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.

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.

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.

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.

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 [...].

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.

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 Approaches to Generating and Identifying Angiotensin-Converting Enzyme I Inhibitory Peptides from Green Macroalga Ulva lactuca.

A protein extract was generated from the macroalga Ulva lactuca, which was subsequently hydrolysed using the food-grade enzyme papain and angiotensin-converting Enzyme I and renin inhibitory peptides identified using a combination of enrichment strategies employing molecular weight cutoff filtration and mass spectrometry analysis. The generated hydrolysates with the most promising in vitro activity were further purified using preparative RP-HPLC and characterised. The 1 kDa hydrolysate (1 kDa-UFH), purified and collected by preparative RP-HPLC at minutes 41‒44 (Fr41‒44), displayed statistically higher ACE-I inhibitory activities ranging from 96.91% to 98.06%. A total of 48 novel peptides were identified from these four fractions by LC-MS/MS. A simulated gastrointestinal digestion of the identified peptide sequences was carried out using in silico enzyme cleavage simulation tools, resulting in 86 peptide sequences that were further assessed for their potential activity, toxicity and allergenicity using multiple predictive approaches. All the peptides obtained in this study were predicted to be non-toxic. However, 28 out of the 86 novel peptides released after the in silico gastrointestinal digestion were identified as potential allergens. The potential allergenicity of these peptides should be further explored to comply with the current labelling regulations in formulated food products containing U. lactuca protein hydrolysates.

Assessment of Cholesterol, Glycemia Control and Short- and Long-Term Antihypertensive Effects of Smooth Hound Viscera Peptides in High-Salt and Fructose Diet-Fed Wistar Rats.

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.

Perspectives in the Use of Peptidomics in Ham.

The use of proteomics in food science has permitted both a better characterization of the food products and the control of their final quality despite the fact that food systems are under continuous changes during processing and consumption. Ham is a very popular meat product frequently consumed in Mediterranean countries after a dry-curing processing that results in the generation of large amounts of peptides that contribute to its texture, flavor, and final quality. Proteolysis is the main biochemical mechanism occurring throughout the dry-cured ham processing and a better knowledge of this phenomenon is essential to give an extra value to the product and produce regular batches. In this regard, peptidomics has become an important tool for the characterization of dry-cured hams in order to identify and quantify bioactive peptides and potential biomarkers, study their stability under processing conditions or gastrointestinal digestion, and search for post-translational modifications that could affect the quality of dry-cured hams. This manuscript is reviewing from the first uses of comparative proteomics in dry-cured ham approaches to the most modern peptidomic studies and their contribution to the advance in meat science.

Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations.

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

Antilisterial peptides from Spanish dry-cured hams: Purification and identification.

The typical Spanish dry-cured ham has a particular sensory quality that makes it a distinctive food, highly appreciated for consumers worldwide. Its particular physicochemical properties, such as high salt content and reduced water activity contribute to their shelf-stability. However, post-processing actions carried out for the commercialization of these products such as slicing may increase the risk of development of pathogenic microorganisms as Listeria monocytogenes. During ripening, muscle proteins are hydrolyzed by muscle peptidases releasing peptides and free amino acids. Some of these peptides have been described to exert biological activities such as antioxidant and ACE-inhibition. In this study, a peptidomic strategy using mass spectrometry techniques has been used to identify and sequence those naturally generated peptides showing antilisterial activity. One hundred and five peptides have been identified in active fractions and some synthesized and their MIC calculated. Ten peptides were able to inhibit the growth of L. monocytogenes, being the pentapeptide RHGYM the most effective showing a MIC value of 6.25 mM. This study proves for the first time the potential antimicrobial action against L. monocytogenes of certain naturally generated peptides obtained from Spanish dry-cured ham.

Gastrointestinal Endogenous Protein-Derived Bioactive Peptides: An in Vitro Study of Their Gut Modulatory Potential.

A recently proposed paradigm suggests that, like their dietary counterparts, digestion of gastrointestinal endogenous proteins (GEP) may also produce bioactive peptides. With an aim to test this hypothesis, in vitro digests of four GEP namely; trypsin (TRYP), lysozyme (LYS), mucin (MUC), serum albumin (SA) and a dietary protein chicken albumin (CA) were screened for their angiotensin-I converting (ACE-I), renin, platelet-activating factor-acetylhydrolase (PAF-AH) and dipeptidyl peptidase-IV inhibitory (DPP-IV) and antioxidant potential following simulated in vitro gastrointestinal digestion. Further, the resultant small intestinal digests were enriched to obtain peptides between 3-10 kDa in size. All in vitro digests of the four GEP were found to inhibit ACE-I compared to the positive control captopril when assayed at a concentration of 1 mg/mL, while the LYS < 3-kDa permeate fraction inhibited renin by 40% (±1.79%). The LYS < 10-kDa fraction inhibited PAF-AH by 39% (±4.34%), and the SA < 3-kDa fraction inhibited DPP-IV by 45% (±1.24%). The MUC < 3-kDa fraction had an ABTS-inhibition antioxidant activity of 150 (±24.79) µM trolox equivalent and the LYS < 10-kDa fraction inhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH) by 54% (±1.62%). Moreover, over 190 peptide-sequences were identified from the bioactive GEP fractions. The findings of the present study indicate that GEP are a significant source of bioactive peptides which may influence gut function.

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.

Development and optimisation of a label-free quantitative proteomic procedure and its application in the assessment of genetically modified tomato fruit.

A key global challenge for plant biotechnology is addressing food security, whereby provision must be made to feed 9 billion people with nutritional feedstuffs by 2050. To achieve this step change in agricultural production new crop varieties are required that are tolerant to environmental stresses imposed by climate change, have better yields, are more nutritious and require less resource input. Genetic modification (GM) and marker-assisted screening will need to be fully utilised to deliver these new crop varieties. To evaluate these varieties both in terms of environmental and food safety and the rational design of traits a systems level characterisation is necessary. To link the transcriptome to the metabolome, quantitative proteomics is required. Routine quantitative proteomics is an important challenge. Gel-based densitometry and MS analysis after stable isotope labeling have been employed. In the present article, we describe the application of a label-free approach that can be used in combination with SDS-PAGE and reverse-phase chromatography to evaluate the changes in the proteome of new crop varieties. The workflow has been optimised for protein coverage, accuracy and robustness, then its application demonstrated using a GM tomato variety engineered to deliver nutrient dense fruit.

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.

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.

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.

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.