Separation of bioactive peptides by electrodialysis with ultrafiltration membranes: membrane characteristics, ex-situ and in-situ digestion and their impact on peptide migration

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Production, isolation and characterization of bioactive peptides with antihypertensive properties from rapeseed and potato protein

Consumers’ increasing awareness of healthiness and sustainability of food presents a great challenge to food industry to develop healthier, biologically active and sustainable food products. Bioactive peptides derived from food proteins are known to possess various biological activities. Among the activities, the most widely studied are antioxidant activities and angiotensin I converting enzyme (ACE) inhibitory activity related to blood pressure regulation and antihypertensive effects. Meanwhile, vast amounts of byproducts with high protein content are produced in food industry, for example potato and rapeseed industries. The utilization of these by-products could be enhanced by using them as a raw material for bioactive peptides. The objective of the present study was to investigate the production of bioactive peptides with ACE inhibitory and antioxidant properties from rapeseed and potato proteins. Enzymatic hydrolysis and fermentation were utilized for peptide production, ultrafiltration and solid-phase extraction were used to concentrate the active peptides, the peptides were fractionated with liquid chromatographic processes, and the peptides with the highest ACE inhibitory capacities were putified and analyzed with Maldi-Tof/Tof to identify the active peptide sequences. The bioavailability of the ACE inhibitory peptides was elucidated with an in vitro digestion model and the antihypertensive effects in vivo of rapeseed peptide concentrates were investigated with a preventive premise in 2K1C rats. The results showed that rapeseed and potato proteins are rich sources of ACE inhibitory and antioxidant peptides. Enzymatic hydrolysis released the peptides effectively whereas fermentation produced lower activities.The native enzymes of potato were also able to release ACE inhibitory peptides from potato proteins without the addition of exogenous enzymes. The rapeseed peptide concentrate was capable of preventing the development of hypertension in vivo in 2K1C rats, but the quality of rapeseed meal used as raw material was found to affect considerably the antihypertensive effects and the composition of the peptide fraction.

Bioactive peptides from food proteins: new opportunities and challenges

Food proteins such as milk and soy are a rich source of bioactive peptides. In the last decade, research into this area has intensified and new bioactive peptide sequences have been discovered with a range of apparent biological functions; for example, antihypertensive, antioxidant, and antimicrobial effects and opiate-like qualities have been reported. These peptides could therefore lead to the development of important functional food products and ingredients for the prevention and even treatment of chronic diseases such as cardiovascular disease and cancer. Peptides can be produced by fermentation with dairy starters for instance, and by enzymatic hydrolysis with pancreatic and microbial enzymes. Further purification is typically carried out by membrane filtration and/or chromatographic methods. The production of novel bioactive peptides and their incorporation into functional food products poses several technological challenges as well as regulatory and marketing issues. Proof of efficacy is of paramount importance; this should be verified by conducting appropriate tests in vivo in animals and in humans. In addition, tests for cytotoxicity and allergenicity must be conducted. Despite all of these hurdles, scientific evidence is increasingly demonstrating the health benefits of diet-based disease prevention, and therefore new developments in this area are likely to continue both at the research and the commercialisation level.

Production of bioactive peptides through sequencial action of Yarrowia lipolytica proteases and chemical glycation

This PhD thesis is aimed at studying the suitability of proteases realised by Yarrowia lipolytica to hydrolyse proteins of different origins available as industrial food by-products. Several strains of Y. lipolytica have been screened for the production of extracellular proteases by zymography. On the basis of the results some strains released only a protease having a MW of 37 kDa, which corresponds to the already reported acidic protease, while other produced prevalently or only a protease with a MW higher than 200 kDa. The proteases have been screened for their "cold attitude" on gelatin, gluten and skim milk. This property can be relevant from a biotechnological point of view in order to save energy consumption during industrial processes. Most of the strains used were endowed with proteolytic activity at 6 °C on all the three proteins. The proteolytic breakdown profiles of the proteins, detected at 27 °C, were different related to the specific strains of Y. lipolytica. The time course of the hydrolysis, tested on gelatin, affected the final bioactivities of the peptide mixtures produced. In particular, an increase in both the antioxidant and antimicrobial activities was detected when the protease of the strain Y. lipolytica 1IIYL4A was used. The final part of this work was focused on the improvement of the peptides bioactivities through a novel process based on the production of glycopeptides. Firstly, the main reaction parameters were optimized in a model system, secondly a more complex system, based on gluten hydrolysates, was taken into consideration to produce glycopeptides. The presence of the sugar moiety reduced the hydrophobicity of the glycopeptides, thus affecting the final antimicrobial activity which was significantly improved. The use of this procedure could be highly effective to modify peptides and can be employed to create innovative functional peptides using a mild temperature process.

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides

Background: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. Results: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. Conclusion: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

Health-promoting properties of bioactive peptides derived from milk proteins in infant food : a review

Peer reviewed

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Effect of a Pool of Peptides Isolated from Crotalus durissus terrificus (South American Rattlesnake) Venom on Glucose Levels of Mice Fed on a High-Fat Diet

This study investigated the effect of a pool of peptides, isolated from venom of Crotalus durissus terrificus (South American rattlesnake) on glucose concentration in C57BL/6 mice fed on a high-fat diet for 6 weeks. The pool of peptides (molecular mass around of 10 kDa) was obtained using a MidJet apparatus with a cartridge of 10 KDa. The peptide pool was injected intraperitoneally in mice in a single dose (0.5 mg/animal) or multiple doses (0.2 mg/dose). After predetermined times (30, 60, 90 and 120 min) post injections, venous blood samples were collected for enzymatic measurement of serum glucose using a commercial glucose kit (glucose oxidase method). High-fat fed mice showed an increase in blood glucose concentration, in comparison with mice fed on the chow diet. Thirty minutes after a single dose of the peptide pool, high-fat fed animals showed a significant decrease (similar to 47%) in glycemia. However, the glucose level increased again at 60 and 120 min. Conversely, after multiple injections of the pool of peptides administered every 30 min, the blood glucose concentration in the high-fat mice was significantly decreased (similar to 37%) and remained at low levels until 120 min. These results suggest that the tested pool of peptides from Crotalus durissus terrificus contained a peptide (or peptides) with a beneficial role on glucose-lowering action of high-fat fed mice.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).