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In this study, the effects of ultrasound-assisted enzymatic hydrolysis on the extraction of anti-inflammatory peptides from porcine bone collagen were investigated. The results showed that ultrasound treatment increased the content of α-helix while decreased β-chain and random coil, promoted generation of small molecular peptides. Ultrasound-assisted enzymatic hydrolysis improved the peptide content, enhanced ABTS+ radical scavenging and ferrous ion chelating ability than non-ultrasound group. At the ultrasonic power of 450 W (20 min), peptides possessed significant anti-inflammatory activity, where the releasing of interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was all suppressed in lipopolysaccharide (LPS) induced RAW264.7 cells. After the analysis with LC-MS/MS, eight peptides with potential anti-inflammatory activities were selected by the PeptideRanker and molecular docking. In general, the ultrasound-assisted enzymatic hydrolysis was an effective strategy to extract the bioactive peptides from porcine bone, and the inflammatory regulation capacity of bone collagen sourced peptides was firstly demonstrated.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐TL(B) by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the modification of fats and oils by interesterification and in the manufacture of enzyme‐modified dairy ingredients. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.057 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,960 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 34,386. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Salting is a crucial step during the production of dry-cured ham and it is not well known whether it has an impact on the generation of taste-active peptides. The present study focused on the quantitation of kokumi γ-glutamyl peptides in low-salted Spanish dry-cured hams with 12 months of processing. By using mass spectrometry, peptides were quantitated from samples obtained after ethanolic deproteinization-based and non-ethanolic deproteinization-based extraction methods. Peptides γ-EA, γ-EE, and γ-EL registered mean values of 0.31, 2.75, and 11.35 µg/g of dry-cured ham, respectively, with no differences observed between both extraction protocols. However, γ-EF, γ-EM, γ-EV, γ-EW, γ-EY, and γ-EVG presented significantly (p < 0.05) higher concentrations in the ethanolic deproteinized samples showing values of 5.58, 4.13, 13.90, 0.77, 3.71, and 0.11 µg/g of dry-cured ham, respectively. These outcomes reflect the importance of protocols for the extraction of peptides to achieve the most feasible results. In addition, potential precursors for the formation of γ-glutamyl peptides are generated during dry-curing under salt restriction. The kokumi activity of these γ-glutamyl peptides could enhance the sensory attributes countering the taste deficiencies caused by the salt restriction.

Abstract High cholesterolemia is a key risk factor for the development of cardiovascular diseases, which are the main cause of mortality in developed countries. Most therapies are focused on the modulation of its biosynthesis through 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) inhibitors. In this sense, food-derived bioactive peptides might act as promising health alternatives through their ability to interact with crucial enzymes involved in metabolic pathways, avoiding the adverse effects of synthetic drugs. Dry-cured ham has been widely described as an important source of naturally-generated bioactive peptides exerting ACEI-inhibitory activity, antioxidant activity, and anti-inflammatory activity between others. Based on these findings, the aim of this work was to assess, for the first time, the in vitro inhibitory activity of HMG-CoAR exerted by dipeptides generated during the manufacturing of dry-cured ham, previously described with relevant roles on other bioactivities. The in vitro inhibitory activity of the dipeptides was assessed by measuring the substrate consumption rate of the 3-hydroxy-3-methylglutaryl CoA reductase in their presence, with the following pertinent calculations. Further research was carried out to estimate the possible interactions of the most bioactive dipeptides with the enzyme by performing in silico analysis consisting of molecular docking approaches. Main findings showed DA, DD, EE, ES, and LL dipeptides as main HMG-CoAR inhibitors. Additionally, computational analysis indicated statin-like interactions of the dipeptides with HMG-CoAR. This study reveals, for the first time, the hypocholesterolemic potential of dry-cured ham-derived dipeptides and, at the same time, converges in the same vein as many reports that experimentally argue the cardiovascular benefits of dry-cured ham consumption due to its bioactive peptide content.

The utilization of animal bones as a protein source could be used as a sustainable pathway for the production of bioactive compounds. In this study, bones were pretreated with pepsin enzyme (PEP) and then sequentially hydrolyzed with Alcalase (PA) and Alcalase, as well as Protana prime (PAPP). The degree of hydrolysis, antioxidant activity, and DPP-IV inhibitory activity were measured. All three hydrolysates showed antioxidant and DPP-IV inhibitory activity; however, the highest result in both bioactivities was obtained with the PAPP hydrolysate. The obtained free amino acid content was 54.62, 88.12, and 668.46 mg/100 mL of hydrolyzed in PEP, PA, and PAPP, respectively. Pepsin pretreatment did not significantly affect the degree of hydrolysis; however, it is suggested that it promoted the cleavage of certain bonds for subsequent protease action. Accordingly, a total of 550 peptides were identified in PEP hydrolysate, 1087 in PA hydrolysate, and 1124 in PAPP hydrolysate using an LC-MS/MS approach. Pepsin pretreatment could be an effective method in the utilization of bone sources for the production of antioxidant and hypoglycemic peptides.

Dry-cured pork products, such as dry-cured ham, undergo an extensive proteolysis during manufacturing process which determines the organoleptic properties of the final product. As a result of endogenous pork muscle endo- and exopeptidases, many medium- and short-chain peptides are released from muscle proteins. Many of them have been isolated, identified, and characterized, and some peptides have been reported to exert relevant bioactivity with potential benefit for human health. However, little attention has been given to di- and tripeptides, which are far less known, although they have received increasing attention in recent years due to their high potential relevance in terms of bioactivity and role in taste development. This review gathers the current knowledge about di- and tripeptides, regarding their bioactivity and sensory properties and focusing on their generation during long-term processing such as dry-cured pork meats.

Sweet dipeptide Ala-Ala (AA) could be one of the most abundant dipeptides naturally generated in meat products due to the high occurrence of its sequence all along endogenous proteins, which means it could play a key role in dry-cured ham flavour development and bioactivity. In this study, a mass spectrometry in tandem methodology was optimised to quantitate the dipeptide AA in dry-cured ham manufactured under different processing conditions. Dipeptide values reached 230 µg/g after 12-month of ripening, but statistically insignificant differences were observed with salt reduction. Regarding its antihypertensive activity, an IC50 value of 110.824 µM was determined from the in vitro inhibition of angiotensin-I converting (ACE-I) enzyme, and the oral administration of 1 mg of AA per kg of body weight showed a significant reduction of the systolic blood pressure (SBP) in spontaneous hypertensive rats (SHR). This study elucidates the importance of AA dipeptide and its potential role in cardiovascular health after dry-cured ham consumption.

The processing of dry-cured ham results in the generation of small peptides by the action of endogenous enzymes on muscle proteins. Common proteomic workflows involve previous separation techniques based on liquid chromatography which are expensive and time-consuming. In this study, a convenient proteomic approach based on MALDI-ToF is proposed for the first time for the detection of dipeptides in Spanish dry-cured ham. Dipeptides AH, AL, DD, EV, and VF were identified in hams of 18 and 24 months of dry-curing. This work provides insights on the efficiency of a new peptidomic workflow for the short peptide identification from a complex food matrix and permits to evaluate the sample in terms of the presence of taste-related and bioactive dipeptides.

This Special Issue of the International Journal of Molecular Sciences is focused on bioactive peptides in foods or hydrolyzates of food by-products, the methods for the extraction and purification of bioactive peptides, their structural and functional characterization, and the mechanisms of action that regulate their activity and support the reported health benefits [...]

Blood is a meat by-product rich in proteins with properties that can be improved after hydrolysis, making it a sustainable alternative for use in the generation of bioactive peptides. The objective of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides obtained from different chicken blood hydrolysates prepared using combinations of four different enzymes. Best results were observed for AP (2% Alcalase + 5% Protana Prime) and APP (2% Alcalase + 5% Protana Prime + 3% Protana UBoost) hydrolysates obtaining inhibition values of 60.55 and 53.61%, respectively, assayed at a concentration of 10 mg/mL. Free amino acids were determined to establish the impact of exopeptidase activity in the samples. A total of 79 and 12 sequences of peptides were identified by liquid chromatography and mass spectrometry in tandem (LC-MS/MS) in AP and APP samples, respectively. Nine of the identified peptides were established as potential DPP-IV inhibitory using in silico approaches and later synthesized for confirmation. Thus, peptides GPF, IGL, and GGGW showed good DPP-IV inhibitory activity with IC50 values of 0.94, 2.22, and 2.73 mM, respectively. This study confirmed the potential of peptides obtained from chicken blood hydrolysates to be used as DPP-IV inhibitors and, therefore, in the control or modulation of type 2 diabetes.

We thank Dr. López-Moreno for the comment [...]

This manuscript is reviewing the presence of substances of safety concern that may be generated in different types of meat products. Such substances include N-nitrosamines that can be generated under certain conditions when using nitrite as preservative, the polycyclic aromatic hydrocarbons generated as a consequence of particular smoking processes, heterocyclic aromatic amines generated under particular cooking conditions, compounds released from the oxidation of lipids and proteins, Maillard reaction products like acrylamide and carboxymethyl lysine, and amines that can be generated and accumulated in fermented meats. These hazardous compounds, their mechanisms of generation, risks for health and ways of preventing its presence in meat products are briefly described in this review. Keywords: Polycyclic aromatic hydrocarbons, Heterocyclic amines, Nitrosamines, Amino acids oxidation, Lipids oxidation, Maillard reaction products, Biogenic amines

Abstract Dry-cured ham has been described as a good source of bioactive peptides and taste-active compounds. Some of them are dipeptides and tripeptides that are released in a large amount from different muscle proteins due to the action of exopeptidases during the dry-cured ham processing. The potential of dipeptides and tripeptides to exert bioactivities and impart taste characteristics to dry-cured ham has been evaluated using the BIOPEP database, since in silico approaches are a time- and cost-effective alternative to empirical approaches. Most of the studied dipeptides and tripeptides showed ACE and DPP inhibitory activities as well as imparted bitter taste. In fact, more than one bioactivity and/or taste could be assigned to a given peptide sequence, and there could be a correlation between both, like ACE inhibitory and bitter EA, EI and LG peptides. Furthermore, several dipeptides such as EK, KP, LA, PL, PP, RG, and VE, among others, were found to be multifunctional (ACE and DPP IV inhibitory) which would be determined by their structure, sequence and amino acid composition. In silico analysis evidences the relevance of dipeptides and tripeptides in the bioactivity and taste of dry-cured hams, but further empirical assays including in vitro and in vivo studies are necessary to confirm such theoretical results. Possible degradation of the small peptides during gastrointestinal digestion and intestinal absorption as well as interactions with the food matrix could reduce their bioavailability and bioaccessibility, and modify their biological activities. Graphical abstract

Chlorpyrifos (CPF) is an organophosphorus pesticide used in poultry to prevent and/or kill insects and such as preserving agents of poultry feed. Exposure continues to CPF can promote its accumulation at trace concentrations in animal tissue. The toxicological effects of these residues (carcinogenicity, genotoxicity, and neurological disorders) have been related to oxidative stress. Nevertheless, it is still unknown if these trace concentrations might promote oxidative stress in muscle proteins since chicken meat proteins are susceptible to undergo oxidation reactions. Moreover, protein oxidation has been related to a decrease in the nutritional value in of meat.To investigate the oxidative effect of CPF on chicken breast proteins, peptidomics and proteomics analysis were used. For this, chicken breast samples were exposed to CPF and subjected to simulated gastrointestinal digestion. The identification of oxidized peptides from digested and undigested proteins were performed by LC MS/MS (nanoESI qQTOF). Prior to mass analyses undigested proteins were trypsinated. Data were analysed using MASCOT and ProteinPilot v 4.5 software. In this study, 90 and 107 oxidized peptides from digested proteins of control and exposed samples were identified, respectively. These peptides corresponding to 12 oxidized proteins. Meanwhile, 260 and 324 oxidized peptides from undigested proteins (control and exposed samples) were identified, which corresponding to 19 and 17 proteins, respectively. Collagen was protein more susceptible to oxidation promoted by chlorpyrifos in digested and undigested proteins. Identification of these oxidized proteins from simulated digestion provides an important insight about the impact of substances like certain veterinary drugs at trace concentrations on the nutritional value of chicken breast meat.

Dry-cured hams are a good source of bioactive peptides, whose stability to gastrointestinal (GI) digestion determines their bioaccessibility and thus their bioavailability to exert beneficial effects. The aim of this study was to evaluate the effect of in vitro GI digestion of dry-cured hams on the peptide profiles and their antioxidant activity. Results showed that the antioxidant activity decreased in the digested samples when measured by DPPH radical scavenging activity and ferric-reducing antioxidant power methods, but increased when assayed by ABTS radical scavenging assay. Chromatographic and mass spectrometry analysis revealed changes in the peptide profiles and evidenced the degradation of proteins, mainly titin, by the digestive enzymes. The study of the peptide profiles before and after digestion of dry-cured hams would help to extend the knowledge about the influence of peptide characteristics on their antioxidant activity and resistance to hydrolysis by digestive enzymes, allowing a better understanding of the bioaccessibility of the bioactive peptides generated in dry-cured hams.

Iberian dry-cured ham is a meat product known as a source of different bioactive peptides due to its high protein content and intense hydrolysis during its processing. In this study, the potential of α-glucosidase inhibitory peptides generated in Iberian dry-cured ham was evaluated for the first time. After size-exclusion chromatography and high-performance liquid chromatography using different stationary phases, the characterization and identification of the peptide sequences contained in the most active fractions was done using MALDI-ToF mass spectrometry and Q-ToF mass spectrometry in tandem. A total of 16 and 47 sequences of peptides were identified in the two most active fractions. On the other hand, the α-glucosidase inhibitory activity of previously identified bioactive peptides in Spanish dry-cured ham such as AEEEYPDL and LGVGG was established, confirming their multifunctionality.

Bioactive peptides have attracted extensive attention worldwide as natural alternatives to promote human health and wellness. Previous studies have shown that chicken hydrolysates could enhance alcohol dehydrogenase, and subsequently they facilitate alcohol metabolism and ameliorate alcohol-induced liver injury. The data presented in this article support the accompanying research article “Isolation and identification of alcohol dehydrogenase stabilizing peptides from Alcalase digested chicken breast hydrolysates”. Present article details all 82 peptides identified from the most active fractions of chicken hydrolysates, and 154 peptides from in silico digestion of the 82 identified peptides, together with the prediction of their potential bioactivity and applicability using several in silico assays. Keywords: Bioactive peptides, Chicken breast, Alcohol dehydrogenase stabilizing, LC-MS/MS, In silico analysis

The effect of chicken hydrolysates (CHs) on alcohol dehydrogenase (ADH) stability was investigated, together with further bioactivity-oriented isolation and identification of CHs. A total of 82 peptides were identified using mass spectrometry in tandem after consecutive separation by size-exclusion chromatography and high-performance liquid chromatography. The identified peptides were then subjected to in silico gastrointestinal digestion and 154 peptides were generated. The potential bioactivity, safety and applicability of the peptides were assessed using multiple predictive programs. A total of 21 among the 154 peptides were predicted to be potentially active with applicability. Four peptides (DPQYPPGPPAF, QKPVL, KPC, and APGH) obtained after in silico digestion were synthesized and validated their activity. Results showed that DPQYPPGPPAF, KPC, and APGH could stabilize ADH in a dose-dependent manner. This study further indicated that chicken hydrolysate could be a novel functional food ingredient in facilitating alcohol metabolism and protection against alcoholic liver injury.

The reduction of salt in meat products influences the natural mechanisms of proteolysis occurring in their processing, and could affect the final characteristics of the product in terms of texture and flavor due to its effect on the activity of enzymes. In the present study, the quantitation of dipeptides PA, GA, VG, EE, ES, DA, and DG in low-salt Spanish dry-cured ham was carried out using a triple quadrupole mass spectrometry instrument. The developed methodology demonstrated the advantages of hydrophilic interaction liquid chromatography in the removal of salt as a clean-up/separation step before ionization. This resulted in a value of 44.88 μg/g dry-cured ham for GA dipeptide, and values ranging from 2 to 8 μg/g dry-cured ham for VG, EE, ES, DA, and DG dipeptides. PA showed the lowest concentration with a value of 0.18 μg/g dry-cured ham. These outcomes prove the remarkable activity of muscular dipeptidyl peptidases during dry-curing as well as confirming the presence of these dipeptides which are related to certain taste attributes (e.g., ‘bitter’ or ‘umami’). Such dipeptides have also been confirmed as anti-inflammatory and potential cardiovascular protectors using in vitro assays, with the advantage of dipeptides small size increases their chance to resist both gastrointestinal digestion and intestinal/bloodstream transport without being degraded or modified.

Background: Evidence suggests that bioactive peptides reduce hypertension and affect certain metabolic pathways. Methods: Fifty-four volunteers with stage 1 prehypertension and/or hypercholesterolemia and/or basal glucose >100 mg/dL were recruited and randomized to pork dry-cured ham (n = 35) or cooked ham (placebo group; n = 19) for 28 days. After a wash-out period, meat products were changed for 28 additional days. Bioactive peptides composition and enzyme inhibitory activities of both products were characterized. Treatment comparisons for the main effects were made using a two (treatment) × two (times) repeated measures minus the effect of cooked ham (placebo). Results: 24 h mean systolic and diastolic pressures decreased up to 2.4 mmHg in the dry-cured ham period (treatment effect, p = 0.0382 y p = 0.0233, respectively) as well as the number of systolic pressure measures > 135 mmHg (treatment effect, p = 0.0070). Total cholesterol levels also decreased significantly after dry-cured ham intake (p = 0.049). No significant differences were observed between the two treatments for basal glucose, HOMA-IR index and insulin levels (p > 0.05). However, a significant rise of ghrelin levels was observed (treatment effect, p = 0.0350), while leptin plasma values slightly decreased (treatment effect, p = 0.0628). Conclusions: This study suggested the beneficial effects of regular dry-cured ham consumption on the improvement of systolic/diastolic blood pressures and facilitated the maintenance of metabolic pathways, which may be beneficial in the primary prevention of cardiovascular disease.

Dry-cured ham-derived dipeptides, generated along a dry-curing process, are of high importance since they play a role in flavor development of dry-cured ham. The objective of this study was to analyze the residues of the less-studied metabotropic glutamate receptor 1 (mGluR1) implicated in the recognition of umami dry-cured ham dipeptides by molecular docking simulation using the AutoDock Suite tool. AH, DA, DG, EE, ES, EV, and VG (and glutamate) were found to attach the enzyme with inhibition constants ranging from 12.32 µM (AH) to 875.75 µM (ES) in the case if Rattus norvegicus mGluR1 and 17.44 µM (VG) to 294.68 µM (DG) in the case of Homo sapiens, in the open–open conformations. Main interactions were done with key receptor residues Tyr74, Ser186, Glu292, and Lys409; and Ser165, Ser186, and Asp318, respectively, for the two receptors in the open–open conformations. However, more residues may be involved in the complex stabilization. Specifically, AH, EE and ES relatively established a higher number of H-bonds, but AH, EV, and VG presented relatively lower Ki values in all cases. The results obtained here could provide information about structure and taste relationships and constitute a theoretical reference for the interactions of novel umami food-derived peptides.

Establishing health effects of bioactive compounds from dry-cured meat is an active area of clinical research. The present study aims to investigate whether consuming dry-cured ham with biopeptides, among other bioactive compounds, modifies blood pressure (BP) and improves other risk factors for cardiovascular disease in humans. This two-arm, cross-over, randomised controlled trial involved 38 healthy subjects with pre-hypertension. Participants received 80 g/day dry-cured pork ham or 100 g/day cooked ham (control product). A daily intake of 80 g dry-cured ham did not impair BP or 24 h sodium excretion. Total cholesterol, LDL and basal glucose levels dropped after dry-cured ham consumption (p = 0.00019, p = 0.021 and p = 0.014, respectively). Cooked ham did not affect any of the clinical and biochemical markers. Dry-cured ham components could exert a plethora of activities over the cardiovascular system including lipid and glucose metabolism. Additional studies are needed to confirm the effects of dry-cured meat biopeptides on diverse risk factors in pathological conditions.

Foods and their industry by-products constitute very good sources of bioactive peptides, which can be naturally generated during processing but are also extensively produced through enzymatic hydrolysis, microbial fermentation, and even during gastrointestinal digestion in the human body [...]

Recently, the use of bioactive compounds in improving human health has received more attention. The aim of the present study was to hydrolyze orange seed proteins using pepsin enzyme to obtain bioactive peptides as well as to study the stability of such activity after simulated gastrointestinal digestion conditions. The method was optimized using different enzyme concentrations from 1% to 3%, hydrolysis times between 2 and 5 h, and an optimal temperature of 33 °C. Biological activities including α-glucosidase inhibition, α-amylase inhibition, Angiotensin I-Converting Enzyme (ACEI) inhibition, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated. According to the results, a significant higher value of the biological activity (p < 0.05) was observed using an enzyme ratio of 0.03 E/S and hydrolysis time of 3.5 h. After size-exclusion chromatography separation, fractions 45–49 and 50–54 showed the highest biological roles such as antioxidant, ACEI inhibitory, and hypoglycemic. Fractions with the highest biological activity were purified using RP-HPLC and analyzed using nano-liquid chromatography and mass spectrometry. The results obtained after simulated gastrointestinal digestion indicated that peptide fractions obtained after chromatographic separation significantly maintain their activity.

Biodiesel constitutes an attractive source of energy because it is renewable, biodegradable, and non-polluting. Up to 20% biodiesel can be blended with fossil diesel and is being produced and used in many countries. Animal fat waste represents nearly 6% of total feedstock used to produce biodiesel through alkaline catalysis transesterification after its pretreatment. Lipase transesterification has some advantages such as the need of mild conditions, absence of pretreatment, no soap formation, simple downstream purification process and generation of high quality biodiesel. A few companies are using liquid lipase formulations and, in some cases, immobilized lipases for industrial biodiesel production, but the efficiency of the process can be further improved. Recent developments on immobilization support materials such as nanoparticles and magnetic nanomaterials have demonstrated high efficiency and potential for industrial applications. This manuscript reviews the latest advances on lipase transesterification and key operational variables for an efficient biodiesel production from animal fat waste.

The agro-food industry generates large amounts of waste that contribute to environmental contamination. Animal fat waste constitutes some of the most relevant waste and the treatment of such waste is quite costly because environmental regulations are quite strict. Part of such costs might be reduced through the generation of bioenergy. Biodiesel constitutes a valid renewable source of energy because it is biodegradable, non-toxic and has a good combustion emission profile and can be blended up to 20% with fossil diesel for its use in many countries. Furthermore, up to 70% of the total cost of biodiesel majorly depends on the cost of the raw materials used, which can be reduced using animal fat waste because they are cheaper than vegetable oil waste. In fact, 6% of total feedstock corresponded to animal fat in 2019. Transesterification with alkaline catalysis is still preferred at industrial plants producing biodiesel. Recent developments in heterogeneous catalysts that can be easily recovered, regenerated and reused, as well as immobilized lipases with increased stability and resistance to alcohol denaturation, are promising for future industrial use. This manuscript reviews the available processes and recent advances for biodiesel generation from animal fat waste.

Angiotensin converting enzyme (ACE) inhibitory peptides have been extensively studied as an alternative to synthetic drugs for the treatment of hypertension. Recent studies have shown that dry-cured ham is an important source of naturally generated bioactive peptides, especially showing ACE inhibitory activity. However, due to their excessive degradation by digestive and brush-border enzymes, it is not clear whether these peptides resist intestinal absorption and reach the blood stream where they may exert their antihypertensive effect. Therefore, dry-cured ham extracts and specific pure peptides naturally generated during the dry-curing process, showing ACE inhibitory activity, have been studied for their stability during transepithelial transport in a Caco-2 cell monolayer. The ACE inhibitory activity of transport samples was assayed, showing the highest values in apical samples at 15 min of incubation. In concentrated basal solutions, ACE inhibition values increased during transport for purified peptides, reaching values close to 70% for AAPLAP and KPVAAP at 60 min of cellular transport. Fragments generated by cellular activity were detected by using tandem mass spectrometry techniques, showing that AAATP, AAPLAP, and KPVAAP were hydrolysed during the transport, although KPVAAP was also absorbed intact. This study highlights the potential of intact dry-cured ham peptides as well as their fragments to be absorbed across the intestinal epithelium and reach the blood stream to exert an antihypertensive action.

In this study, orange seed proteins were hydrolyzed by Alcalase enzyme at different enzyme concentrations 1–3% (v/w) and hydrolysis times (2–5 h), to obtain bioactive peptides showing antioxidant, Angiotensin-converting enzyme (ACE) -inhibitory, and hypoglycemic activities. The highest biological activities (p < 0.05) were achieved by using a hydrolysis time of 5 h and an enzyme concentration of 2%. Orange seed protein hydrolysate (OSPH) was prepared under these conditions, and peptides were isolated and purified by using size-exclusion chromatography and high-performance liquid chromatography, respectively. The fractions that showed the highest biological activities were analyzed by mass spectrometry in tandem, and a total of 63 peptide sequences were found. Moreover, the effect of simulated gastrointestinal digestion on the bioactivity of the fractions was studied, and the novel peptide sequences generated were also identified. Overall, despite there being some differences in the profile of peptide sequences obtained, the main results showed non-significant differences in the analyzed bioactivities after simulated gastrointestinal digestion.

The use of edible biopolymers and natural additives obtained from food processing by-products is a sustainable strategy for food packaging applications. Gelatin is a biopolymer with great potential as a coating due to its low cost, high availability, and technological and functional properties. Among them, gelatin can be used as a carrier of bioactive compounds such as antioxidants, which can retard oxidation processes and thus extend the shelf-life of highly-perishable products. This study evaluated the effect of gelatin coating enriched with antioxidant tomato by-products hydrolysate (TBPH) on the quality of pork meat during cold storage. Results showed that TBPH obtained from Alcalase hydrolysis presented antioxidant activity with good stability against cooking. Additionally, chromatographic and mass spectrometry techniques, as well as in silico analysis, were used for the peptidomic characterisation of TBPH. The application of enriched gelatin coating on meat led to some physicochemical changes including increased weight loss and colour differences; however, the pH and water activity, which control meat spoilage, were maintained during storage. Moreover, coating prevented lipid oxidation of meat, and enriched-coated meat presented high antioxidant activity after cooking. These results suggest the positive role of gelatin coating enriched with TBPH in extending the shelf-life of meat during storage.

The angiotensin I-converting enzyme (ACE) inhibitory activities and antioxidant properties of thornback ray gelatin hydrolysates (TRGHs) prepared by treatment with proteolytic proteases from Bacillus subtilis A26, Raja clavata crude alkaline protease extract, Alcalase and Neutrase were investigated. All gelatin hydrolysates showed different degrees of hydrolysis and hydrophobic/hydrophilic peptides ratio. Moreover, they possess high protein content (70.04 ± 0.55–74.14 ± 0.28%). The antioxidant activity was assayed using various in vitro tests. The highest antioxidant activity was observed with hydrolysate obtained by treatment with A26 proteases (TRGH-A26) which exhibited a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity with a concentration that produces 50% of inhibition (IC50) of 1.98 ± 0.02 mg/ml of sample, reduced the ferric ions with an absorbance at 700 nm of 0.962 ± 0.07, prevents bleaching of β-carotene with 73.02 ± 1.90% inhibition and gave an antioxidative efficacy of 180 ± 0.08 µmol/ml α-tocopherol equivalents at 5 mg/ml in the phosphomolybdenum assay. However, gelatin hydrolysate treated with Alcalase (TRGH-Alcalase) was the most potent to prevent DNA oxidation. For the ACE inhibitory activity, all hydrolysates displayed ACE-inhibitory activity. TRGH-A26 and TRGH-Alcalase exhibited the highest activity with 85 ± 0.65 and 82 ± 0.49%, respectively, at 5 mg/ml. The results revealed that TRGHs could be used as ingredients to formulate functional foods.

In this study, the antihypertensive activity of Purafect®-smooth hound viscera protein hydrolysate (VPH) and its peptide fraction with molecular weight (MW) below 1 kDa (VPH-I) was investigated. In addition, the lipase inhibitory activity, as well the anticoagulant potential, in vitro, were assessed. The antihypertensive effects of VPH and VPH-I were studied during 24 h (short-term effect) and 30 days (long-term effect) using high-salt (18% NaCl) and -fructose (10%) diet (HSFD)-induced hypertension. Data showed that, 4 h post-administration of VPH and VPH-I (200 mg/kg BW), the systolic blood pressure of rats was reduced by about 6 and 9 mmHg, respectively. These effects were similar to that obtained with Captopril (~9 mmHg at t = 4 h). On the other hand, exposing the rats to daily to HSFD, coupled to the administration of viscera peptides, was found to attenuate hypertension. In addition, the proteins’ treatments were able to correct lipid and glycemic disorders, by reducing the total cholesterol and triglyceride contents and resorting to the plasma glucose level, compared to the HSFD group. Overall, the present findings demonstrated the preventive effect of VPH-peptides from hypertension complications, as a result of their biological properties.

Myoglobin is a very important sarcoplasmic protein responsible for the colour of meat and meat products. This protein is degraded during post-mortem period (ageing) as well as during longer processes such as dry curing. In the present study, a total of eleven naturally generated fragments of myoglobin detected at the end of the processing of dry-cured ham have been identified for the first time and the intense proteolysis of myoglobin during dry curing has been confirmed. This study was carried out using one-dimensional reversed-phase liquid chromatography combined with nano liquid chromatography and an electrospray ionization source coupled to a quadrupole time-of-flight mass spectrometer. Paragon and Mascot search engines with the databases NCBInr and Swiss-Prot were used for the identification based on raw MS/MS data from one or more peptides, and the possible enzymes involved in myoglobin hydrolysis have also been discussed.

Meat and meat products have been described as a very good source of angiotensin I converting enzyme (ACEI)-inhibitory peptides. The generation of bioactive peptides can occur through the action of endogenous muscular enzymes during processing, gastrointestinal digestion, or by using commercial enzymes in laboratory or industry under controlled conditions. Studies of bioavailability are necessary in order to prove the positive health effect of bioactive peptides in the body as they should resist gastrointestinal digestion, cross the intestinal barrier, and reach blood stream and target organs. However, in order to better understand their effect, interactions, and bioavailability, it is necessary to consider food matrix interactions and continue the development of quantitative methodologies in order to obtain more data that will enable advances in the field of bioactive peptides and the determination of their influence on health.

Potassium and sodium salts of nitrite (E 249 and E 250) and nitrate (E 251 and E 252) are authorised for use under certain levels in several foodstuffs such as non-heat-treated, cured and dried meat products, other cured meat products, canned meat products and bacon. The key point in the use of nitrate and nitrite as preservatives is to find a balance between ensuring the microbiological safety of the ham and keeping as low as possible the level of nitrosamines in the final product. Nitrites and nitrates are authorised as additives for dry-cured ham in the Directive 2006/52/EC of 5 July 2006 that modifies previous Council Directive 95/2/EC on food additives other than colours and sweeteners. The effect of nitrate and its reduction to nitrite in controlling the lipid oxidation process during the ham ripening is very important for the development of the characteristic cured flavour. The main benefits and drawbacks of the use of nitrites and nitrates in dry-cured ham and how these levels may affect its flavour are discussed in this manuscript.

This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1224/full