Development of Fortified Breads Enriched with Plant-Based Bioactive Peptides Derived from the Chia (Salvia hispanica L.) Expeller.

By-products from the industrialization of oilseeds, particularly chia, can be sustainably used for the development of new functional products. In this work, wheat breads supplemented with up to 10 mg of chia expeller hydrolysate/g of flour were prepared, obtaining fortified breads with acceptability for consumption, according to a preliminary consumer research study based on an affective test employing a five-point hedonic scale of global acceptance. In this context, protein hydrolysates of the chia expeller were produced using Alcalase, reaching a degree of hydrolysis of 54.3 ± 1.6% with an antioxidant activity of 55.8 ± 0.4% after 6 h incubation at 25 °C in the presence of the enzyme. These peptides showed appropriate techno-functional properties and chemical compositions suitable for the further development of bakery products. Taken together, our approach and the development of a fortified bread with plant-based bioactive peptides provide a novel and eco-friendly alternative for the recovery of nutrients from agro-industrial waste. More importantly, these enriched breads could exert beneficial effects on human health by exploiting the antioxidant properties of functional peptides derived from the chia expeller.

A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules.

Chia expeller: A promising source of antioxidant, antihypertensive and antithrombotic peptides produced by enzymatic hydrolysis with Alcalase and Flavourzyme.

Chia expeller is a promising source of bioactive compounds suitable for the development of nutraceutical ingredients due to its functional, biological, and nutritional properties. In this work, chia expeller was hydrolysed with Alcalase-Flavourzyme sequential system and compared to the individual enzymes. A higher degree of hydrolysis (57.63 ± 6.08%) was obtained after 90 min-Alcalase and 90 min-Flavourzyme (H-A90-F90), with the development of low molecular weight peptides as observed by SDS-PAGE. H-A90-F90 exhibited antiradical activity with ABTS (TE = 4.87 ± 0.13 mmol L-1 mg-1), DPPH (TE = 1.55 ± 0.02 mmol L-1 mg-1), antihypertensive activity (45% ACE-I inhibition), and antithrombotic activity against both intrinsic and extrinsic coagulation pathways. These results represent the first report of antithrombotic peptides from Salvia hispanica, highlighting the relevant use of chia seed by-products to obtain potentially antioxidant, antihypertensive, and anticoagulant peptides by enzymatic hydrolysis with Alcalase and Flavourzyme, enhancing this agro-industrial by-product.

Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

Purification and Identification of Novel Antioxidant Peptides Isolated from Geoffroea decorticans Seeds with Anticoagulant Activity.

Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory concentration (IC50) of 35.5 ± 0.3 µg/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of ~82 µg/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid sequence of GdAPs revealed their unique structural features, exhibiting homology with various proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability of GdAPs for pharmaceutical purposes.

Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties.

Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules.

Purification and characterization of a novel trypsin inhibitor from Solanum tuberosum subsp. andigenum var. overa: Study of the expression levels and preliminary evaluation of its antimicrobial activity.

Protease inhibitors (PIs) have been traditionally recognized by their potential biomedical application in events with exacerbation of endogenous proteases activity. Plant PIs have gained interest as naturally occurring molecules, which usually show lower environmental impact residual toxicity than synthetic compounds. In this work, we isolated, cloned, expressed and purified a novel trypsin inhibitor from S. tuberosum subsp. andigenum var. overa, named oPTI. A significant over-expression of the oPTI coding gene after 48 h exposure of methyl jasmonate compared to the gene of reference. This inhibitor showed a molecular mass of 12 kDa and a Ki of 7.3 × 10-7 M. Finally, we evaluated the antimicrobial activity of oPTI against different pathogenic microorganisms. The oPTI demonstrated inhibitory effect on the growth of Acinetobacter baumannii S-1, Acinetobacter baumannii R, Acinetobacter calcoaceticus R, Acinetobacter calcoaceticus S, Bacillus stearothermophilus, Escherichia coli, Pseudomonas aeruginosa, Salmonella braenderup, Salmonella enteritidis, Salmonella typhimurium and Yersinia enterocolitica strains. This study represents the first report for the antimicrobial activity of a plant PI over a wide range of microorganisms. Our studies reinforce the importance of natural PIs as promising molecules for their potential application in the biomedical field and/or in the food industry as natural food preservatives.

Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review.

Protease inhibitors (PIs) are regulatory proteins found in numerous animal tissues and fluids, plants, and microorganisms that reduce and inhibit the exacerbated and uncontrolled activity of the target proteases. Specific PIs are also effective tools for inactivating proteases involved in human diseases like arthritis, pancreatitis, hepatitis, cancer, AIDS, thrombosis, emphysema, hypertension, and muscular dystrophy among others. Plant PIs-small peptides with a high content of cystine residues in disulfide bridges-possess a remarkable resistance to heat treatment and a high stability against shifts in pH, denaturing agents, ionic strength, and proteolysis. In recent years, novel biologic activities have been reported for plant PIs, including antimicrobial, anticoagulant, antioxidant action plus inhibition of tumor-cell growth; thus pointing to possible applications in medicine, agriculture, and biotechnology. In this review, we provide a comparative overview of plant-PIs classifying them in four groups according of their thermal and pH stability (high stability and hyperstable -to temperature and to pHs-, respectively), then emphasizing the relevance of the physicochemical characteristics of these proteins for potential biotechnological and industrial applications. Finally, we analyze the biologic activities of the stable protease inhibitors previously characterized that are the most relevant to potential applications in biomedicine, the food industry, and agriculture.

GdTI, the first thermostable trypsin inhibitor from Geoffroea decorticans seeds. A novel natural drug with potential application in biomedicine.

A novel thermostable trypsin inhibitor was obtained from Geoffroea decorticans seeds. G. decorticans trypsin inhibitor (GdTI) is a protein with molecular mass of 6743.7 Da, with a potent inhibitory activity (Ki of 2.1 nM) even at high temperatures and extreme pHs (100% after 5 h at 100 °C and 80% after 60 min at pH 2-12) constituting one of the most powerful serine protease inhibitors isolated from a plant source. GdTI displays anticoagulant activity against both extrinsic and intrinsic coagulation pathways, representing the first report of a plant serine protease inhibitor with anticoagulant activity against the extrinsic pathway. Finally, GdTI showed inhibitory activity against α-glucosidase (IC50 of 0.18 µM) evidencing the hypoglycemic effect of this inhibitor. Our results evidence the discovery of a natural molecule with unique features: i) GdTI is one of the most potent serine protease inhibitors founded to date, ii) with the most powerful thermostability reported in literature, iii) with anticoagulant effect against both coagulation pathways and hypoglycemic activity. This report suggest that GdTI could be exploited as a natural and hyperstable antidiabetic drug, in behalf of its antithrombotic and hypoglycemic activities, encouraging future studies with high impact on biomedical research and potential pharmaceutical applications.

Adding value to the chia (Salvia hispanica L.) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain.

Chia expeller is a by-product of the extrusion process of chia seeds generated during oil production. Typically, this material is non-utilized or used for non-valuable applications. In the present work, the chia expeller was hydrolysed with Papain and the antioxidant properties of the resultant peptides were evaluated. Papain treatment of the chia seed expeller demonstrated an enrichment of low molecular weight peptides (molecular weight <15 kDa) as determined by SDS-PAGE and MALDI-TOF/MS analyses. Such peptides showed a potent radical scavenging effect in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals in comparison with those non-hydrolysed samples. Taken together our results demonstrate the generation of functional peptides from the chia expeller by enzymatic hydrolysis with Papain. This value-added hydrolysate can be potentially included as a supplement in functional food and nutraceutical products.

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

Biochemical characterization of the YBPCI miniprotein, the first carboxypeptidase inhibitor isolated from Yellow Bell Pepper (Capsicum annuum L). A novel contribution to the knowledge of miniproteins stability.

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 µg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance.

Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.