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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.

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 [...]

The ultrasound-induced impacts on the peptide characteristics and taste of unsmoked bacon have been evaluated through the use of peptidomics and bioinformatics approaches. Furthermore, the effect of such ultrasound-induced changes on the main endogenous proteases responsible for peptide generation was also investigated. In fact, the activity of main endogenous proteases was significantly increased after ultrasonic treatment during the processing of unsmoked bacon, and contributed to an increased number and an enhanced LFQ intensity of peptides. Besides, such increased amount of peptides and LFQ intensity with up to 500 W ultrasonic treatment were beneficial for the taste improvement of the final products as shown by taste prediction analysis. Nevertheless, an excessive ultrasonic power like 750 W hindered protein hydrolysis and further exerted a negative effect on peptide generation. Therefore, ultrasound under controlled conditions could be considered as a promising way to improve the taste of unsmoked bacon., This work was financially supported by the earmarked fund for China Agriculture Research System (CARS-35) and the Kekedala City of the 4th Division Science and Technology Plan (2022GY03).

The aim of this study was to investigate the modifications of the proteome and flavor-related proteins in unsmoked bacon resulting from ultrasound treatment with the application of label-free quantitation technology together with bioinformatics analysis. Results showed that the expression levels of 137 proteins were markedly affected by ultrasound with most of them being significantly upregulated. The proteins distributed in the cytoplasm and the cytosol, the mitochondrion, and the nucleus were more susceptible to ultrasound treatment. Meanwhile, 20 flavor-related proteins, mostly myofibrillar proteins and metabolic enzymes mainly involved in the metabolic pathways of signaling and cellular processes and environmental information processing, were screened out. In addition, the differential expressions of flavor-related proteins induced by ultrasound were verified by western blotting. This study displayed insightful information from the proteomics perspective for a better understanding of the influential effect of ultrasound treatment on meat flavor., This work was financially supported by the China Agriculture Research System of MOF and MARA (CARS-35), the earmarked fund for the Jiangsu Agricultural Industry Technology System (JATS(2021)476), and the Kekedala City of the 4th Division Science and Technology Plan (2022GY03).

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

There is an intense research activity on bioactive peptides derived from food proteins in view of their health benefits for consumers. However, their identification is quite challenging as a consequence of their small size and low abundance in complex matrices such as foods or hydrolyzates. Recent advances in peptidomics and bioinformatics are getting improved sensitivity and accuracy and therefore such tools are contributing to the development of sophisticated methodologies for the identification and quantification of peptides. These developments are very useful for the follow-up of peptides released through proteolysis either in the food itself through the action of endogenous peptidases during processing stages like fermentation, drying or ripening, or from food proteins hydrolyzed by commercial peptidases or microorganisms with proteolytic activity. This chapter is presenting the latest advances in peptidomics and its use for the identification and quantification of peptides, and as a useful tool for controlling the proteolysis phenomena in foods and protein hydrolyzates.

Fat, composed mainly of triacylglycerols and phospholipids, and protein, composed of amino acids, constitute major components of meat. Carbohydrate, in the form of glycogen, is present in very low amounts. This article describes the main methodologies available for the analysis of these substances, discusses their advantages and disadvantages, and gives recommendations for the choice of methods.

Alcohol hydydrogenase (ADH) is a key enzyme that influences alcohol metabolism in vivo. Our previous study showed that chicken-derived tripeptide KPC could stabilize ADH in a dose-dependant manner. In this study, possible mechanism underlying how KPC could stabilize ADH was further investigated. Fluorescence quenching data showed that KPC induced a dynamic fluorescence quenching with a quenching rate constant value of 1.074 × 1010 M−1s−1, indicating a conformational change. Circular dichroism further suggested a possible transformation from α-helix (14.03%–13.90%) to random coil (31.98–33.04%) in ADH structure as affected by KPC (5 mmol/L). Molecular docking analysis predicted that KPC may bind to ADH through hydrophobic interaction and hydrogen bonds. Validation of ADH stabilizing activity of fragment peptides and amino acids from KPC implied the vital role of Cys residue to the bio-activity of KPC. These results indicated that KPC may stabilize ADH through peptide-enzyme interactions, and protect ADH against oxidative modification. Besides, a favorable absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of KPC was obtained using in silico assays. Overall, this study provided a comprehensive understanding on the interaction mechanism between KPC and ADH, and illustrated the potential applicability of KPC as drug or functional food ingredient., This work was supported by the National Natural Science Foundation of China (No. 32101896 and No. 31871746), Key-Area Research and Development Program of Guangdong Province (2021B0707060001), the Self-innovation Research Funding Project of Hanjiang Laboratory (HJL202010B002, HJL202101B001, HJL202101B004,and HJL202101B005), and the Chaozhou Science and Technology Project (2020PT01).

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., Grant AGL2017-89381-R funded by MCIN/AEI/10.13039/501100011033 by “ERDF A way of making Europe” is acknowledged. Grant PRE2018-083367 (A.H.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future” are also acknowledged.

Chicken hydrolysates (CHs) have been reported to protect mice against alcoholic liver injury possibly through oxidative stress reduction. In this study, the antioxidant activity of CHs was studied. Results showed that CHs exhibited significant antioxidant activity (around 600 and 400 μM TEAC/g in ORAC and ABTS assay, respectively) and could resist simulated gastrointestinal digestion. A total of 22 peptides were identified after antioxidant activity-oriented isolation using size-exclusion chromatography and high-performance liquid chromatography. Further in silico analysis and the validation of antioxidant activity revealed that novel peptides (RWGG and YYCQ) exhibited strong antioxidant activity. The most active peptide YYCQ displayed a TEAC value of 3.54 and 4.28 μM TEAC/μM in ORAC and ABTS assay, respectively. These peptides could contribute to reduce oxidative stress and protect against alcohol-induced liver injury. However, further studies understanding the bioactivity of such peptides in vivo are necessary before further applying them as functional food ingredient., This work was supported by the National Natural Science Foundation of China (No. 32101896 and No. 31871746), Key-Area Research and Development Program of Guangdong Province (2021B0707060001), and Self-innovation Research Funding Project of Hanjiang Laboratory (HJL202010B002). Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 by “ERDF A way of making Europe” is acknowledged and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” is also acknowledged.

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., This research was funded by Grant PID2020-119684RB-I00 funded by MCIN/AEI/10.13039/501100011033. Grant GRISOLIAP/2020/021 de la Consellería de Innovació, Universitats, Ciència i Societat Digital de la Generalitat Valenciana (GCA) is also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to Proteored.

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

Meat and meat products constitute an important source of nutrients and play vital roles for growth, maintenance and repair of the body. In addition to the high quality of proteins, meat is also regarded as a major resource to produce bioactive peptides. Meat processing industry also produces by-products such as bones, blood and viscera, which could be further used for the production of bioactive compounds. In the physiological analysis, meat bioactive peptides have been reported to exert antioxidant, anti-hypertensive, anti-inflammatory, anti-microbial and antitumoral activities, which endow nutritional and functional value of meat. With the objective to exert the functional effect, the bioavailability should also be considered due to the degradation by digestion enzymes and the absorption process in intestinal mucosa. In this chapter, the general source, the enzymatic hydrolysis, the physiological effects as well as the bioavailability of bioactive peptides in meat are discussed.

A systematic insight into the structure-function properties of small bioactive peptides is of great importance. Herein, peptidomics and computational methodology were adopted to investigate the stabilization patterns and building blocks of antioxidant peptides resulting from proteolysis during the ripening of Spanish dry-cured ham (9-24 months of processing). The results showed that native peptides underwent manufacture-induced steric/redox stress, while homogeneous/heterogeneous p-π/π-π interaction significantly improved the ABTS+ inhibition activity of hydrophobic peptides. However, for more hydrophilic peptides, the intrinsic π-interaction system (i.e., cation-π and π-π packing) substantially interfered with ABTS+/DPPH scavenging events when compared to the aromatic residues. Semi-quantitative peptidomics and molecular simulation/docking revealed that VFSSQGQSELILLQK and LCPSPDGLYL are two potential antioxidant peptides at the late ripening-drying. They had distinctive self-folding destinies following solvation owing to varying charged/hydrophobic properties of termini and hydrogen atom donor, allowing different flexibility of backbone and interactive surface towards free radicals ex situ followed by electron/proton transfer., Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 and “ERDF A way of making Europe” and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” are also acknowledged. C.L. appreciated the financial support from China Scholarship Council (CSC), China through the State Scholarship Fund for Overseas Study (No. 201806760050).

Royal jelly (RJ) has been known for centuries, but in the last 5-6 decades its systematic production and consumption has increased. RJ is secreted by the hypopharyngeal and mandibular glands of worker honeybees (Apis mellifera). This thick and milky substance contains water, proteins, carbohydrates, lipids, minerals, vitamins and such bio-active compounds as acetylcholine, peptides, the hormones testosterone, progesterone, prolactin, estradiol, (hydroxydecanoic acid) (HAD), adenosine monophosphate (AMP)-N1Oxide, polyphenols, flavonoids and adenosine. Because of its bioactive compounds, RJ can be considered as a functional and nutraceutical food. The main goal of this review is to summarize and update its physicochemical properties, bio-active ingredients, storage stability and shelf life. The functional properties are antioxidative activity, insulin-like action, improvement against diabetes, liver protection, antitumoral action, neurotrophic action, antibiotic effect, anti-inflammatory action and wound healing, hypotensive effect and blood regulatory actions, anti-aging effect and skin protection, effects on the reproductive system and fertility and also fortifying, tonic action and immunomodulating and anti-alergic activity. RJ may cause allergic reactions, asthma and even fatal anaphylaxis in some humans. Therefore, RJ should be orally ingested as nutreaceutical agent or food-ingredient only after an allergy test.

Background: Dry-cured ham is one of the most valued food products by Mediterranean consumers. In this sense, the appropriate development of its different production stages is essential to ensure the quality requirements. For this reason, non-invasive technologies have gained popularity and have been reported as useful not only to ensure the food safety of different products, but also to monitor fundamental stages in the production process, such as the salting stage, to analyze the content of different compounds without sample losses, and to correct possible defects in the final product. Scope and approach: This work has been focused on summarizing the studies that describe and have successfully applied these techniques, as well as on mentioning other technologies with potential use in dry-cured ham manufacture which have not been studied enough. Finally, the potential next steps to improve and optimize the process, as well as the suitability of creating new products with added value based on the new quality standards, have also been evaluated. Key findings and conclusions: Innovative non-invasive technologies such as high pressure (HP), ultrasound (US), pulsed electric fields (PEF), microwaves, irradiation, etc. can be used as promising tools to effectively control salting and curing stages as well as for checking defects of the final product and/or ensuring food safety. HP and US are useful tools for the determination of salt and fat content, and for monitoring the salting process. Moreover, HP enhances salty taste perception, which makes it a useful tool to reduce salt addition. Both, HP and US, can correct texture defects. In addition, NIRS allows predicting the state of the meat to remove those pieces that could result in defective products. Moreover, RAMAN or MRI are able to detect anomalous textures at the end of the process. Microwaves could be useful for the online estimation of salt, water and fat contents easily with portable equipment. Finally, data mining, that allows to make predictions based on an immense data file, is the most promising discovery in recent years for detecting defects or classifying products according to sensory attributes., This research was supported by Grant RTA 2013-00030-CO3-03 from INIA (Spain). Acknowledgements to INIA for granting Cristina Pérez Santaescolástica with a predoctoral scholarship (grant number CPD2015-0212). José M. Lorenzo is member of the MARCARNE network, funded by CYTED (ref. 116RT0503).

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 [...]

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., Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 by “ERDF A way of making Europe”,is acknowledged. Grant PRE2018-083367 (A.H.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” are also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF.

Proteins and peptides are very important nutritional and functional constituents of foods that may be subject to changes derived from either food processing or gastrointestinal digestion. Some of these changes may affect their functional properties and also their biological activities and, therefore, it is important for them to be protected. There have been numerous recent developments to ensure the delivery of bioactive peptides with good stability. Such techniques are based either on the encapsulation of bioactive peptides or on the modification of the peptide structure in order to increase their stability against any food processing treatment such as heating, cooling, pH changes, NaCl treatment and also hydrolysis during gastrointestinal digestion.

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., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness, and Ramón y Cajal postdoctoral contract RYC-2016-19635 by L.M. are acknowledged.

Meat and meat products constitute an important source of nutrients and play vital roles for growth, maintenance and repair of the body. In addition to the high quality of proteins, meat is also regarded as a major resource to produce bioactive peptides. Meat processing industry also produces by-products such as bones, blood and viscera, which could be further used for the production of bioactive compounds. In the physiological analysis, meat bioactive peptides have been reported to exert antioxidant, anti-hypertensive, anti-inflammatory, anti-microbial and antitumoral activities, which endow nutritional and functional value of meat. With the objective to exert the functional effect, the bioavailability should also be considered due to the degradation by digestion enzymes and the absorption process in intestinal mucosa. In this chapter, the general source, the enzymatic hydrolysis, the physiological effects as well as the bioavailability of bioactive peptides in meat are discussed.

A large number of bioactive peptides have been reported with a variety of benefits for human health like angiotensin-converting enzyme inhibition, antioxidant, antiinflammatory, hypoglycemic, or antithrombotic activity, among others. Bioactive peptides are generated through the enzymatic breakdown of proteins either in the food itself by endogenous or microbial peptidases, or in reactors with commercial peptidases. Once consumed, gastrointestinal digestion may also contribute to further generation of bioactive peptides. Endopeptidases are those acting first on proteins followed by the action of exopeptidases on the generated polypeptides and peptides. This chapter is reporting the enzymes and mechanisms involved in the hydrolysis of food proteins for the generation of bioactive peptides.