Generation, Characterisation and Identification of Bioactive Peptides from Mesopelagic Fish Protein Hydrolysates Using In Silico and In Vitro Approaches.

This study generated bioactive hydrolysates using the enzyme Alcalase and autolysis from mesopelagic fish, including Maurolicus muelleri and Benthosema glaciale. Generated hydrolysates were investigated for their bioactivities using in vitro bioassays, and bioactive peptides were identified using mass spectrometry in active hydrolysates with cyclooxygenase, dipeptidyl peptidase IV and antioxidant activities. In silico analysis was employed to rank identified peptide sequences in terms of overall bioactivity using programmes including Peptide Ranker, PrepAIP, Umami-MRNN and AntiDMPpred. Seven peptides predicted to have anti-inflammatory, anti-type 2 diabetes or Umami potential using in silico strategies were chemically synthesised, and their anti-inflammatory activities were confirmed using in vitro bioassays with COX-1 and COX-2 enzymes. The peptide QCPLHRPWAL inhibited COX-1 and COX-2 by 82.90% (+/-0.54) and 53.84%, respectively, and had a selectivity index greater than 10. This peptide warrants further research as a novel anti-inflammatory/pain relief peptide. Other peptides with DPP-IV inhibitory and Umami flavours were identified. These offer potential for use as functional foods or topical agents to prevent pain and inflammation.

Sequential Enzymatic Hydrolysis and Ultrasound Pretreatment of Pork Liver for the Generation of Bioactive and Taste-Related Hydrolyzates.

In the study of protein-rich byproducts, enzymatic hydrolysis stands as a prominent technique, generating bioactive peptides. Combining exo- and endopeptidases could enhance both biological and sensory properties. Ultrasound pretreatment is one of the most promising techniques for the optimization of enzymatic hydrolysis. This research aimed to create tasteful and biologically active pork liver hydrolyzates by using sequential hydrolysis with two types of enzymes and two types of ultrasound pretreatments. Sequential hydrolyzates exhibited a higher degree of hydrolysis than single ones. Protana Prime hydrolyzates yielded the largest amount of taste-related amino acids, enhancing sweet, bittersweet, and umami amino acids according to the Taste Activity Value (TAV). These hydrolyzates also displayed significantly higher antioxidant activity. Among sequential hydrolyzates, Flavourzyme and Protana Prime hydrolyzates pretreated with ultrasound showed the highest ferrous ion chelating activity. Overall, employing both Alcalase and Protana Prime on porcine livers pretreated with ultrasound proved to be highly effective in obtaining potentially tasteful and biologically active hydrolyzates.

Effect of thermal pretreatment and gastrointestinal digestion on the bioactivity of dry-cured ham bone enzymatic hydrolyzates.

This study reports the effect of thermal pretreatment and the use of different commercial proteolytic enzymes (Protamex, Flavourzyme, Protana prime, and Alcalase) on the free amino acid content (FAA), peptide profile, and antioxidant, antidiabetic, antihypertensive, and anti-inflammatory potential (DPPH, FRAP, and ABTS assay, DPP-IV, ACE-I, and NEP inhibitory activities) of dry-cured ham bone hydrolyzates. The effect of in vitro digestion was also determined. Thermal pretreatment significantly increased the degree of hydrolysis, the FAA, and the DPP-IV and ACE-I inhibitory activities. The type of peptidase used was the most significant factor influencing antioxidant activity and neprilysin inhibitory activity. Protana prime hydrolyzates failed to inhibit DPP-IV and neprilysin enzymes and had low values of ACE-I inhibitory activity. After in vitro digestion, bioactivities kept constant in most cases or even increased in ACE-I inhibitory activity. Therefore, hydrolyzates from dry-cured ham bones could serve as a potential source of functional food ingredients for health benefits.

Pork organs as a potential source of flavour-related substances.

The increase in world population has generated a higher demand for quality proteins, increasing the production in meat industry but also the generation of thousands of tons of by-products, with a negative economic and environmental impact. The valorisation of slaughterhouse by-products by giving by-products a new use as food ingredient is one of the best strategies to add value while reducing environmental damage. Flavour is one of the most influential parameters in the purchasing decision of consumers, and in meat products it is mostly influenced by the content in free amino acids and nucleotides. In this study, the potential of 4 pork organs (liver, kidney, lung, and brain) as a source of flavour-related substances was investigated. Liver proved to be the organ showing the highest content of free and total amino acids related to taste, while kidney was the organ with the highest content of umami nucleotides. The results of the Taste Activity Value indicated that umami, sweet, and bittersweet amino acids are main responsible for the taste of the organs. On the other hand, the synergy between amino acids and nucleotides in relation with umami taste was determined, showing liver and kidney the best values in Equivalent Umami Content. In addition, the antioxidant activity of the organs was determined, and liver and kidney showed the highest antioxidant activity in all assays (p < 0.05). In conclusion, pork organs, especially liver and kidney, may be good candidates to be used as raw materials to produce functional flavouring ingredients.

Generation of kokumi γ-glutamyl short peptides in Spanish dry-cured ham during its processing.

The typical dry-cured ham flavor is rich in umami and brothy perceptions, for which short peptides may contribute. Particularly, γ-glutamyl peptides could be the responsible of these previously reported attributes, as they exert a synergistic interaction with other basic tastes and modify the intensity of salty, sweet, and umami tastes. The content of peptides has been reported to evolve along the processing, but no kokumi γ-glutamyl peptides have been identified in Spanish dry-cured hams yet. In this research, nine γ-glutamyl dipeptides (γ-EA, γ-EC, γ-EE, γ-EF, γ-EL, γ-EM, γ-EV, γ-EW, and γ-EY) and two γ-glutamyl tripeptides (GSH and γ-EVG) have been quantitated at 6, 12, 18 and 24 months of traditional processing of Spanish dry-cured ham by performing a Q Exactive Orbitrap-based tandem mass spectrometry. The results show an increase of γ-EA, γ-EE, γ-EF, γ-EL, γ-EM and γ-EVG, obtaining maximums at 24 months of curing ranging from 0.14 (γ-EVG) to 18.86 (γ-EL) µg/g dry-cured ham. Otherwise, γ-EV, γ-EW and γ-EY accumulated until the 18th month of storage to 15.10, 0.54 and 3.17 µg/g dry-cured ham, respectively; whereas γ-EC and GSH amounts decreased starting from 0.0676 and 4.41 µg/g dry-cured ham, respectively at earlier stages. The concentration dynamics of these compounds may be linked with proteolytic and oxidative reactions during processing. In addition, due to their synergistic effect on kokumi activity, this could constitute insights of the brothy perceptions of dry-cured ham, and these peptides probably contribute to the sensory differences existing in long processed Spanish dry-cured hams.

Generation of Bioactive Peptides from Porphyridium sp. and Assessment of Their Potential for Use in the Prevention of Hypertension, Inflammation and Pain.

Inflammation, hypertension, and negative heart health outcomes including cardiovascular disease are closely linked but the mechanisms by which inflammation can cause high blood pressure are not yet fully elucidated. Cyclooxygenase (COX) enzymes play a role in pain, inflammation, and hypertension development, and inhibition of these enzymes is currently of great interest to researchers and pharmaceutical companies. Non-steroidal anti-inflammatory drugs are the drug of choice in terms of COX inhibition but can have negative side effects for consumers. Functional food ingredients containing cyclooxygenase inhibitors offer a strategy to inhibit cyclooxygenases without negative side effects. Several COX inhibitors have been discovered, to date, from marine and other resources. We describe here, for the first time, the generation and characterization of a bioactive hydrolysate generated using Viscozyme® and Alcalase from the red microalga Porphyridium sp. The hydrolysate demonstrates in vitro COX-1 inhibitory activity and antihypertensive activity in vivo, assessed using spontaneously hypertensive rats (SHRs). Peptides were identified and sequenced using MS and assessed using an in silico computational approach for potential bioactivities. The peptides predicted to be bioactive, including GVDYVRFF, AIPAAPAAPAGPKLY, and LIHADPPGVGL were chemically synthesized and cyclooxygenase inhibition was confirmed. Peptides AIPAAPAAPAGPKLY and LIHADPPGVGL had COX-1 IC50 values of 0.2349 mg/mL (0.16 µM) and 0.2193 mg/mL (0.2 µM), respectively. The hydrolysate was included in a food carrier (jelly candies) and an antihypertensive effect was observed in SHRs.

Comparative Quantitation of Kokumi γ-Glutamyl Peptides in Spanish Dry-Cured Ham under Salt-Reduced Production.

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.

Potential of Dry-Cured Ham Bones as a Sustainable Source to Obtain Antioxidant and DPP-IV Inhibitory Extracts.

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.

Antioxidant peptides generated from chicken feet protein hydrolysates.

BACKGROUND: As major industrial poultry by-products, chicken feet are considered as notable sources of several bioactive molecules. The current work covers the processing of chicken feet proteins as substrates to be hydrolysed by combinations of three commercial enzymes (Alcalase®, Flavourzyme® and Protana® Prime) during different hydrolysis periods and the evaluation of the identified peptides having antioxidant activity after simulated gastrointestinal digestion. RESULTS: Enzymatic hydrolysis with Alcalase® and Protana® Prime combination for 4 h resulted in the highest activities. Reversed-phase high-performance liquid chromatographic separation of the purified hydrolysate yielded three active fractions that were further identified by nano-liquid chromatography-tandem mass spectrometry. The bioactivities of over 230 identified peptide sequences were estimated after simulated gastrointestinal digestion, and those peptides with the highest chance of exerting antioxidant activity were selected to be further synthesised and tested. In this sense, the synthesised dipeptides CF and GY showed the highest antioxidant capacity. CF presented IC50 values of 69.63 and 145.41 µmol L-1 in 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays, respectively. In contrast, GY IC50 values were 15.27 and 10.06 µmol L-1 in ABTS and ORAC assays, respectively. Significant differences (P < 0.05) were registered between peptides in the same antioxidant assays. CONCLUSION: Overall, the findings emphasised the favourable impact of enzymatic hydrolysis with the obtaining of antioxidant peptides from poultry by-products that could be evaluated as a safe and economical source to retard oxidation in food systems. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Vacuum impregnation as a sustainable technology to obtain iron-fortified broad bean (Vicia faba) flours.

Iron-fortified broad bean flours were obtained by vacuum impregnation during soaking. The impact of vacuum impregnation and iron fortification on the hydration kinetics of broad beans, as well as the processing (soaking, autoclaving, and dehulling) on the iron-absorption inhibitors (phytic acid and tannins), iron content, iron bioaccessibility, and physicochemical and techno-functional properties of flours was investigated. Results showed that the use of vacuum impregnation during soaking reduced the broad beans' soaking time by 77%, and using iron solution instead of water did not affect the hydration kinetics. After soaking, iron-fortified broad bean flours increased twice (without hull) or more (with hull) the iron and bioaccessible iron content regarding non-fortified flours. Cooking broad beans by autoclaving modified the tannin content, the iron content and its bioaccessible fraction, and the physicochemical and techno-functional properties of the flours. Autoclaving increased the water holding capacity and absorption rate, swelling capacity, bulk density, and particle size, while decreased the solubility index, whiteness index, emulsifying capacity, emulsion stability, and gelling capacity. Finally, dehulling did not practically affect the physicochemical and techno-functional properties of flours, but showed a decrease in iron content, although increased iron bioaccessibility was observed, occurred mainly due to the reduction in tannin concentrations. The results obtained in this study demonstrated that vacuum impregnation is a useful technology for obtaining iron-fortified broad bean flours with different physicochemical and techno-functional properties depending on the production process used.

Impact of thermal treatments and simulated gastrointestinal digestion on the α-amylase inhibitory activity of different legumes.

Legumes are excellent sources of proteins that can be hydrolysed to generate antidiabetic peptides, which inhibit carbohydrate digestive enzymes. The degree of protein hydrolysis depends on the thermal treatment applied and how it impacts protein denaturation and thus accessibility to enzymes. In this study, α-amylase inhibitory activities of cooked (conventional, pressure, and microwave cooking) and digested (simulated gastrointestinal digestion, GID) green pea, chickpea, and navy beans were evaluated, together with the impact of thermal treatments on peptide profiles after GID. All peptides extracts inhibited α-amylase after cooking and GID, and the peptide fraction <3 kDa was responsible for main activity. In green peas and navy beans, microwave cooking showed the highest impact whereas none thermal treatment highlighted in chickpeas. The peptidomics analysis of the fractions <3 kDa identified a total of 205 peptides, 43 of which were found to be potentially bioactive according to in silico analysis. Also quantitative results evidenced differences in the peptide profile between the type of legume and thermal treatment.

Bioactive and Sensory Di- and Tripeptides Generated during Dry-Curing of Pork Meat.

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.

Identification of Bioactive Peptides from Nannochloropsis oculata Using a Combination of Enzymatic Treatment, in Silico Analysis and Chemical Synthesis.

In vitro ACE-1 inhibitory peptides were characterised previously from a number of microalgal species including Spirulina platensis (peptide IAPG), Chlorella vulgaris (peptides FDL, AFL, VVPPA), Isochrysis galbana (peptide YMGLDLK), Chlorella sorokiniana (peptides IW and LW) and indeed Nannochloropsis oculata (peptides GMNNLTP and LEQ). The isolation of protein from Nannochloropsis oculata using a combination of ammonium salt precipitation and xylanase treatment of resulting biomass combined with molecular weight cut off filtration to produce a permeate and characterisation of bioactive peptides is described. The Angiotensin-1-converting enzyme (ACE-1) IC50 value for the generated permeate fraction was 370 µg/mL. Ninety-five peptide sequences within the permeate fraction were determined using mass spectrometry and eight peptides were selected for chemical synthesis based on in silico analysis. Synthesized peptides were novel based on a search of the literature and relevant databases. In silico, simulated gastrointestinal digestion identified further peptides with bioactivities including ACE-1 inhibitory peptides and peptides with antithrombotic and calcium/calmodulin-dependent kinase II (CAMKII) inhibition. This work highlights the potential of Nannochloropsis oculata biomass as both a protein and bioactive peptide resource, which could be harnessed for use in the development of functional foods and feeds.

Impact of oxidation on the cardioprotective properties of the bioactive dipeptide AW in dry-cured ham.

Unbalanced oxidative reactions occurred during the dry-curing period of ham can trigger unpleasant taste. Additionally, salt might mediate in these reactions that cause the oxidation of some of the generated peptides acting as a pro-oxidant. The influence of the processing and oxidation on the release of peptides and bioactivity have been dimly investigated. In this study, the dipeptide AW, and its oxidized form AWox were quantitated in dry-cured ham. AW concentration reached 4.70 mg/g of dry-cured ham at 24 months of traditional dry-curing. The intact and the oxidized peptide forms accumulated to 5.12 and 6.80 µg/g dry-cured ham in 12-months low-salted hams, respectively, while they were undetectable in 12 months-traditionally elaborated hams. Moreover, oxidation affected the antioxidant properties depending on the in vitro assay and reduced the AW potential as antihypertensive. This study reports the potential role of the dry-cured ham-derived peptide AW on cardiovascular health and the relevance of post-oxidation on its bioactivity.

Special Issue: Food Bioactive 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 [...].

DPP-IV Inhibitory Peptides GPF, IGL, and GGGW Obtained from Chicken Blood Hydrolysates.

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.

In vitro and in silico analysis of potential antioxidant peptides obtained from chicken hydrolysate produced using Alcalase.

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.

Peptidomics as a useful tool in the follow-up of food bioactive peptides.

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.

Identification and Quantitation of Bioactive and Taste-Related Dipeptides in Low-Salt Dry-Cured Ham.

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.