Tomas Lafarga

Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients.

Bioactive peptides are sequences of between 2-30 amino acids in length that impart a positive health effect to the consumer when ingested. They have been identified from a range of foods, including milk and muscle sources including beef, chicken, pork and marine muscles. The myriad of peptides identified from these sources have known antihypertensive, opioid, antioxidant, antithrombotic and other bioactivities. Indeed, bioactive peptides could play a role in the prevention of diseases associated with the development of metabolic syndrome and mental health diseases. The aim of this work is to present an overview of the bioactive peptides identified in muscle proteins and by-products generated during the processing of meat. The paper looks at the isolation, enrichment and characterisation strategies that have been employed to date to generate bioactive peptides and the potential future applications of these peptides in functional foods for the prevention of heart and mental health problems and obesity.

Identification of novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides from meat proteins using in silico analysis

Angiotensin-I-converting enzyme (ACE-I, EC 3.4.15.1), renin (EC 3.4.23.15), and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) play key roles in the control of hypertension and the development of type-2 diabetes and other diseases associated with metabolic syndrome. The aim of this work was to utilize known in silico methodologies, peptide databases and software including ProtParam (http://web.expasy.org/protparam/), Basic Local Alignment Tool (BLAST), ExPASy PeptideCutter (http://web.expasy.org/peptide_cutter/) and BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) to assess the release of potentially bioactive DPP-IV, renin and ACE-I inhibitory peptides from bovine and porcine meat proteins including hemoglobin, collagen and serum albumin. These proteins were chosen as they are found commonly in meat by-products such as bone, blood and low-value meat cuts. In addition, the bioactivities of identified peptides were confirmed using chemical synthesis and in vitro bioassays. The concentration of peptide required to inhibit the activity of ACE-I and DPP-IV by 50% was determined for selected, active peptides. Novel ACE-I and DPP-IV inhibitory peptides were identified in this study using both in silico analysis and a literature search to streamline enzyme selection for peptide production. These novel peptides included the ACE-I inhibitory tri-peptide Ile-Ile-Tyr and the DPP-IV inhibitory tri-peptide Pro-Pro-Leu corresponding to sequences f (182-184) and f (326-328) of both porcine and bovine serum albumin which can be released following hydrolysis with the enzymes papain and pepsin, respectively. This work demonstrates that meat proteins are a suitable resource for the generation of bioactive peptides and further demonstrates the usefulness of in silico methodologies to streamline identification and generation of bioactive peptides.

In silico methods to identify meat-derived prolyl endopeptidase inhibitors.

According to the World Health Organization (WHO), approximately 450 million people suffer from mental or neurological disorders and five of the ten leading causes of disability and premature death worldwide are psychiatric conditions. Social, biological and neurological sciences provided extensive understanding into the role of risk and protective factors in the development of mental disorders and poor mental health. Altered activity of a number of enzymes, such as prolyl endopeptidase (PEP, EC 3.4.21.26), has been linked to the prevention and treatment of a number of mental disorders, including anxiety, depression and Alzheimers disease. The inhibition of PEP has potential for use in the prevention and in the treatment of mental disorders. The objective of this work was to identify PEP-inhibitory peptides from meat proteins using in silico methods. In this paper, five proteins commonly found in meat by-products were evaluated as a substrate for use in the generation of PEP inhibitory peptides. These include serum albumin, collagen and myosin. These proteins were cleaved in silico using BIOPEP and ExPASy PeptideCutter and the generated peptides were compared to known PEP-inhibiting peptides in the database of BIOPEP. A number of novel PEP inhibitory peptide sequences were identified in this study, including PPL, APPH, IPP and PPG with corresponding IC50 values of 2.86, 3.95, 4.02 and 2.70 mM, respectively. This work demonstrates the usefulness of in silico analysis for predicting the release of PEP-inhibiting peptides from meat proteins.

Bioactive protein hydrolysates in the functional food ingredient industry: Overcoming current challenges

ABSTRACT Meat proteins and associated by-products can be used as a source of bioactive hydrolysates and peptides with potential for use as functional food ingredients. Functional foods are foods that have a potentially positive effect on health, beyond basic nutrition. Numerous bioactive peptides, including angiotensin-I-converting enzyme (ACE-I, EC 3.4.15.1) and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) inhibitors, have been generated from meat by-product proteins to date. However, in order to use and commercialize bioactive hydrolysates and peptides as food ingredients, a number of significant challenges must first be overcome. This article gives an overview of the current state-of-the-art of meat-derived bioactive hydrolysate and peptide uses in the food industry. It also reviews frequent challenges faced when developing biologically active hydrolysates and peptides as food ingredients. These challenges include, but are not limited to, high production costs, negative sensory attributes in end products, taste modifications of carrier food products and compliance with, for example, the European Food Safety Authority (EFSA), the Food and Drug Administration (FDA), and other regulatory bodies in China, or Japan, as well as potential toxicity or allergenicity. We suggest strategies that may assist in overcoming these challenges, focusing on those that may be used to improve the taste attributes of the end products.

A Bovine Fibrinogen-Enriched Fraction as a Source of Peptides with in Vitro Renin and Angiotensin-I-Converting Enzyme Inhibitory Activities

Bovine fibrinogen is currently used in the food industry as a binding agent in restructured meat products. However, this protein is underused as a source of bioactive peptides. In this study, a number of novel angiotensin-I-converting enzyme (ACE-I) and renin inhibitory peptides were identified and enriched from a bovine fibrinogen fraction. Fibrinogen was isolated and enriched from bovine blood and hydrolyzed with the food-grade enzyme papain, which was selected for use using in silico analysis. The generated hydrolysate was subjected to ultrafiltration and its peptide profile characterized by liquid chromatography-tandem mass spectrometry. A number of peptides were identified and chemically synthesized to confirm their bioactivity in vitro. Identified peptides included the multifunctional tripeptide SLR, corresponding to f(35-37) of the β-chain of bovine fibrinogen with ACE-I and renin IC50 values of 0.17 and 7.2 mM, respectively. Moreover, the resistance of identified peptides to gastrointestinal degradation and their bitterness were predicted using in silico methods.

Addition of an Enzymatic Hydrolysate of Bovine Globulins to Bread and Determination of Hypotensive Effects in Spontaneously Hypertensive Rats

The aim of this study was to develop bread containing a papain hydrolysate of bovine α- and β-globulins (GPH) with in vitro and in vivo antihypertensive activities. The physical characteristics of the formulated bread were assessed over a six day period and results suggested that the overall quality and acceptance of bread was not affected by the inclusion of GPH at a concentration of 4% (w/w). Bright field light microscopy and confocal scanning laser microscopy images were used to visualize the main ingredients of the bread. In addition, the antihypertensive activity of the bread was assessed in spontaneously hypertensive rats (SHRs) over a 24 h period where a maximum significant decrease in systolic blood pressure of 36.2 ± 1.9 mmHg was observed 8 h after oral administration. Results demonstrate that the antihypertensive activity of GPH was resistant to the baking process and shows potential for use as a functional antihypertensive ingredient.

Chitosan-Containing Bread Made Using Marine Shellfishery Byproducts: Functional, Bioactive, and Quality Assessment of the End Product

Chitosan is natures second most abundant polymer after cellulose and forms the structural support in crustacean shell material and Basidomycete mushroom stalks. Chitosan is a known antimicrobial agent but, to date, was not examined as an antimicrobial agent in bread formulations for the prevention of mold or rope formation. The aim of this work was to investigate the effects of chitosan generated from prawn shell byproducts on the color, moisture, and texture and crumb formation of bread. A secondary aim of this work was to determine the antimicrobial effect of chitosan added to bread at a rate of 1% against the rope spoilage pathogen Bacillus cereus along with natural molds. The addition of chitosan to bread with a molecular mass of 124000 ± 10000 g/mol and 19% deacetylated was found to inhibit B. cereus growth and rope formation in bread when monitored over 3-5 days. Natural mold growth was also significantly delayed in bread made using chitosan substitution of flour at 1% compared to the control bread, where mold was observed growing on the bread surface after 72 h when bread was incubated at 30 °C.

Anti-proliferative activity of bovine blood hydrolysates towards cancer cells in culture

Four proteins, α/β globulin, serum albumin, γ-globulin and fibrinogen, were isolated from bovine blood and hydrolysed using papain. Hydrolysates were assessed for non-cellular and cellular antioxidant activity. The anti-proliferative activity of hydrolysed fractions was assessed in a number of cancer cell lines including U937 lymphoma cells, MCF-7 breast cancer cells, HepG2 hepatocytes and Caco-2 epithelial colorectal adenocarcinoma cells. Anti-inflammatory activity of the hydrolysates was also assessed. Hydrolysates generated from γ-globulin or fibrinogen had significant antioxidant activity in non-cellular assays. Hydrolysates were also found to be highly toxic to different cancer cell lines, in particular U937 lymphoma cells when assessed using the MTT assay. The fibrinogen hydrolysate was the most toxic sample and toxicity appeared to correlate with its non-cellular antioxidant activity. None of the hydrolysates had significant anti-inflammatory activity. The high cytotoxicity of the γ-globulin and the fibrinogen hydrolysates towards cancer cells may indicate a potential use as anti-proliferative agents.

Predicted Release and Analysis of Novel ACE-I, Renin, and DPP-IV Inhibitory Peptides from Common Oat (Avena sativa) Protein Hydrolysates Using in Silico Analysis

The renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating hypertension by controlling vasoconstriction and intravascular fluid volume. RAAS itself is largely regulated by the actions of renin (EC 3.4.23.15) and the angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1). The enzyme dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5) also plays a role in the development of type-2 diabetes. The inhibition of the renin, ACE-I, and DPP-IV enzymes has therefore become a key therapeutic target for the treatment of hypertension and diabetes. The aim of this study was to assess the bioactivity of different oat (Avena sativa) protein isolates and their ability to inhibit the renin, ACE-I, and DPP-IV enzymes. In silico analysis was carried out to predictthe likelihood of bioactive inhibitory peptides occurring from oat protein hydrolysates following in silico hydrolysis with the proteases papain and ficin. Nine peptides, including FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA, FLLA, and FEPL were subsequently chemically synthesised, and their bioactivities were confirmed using in vitro bioassays. The isolated oat proteins derived from seven different oat varieties were found to inhibit the ACE-I enzyme by between 86.5 ± 10.7% and 96.5 ± 25.8%, renin by between 40.5 ± 21.5% and 70.9 ± 7.6%, and DPP-IV by between 3.7 ± 3.9% and 46.2 ± 28.8%. The activity of the synthesised peptides was also determined.

Effects of thermal and non-thermal processing of cruciferous vegetables on glucosinolates and its derived forms

Brassica vegetables, which include broccoli, kale, cauliflower, and Brussel sprouts, are known for their high glucosinolate content. Glucosinolates and their derived forms namely isothiocyanates are of special interest in the pharmaceutical and food industries due to their antimicrobial, neuroprotective, and anticarcinogenic properties. These compounds are water soluble and heat-sensitive and have been proved to be heavily lost during thermal processing. In addition, previous studies suggested that novel non-thermal technologies such as high pressure processing, pulsed electric fields, or ultraviolet irradiation can affect the glucosinolate content of cruciferous vegetables. The objective of this paper was to review current knowledge about the effects of both thermal and non-thermal processing technologies on the content of glucosinolates and their derived forms in brassica vegetables. This paper also highlights the importance of the incorporation of brassica vegetables into our diet for their health-promoting properties beyond their anticarcinogenic activities.