Leticia Mora

Innovations in value-addition of edible meat by-products

While muscle foods are the more commonly consumed portion of an animal, meat by-products such as the entrails and internal organs are also widely consumed. Considered high-priced delicacies or waste material to be tossed away, the use and value of offal-edible and inedible meat by-products depend entirely on the culture and country in question. The skin, blood, bones, meat trimmings, fatty tissues, horns, hoofs, feet, skull, and internal organs of harvested animals comprise a wide variety of products including human or pet food or processed materials in animal feed, fertilizer, or fuel. Industry is using science and innovation to add value to animal by-products far beyond its usual profitability. Regardless of the final products destination, it is still necessary to employ the most up-to-date and effective tools to analyze these products for nutritional properties, to search for key active molecules in nutrition like bioactive peptides, food safety (antimicrobial peptides), medicine, cosmetics or other fields, to develop new technological applications and to continue innovation towards advanced value-addition of meat by-products.

Purification and Identification of antihypertensive peptides in Spanish dry-cured ham.

Novel sequences exhibiting in vitro ACE inhibitory activity as well as in vivo antihypertensive activity were identified from Spanish dry-cured ham. Water soluble peptide extracts from dry-cured ham were purified by size-exclusion chromatography and reversed-phase high performance liquid chromatography and then, further identification of sequences was carried out by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 73 peptide sequences were identified from active fractions presenting 100% homology with different Sus scrofa skeletal muscle proteins. All identified peptides showed Mr between 374 and 1610 and amino acid sequences between 5 and 14 amino acids in length. Considering the low molecular mass and structural requirements for ACE inhibition some of the identified peptides were synthesised and their IC(50) calculated. The most potent peptide was found to be AAATP (IC(50) value of 100 μM). This peptide also showed good in vivo activity because it decreased systolic blood pressure by -25.62 ± 4.5 mmHg (p<0.05) in spontaneous hypertensive rats after 8 h administration. Other sequences yielded a moderate ACE inhibition. Results from this study show that Spanish dry-cured ham may represent a source of natural peptides with potential benefit for human health.

Dry‐Cured Ham

The processing of dry-cured ham includes many biochemical reactions that are responsible for its final characteristic properties. Proteolysis is one of the most important biochemical phenomena occurring during this period and results in the generation of a large number of peptides. Some of them could be used as biomarkers of any specific quality parameter of the process and others could be bioactive peptides. Advanced techniques of proteomics such as tandem mass spectrometry play a crucial role for a better understanding of the mechanisms that control the generation of these natural peptides during the post-mortem proteolysis and their possible biological activity that will facilitate the use of these peptides in the development of novel meat products and as ingredients of healthier foods.

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.

Identification of novel antioxidant peptides generated in Spanish dry-cured ham

The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5 units of absorbance at 700 nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.

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.

Subchromoplast Sequestration of Carotenoids Affects Regulatory Mechanisms in Tomato Lines Expressing Different Carotenoid Gene Combinations

Pathway engineering has been the predominant strategy used to enhance valuable natural products in plants. This study identifies sequestration mechanisms associated with pathway engineering outputs that enable the plastid organelle to adapt and accommodate newly synthesized precursors and products. Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.

Stability of ACE inhibitory ham peptides against heat treatment and in vitro digestion.

Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from Spanish dry-cured ham have been examined for their stability during processing and after in vitro digestion. Results indicate that peptides preserved almost the same ACE inhibitory activity before and after applying diverse heating (from 50 to 117°C), times of processing (from 3 to 60min) and simulated in vitro digestion with gastrointestinal proteases. Peptides KAAAAP, AAPLAP, KPVAAP, IAGRP, and KAAAATP were the most potent peptides with IC50 values ranging from 12.37 to 25.94μM. Peptides IAGRP and PTPVP have also been identified in the processed sample (6min at 117°C), and in the in vitro digested sample. This study proves the high stability of ACE inhibitory peptides derived from Spanish dry-cured ham against temperature of processing and gastrointestinal digestion as well as the powerful ACE inhibitory activity of some of the peptides identified in Spanish dry-cured ham.

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.

Bioactive peptides identified in thornback ray skin's gelatin hydrolysates by proteases from Bacillus subtilis and Bacillus amyloliquefaciens

UNLABELLED Thornback ray skin gelatin has been hydrolyzed with two different proteases in order to obtain peptides with ACE inhibitory and antioxidant activity. Hydrolysates with protease from Bacillus subtilis A26 (TRGH-A26) displayed ACE inhibitory activity with an IC50 value of 0.94 μg/μL whereas Neutrase® hydrolysate from Bacillus amyloliquefaciens (TRGH-Neutrase) showed an IC50 value of 2.07 μg/μL. Regarding antioxidant activity, IC50 values of 1.98 and 21.2 μg/μL in TRGH-A26 and TRGH-Neutrase, respectively, were obtained using the DPPH radical-scavenging assay. The most active fractions identified by size-exclusion chromatography were further purified by RP-HPLC and analysed using nanoESI-LC-MS/MS to identify the sequence of the peptides. APGAP was the most active peptide inTRGH-A26 for ACE inhibitory activity with an IC50 value of 170 μM, whereas GIPGAP showed the best ACE inhibitory activity in TRGH-Neutrase sample with an IC50 value of 27.9 μM. The highest antioxidant activity was identified in peptide AVGAT, showing a 33% of activity at 3mg/mL using the DPPH radical-scavenging assay. The obtained results proved the potential of thornback ray skin gelatin hydrolysates as a source of bioactive peptides. STATEMENT OF SIGNIFICANCE This study describes a peptidomic approach for the identification of ACE-inhibitory and antioxidant peptides generated from thornback ray gelatin (Raja clavata) hydrolysates from Bacillus subtilis A26 and Bacillus amyloliquefaciens Neutrase® enzymes and expose the potential of thornback ray gelatin hydrolysate as a source of bioactive peptides. In this sense, the decrease of systolic blood pressure is one of the main measurements considered in public health for the treatment of cardiovascular diseases, stroke and even end-stage renal disease. Traditionally, synthetic drugs such as captopril and enalapril have been used as ACE inhibitors despite their secondary effects, but the finding of new sources for the generation of natural bioactive peptides such as thornback ray muscle results is very important in the knowledge of less hostile but highly effective antihypertensive peptides as well as the development of new uses for waste and by-products generated from marine products, helping to solve the already existing environmental problem affecting this industry.