Functional properties of whey,whey components,and essential amino acids: mechanisms underlying health benefits for active people (review)

Whey proteins and amino acid supplements have a strong position in the sports nutrition market based on the purported quality of proteins and amino acids they provide. Recent studies employing stable isotope methodology demonstrate the ability of whey proteins or amino acid mixtures of similar composition to promote whole body and muscle protein synthesis. Other developing avenues of research explore health benefits of whey that extend beyond protein and basic nutrition. Many bioactive components derived from whey are under study for their ability to offer specific health benefits. These functions are being investigated predominantly in tissue culture systems and animal models. The capacity of these compounds to modulate adiposity, and to enhance immune function and anti-oxidant activity presents new applications potentially suited to the needs of those individuals with active lifestyles. This paper will review the recent literature that describes functional properties of essential amino acids, whey proteins, whey-derived minerals and other compounds and the mechanisms by which they may confer benefits to active people in the context that exercise is a form of metabolic stress. The response to this stress can be positive, as with the accretion of more muscle and improved functionality or greater strength. However, overall benefits may be compromised if immune function or general health is challenged in response to the stress. From a mechanistic standpoint, whey proteins, their composite amino acids, and/or associated compounds may be able to provide substrate and bioactive components to extend the overall benefits of physical activity.     

Bioactive peptides from marine organisms: a short overview

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.     

Biological activities and potential health benefits of bioactive peptides derived from marine organisms

Marine organisms have been recognized as rich sources of bioactive compounds with valuable nutraceutical and pharmaceutical potentials. Recently, marine bioactive peptides have gained much attention because of their numerous health beneficial effects. Notably, these peptides exhibit various biological activities such as antioxidant, anti-hypertensive, anti-human immunodeficiency virus, anti-proliferative, anticoagulant, calcium-binding, anti-obesity and anti-diabetic activities. This review mainly presents biological activities of peptides from marine organisms and emphasizing their potential applications in foods as well as pharmaceutical areas.     

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.     

The macromolecular composition of noncalcified marine macroalgae

The macromolecular composition of macroalgae influences nutrient flow and food quality in aquatic ecosystems and the value of macroalgae species for human consumption, aquaculture, biofuels, and other applications. We used literature data (125 publications, 1,117 observations) and a hierarchal Bayesian statistical model to estimate the average macromolecular composition, protein, lipid, and carbohydrate of macroalgae as a whole and at the phylum level. Our focus was on marine, noncalcified macroalgae sampled from wild‐grown populations in the field. We found that the median macromolecular composition is 9.98% protein, 2.7% lipid, 48.5% carbohydrate, and 31.8% ash as percent dry weight. We compared the median macromolecular content of macroalgae to microalgae and herbaceous plants and test for differences in macromolecular content across macroalgal phyla. Macroalgae were much more enriched in carbohydrate and minerals than the microalgae and lower in protein and lipid than many herbaceous plants. Rhodophyte macroalgae have significantly less lipid and more protein and the Ochrophyte macroalgae have significantly less protein than the average.     

Detection of small bioactive peptides from Atlantic herring (Clupea harengus L.)

Recent research has shown that fish residual materials contain a range of components with interesting biological activity. Therefore, there is a great potential in the marine bioprocess industry to utilize these by-products as starting material for generating more valuable products. The aim of the present study was to search for bioactive peptides (in particular small natural bioactive peptides with molecular weight lower than 10 kDa) in Atlantic herring (Clupea harengus L.) by-products such as skin and more general residual materials. By such means a range of peptides with claimed interesting biological activities was found. Herein the activity of the detected bioactive peptides and strategies for isolating peptide fragments containing the bioactive motif is discussed. Identification of bioactive peptides in crude peptide/protein sources (skin and residual materials) was performed directly using a combination of mass spectrometry (Orbitrap), bioinformatics and database search. This method was a good angle of approach in order to map the potential in new species and species that have been very little studied.     

A label‐free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC‐Chip coupled with Ion Trap mass spectrometry

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label‐free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label‐free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC‐Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (β‐conglutin precursor and vicilin‐like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin‐encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the “normalized protein average of common reproducible peptides” (N‐ACRP) for γ‐conglutin, which is a homogeneous protein, and the “normalized protein mean peptide spectral intensity” (N‐MEAN) for the highly heterogenous class of the vicilins.     

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05 mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

     

Design of a minimized cyclic tetrapeptide that neutralizes bacterial endotoxins.

Septic shock is a leading cause of mortality in intensive care patients, and no specific drugs are as yet available for its treatment. Therefore, new leads are required in order to increase the number of active molecules that may develop into efficacious and safe LPS-neutralizing molecules during pre-clinical stages. We used peptides, derived from the binding regions of known LPS-binding proteins, as scaffolds to introduce modifications at the amino acid level. Structure-activity relationship studies have shown that these modifications generate highly active peptides. Thus, from a bioactive peptide with an initial 16 amino acid residues, a tetrapeptide sequence was determined. After inserting this sequence in a Cys cyclic peptide, it showed the same biological activity as the parent peptide. This sequence could provide the basis for the design of small molecules with LPS-binding properties.     

The importance of effective sampling for exploring the population dynamics of haploid–diploid seaweeds

The mating system partitions genetic diversity within and among populations and the links between life history traits and mating systems have been extensively studied in diploid organisms. As such most evolutionary theory is focused on species for which sexual reproduction occurs between diploid male and diploid female individuals. However, there are many multicellular organisms with biphasic life cycles in which the haploid stage is prolonged and undergoes substantial somatic development. In particular, biphasic life cycles are found across green, brown and red macroalgae. Yet, few studies have addressed the population structure and genetic diversity in both the haploid and diploid stages in these life cycles. We have developed some broad guidelines with which to develop population genetic studies of haploid‐diploid macroalgae and to quantify the relationship between power and sampling strategy. We address three common goals for studying macroalgal population dynamics, including haploid‐diploid ratios, genetic structure and paternity analyses.     

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.     

Potential Applications of Food Derived Bioactive Peptides in Management of Health

Recently the rise in noncommunicable diseases and side effects of drugs has promoted the research in food components with biologically active molecules. These bioactive components are vital in reducing and regulating the onset of such chronic degenerative diseases. Many food derived peptides are biologically active fragments encrypted within the primary protein sequence in nascent (inactive) form, hence also called ‘cryptides’. These bioactive peptides range in size from 2 to 50 amino acids. They function beyond their basic nutritional benefits. Upon oral administration, these peptides play various roles such as opiate like, antioxidative, immunomodulatory, antihypertensive, hypocholesterolemic, mineral binding, antiobesity and antimicrobial. Both animal and plant proteins are rich sources of bioactive peptides having specific physiological and biochemical functions. Digestion of proteins in vivo or in vitro produces free amino acids and peptides which enter circulatory system and exert systemic effect. Bioactive peptides can be produced in vivo through gastrointestinal digestion whereas in vitro through chemical processing of food proteins with acid, alkali, heat and enzymatic hydrolysis either by digestion or fermentation. Protein hydrolysates being rich source of bioactive peptides can serve as an alternative to intact protein and elemental formula in the development of functional foods.     

Metabolomic assessment of induced and activated chemical defence in the invasive red alga Gracilaria vermiculophylla

In comparison with terrestrial plants the mechanistic knowledge of chemical defences is poor for marine macroalgae. This restricts our understanding in the chemically mediated interactions that take place between algae and other organisms. Technical advances such as metabolomics, however, enable new approaches towards the characterisation of the chemically mediated interactions of organisms with their environment. We address defence responses in the red alga Gracilaria vermiculophylla using mass spectrometry based metabolomics in combination with bioassays. Being invasive in the north Atlantic this alga is likely to possess chemical defences according to the prediction that well-defended exotics are most likely to become successful invaders in systems dominated by generalist grazers, such as marine macroalgal communities. We investigated the effect of intense herbivore feeding and simulated herbivory by mechanical wounding of the algae. Both processes led to similar changes in the metabolic profile. Feeding experiments with the generalist isopod grazer Idotea baltica showed that mechanical wounding caused a significant increase in grazer resistance. Structure elucidation of the metabolites of which some were up-regulated more than 100 times in the wounded tissue, revealed known and novel eicosanoids as major components. Among these were prostaglandins, hydroxylated fatty acids and arachidonic acid derived conjugated lactones. Bioassays with pure metabolites showed that these eicosanoids are part of the innate defence system of macroalgae, similarly to animal systems. In accordance with an induced defence mechanism application of extracts from wounded tissue caused a significant increase in grazer resistance and the up-regulation of other pathways than in the activated defence. Thus, this study suggests that G. vermiculophylla chemically deters herbivory by two lines of defence, a rapid wound-activated process followed by a slower inducible defence. By unravelling involved pathways using metabolomics this work contributes significantly to the understanding of activated and inducible defences for marine macroalgae.     

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix–loop–helix peptides

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix–loop–helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.     

WATER MOTION, MARINE MACROALGAL PHYSIOLOGY, AND PRODUCTION

Water motion is a key determinant of marine macroalgal production, influencing directly or indirectly physiological rates and community structure. Our understanding of how marine macroalgae interact with their hydrodynamic environment has increased substantially over the past 20 years, due to the application of tools such as flow visualization to aquatic vegetation, and in situ measurements of seawater velocity and turbulence. This review considers how the hydrodynamic environment in which macroalgae grow influences their ability to acquire essential resources and how macroalgae might respond physiologically to fluctuations in their hydrodynamic regime with a focus on: (1) the biochemical processes occurring within the diffusion boundary layer (DBL) that might reduce rates of macroalgal production; (2) time scales over which measurements of velocity and DBL processes should be made, discussing the likelihood of in situ mass transfer limitation; (3) if and how macroalgal morphology influences resource acquisition in slow flows; and (4) ecobiomechanics and how hydrodynamic drag might influence resource acquisition and allocation. Finally, the concept that macroalgal production is enhanced in wave-exposed versus sheltered habitats is discussed.     

Biochemical and metabolic mechanisms by which dietary whey protein may combat obesity and Type 2 diabetes

Consumption of milk and dairy products has been associated with reduced risk of metabolic disorders and cardiovascular disease. Milk contains two primary sources of protein, casein (80%) and whey (20%). Recently, the beneficial physiological effects of whey protein on the control of food intake and glucose metabolism have been reported. Studies have shown an insulinotropic and glucose-lowering properties of whey protein in healthy and Type 2 diabetes subjects. Whey protein seems to induce these effects via bioactive peptides and amino acids generated during its gastrointestinal digestion. These amino acids and peptides stimulate the release of several gut hormones, such as cholecystokinin, peptide YY and the incretins gastric inhibitory peptide and glucagon-like peptide 1 that potentiate insulin secretion from β-cells and are associated with regulation of food intake. The bioactive peptides generated from whey protein may also serve as endogenous inhibitors of dipeptidyl peptidase-4 (DPP-4) in the proximal gut, preventing incretin degradation. Indeed, recently, DPP-4 inhibitors were identified in whey protein hydrolysates. This review will focus on the emerging properties of whey protein and its potential clinical application for obesity and Type 2 diabetes.