Enzyme-Assisted Release of Antioxidant Peptides from Porphyra dioica Conchocelis.

The conchocelis life cycle stage of P. dioica represents an unexplored source of bioactive compounds. The aim of this study was to generate and characterise, for the first time, hydrolysates of conchocelis using a specific combination of proteases (Prolyve® and Flavourzyme®). Hydrolysate molecular mass distribution and free amino acid contents were assessed, and the antioxidant activity was determined using a range of in vitro assays. The protein content and the total amino acid profiles of conchocelis were also studied. Conchocelis contained ~25% of protein (dry weight basis) and had a complete profile of essential amino acids. Direct sequential enzymatic treatment modified the profile of the generated compounds, increasing the amount of low molecular weight peptides (<1 kDa). There was a significant improvement in the antioxidant activity of the hydrolysates compared with the control (up to 2.5-fold), indicating their potential as a novel source of antioxidant ingredients.

Amino Acid Profile and Protein Quality Assessment of Macroalgae Produced in an Integrated Multi-Trophic Aquaculture System.

Seaweeds are a recognized source of bioactive compounds and techno-functional ingredients. However, its protein fraction is still underexplored. The aim of this study was to determine the total and free amino acid profile and protein content of four seaweeds species (Porphyra dioica, Porphyra umbilicalis,Gracilaria vermiculophylla, and Ulva rigida) produced in an integrated multi-trophic aquaculture system, while assessing their protein quality. Samples were submitted to acid and alkaline hydrolysis (total amino acids) and to an aqueous extraction (free amino acids) followed by an automated online derivatization procedure, and analyzed by reverse phase-high performance liquid chromatography. Protein-, non-protein and total-nitrogen were quantified by the Kjeldahl method. Crude and true protein contents were estimated based on the nitrogen and amino acid composition. Protein quality was assessed based on the amino acids profile. Porphyra species presented the highest protein content compared to the remaining three seaweed species tested. All samples presented a complete profile of essential amino acids and a high quality protein profile, according to World Health Organization and Food and Agriculture Organization standards. Methionine and tryptophan were the first limiting amino acids in all species. Red species (Porphyra and Gracilaria) presented high levels of free alanine, glutamic, and aspartic acids. The results highlight the potential of using seaweeds as an alternative and sustainable source of protein and amino acids for human nutrition and industrial food processing.

Effect of in vitro simulated gastrointestinal digestion on the antioxidant activity of the red seaweed Porphyra dioica.

Porphyra sp. is one of the most cultivated and commercially valuable species, recognized for its high protein content (up to 47% dry weight) and complete amino acids profile. Based on these characteristics, P. dioica produced in an integrated multitrophic aquaculture system was selected for this study. The aim was to evaluate the effect of in vitro simulated gastrointestinal digestion (SGID) on the antioxidant activity of the hydrolysates generated from dried blades and from the protein isolate (PI) extracted from them. The alkali extraction and isoelectric precipitation (pH 4.5) of P. dioica protein prior SGID allowed isolating/enriching protein, while direct SGID of blades allowed assessing the potential influence of other constituents of the sample on the bioactive properties. Overall, SGID promoted the release of smaller bioactive peptides and their in vitro antioxidant activity, which was assessed by different methods (DPPH and ABTS+ scavenging capacity, ORAC and FRAP), was improved compared to the intact samples. Blades submitted to direct SGID presented significantly higher ORAC values compared to PI (2010 ± 136 vs 542 ± 21 µmol TE/g FDS, respectively). For the remaining assays, PI presented more potent antioxidant activity, especially FRAP (131 ± 2 vs 16 ± 1 µmol TE/g FDS) and ABTS+ (1244 ± 157 vs 230 ± 15 µmol TE/g FDS). The results indicated that gastrointestinal digestion improved the antioxidant activity of P. dioica-derived hydrolysates, as they presented effective activity against different oxidative mechanisms, thus suggesting health-protecting effects.

Enzymatic Modification of Porphyra dioica-Derived Proteins to Improve their Antioxidant Potential.

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve® and Prolyve® plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components <1 kDa. Antioxidant activity was assessed using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) inhibition, and reactive oxygen species scavenging ability, i.e., oxygen radical absorbance capacity (ORAC) and hypochlorous acid (HOCl) scavenging assays. In general, enzymatic hydrolysis of P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 µmol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS·+ assays). Such hydrolysates may be of value as functional food ingredients.

The phytochemical and bioactivity profiles of wild Calluna vulgaris L. flowers.

The nutritional composition of Calluna vulgaris flowers as well as the phytochemical profile, antioxidant (DPPH and FRAP assays), antimicrobial and cytotoxic (in human immortalized non-tumorigenic keratinocyte and fibroblasts) activities of aqueous, hydroalcoholic and ethanolic extracts were evaluated. A high content of fiber and carbohydrates (75%) and the prevalence of α-tocopherol as vitamer deserves attention. Linolenic (35%), linoleic (27%) and palmitic (21%) acids were the most abundant fatty acids. Qualitative and quantitative analysis by LC-MS and 1NMR indicated high levels of quercetin, kaempferol and myricetin derivatives as well as procyanidins. The hydro-alcoholic extract displayed the highest antioxidant activity and total phenolics (TPC) and flavonoid contents (TFC). No adverse effects on cells were observed until a concentration of 100 µg/mL and a good antimicrobial activity was reported against S. epidermidis and S. aureus with the hydro-alcoholic extract. The data obtained demonstrated that wild plants like heather, although not being a common nutritional reference, can be used in an alimentary base as a source of bioactive compounds, namely antioxidants.

Evaluation of the cytotoxicity (HepG2) and chemical composition of polar extracts from the ruderal species Coleostephus myconis (L.) Rchb.f.

Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a highly disseminated plant species with ruderal and persistent growth. Owing to its advantageous agronomic properties, C. myconis might have industrial applications. However, this species needs to be comprehensively characterized before any potential use. In a previous study, the phenolic composition and antioxidant activity of different C. myconis tissues were characterized. This investigation was extended to examine the cytotoxic potential of selected plant tissues (flowers and green parts) using a HepG2 cell line by utilizing the lysosomal neutral red uptake assay or mitochondrial (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. In addition, the macronutrients content, lipophilic compounds (fatty acids, tocopherols), and amino acids were also determined. C. myconis flowers were used in the senescence stage, which was previously identified as the stage that presented maximal phenolic content and highest antioxidant activity. In contrast, stems and leaves were employed due to their high biomass proportion. Regarding cytotoxicity, mitochondrial and lysosomal damage was only significant when HepG2 cells were exposed to the highest extract concentrations (stems and leaves, 0.9 mg/ml; senescent flowers, 0.3 mg/ml). Chemically, the senescent flowers were mostly characterized by their high levels of fat, amino acids (especially threonine), oleic acid, ß-, and γ-tocopherol, while stems and leaves contained high concentrations of carbohydrates, linolenic acid, and α-tocopherol. In general, these results provide information regarding the threshold concentrations of C. myconis extracts that might be used in different applications without toxicity hazards.

The Castanea sativa bur as a new potential ingredient for nutraceutical and cosmetic outcomes: preliminary studies.

Chestnuts are a common food product in Mediterranean countries, being recognized also for their beneficial effects on human health. Nevertheless, during processing, these fruits generate a large amount of food by-products, such as shells and burs. In the present work, the macronutrient composition, vitamin E profile and amino acid content of the burs were determined in samples from three different Portuguese regions (Minho, Trás-os-Montes and Beira-Alta). The nutritional composition was similar for all samples, being characterised by a high moisture content and low fat amounts. All essential amino acids were present in considerable amounts. Concerning vitamin E, the predominant vitamer was α-tocopherol for the Minho and Beira-Alta samples. The total phenolic compounds were quantified, and the antioxidant activity evaluated in different extracts using two biochemical assays (DPPH˙ and FRAP). All bur extracts showed a high total phenolic content, the highest obtained being that for the Beira-Alta samples. The chestnut bur from Minho showed the highest antioxidant activity in both assays. This study aims to demonstrate the potential of the Castanea sativa bur as a cosmetic and nutraceutical ingredient.

Improving the extraction of Ara h 6 (a peanut allergen) from a chocolate-based matrix for immunosensing detection: Influence of time, temperature and additives.

The extraction of Ara h 6 (a peanut allergen) from a complex chocolate-based food matrix was optimized by testing different temperatures, extraction times, and the influence of additives (NaCl and skimmed milk powder) in a total of 36 different conditions. Analyses were carried out using an electrochemical immunosensor. Three conditions were selected since they allowed the extraction of the highest levels of Ara h 6. These extractions were performed using 2g of sample and 20ml of Tris-HNO3 (pH=8) containing: a) 0.1M NaCl and 2g of skimmed milk powder at 21°C for 60min; b) 1M NaCl and 1g of skimmed milk powder at 21°C for 60min; and c) 2g of skimmed milk powder at 60°C for 60min. Recoveries were similar or higher than 94.7%. This work highlights the importance to adjust extraction procedures regarding the target analyte and food matrix components.

The isoflavone content of two new alfalfa-derived products for instant beverage preparation.

The frequent use of plant-based products to promote health leads to the search for scientific information related to efficacy and safety of those products for human consumption. Two alfalfa-derived products (ADP), freeze-dried juice (FDJ) and dehydrated powder (DP), from alfalfa harvested in Mexico, are being developed as new possible nutraceuticals. To the best of our knowledge, any study reports the real composition of such products used to prepare instant beverages in what concerns isoflavone contents. Seven isoflavones (glycitein, formononetin, biochanin A, daidzein, genistein, daidzin and genistin) were assessed by HPLC-DAD analysis as well as its variation in five different batches of these products. Different solvents were tested in order to choose the best one to extract isoflavones. The results showed the presence of daidzein, genistein, genistin and daidzin in most samples while glycitein, formononetin and biochanin A were not detected. Significant differences between isoflavone contents were found with different solvent systems. Water was the best option to extract daidzein (0.40-1.08 mg per unit and 1.30-4.90 mg per unit for DP and FDJ, respectively) whereas the water-methanol-formic acid mixture was efficient to extract genistein (0.19-0.43 mg per unit and 0.15-0.72 mg per unit for DP and FDJ, respectively). In all cases, the total isoflavone content was higher in freeze-dried juices than in dehydrated powders. Genistein and daidzein were the more abundant isoflavones quantified. Further physiological and nutritional studies are needed to complete the validation of effectiveness and safety of these products.

Detection of the peanut allergen Ara h 6 in foodstuffs using a voltammetric biosensing approach.

A voltammetric biosensor for Ara h 6 (a peanut allergen) detection in food samples was developed. Gold nanoparticle-modified screen-printed carbon electrodes were used to develop a sandwich-type immunoassay using two-monoclonal antibodies. The antibody-antigen interaction was detected through the electrochemical detection of enzymatically deposited silver. The immunosensor presented a linear range between 1 and 100 ng/ml, as well as high precision (inter-day RSD ≤9.8%) and accuracy (recoveries ≥96.7%). The detection and quantification limits were 0.27 and 0.88 ng/ml, respectively. It was possible to detect small levels of Ara h 6 in complex food matrices.

Promising new applications of Castanea sativa shell: nutritional composition, antioxidant activity, amino acids and vitamin E profile.

The present study was aimed to assess the macronutrient composition and the amino acid and vitamin E profiles of Castanea sativa shell from different production regions of Portugal (Minho, Trás-os-Montes and Beira-Alta). The nutritional composition was similar for all samples, with a high moisture content and low fat amounts. Arginine and leucine were the predominant essential amino acids (EAA) accounting for 3.55-7.21% and 1.59-2.08%, respectively, for samples of the different production zones. All the shells presented high contents of vitamin E (481.5 mg per 100 g sample, 962.8 mg per 100 g sample and 567.5 mg per 100 g sample, respectively, for Minho, Trás-os-Montes and Beira-Alta). The predominant vitamer was γ-tocopherol (670 mg per 100 g sample for Trás-os-Montes). The antimicrobial and antioxidant activities of C. sativa shell were also determined. Trás-os-Montes extracts displayed the highest antioxidant activity (EC50 = 31.8 ± 1.3 µg mL(-1) for DPPH; 8083.5 ± 164.8 µmol per mg db for FRAP). The total phenolic content (TPC) varied from 241.9 mg to 796.8 mg gallic acid equivalents (GAE) per g db sample, the highest TPC being obtained for Trás-os-Montes. The total flavonoid content (TFC) varied from 31.4 to 43.3 mg of catechin equivalents (CEQ) per g db sample. No antimicrobial activity was observed. The results showed the potentialities of C. sativa shell extracts.

Detection of Ara h 1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor.

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter = 32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody-antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut.

Targeting specific nutrient deficiencies in protein-restricted diets: some practical facts in PKU dietary management.

Among aminoacidopathies, phenylketonuria (PKU) is the most prevalent one. Early diagnosis in the neonatal period with a prompt nutritional therapy (low natural-protein and phenylalanine diet, supplemented with phenylalanine-free amino acid mixtures and special low-protein foods) remains the mainstay of the treatment. Data considering nutrient contents of cooked dishes is lacking. In this study, fourteen dishes specifically prepared for PKU individuals were analysed, regarding the lipid profile and iron and zinc contents. These dishes are poor sources of essential nutrients like Fe, Zn or n-3 fatty acids, reinforcing the need for adequate supplementation to cover individual patients' needs. This study can contribute to a more accurate adjustment of PKU diets and supplementation in order to prevent eventual nutritional deficiencies. This study contributes to a better understanding of nutrient intake from PKU patients' meals, showing the need for dietary supplementation.

Phenylketonuria: protein content and amino acids profile of dishes for phenylketonuric patients. The relevance of phenylalanine.

Phenylketonuria is an inborn error of metabolism, involving, in most cases, a deficient activity of phenylalanine hydroxylase. Neonatal diagnosis and a prompt special diet (low phenylalanine and natural-protein restricted diets) are essential to the treatment. The lack of data concerning phenylalanine contents of processed foodstuffs is an additional limitation for an already very restrictive diet. Our goals were to quantify protein (Kjeldahl method) and amino acid (18) content (HPLC/fluorescence) in 16 dishes specifically conceived for phenylketonuric patients, and compare the most relevant results with those of several international food composition databases. As might be expected, all the meals contained low protein levels (0.67-3.15 g/100 g) with the highest ones occurring in boiled rice and potatoes. These foods also contained the highest amounts of phenylalanine (158.51 and 62.65 mg/100 g, respectively). In contrast to the other amino acids, it was possible to predict phenylalanine content based on protein alone. Slight deviations were observed when comparing results with the different food composition databases.