Peptide Hydrolysis Research Articles

Toxicological evaluation of peptide hydrolysates from bovine blood meal with antioxidant and antifungal activities

This study addresses the underutilized potential of bovine blood-derived bioactive peptides. With approximately 22 million metric tons of bovine blood waste generated annually, sustainable utilization is essential. Our research focuses on exploring antifungal and antioxidant properties and includes proximate analysis of blood meal, enzymatic activity determination, and optimization of bovine blood protein hydrolysis. Optimal conditions were identified at 70 °C and pH 9, achieving a remarkable degree of hydrolysis (25.75 %). Key results demonstrate that lower molecular weight peptides exhibit enhanced antioxidant capacity, making them suitable for functional foods and nutraceuticals. While antimicrobial activity against specific strains had limitations, significant antifungal efficacy against Fusarium keratoplasticum ATCC 36031 was observed. Electrophoresis analysis confirmed the presence of peptides with a molecular weight below 14 kDa in optimally hydrolyzed samples, aligning with their bioactivity. A maximum tolerated concentration of 100 µg/mL was established through toxicity assessment using a zebrafish model, ensuring safety. This research reorganizes the abstract to address underutilized bovine blood, explores bioactive potential, and presents significant results concerning antioxidant and antifungal activity. Significantly, this study has implications for food safety, environmental sustainability, and health-related product development. The enzyme used was obtained from fermenting soybean cake with B. subtilis, and treatment selection was based on optimization studies. This research offers insights into industrial applications, environmental impact reduction, and health-related advancements.

Optimization of Hydrolysis and Identification of Bioactive Peptides in Brewery Yeast Residuals

Industrial waste products are of biotechnological interest due to their abundance and can be utilized as protein sources for the production of bioactive peptides through hydrolysis. The utilization of yeast residues from the brewing industry has been demonstrated to be a viable method for obtaining protein hydrolysates. The degree of hydrolysis and soluble total protein of the hydrolysates were evaluated for optimization purposes to achieve a product with a high proportion of soluble organic nitrogen, increased levels of bioactive peptides and amino acids. Both alkaline and enzymatic hydrolysis processes were tested. The hydrolyses were carried out with the aim of large-scale implementation. The treatments were effective in liberating intracellular content and producing biologically functional hydrolysates, with degree of hydrolysis values ranging from 28 to 63% and total protein from 2.95 to 8.28% at the optimum points confirmed by statistical analysis (analysis of variance (ANOVA)). The hydrolysate produced with Alcalase® exhibited the highest peptide intensity, while the hydrolysate produced with Flavourzyme® showed the highest concentration of free amino acids. The peptides were identified by mass spectrometry and were found to have biological activities according to the Biopep database. This study presents a quick and economically feasible approach for the implementation of a pilot project for the reuse of this waste.

Investigation of umami peptides and taste mechanisms in Agrocybe aegerita: based on sensory evaluation and molecular docking techniques.

In the present study, sensory orientation and instrumental analysis were employed to separate, purify, and identify umami peptides in Agrocybe aegerita hydrolysate. Using UPLC-ESI-Q-TOF MS, 11 potential umami peptides (EY, EG, EV, ENG, PEG, DEL, ECG, DDL, PEEL, EDCS and DGPL) were identified from the screened fractions. Moreover, sensory evaluation and E-tongue results showed that the identified umami peptides had umami attributes, within an umami threshold range of 0.0625-0.25 mg mL-1. In addition, DDL and DEL exhibited the highest umami flavor intensity. Molecular docking analysis further showed that 4 umami peptides (namely, EY, EG, ECG, and DGPL) entered the T1R1 cavity of the umami receptor. Additionally, 4 umami peptides (namely, EV, ENG, DEL, and EDCS) could be embedded in the binding pocket of the T1R3 cavity. Furthermore, 3 umami peptides (PEG, DDL, and PEEL) strongly interacted with T1R1/T1R3. Thus, the findings collectively indicated that the predominant interacting forces between umami peptide and umami receptor are hydrogen bonding and hydrophobic interactions. Finally, it was shown that the primary binding sites of T1R1 were residues Ser109, Gln52 and Ser148, while the primary binding sites of T1R3 were residues Ser172, Arg277 and Ala170. The study identified the umami peptides in A. aegerita for the first time, which provided more information for the umami research of A. aegerita and provided the theoretical basis for the further development and utilization of A. aegerita.

Molecular mechanisms of the effect of standardized placental hydrolysate peptides on mitochondria functioning

Human placenta hydrolysates (HPH), the study of which was initiated by the scientific school of Vladimir P. Filatov, are currently being investigated using modern proteomic technologies. HPH is a promising tool for maintaining the function of mitochondria and regenerating tissues and organs with a high content of mitochondria (liver, heart muscle, skeletal muscles, etc.). The molecular mechanisms of action of HPH are practically not studied. Identification of mitochondrial support mitochondrial function-supporting peptides in HPH (Laennec, produced by Japan Bioproducts). Data on the chemical structure of the peptides were collected through a mass spectrometric experiment. Then, to establish the amino acid sequences of the peptides, de novo peptide sequencing algorithms based on the mathematical theory of topological and metric analysis of chemographs were applied. Bioinformatic analysis of the peptide composition of HPH was carried out using the integral protein annotation method. The biological functions of 41 peptides in the composition of HPH have been identified and described. Among the target proteins, the activity of which is regulated by the identified peptides and significantly affects the function of mitochondria, are caspases (CASP1, CASP3, CASP4) and other proteins regulating apoptosis (BCL2, CANPL1, PPARA), MAP kinases (MAPK1, MAPK3, MAPK4, MAPK8, MAPK9 , MAPK10, MAPK14), AKT1/GSK3B/MTOR cascade kinases, and a number of other target proteins (ADGRG6 receptor, inhibitor of NF-êB kinase IKKE, pyruvate dehydrogenase 2/3/4, SIRT1 sirtuin deacetylase, ULK1 kinase). HPH peptides have been identified that promote inhibition of mitochondrial pore formation, apoptosis, and excessive mitochondrial autophagy under conditions of oxidative/toxic stress, chronic inflammation, and/or hyperinsulinemia.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.

Hydrolysis of Antimicrobial Peptides by Extracellular Peptidases in Wastewater.

Several antimicrobial peptides (AMPs) are emerging as promising novel antibiotics. When released into wastewater streams after use, AMPs might be hydrolyzed and inactivated by wastewater peptidases─resulting in a reduced release of active antimicrobials into wastewater-receiving environments. A key step towards a better understanding of the fate of AMPs in wastewater systems is to investigate the activity and specificity of wastewater peptidases. Here, we quantified peptidase activity in extracellular extracts from different stages throughout the wastewater treatment process. For all four tested municipal wastewater treatment plants, we detected highest activity in raw wastewater. Complementarily, we assessed the potential of enzymes in raw wastewater extracts to biotransform 10 selected AMPs. We found large variations in the susceptibility of AMPs to enzymatic transformation, indicating substantial substrate specificity of extracted enzymes. To obtain insights into peptidase specificities, we searched for hydrolysis products of rapidly biotransformed AMPs and quantified selected products using synthetic standards. We found that hydrolysis occurred at specific sites and that these sites were remarkably conserved across the four tested wastewaters. Together, these findings provide insights into the fate of AMPs in wastewater systems and can inform the selection and design of peptide-based antibiotics that are hydrolyzable by wastewater peptidases.

Comparison of lignocellulosic enzymes and CAZymes between ascomycetes (Trichoderma) and basidiomycetes (Ganoderma) species: a proteomic approach

Abstract Wood decomposing ascomycetes and basidiomycetes group of fungi are the most valuable microbes on the earth’s ecosystem that recycles the source of carbon; therefore, they are essential for the biorefinery industries. To understand the robustness of the enzymes and their metabolic pathways in the fungal system, label-free quantification of the total proteins was performed. The fungi showed a comparable quantity of protein abundance [Trichoderma citrinoviride (285), Thermoascus aurantiacus (206), Ganoderma lucidum MDU-7 (102), G. lucidum (242)]. Differentially regulated proteins of ascomycetes and basidiomycetes were analyzed, and their heatmap shows upregulated and downregulated proteins [25 differentially expressed proteins in T. citrinoviride (8.62 % up-regulated and 91.37 % down-regulated) and G. lucidum (5.74 % up-regulated and 94.25 % down-regulated)] by using the normalized peptide-spectrum match (PSMs) and log2fold change. These proteins were similarly matched to the carbohydrate active enzymes family (CAZymes) like glycoside hydrolase (GH family), carbohydrate-binding module (CBM family) with auxiliary activities, and also involved in the hydrolysis of carbohydrate, lignin, xylan, polysaccharides, peptides, and oxido-reductase activity that helps in antioxidant defense mechanism. The lignocellulolytic enzymes from two different divisions of fungi and proteomics studies gave a better understanding of carbon recycling and multi-product lignocellulosic biorefinery processes.

Anti-diabetic mechanism and potential bioactive peptides of casein hydrolysates in STZ/HFD-induced diabetic rats.

Casein hydrolysates have attracted much interest as anti-diabetic food, but their hypoglycemic mechanism and biopeptides are not well understood. This study aimed to explore the anti-diabetic mechanism and potential biopeptides of casein hydrolysates in streptozotocin/high-fat-diet-induced diabetic rats and HepG2 cells. Oral administration of casein hydrolysate prepared with papain-Flavourzyme combination (P-FCH) decreased fasting blood glucose, improved oral glucose tolerance, and reduced HbA1c values in diabetic rats. P-FCH was ineffective in alleviating insulin resistance (homeostasis model assessment and insulin sensitivity index) and enhancing hepatic insulin signaling transduction (phosphorylated Akt, hexokinase activity, and pyruvate kinase activity) in diabetic rats. However, P-FCH significantly upregulated adenosine monophosphate-activated protein kinase phosphorylation and glucose transporter-2 expression, inhibited phosphoenolpyruvate carboxylase kinase activity, and elevated glycogen content in liver tissue of diabetic rats. Furthermore, P-FCH increased glucose consumption independently in normal and insulin-resistant HepG2 cells without the presence of insulin. The peptide composition of P-FCH was characterized. The potential biopeptides in P-FCH showed the sequence characteristic of a Val at the N-terminal or a Pro at the P2 position, and the hypoglycemic activity of Val-Pro-Leu-Gly (the most potential biopeptide in P-FCH) was verified by oral glucose tolerance test in mice. These results suggested that activation of the non-insulin-mediated AMPK pathway might be the determinant mechanism of P-FCH on the hypoglycemic effect. The novel peptide Val-Pro-Leu-Gly in P-FCH was effective in reducing blood glucose levels when orally administered to mice. © 2023 Society of Chemical Industry.

Xanthan gum and pectin as beverage stabilizers reduce the digestive enzyme hydrolysis of antioxidant and antihypertensive peptides obtained from a brewery byproduct

An acidic beverage was formulated with xanthan gum (XG), pectin (P) and brewer spent grain (BSG) peptides with antioxidant and antihypertensive properties. The impact of hydrocolloids levels on peptide bioaccessibility was studied. Peptides were obtained from BSG using Purazyme and Flavourzyme enzymes. BSG peptides were fractionated by ultrafiltration (UF) and four fractions were obtained: F1 (>10 kDa), F2 (10–5 kDa), F3 (1–5 kDa), and F4 (<1 kDa). F3 showed the highest protein purity, ferulic acid content, proportion of amphipathic peptides, and bioactive properties (ABTS+ radical scavenging and ACE-I inhibitory activity). The identified peptides from F3 by tandem mass spectrometry were 138. In silico analysis showed that 26 identified peptides had ABTS+ inhibitory activity, while 59 ones presented good antihypertensive properties. The effect of XG and P levels on bioaccessibility of F3 peptides in the formulated beverages was studied by a central composite experimental design. It was observed that F3 peptides interacted with hydrocolloids by electrostatic forces at pH of formulated beverages. The addition of hydrocolloids to formulation modulated the release of the antioxidant peptides and protected the degradation of ACE-I inhibitory peptides from F3 during simulated gastrointestinal digestion. Finally, the level of hydrocolloids that produced intermediate viscosities in the formulated beverages improved the bioaccessibility of the F3 peptides.

Identification, characterization and hypolipidemic effect of novel peptides in protein hydrolysate from Protaetia brevitarsis larvae

The proteins were mainly derived from Protaetia brevitarsis larval extracts obtained using two empty intestine methods (traditional static method: TSM or salt immersion stress method: SISM) and extraction solvents (water: W or 50 % water–ethanol: W:E), and the proteins were used as objects to investigate the effect of emptying intestine methods on hypolipidemic peptides. The results revealed that the F-2 fractions of protein hydrolysate had stronger in vitro hypolipidemic activity, with the peptides obtained by SISM possessing a stronger cholesterol micelle solubility inhibition rate, especially in SISM-W:E-P. Moreover, a total of 106 peptides were tentatively identified, among which SISM identified more peptides with an amino acid number < 8. Meanwhile, five novel peptides (YPPFH, YPGFGK, KYPF, SPLPGPR and VPPP) exhibited good hypolipidemic activity in vitro and in vivo, among which YPPFH, VPPP and KYPF had strong inhibitory activities on pancreatic lipase (PL) and cholesteryl esterase (CE), and KYPF, SPLPGPR and VPPP could significantly reduce the TG content in Caenorhabditis elegans. Thus, P. brevitarsis can be developed as a naturally derived hypolipidemic component for the development and application in functional foods.

Oral administration of tilapia hydrolysate peptides ameliorates the immune-related side effects of cyclophosphamide in BALB/c mice

Bioactive peptide derived from fish is a novel and promising immunomodulating agent. However, the use of tilapia peptides as adjuvant therapy to ameliorate chemotherapy-induced immunosuppression in vivo has not been previously studied. In this study, tilapia hydrolysate peptides (THP) with a low molecular weight distribution (<3 kDa) were obtained from tilapia processing byproducts using Flavourzyme hydrolysis. These peptides showed no cytotoxicity to RAW264.7 macrophage cells and splenocytes. THP promoted the proliferation of RAW264.7 macrophage cells under basal conditions and enhanced mitogen-induced splenocyte proliferation. When treated with LPS-induced RAW264.7 macrophage cells, THP exhibited anti-inflammatory properties by inhibiting iNOS expression and NO production. To evaluate the in vivo immunomodulatory activity of THP, BALB/c mice were assigned to four groups (n = 7) and received either water (Control and CY group) or THP at doses of 400 mg/kg (Low-THP) and 800 mg/kg (High-THP) body weight through oral gavage for total 12 days. All groups, except the control, were injected with 100 mg/kg BW of cyclophosphamide (CY) on day 7. The high dose of THP positively influenced the recovery of peripheral white blood cell counts. Both doses of THP improved the proliferation ability of splenic T cells and immune organ indices. Additionally, the results of pro-inflammatory gene expression and histological examination suggested that both doses of THP alleviated CY-induced spleen damage. Consequently, oral administration of THP effectively ameliorated the immune-related side effects of CY, including reductions of peripheral white blood cell count, inhibition of splenic lymphocytes proliferation, atrophy of the spleen and thymus, and spleen damage.

Safety evaluation, hydrolysis activity of Kluyveromyces marxianus JY-1 and its application to obtain immunomodulatory peptide from bovine casein

Except in vitro enzymolysis, there are few studies on the production of casein-derived immunomodulatory peptides by microbial fermentation technology. In this study, the ability of a strain of Kluyveromyces marxianus JY-1 isolated from traditional fermented milk in Xinjiang District (China) to release casein-derived immunomodulatory peptides was evaluated. Results showed that this strain was safe and with high protease activity, which could highly hydrolyze casein. The hydrolysate of casein hydrolyzed by Kluyveromyces marxianus JY-1 could promote the proliferation of RAW264.7 macrophages and the production of cytokines (IL-6, IL-1β, and TNF-α) compared with the control group, showing obvious immunomodulatory activity. A nanoscale liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (Nano LC-Q-TOF-MS/MS) was used to determine the amino acid sequences of bioactive peptides in casein hydrolysate of Kluyveromyces marxianus. According to the structural characteristics of immunomodulatory peptides, 3 peptides (HPHPHLSF, PFPEVFG, and SPAQILQW) with immunomodulatory activity were selected from 55 identified peptides by using in silico methods. This study demonstrates that Kluyveromyces marxianus JY-1 has a wide range of potential applications in the production of immunomodulatory peptides from casein and other food proteins.

Insects as Valuable Sources of Protein and Peptides: Production, Functional Properties, and Challenges.

As the global population approaches 10 billion by 2050, the critical need to ensure food security becomes increasingly pronounced. In response to the urgent problems posed by global population growth, our study adds to the growing body of knowledge in the field of alternative proteins, entomophagy, insect-based bioactive proteolysates, and peptides. It also provides novel insights with essential outcomes for guaranteeing a safe and sustainable food supply in the face of rising global population demands. These results offer insightful information to researchers and policymakers tackling the intricate relationship between population expansion and food supplies. Unfortunately, conventional agricultural practices are proving insufficient in meeting these demands. Pursuing alternative proteins and eco-friendly food production methods has gained urgency, embracing plant-based proteins, cultivated meat, fermentation, and precision agriculture. In this context, insect farming emerges as a promising strategy to upcycle agri-food waste into nutritious protein and fat, meeting diverse nutritional needs sustainably. A thorough analysis was conducted to evaluate the viability of insect farming, investigate insect nutrition, and review the techniques and functional properties of protein isolation. A review of peptide generation from insects was conducted, covering issues related to hydrolysate production, protein extraction, and peptide identification. The study addresses the nutritional value and global entomophagy habits to elucidate the potential of insects as sources of peptides and protein. This inquiry covers protein and hydrolysate production, highlighting techniques and bioactive peptides. Functional properties of insect proteins' solubility, emulsification, foaming, gelation, water-holding, and oil absorption are investigated. Furthermore, sensory aspects of insect-fortified foods as well as challenges, including Halal and Kosher considerations, are explored across applications. Our review underscores insects' promise as sustainable protein and peptide contributors, offering recommendations for further research to unlock their full potential.

Non-avian dinosaur eggshell calcite can contain ancient, endogenous amino acids

Proteins are the most stable of the macromolecules that carry genetic information over long periods of time. Closed systems are more likely to retain endogenous proteins or their degradation products. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, Late Cretaceous (Campanian–Maastrichtian) Argentine titanosaurian sauropod eggshells show dark, organic stains under light microscopy/photography and fluorescence imaging. Raman spectroscopy can yield bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, consistent with kerogen formation deriving from eggshell lipids. High-performance liquid chromatography reveals that their intra-crystalline fraction can be enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and are fully racemic (despite being some of the slowest racemising amino acids), indicating ancient origin. This preservation varies across localities, but similar ancient amino acid profiles were also observed in Late Cretaceous Spanish titanosaurians from several localities and Chinese putative hadrosaurid eggshell. These amino acid results are consistent with previous studies on degradation trends deduced from modern, thermally matured, sub-fossil, and ∼3.8–6.5 Ma avian eggshell, as well as ∼30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, and the concentration of free amino acids suggests likely complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation, even if such reactions might be slowed in exceptional circumstances. Future work should survey fossil biocalcite to determine variability in amino acid preservation.

Soil aminopeptidase activities under 145-year crop rotation and fertility practices in the North Central US

Aminopeptidases catalyze the hydrolysis of peptides into constituent amino acids and are therefore a key step in nitrogen (N) depolymerization and downstream mineralization. Differences in carbon (C):N as well as sulfur (S) content of amino acids present the possibility of amino acid-specific aminopeptidase activities revealing distinct information on N and non-N processes in soils. However, chromogenic assays of soil aminopeptidases using para-nitroaniline (pNA) substrates are underutilized and have largely focused on just two aminopeptidases to-date. We evaluated activities of eight amino acid-specific aminopeptidases using pNA substrates for arginine (ARG), glycine (GLY), alanine (ALA), lysine (LYS), glutamic acid (GLU), methionine (MET), proline (PRO) and leucine (LEU) aminopeptidases in a high-resolution sampling (30 samples per 0.01 ha−1 treatment plot) of nine 145-year treatment plots resulting from a factorial combination of three crop rotations and three fertility treatments on a Argiudoll in North Central United States. Across treatments, we identified non-detectable activities of ARG and PRO, and detectable activities of GLY, ALA, LYS, GLU, MET, and LEU ranging 0.2 to 400 nmol pNA h−1 g−1. Except for LYS and GLU activities, aminopeptidase activities were homogeneously distributed within treatments (n=30 locations per plot), and exhibited a mean coefficient of variation (CV) of 9 to 30 % and spatial range of 4.1 to 9.0 m. Activities of ALA and MET had the lowest CV and greatest contribution in explaining overall variability of aminopeptidase activities and were more sensitive to treatments compared to GLY and LEU activity. Multivariate analysis revealed a positive correlation among aminopeptidase activities, with major variation explained by β-glucosidase activity followed by pH and total N (TN). Absolute aminopeptidase activities generally reflected soil organic C (SOC) and TN concentrations, with greater activities under diverse rotation with organic fertilization (manure) and lower activities under continuous maize without fertilization. However, aminopeptidase activities normalized to SOC concentration were greater in soils without fertilization relative to organic fertilization. This study provides benchmark values of soil aminopeptidase activities assayed by pNA substrates using the world’s second oldest continuous agricultural experiment, including six aminopeptidases that have not been previously assayed with these substrates. Given their low magnitude of variability, high spatial correlation, and sensitivity to management, assays of aminopeptidase activities – particularly MET and ALA activities – could be fruitful tools for better understanding soil N cycling. Future work should evaluate assay optimization and expand to additional amino acid-specific substrates using pNA-based substrates.

Investigation of active peptides from natural products that induce ER-stress-mediated apoptosis in cancer as potential therapeutics for kidney cancer, common in some genetic diseases: An in silico approach

Background: Endoplasmic reticulum (ER) stress initiates an unfolded protein response (UPR) to re-establish ER homeostasis as an adaptive pathway in cancer. However, persistent ER stress triggers the apoptotic pathway usually observed in kidney cancer (KC). The term KC actually refers to a number of distinct cancers. Kidney cancer is generally observed in a number of genetic diseases. Treatment options for KC have changed greatly over the years. The most commonly used tyrosine kinase inhibitors, TKI, show conflicting results regarding their beneficial effects on patients, as demonstrated in different KC patient cohort studies, indicating that the underlying molecular mechanisms involved in KC are more complex and likely need combined therapies able to modulate the activity of endoplasmic reticulum. Methods: The purpose of this research was to generate peptides from natural products in sillico that might be used as kidney cancer potential modulators. For this aim, several methods were used: Target prediction, protein hydrolysis, and protein-peptide molecular docking have all been used as techniques. Results: The network of critical KC genes is composed of C3AR1, CSNK2A2, ACE, DPP4, CAPN1, FPR2, HLA-A, and MMP2, together with predicted kinases such as RPS6KA5, MAPK14, CSNK2A1, PRKCD, CDK1, and HIPK2, in addition to transcription factors such as IRF8, TCF3, ERG, CREB1, EZH2, SPI1, IRF1, and SUZ12. The identified molecular targets of the isolated peptides are: HLA class I histocompatibility antigen A-3, lipoxin A4, dipeptidyl peptidase IV, angiotensinconverting enzyme, cyclooxygenase-2, C3a anaphylatoxin chemotactic receptor, melanocortin receptor 4, neurotensin receptor 1, mu opioid receptor, delta opioid receptor, and calpain 1. Conclusion: Overall, the results showed that GVSK, PGP, WQR, YGGF, and IF peptides are promising candidates for further study. Future work would be needed to test the therapeutic properties of these hydrolysate peptides using in vitro and in vivo approaches.

A novel alcalase-hydrolyzed soybean meal hydrolysates prepared using by-product material: Structure, function property, sensory property, and biological activity

This work investigated the effects of different hydrolysis times (0.5, 1 and 2 h) on the structure, interface and sensory properties, and biological activity of soybean meal peptides obtained by alcalase protease hydrolysis. After hydrolyzation, the molecular weight of soybean meal hydrolysates (SMHs) was below 10 kDa. An increase in the number of β-turns and a decrease in α-helices suggested the unfolding of soybean meal proteins. The ultraviolet absorption and fluorescence intensities of SMHs increased, suggesting an increase in the content of free amino acids, which likely contributed to the biological activity of SMHs. Additionally, after hydrolyzation for 1 h, the SMHs showed the highest antioxidant and ACE inhibitory activity. LC-MS/MS-based peptidomics analysis showed 101 differential peptides in hydrolysates among different hydrolysis times with reliable potential biological activities. However, the emulsification of the SMHs decreased due to the exposure of hydrophilic amino acids. All in all, hydrolysis time is a factor that influences the peptide sequence and properties, resulting in different antioxidant and ACE inhibitory activities.

Hydrolysis of Gluten-Derived Celiac Disease-Triggering Peptides across a Broad pH Range by RmuAP1: A Novel Aspartic Peptidase Isolated from Rhodotorula mucilaginosa.

An aspartate peptidase with proteolytic activity toward gluten was identified from an isolated red yeast Rhodotorula mucilaginosa strain. This peptidase consists of 425 amino acids, comprising an N-terminal signal peptide, a propeptide, and a C-terminal catalytic domain. The catalytic domain, termed RmuAP1CD, could be secreted by the recombinant oleaginous yeast Yarrowia lipolytica, whose genome contains the expression cassette for RmuAP1CD. RmuAP1CD exhibited optimum activity at pH 2.5 when acting on bovine serum albumin. Moreover, it facilitated the hydrolysis of gluten-derived immunogenic peptides (GIPs), which are responsible for triggering celiac disease symptoms, across a pH range of 3.0-6.0. The preferred cleavage sites are P-Q-Q-↓-P-Q in the 26-mer and P-Q-L-↓-P-Y in the 33-mer GIPs. Conversely, porcine pepsin cannot hydrolyze these two GIPs. The ability of RmuAP1CD to degrade GIPs under acidic conditions of the stomach indicates its potential as a viable oral enzyme therapy for celiac disease.

Lactococcus lactis subsp. lactis bv. diacetylactis Q5C6 strain as debittering adjunct culture for vegetable clotted cheese

One of the main challenges associated to the use of vegetable coagulants in cheesemaking is represented by the bitter taste conferred to the final product. In this context, the use of aminopeptidases positive strains could lead the hydrolysis of bitter peptides. The present study aimed to setup an experimental cheese clotted by using a kiwifruit enzymatic extract and inoculated with the Lactococcus lactis subsp. lactis bv. diacetylactis Q5C6 strain as debittering adjunct culture. The optimal amount of kiwifruit enzymatic extract, to be used in cheesemaking, was determined by performing laboratory-scale coagulation tests. Two experimental cheesemaking trials (A and B) were performed and the obtained cheeses were subjected to physico-chemical, microbiological, and sensory analysis. Results showed that, compared to cheeses clotted by animal rennet, the use of the kiwifruit enzymatic extract determined changes in fat, ash, and protein content. The L. lactis subsp. lactis bv. diacetylactis Q5C6 strain, inoculated at 1%, was able to reduce the bitter taste obtaining a final product with a sensory profile comparable to cheese clotted by animal rennet.

Obtaining New Candidate Peptides for Biological Anticancer Drugs from Enzymatic Hydrolysis of Human and Bovine Hemoglobin.

Enzymatic hydrolysis of bovine and human hemoglobin generates a diversity of bioactive peptides, mainly recognized for their antimicrobial properties. However, antimicrobial peptides stand out for their ability to specifically target cancer cells while preserving rapidly proliferating healthy cells. This study focuses on the production of bioactive peptides from hemoglobin and evaluates their anticancer potential using two distinct approaches. The first approach is based on the use of a rapid screening method aimed at blocking host cell protein synthesis to evaluate candidate anticancer peptides, using Lepidium sativum seed germination as an indicator. The results show that: (1) The degree of hydrolysis (DH) significantly influences the production of bioactive peptides. DH levels of 3 to 10% produce a considerably stronger inhibition of radicle growth than DH 0 (the native form of hemoglobin), with an intensity three to four times greater. (2) Certain peptide fractions of bovine hemoglobin have a higher activity than those of human hemoglobin. (3) The structural characteristics of peptides (random coil or alpha helix) play a crucial role in the biological effects observed. (4) The α137-141 peptide, the target of the study, was the most active of the fractions obtained from bovine hemoglobin (IC50 = 29 ± 1 µg/mL) and human hemoglobin (IC50 = 48 ± 2 µg/mL), proving to be 10 to 15 times more potent than the other hemoglobin fractions, attributed to its strong antimicrobial potential. The second approach to assessing anticancer activity is based on the preliminary in vitro analysis of hydrolysates and their peptide fractions, with a focus on the eL42 protein. This protein is of major interest due to its overexpression in all cancer cells, making it an attractive potential target for the development of anticancer molecules. With this in mind, astudy was undertaken using a method for labeling formylase (formyl-methionyl-tRNA transformylase (FMTS)) with oxidized tRNA. This approach was chosen because of the similarities in the interaction between formylase and the eL42 protein with oxidized tRNA. The results obtained not only confirmed the previous conclusions but also reinforced the hypothesis that the inhibition of protein synthesis plays a key role in the anticancer mechanism of these peptides. Indeed, the data suggest that samples containing α137-141 peptide (NKT) and total hydrolysates may have modulatory effects on the interaction between FMTS and oxidized tRNA. This observation highlights the possibility that the latter could influence molecular binding mechanisms, potentially resulting in a competitive situation where the ability of substrate tRNA to bind efficiently to ribosomal protein is compromised in their presence. Ultimately, these results suggest the feasibility of obtaining candidate peptides for biological anticancer drugs from both human and bovine hemoglobin sources. These scientific advances show new hope in the fight against cancer, which affects a large number of people around the world.