e 71,6 por cento de redução dos índices de lesões ulcerativas, respectivamente). Não foi constatada atividade protetora em modelos de úlcera induzidos por estresse.

Effect of different hydrolysates of whey protein on hepatic glutathione content in mice.

This study was designed to compare the effects of diets prepared with enzymatic hydrolysate of a whey protein concentrate (WPC) by pancreatin, protamex (Novo Nordisk, Bagsvaerd, Denmark), and alcalase proteases on the hepatic glutathione content in mice. The undenatured WPC was produced in a pilot plant by membrane technology (microfiltration/diafiltration) after separation of the casein clot through a conventional process. All three hydrolysates with 20% degree of hydrolysis showed an amino acid profile similar to WPC. Male A/J mice were fed on diets containing 20% WPC or hydrolysates. Commercial casein was used as a reference protein in the biological assays. The glutathione content was determined after liver extraction through high-performance capillary electrophoresis. WPC and its pancreatin and protamex hydrolysates showed higher ability to stimulate liver glutathione synthesis than alcalase hydrolysate. This difference was probably related to an amino acid sequence in the peptides that were formed during hydrolysis of whey proteins. Commercial casein and WPC alcalase hydrolysate produced lower stimulation of liver glutathione synthesis (7.09 and 5.66 micromol/g of wet weight) compared with WPC and pancreatin and protamex hydrolysates (8.72, 8.71, and 8.45 micromol/g of wet weight, respectively). These results indicate that the hydrolysates obtained by treatment with pancreatin and protamex are good sources of peptides with activity to stimulate glutathione synthesis.