Miguel Angel Sentandreu

A Proteomic-Based Approach for Detection of Chicken in Meat Mixes

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Angiotensin I-Converting Enzyme Inhibitory Peptides Generated from in Vitro Gastrointestinal Digestion of Pork Meat

The main purpose of this work was to study the generation of Angiotensin I-converting enzyme inhibitory (ACEI) peptides after gastrointestinal digestion of pork meat by the action of pepsin and pancreatin at simulated gut conditions. The hydrolysate was further subjected to reverse phase chromatography in order to separate the fractions with ACEI activity. Using MALDI-TOF/TOF mass spectrometry, 12 peptides were identified in these fractions. It is worth highlighting the novel peptides ER, KLP, and RPR with IC(50) values of 667 microM, 500 microM, and 382 microM, respectively. Results obtained by MALDI-TOF/TOF mass spectrometry were complemented by a second approach consisting of the analysis of the hydrolysate directly by nanoLC-ESI-MS/MS followed by a study of the obtained sequences and comparison with known ACEI peptide sequences. By using these two approaches, a total of 22 peptides were selected for its synthesis and further in vitro assay of ACEI activity. The strongest ACE inhibition was observed for peptide KAPVA (IC(50) = 46.56 microM) followed by the sequence PTPVP (IC(50) = 256.41 microM). Sequence similarity searches revealed that these two peptides derive from muscle titin, constituting the first identified ACEI peptides coming from this protein. This is also the first time that ACEI sequences MYPGIA and VIPEL have been reported. Other identified and synthesized sequences showed less ACEI activity. The obtained results evidence the potential of pork meat proteins as a source of antihypertensive peptides after gastrointestinal digestion.

Contents of creatine, creatinine and carnosine in porcine muscles of different metabolic types

Creatine, creatinine and carnosine have been analyzed by hydrophilic interaction chromatography (HILIC) in seven different pork muscles of different metabolic type (Semimembranosus, Biceps femoris, Gluteus maximus, Longissimus dorsi, Gluteus medius, Trapezius and Masseter). As reported in previous literature, carnosine contents are related with the type of metabolism, being higher in those muscles with glycolytic metabolism. Creatine and creatinine also showed significantly higher concentrations in glycolytic muscles such as Semimembranosus, Biceps femoris, Gluteus maximus and Longissimus dorsi. Masseter, a red oxidative muscle, was characterized by the lowest contents of creatine, creatinine and carnosine and, finally, Gluteus medius and Trapezius, both intermediate muscles, had also intermediate contents of these studied compounds. Finally, a correlation between initial content of creatine and creatinine formation after cooking has been verified using pure standards and two different metabolic type muscles, Longissimus dorsi and Masseter, obtaining slightly higher creatinine amounts in Longissimus dorsi, probably due to its higher initial creatine content and its lower pH.

Naturally Generated Small Peptides Derived from Myofibrillar Proteins in Serrano Dry-Cured Ham

A proteomic approach has been carried out to investigate the extensive proteolysis occurring in the processing of Serrano ham. In this study, a total of 14 peptide fragments derived from myosin light chain I and titin have been identified for the first time. Nine of these peptides originated from myosin light chain I protein, with the loss of dipeptides at the N-terminal position observed in some of them. This suggests that dipeptidyl peptidases are involved in the generation of dipeptides, which contribute to the generation of the characteristic taste associated with Serrano ham. The other five peptides came from the PEVK region of the titin protein. This region is believed to confer elasticity to the sarcomere as well as the ability to bind calpains. The hypothetical action of mu-calpain and calpain 3 enzymes over this region would make these enzymes potentially responsible for protein breakdown during the early dry-curing stage.

Characterization of Peptides Released by in Vitro Digestion of Pork Meat

The main objective of this work was to identify and characterize the peptides generated by simulated gastrointestinal digestion of pork meat (longissimus dorsi) by the sequential action of pepsin and pancreatin. The obtained hydrolysate was analyzed by liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization source (nano LC-ESI-MS/MS). Using this technique 51 different peptides were identified in the hydrolysate, corresponding to fragments of the main structural muscle proteins and some well-known sarcoplasmic proteins. To the best of our knowledge, this constitutes the highest number of peptides identified in pork meat digests. Peptide fragment size ranged from six to sixteen amino acids, being rich in proline residues and thus making them more resistant to further degradation by digestive enzymes. The present study constitutes a clear evidence of the extensive degradation that pork muscle proteins would undergo after gastrointestinal digestion, giving rise to a wide variety of short peptides. So, the use of in vitro digestion contributes to a better knowledge about the generation of peptides from diets with high protein quality.

A fluorescence-based protocol for quantifying angiotensin-converting enzyme activity

The determination of angiotensin-converting enzyme (ACE) activity represents a useful tool in the study of different health pathologies, such as hypertension. This protocol describes a fluorescent assay for measuring ACE activity in vitro with high precision and sensitivity. The method relies on the ability of ACE to hydrolyse the internally quenched fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline. The generation of the fluorescent product o-aminobenzoylglycine can be continuously monitored, preferably using a microtiter-plate fluorometer, though the use of a conventional cuvette fluorometer would also be possible. The method has important advantages with respect to other assays, because it involves only a one-step reagent, is easy to carry out and allows the analysis of an elevated number of samples in shorter times. It can be completed in one and a half hours. In addition, the fact that all reagents are commercially available allows the rapid introduction of the assay into the laboratory.

Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat.

This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals.

Peptides with angiotensin I converting enzyme (ACE) inhibitory activity generated from porcine skeletal muscle proteins by the action of meat-borne Lactobacillus

UNLABELLED Angiotensin I converting enzyme (ACE) inhibitory activity of peptides derived from the hydrolysis of sarcoplasmic and myofibrillar porcine proteins by the action of Lactobacillus sakei CRL1862 and Lactobacillus curvatus CRL705 (whole cells+cell free extracts) was investigated at 30°C for 36 h. The protein hydrolysates were subjected to RP-HPLC in order to fractionate the extracts for further evaluation of ACE inhibitory activity. Bioactive fractions were only found from the hydrolysis of sarcoplasmic proteins by both assayed lactobacilli strains. Identification of peptides contained in the bioactive fractions was carried out by tandem mass spectrometry using a nanoLC-ESI-QTOF instrument and the mascot search engine. From the four most active fractions obtained, a total of eighteen and fifty peptides were characterized from L. sakei CRL1862 and L. curvatus CRL705 protein hydrolysates, respectively. The sequence FISNHAY was generated by the proteolytic activity of the two lactobacilli species. Sequence similarity analyses between the peptides identified in this study and those previously identified as ACE inhibitory peptides and detailed in the BIOPEP database were outlined. Results suggest that meat-borne Lactobacillus were able to generate peptides with ACE inhibitory activity, highlighting their potential to be used in the development of functional fermented products. BIOLOGICAL SIGNIFICANCE The results of this study would enable the obtention of porcine functional foods by applying lactic acid bacteria generating bioactive peptides. ACE inhibitory peptides obtained by the hydrolytic action of L. curvatus CRL705 and L. sakei CRL1862 on sarcoplasmic proteins were analyzed. Among them, the peptide FISNHAY exhibited the highest activity and its sequence has not yet been reported.

Small peptides released from muscle glycolytic enzymes during dry-cured ham processing.

Glycolytic enzymes are a group of sarcoplasmic enzymes responsible for the extraction of the energy available from carbohydrates. The glycolytic pathway consists of 10 enzyme-catalyzed steps. Fragments identified in this study, within the range 1100-2600 Da, correspond to glycogen phosphorylase enzyme, which catalyzes the rate limiting step in the degradation of glycogen, enzymes that catalyze steps 6-10 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, respectively), and lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate. A total of 45 specific fragments of these enzymes resulting from the processing of dry-cured ham are reported for the first time in this work. This study evidences the intense proteolysis occurring in the sarcoplasmic fraction of dry-cured ham as well as facilitates the choice of the most adequate tools in the identification of naturally generated peptides through comparison between Paragon and Mascot search engines, together with UniProt and NCBInr databases.

Lactobacillus sakei CRL1862 improves safety and protein hydrolysis in meat systems

The capacity of Lactobacillus sakei CRL1862 to prevent the growth of pathogens and its ability to degrade sarcoplasmic and myofibrillar proteins in pork meat systems was evaluated. In addition, basic safety aspects of Lact. sakei CRL1862 such as production of biogenic amines and antibiotic susceptibility were addressed.