A review on bioactive peptides derived from meat and by-products: Extraction methods, biological activities, applications and limitations.

Meat and its by-products offer a rich source of bioactive compounds which have potential applications in both the food and pharmaceutical industries. In this review, we present several extraction methods and report the identification and properties of bioactive peptides. We also examine the challenges and limitations associated with their use in food applications. Enzymatic hydrolysis and fermentation using starts cultures are common methods for generating bioactive peptides from meat proteins. Additionally, natural gastrointestinal digestion can also produce bioactive peptides. However, emerging technologies like high hydrostatic pressure, subcritical extraction and pulsed electric fields can improve hydrolysis and increase the yield of bioactive peptides. Online bioinformatics applications have emerged as an established method for identifying potentially bioactive peptides. These tools reduce the cost and time required for traditional methods of research. Finally, incorporating bioactive peptides into diets for specific purposes such as supporting vulnerable populations like children and the elderly ensures safety and efficacy.

Advanced Proteomic and Bioinformatic Tools for Predictive Analysis of Allergens in Novel Foods.

In recent years, novel food is becoming an emerging trend increasingly more demanding in developed countries. Food proteins from vegetables (pulses, legumes, cereals), fungi, bacteria and insects are being researched to introduce them in meat alternatives, beverages, baked products and others. One of the most complex challenges for introducing novel foods on the market is to ensure food safety. New alimentary scenarios drive the detection of novel allergens that need to be identified and quantified with the aim of appropriate labelling. Allergenic reactions are mostly caused by proteins of great abundance in foods, most frequently of small molecular mass, glycosylated, water-soluble and with high stability to proteolysis. The most relevant plant and animal food allergens, such as lipid transfer proteins, profilins, seed storage proteins, lactoglobulins, caseins, tropomyosins and parvalbumins from fruits, vegetables, nuts, milk, eggs, shellfish and fish, have been investigated. New methods for massive screening in search of potential allergens must be developed, particularly concerning protein databases and other online tools. Moreover, several bioinformatic tools based on sequence alignment, motif identification or 3-D structure predictions should be implemented as well. Finally, targeted proteomics will become a powerful technology for the quantification of these hazardous proteins. The ultimate objective is to build an effective and resilient surveillance network with this cutting-edge technology.

Valorisation of pork by-products to obtain antioxidant and antihypertensive peptides.

The porcine liver could be used for the extraction of zinc-protoporphyrin (ZnPP) as a natural red meat pigment. During the autolysis process, porcine liver homogenates was incubated at pH 4.8 and 45 °C under anaerobic conditions to obtain insoluble ZnPP. After incubation, the homogenates were readjusted at pH 4.8, and at pH 7.5 before being centrifuged at 5500 × g for 20 min at 4 °C and the resulting supernatant were compared with the obtained at pH 4.8 at the beginning of the incubation. The molecular weight distributions of the porcine liver fractions at both pHs were very similar, however, eight essential amino acids were more abundant in fractions obtained at pH 4.8. Regarding the ORAC assay, porcine liver protein fraction at pH 4.8 showed the highest antioxidant capacity but antihypertensive inhibition was similar for both pHs. Peptides with strong bioactivity potential from aldehyde dehydrogenase, lactoylglutathione lyase, SEC14-like protein 3 and others were identified. The findings have demonstrated the potential of the porcine liver to extract natural pigments and bioactive peptides.

Towards the discovery of goat meat quality biomarkers using label-free proteomics.

This study aimed to identify for the first time protein biomarkers of meat quality traits from Longissimus thoracis (LT) muscle of goats (Capra hircus). Male goats of similar age and weight reared under extensive rearing conditions were used to relate the LT muscle proteome with multiple meat quality traits. The early post-mortem muscle proteome analyzed using label-free proteomics was compared among three texture clusters built using hierarchical clustering analysis. Twenty-five proteins were differentially abundant and their mining using bioinformatics revealed three major biological pathways to be involved: 10 muscle structure proteins (MYL1, MYL4, MYLPF, MYL6B, MYH1, MYH2, ACTA1, ACTBL2, FHL1 and MYOZ1); 6 energy metabolism proteins (ALDOA, PGAM2, ATP5F1A, GAPDH, PGM1 and ATP5IF1), and two heat shock proteins: HSPB1 (small) and HSPA8 (large). Seven other miscellaneous proteins belonging to pathways such as regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding were further identified to play a role in the variability of goat meat quality. The differentially abundant proteins were correlated with the goat meat quality traits in addition to multivariate regression models built to propose the first regression equations of each quality trait. This study is the first to highlight in a multi-trait quality comparison the early post-mortem changes in the goat LT muscle proteome. It also evidenced the mechanisms underpinning the development of several quality traits of interest in goat meat production along the major biochemical pathways at interplay. SIGNIFICANCE: The discovery of protein biomarkers in the field of meat research is an emerging topic. In the case of goat meat quality, very few studies using proteomics have been conducted with the aim of proposing biomarkers. Therefore, this study is the first to quest for biomarkers of goat meat quality using label-free shotgun proteomics with a focus on multiple quality traits. We identified the molecular signatures underlying goat meat texture variation, which were found to belong to muscle structure and related proteins, energy metabolism and heat shock proteins along with other proteins involved in regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding. We further evaluated the potential of the candidate biomarkers to explain meat quality using the differentially abundant proteins by means of correlation and regression analyses. The results allowed the explanation of the variation in multiple traits such as pH, color, water-holding capacity, drip and cook losses traits and texture.

A proteomic approach to identify biomarkers of foal meat quality: A focus on tenderness, color and intramuscular fat traits.

Foal meat is considered a healthy alternative to other meat sources and more environmentally sustainable. However, its quality is highly variable and there is lack of knowledge about the molecular mechanisms underlying its determination. Genotype and diet play a relevant role as the main factors that can allow a control of the final quality and the use of high-throughput analytical methods such as proteomics is a way to achieve this lofty goal. This research aimed to study-two breeds (Burguete and Jaca Navarra) supplemented with two different finishing diets: conventional concentrate and straw (C) vs silage and organic feed (S). The proteomic approach built a library of 294 proteins that were subjected to several statistical and bioinformatic analyses. Burguete breed finished with concentrate produced higher meat quality in terms of tenderness, intramuscular fat and color lightness mainly due to the high abundance of energy metabolic proteins. Tenderness was correlated to myofibrillar proteins (ACTA1, MYBPH, MYL1 and TNNC1) and energy metabolic proteins (ALDOA, CKM, TPI1 and PGMA2). Regarding color, the main pathways were energy metabolism, involving several glycolytic enzymes (ALDOA, PKM, PFKM and CKM). Oxidative stress and response to stress proteins (HSPA1A, SOD2 and PRDX2) were further involved in color variation. Moreover, we revealed that several proteins were related to the intramuscular fat accordingly to the breed. This study proposed several candidate protein biomarkers for foal meat quality that are worthy to evaluate in the future.

Advances in Natural Antioxidants for Food Improvement.

In the food industry, antioxidants are natural and synthetic compounds added to neutralize free radicals that deteriorate fats, proteins and cellular DNA, causing rancidity of fats and accelerating the ageing process, which lead to undesirable smells and tastes [...].

Role of Food Hydrocolloids as Antioxidants along with Modern Processing Techniques on the Surimi Protein Gel Textural Properties, Developments, Limitation and Future Perspectives.

Texture is an important parameter in determining the quality characteristics and consumer acceptability of seafood and fish protein-based products. The addition of food-based additives as antioxidants (monosaccharides, oilgosaccharides, polysaccharides and protein hydrolysates) in surimi and other seafood products has become a promising trend at an industrial scale. Improvement in gelling, textural and structural attributes of surimi gel could be attained by inhibiting the oxidative changes, protein denaturation and aggregation with these additives along with new emerging processing techniques. Moreover, the intermolecular crosslinking of surimi gel can be improved with the addition of different food hydrocolloid-based antioxidants in combination with modern processing techniques. The high-pressure processing (HPP) technique with polysaccharides can develop surimi gel with better physicochemical, antioxidative, textural attributes and increase the gel matrix than conventional processing methods. The increase in protein oxidation, denaturation, decline in water holding capacity, gel strength and viscoelastic properties of surimi gel can be substantially improved by microwave (MW) processing. The MW, ultrasonication and ultraviolet (UV) treatments can significantly increase the textural properties (hardness, gumminess and cohesiveness) and improve the antioxidative properties of surimi gel produced by different additives. This study will review potential opportunities and primary areas of future exploration for high-quality surimi gel products. Moreover, it also focuses on the influence of different antioxidants as additives and some new production strategies, such as HPP, ultrasonication, UV and MW and ohmic processing. The effects of additives in combination with different modern processing technologies on surimi gel texture are also compared.

A proteomic approach for in-depth characterization and understanding the impact of immunocastration on dry-cured ham of male and female pigs.

Dry-cured ham is a high-quality product elaborated through a long and complex process. To ensure the success of the process, it is necessary to select the most suitable pork leg and one of the major factors is pig castration. Due to animal welfare, pig castration is becoming a paramount issue in recent years. The proteomic differences of dry-cured ham from immunecastrated pigs against entire females, as well as, between immunecastrated pigs and surgically castrated males were analysed. The identification and quantification of proteins were carried out by sequential window acquisition of all theoretical mass spectra (SWATH-MS). A total of 249 proteins were identified across the samples of dry-cured ham, resulting in 17 and 37 differentially abundant proteins in the case of males and females, respectively. In the case of males, a high abundance of structural proteins in dry-cured ham from surgically castrated animals as well as a high abundance of trypsinogen and proteosome subunit C9-like protein with protease activity in samples from immunocastrated males suggests that immunocastration impact on myofibrils of dry-cured ham. Regarding females, the immunocastration provoked an increase of abundance in several structural proteins of the myosin heavy chain (MYH7, MYH7B and MYH4) and a decrease in others (ACTN2, TNNT3, MYL3 and TCAP) concerning entire. Overall, MYH4 and ACT were found to a greater degree in immunocastrated males and females indicating a potential for biomarkers.

Finding Biomarkers in Antioxidant Molecular Mechanisms for Ensuring Food Safety of Bivalves Threatened by Marine Pollution.

Aquaculture production as an important source of protein for our diet is sure to continue in the coming years. However, marine pollution will also likely give rise to serious problems for the food safety of molluscs. Seafood is widely recognized for its high nutritional value in our diet, leading to major health benefits. However, the threat of marine pollution including heavy metals, persistent organic pollutants and other emerging pollutants is of ever-growing importance and seafood safety may not be guaranteed. New approaches for the search of biomarkers would help us to monitor pollutants and move towards a more global point of view; protocols for the aquaculture industry would also be improved. Rapid and accurate detection of food safety problems in bivalves could be carried out easily by protein biomarkers. Hence, proteomic technologies could be considered as a useful tool for the discovery of protein biomarkers as a first step to improve the protocols of seafood safety. It has been demonstrated that marine pollutants are altering the bivalve proteome, affecting many biological processes and molecular functions. The main response mechanism of bivalves in a polluted marine environment is based on the antioxidant defense system against oxidative stress. All these proteomic data provided from the literature suggest that alterations in oxidative stress due to marine pollution are closely linked to robust and confident biomarkers for seafood safety.

SWATH-MS Quantitative Proteomic Analysis of Deer Antler from Two Regenerating and Mineralizing Sections.

Antlers are the only organ in the mammalian body that regenerates each year. They can reach growth rates of 1-3 cm/day in length and create more than 20 cm2/day of skin in the antler tips (their growth centers). Previous proteomic studies regarding antlers have focused on antler growth centers (tips) compared to the standard bone to detect the proteins involved in tissue growth. However, proteins of cell differentiation and regeneration will be more accurately detected considering more growing tissues. Thus, we set out to compare proteins expressed in antler tips (the highest metabolism rate and cell differentiation) vs. middle sections (moderate cell growth involving bone calcification), using ribs as controls. Samples were obtained in mid-June with antlers' phenology corresponding to the middle of their growth period. Quantitative proteomic analysis identified 259 differentially abundant proteins mainly associated with antioxidant metabolic mechanisms, protein formation and Wnt signalling pathway, meanwhile, the mid antler section was linked to blood proteins. The high metabolic rate and subsequent risk of oxidative stress also seem to have resulted in strong antioxidant mechanisms. These results suggest that redox regulation of proteins is a key factor in the model of deer antler regeneration.

Proteomic Advances in Cereal and Vegetable Crops.

The importance of vegetables in human nutrition, such as cereals, which in many cases represent the main source of daily energy for humans, added to the impact that the incessant increase in demographic pressure has on the demand for these plant foods, entails the search for new technologies that can alleviate this pressure on markets while reducing the carbon footprint of related activities. Plant proteomics arises as a response to these problems, and through research and the application of new technologies, it attempts to enhance areas of food science that are fundamental for the optimization of processes. This review aims to present the different approaches and tools of proteomics in the investigation of new methods for the development of vegetable crops. In the last two decades, different studies in the control of the quality of crops have reported very interesting results that can help us to verify parameters as important as food safety, the authenticity of the products, or the increase in the yield by early detection of diseases. A strategic plan that encourages the incorporation of these new methods into the industry will be essential to promote the use of proteomics and all the advantages it offers in the optimization of processes and the solution of problems.

Proteomic Advances in Milk and Dairy Products.

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.

Dark-cutting beef: A brief review and an integromics meta-analysis at the proteome level to decipher the underlying pathways.

Comprehensive characterization of the post-mortem muscle proteome defines a fundamental goal in meat proteomics. During the last decade, proteomics tools have been applied in the field of foodomics to help decipher factors underpinning meat quality variations and to enlighten us, through data-driven methods, on the underlying mechanisms leading to meat quality defects such as dark-cutting meat known also as dark, firm and dry (DFD) meat. In cattle, several proteomics studies have focused on the extent to which changes in the post-mortem muscle proteome relate to dark-cutting beef development. The present data-mining study firstly reviews proteomics studies which investigated dark-cutting beef, and secondly, gathers the protein biomarkers that differ between dark-cutting versus beef with normal-pH in a unique repertoire. A list of 130 proteins from eight eligible studies was curated and mined through bioinformatics for Gene Ontology annotations, molecular pathways enrichments, secretome analysis and biological pathways comparisons to normal beef color from a previous meta-analysis. The major biological pathways underpinning dark-cutting beef at the proteome level have been described and deeply discussed in this integromics study.

Quantitative proteomic analysis of beef tenderness of Piemontese young bulls by SWATH-MS.

Quantitative proteomic approach is a suitable way to tackle the beef tenderness. Ten aged-beef samples from Longissimus thoracis of Piemontese breed classified as tender (n = 5) and tough (n = 5) meat were evaluated using SWATH-MS and bioinformatic tools for the identification of the proteins and pathways most influencing tenderness variability. Between the two textural groups, proteomic changes were mainly caused by 43 differentially abundant proteins (DAPs) arranged in reference patterns as displayed by the heat map analysis. Most of these DAPs were associated with energy metabolism. From the functional proteomic analysis, two clusters of proteins, including ACO2, MDH1, MDH2 and CS in one cluster and FBP2, PFKL, LDHA, TPI1 and GAPDH/S in the other cluster, suggest gluconeogenesis, glycolysis and citrate cycle as key pathways for Piemontese breed beef tenderness. These findings contribute to a deeper insight into molecular pathways related to beef tenderness.

In Search of Antioxidant Peptides from Porcine Liver Hydrolysates Using Analytical and Peptidomic Approach.

The search for antioxidant peptides as health-promoting agents is of great scientific interest for their biotechnological applications. Thus, the main goal of this study was to identify antioxidant peptides from pork liver using alcalase, bromelain, flavourzyme, and papain enzymes. All liver hydrolysates proved to be of adequate quality regarding the ratio EAA/NEAA, particularly flavourzyme hydrolysates. The peptidomic profiles were significantly different for each enzyme and their characterizations were performed, resulting in forty-four differentially abundant peptides among the four treatments. Porcine liver hydrolysates from alcalase and bromelain are demonstrated to have the most antioxidant capacity. On the other hand, hydrophobic amino acid residues (serine, threonine, histidine and aspartic acid) might be reducing the hydrolysates antioxidant capacity. Seventeen peptides from collagen, albumin, globin domain-containing protein, cytochrome ß, fructose-bisphosphate aldolase, dihydropyrimidinase, argininosuccinate synthase, and ATP synthase seem to be antioxidant. Further studies are necessary to isolate these peptides and test them in in vivo experiments.

Peptidomic analysis of antioxidant peptides from porcine liver hydrolysates using SWATH-MS.

There is a growing interest in the production and identification of bioactive peptides as health-promoting agents. A relevant method to produce biopeptides is enzymatic hydrolysis from protein-rich meat by-products. Pork liver proved to be a good source of protein (18.54%) with a low-fat content (3.38%). After hydrolysis at different times (4,6,8 and 10 h) with Alcalase, relevant amino acids such as hydrophobic (leucine, valine and isoleucine) and aromatic (tyrosine and phenylalanine) involved in antioxidant capacity were strongly increased. For the peptidomic analysis, a novel technique called sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used. Regarding the effect of hydrolysis time, PCA demonstrated a great differentiation among the peptidomic pattern. Fifty-one peptides were correlated with antioxidant activity measured by DPPH, ABTS, FRAP and ORAC assays. SWATH-MS allowed the identification and quantification of six peptides from trypsinogen, ferritin, keratin, carboxylic ester hydrolase and globin domain-containing protein as potential antioxidant compounds. SIGNIFICANCE: The pork liver tissue contains a substantial amount of proteins whose enzymatic hydrolysis might generate antioxidant peptides. The bioactive peptides from pork liver would contribute to harnessing by-products of the swine industry as well as added-value products will be produced. The antioxidant activity of the mixtures revealed potential antioxidant peptides which could be used in the development of nutraceutical and functional food products.

Application of Proteomic Technologies to Assess the Quality of Raw Pork and Pork Products: An Overview from Farm-To-Fork.

The quality assurance of pork meat and products includes the study of factors prior to slaughter such as handling practices, diet and castration, and others during the post-mortem period such as aging, storage, and cooking. The development over the last two decades of high-throughput techniques such as proteomics offer great opportunities to examine the molecular mechanisms and study a priori the proteins in the living pigs and main post-mortem changes and post-translational modifications during the conversion of the muscle into the meat. When the most traditional crossbreeding and rearing strategies to improve pork quality were assessed, the main findings indicate that metabolic pathways early post-mortem were affected. Among the factors, it is well documented that pre-slaughter stress provokes substantial changes in the pork proteome that led to defective meat, and consequently, novel protein biomarkers should be identified and validated. Additionally, modifications in pork proteins had a strong effect on the sensory attributes due to the impact of processing, either physical or chemical. Maillard compounds and protein oxidation should be monitored in order to control proteolysis and volatile compounds. Beyond this, the search of bioactive peptides is becoming a paramount goal of the food and nutraceutical industry. In this regard, peptidomics is a major tool to identify and quantify these peptides with beneficial effects for human health.

Antioxidant activity and peptidomic analysis of porcine liver hydrolysates using alcalase, bromelain, flavourzyme and papain enzymes.

Antioxidant peptides are increasingly being recognized as food additives due to their effects on body human, regulating in vivo oxidative stress against oxidation of lipids and proteins. Meat by-products are rich sources of protein that can be employed for this purpose. Specifically, porcine liver can be used to prepare hydrolysates with antioxidant activity employing proteolytic enzymes such as alcalase, bromelain, papain and flavourzyme. In this study, the antioxidant activity of these four porcine liver hydrolysates was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ((2,2-azinobis-(3-ethyl-benzothiazoline-6-sulphonate) (ABTS), Ferric reducing antioxidant power assay (FRAP) and Oxygen radical absorbance capacity assay (ORAC) assays and the identification of bioactive peptides was carried out by SWATH-MS technology. According to the SDS-PAGE pattern, the proteolysis index and the free amino acids amount, the protein degradation was clearly different among the studied enzymes. Indeed, alcalase enzyme produced the release of small peptides, meanwhile flavourzyme produced higher level of free amino acids. The heatmap analysis showed a peptidomic pattern more differentiated for alcalase than for the other enzymes. The peptides most abundant and correlated with antioxidant capacity were APAAIGPYSQAVLVDR from uncharacterized protein, GLNQALVDLHALGSAR, ALFQDVQKPSQDEWGK and LSGPQAGLGEYLFER from ferritin and LGEHNIDVLEGNEQFINAAK from trypsinogen. The production and characterization of biopeptides is a new merging challenge of meat industry.

Current Trends in Proteomic Advances for Food Allergen Analysis.

Food allergies are a global food challenge. For correct food labelling, the detection and quantification of allergens are necessary. However, novel product formulations and industrial processes produce new scenarios, which require much more technological developments. For this purpose, OMICS technologies, especially proteomics, seemed to be relevant in this context. This review summarises the current knowledge and studies that used proteomics to study food allergens. In the case of the allergenic proteins, a wide variety of isoforms, post-translational modifications and other structural changes during food processing can increase or decrease the allergenicity. Most of the plant-based food allergens are proteins with biological functions involved in storage, structure, and plant defence. The allergenicity of these proteins could be increased by the presence of heavy metals, air pollution, and pesticides. Targeted proteomics like selected/multiple reaction monitoring (SRM/MRM) have been very useful, especially in the case of gluten from wheat, rye and barley, and allergens from lentil, soy, and fruit. Conventional 1D and 2-DE immunoblotting have been further widely used. For animal-based food allergens, the widely used technologies are 1D and 2-DE immunoblotting followed by MALDI-TOF/TOF, and more recently LC-MS/MS, which is becoming useful to assess egg, fish, or milk allergens. The detection and quantification of allergenic proteins using mass spectrometry-based proteomics are promising and would contribute to greater accuracy, therefore improving consumer information.

Aquaculture and by-products: Challenges and opportunities in the use of alternative protein sources and bioactive compounds.

There is a growing concern about chronic diseases such as obesity, diabetes, hypertension, hypercholesterolemia, cancer and cardiovascular diseases resulting from profound changes in the western lifestyle. Aquaculture by-products are generated in large quantities and they can be profitably recycled through their bioactive compounds used for health or food supplements. Improving waste utilization in the field of aquaculture is essential for a sustainable industry to prevent or minimize the environmental impact. In this sense fish by-products are a great source of protein and omega-3 polyunsaturated fatty acids which are particularly studied on Atlantic salmon or rainbow trout. Fish protein hydrolysate (FPH) obtained from chemical, enzymatical and microbial hydrolysis of processing by-products are being used as a source of amino acids and peptides with high digestibility, fast absorption and important biological activities. Omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) from fish discards have been reported to decrease postprandial triacylglycerol levels, reduction of blood pressure, platelet aggregation and the inflammatory response. Crustacean by-products can also be used to produce chitosan with antioxidant and antimicrobial activity for food and pharmaceutical industries and carotenoids with important biological activity. Seaweeds are rich in bioactive compounds such as alginate, carrageenan, agar, carotenoids and polyphenols with different biological activities such as antioxidant, anticancer, antidiabetic, antimicrobial or anti-inflammatory activity. Finally, regarding harvest microalgae, during the past decades, they were mainly used in the healthy food market, with >75% of the annual microalgal biomass production, used for the manufacture of powders, tablets, capsules or pills. We will report and discuss the present and future role of aquaculture by-products as sources of biomolecules for the design and development of functional foods/beverages. This chapter will focus on the main bioactive compounds from aquaculture by-products as functional compounds in food and their applications in biomedicine for the prevention and treatment of diseases.