Search Results (21)

A rising demand for low-gluten beer fuels research into enzymatic solutions. This study optimized Aspergillus niger prolyl endopeptidase (AN-PEP) application timing during brewing to reduce gluten while preserving physicochemical quality. Ale-type beers were produced with AN-PEP (2% v/v) added at mashing, boiling, post-boiling, or post-fermentation, plus a control. Three mashing profiles (Mash A, B, C) were also tested. Gluten was quantified by R5 ELISA (LOQ > 270 mg/L). Color, bitterness, ABV, and foam stability were assessed. Statistical analysis involved ANOVA and Tukey’s HSD (p < 0.05). Enzyme activity and thermal inactivation were also evaluated. Initial gluten levels consistently exceeded LOQ. Significant gluten reduction occurred only post-fermentation. Mashing, boiling, and post-boiling additions effectively lowered gluten to below 20 mg/L. Post-fermentation addition resulted in significantly higher residual gluten (136.5 mg/L). Different mashing profiles (A, B, C) with early enzyme addition achieved similar low-gluten levels. AN-PEP showed optimal activity at 60–65 °C, inactivating rapidly at 100 °C. Physicochemical attributes (color, extract, bitterness, ABV) were largely unaffected. However, foam stability was significantly compromised by mashing and post-fermentation additions, while preserved with boiling and post-boiling additions. AN-PEP effectively produces low-gluten beers. Enzyme addition timing is critical: while mashing, boiling, or post-boiling additions reduce gluten to regulatory levels, only the beginning of boiling or post-boiling additions maintain desirable foam stability. These findings offer practical strategies for optimizing low-gluten beer production. Full article

Type 1 diabetes (T1D) is a chronic metabolic disease defined by sustained hyperglycemia, leading to oxidative stress (OS) and systemic complications, including male subfertility. This study investigates the potential impact of T1D-induced OS on microtubule (MTs) dynamics and microtubule-associated proteins (MAPs) in the testis and spermatozoa (SPZ). Using a streptozotocin-induced T1D rat model, we examined the expression and localization of key MAPs, including Microtubule Affinity-Regulating Kinase 4 (MARK4), Microtubule-Associated Protein 1A (MAP1A), Dynein Light Chain LC8-Type 1 (DYNLL1), Prolyl Endopeptidase (PREP), and Radial Spoke Head 6 Homolog A (RSPH6A), alongside sperm functional parameters. Our findings showed that T1D significantly impaired the expression and distribution of these proteins, which may affect MTs organization and be associated with cytoskeletal disorganization, and impaired germ cell differentiation. Moreover, T1D rats exhibited reduced sperm count, viability, and motility, accompanied by increased DNA fragmentation and chromatin defects. Elevated levels of 4-hydroxy-2-nonenal (4-HNE), a marker of OS, were detected in SPZ, particularly in the acrosome and flagellum, correlating with mitochondrial dysfunction and ATP depletion. Additionally, decreased intracellular Ca²⁺ levels, downregulation of Cation Channel of Sperm (CATSPER) and Voltage-Dependent Anion Channel 3 (VDAC3), and altered tubulin acetylation, possibly due to imbalanced Alpha-Tubulin N-Acetyltransferase 1 (ATAT1) and Histone Deacetylase 6 (HDAC6) expression, were also associated with impaired sperm motility. The combined data suggest that T1D-induced OS is linked to disrupted MTs dynamics, which may contribute to testicular dysfunction and reduced sperm quality, potentially affecting male fertility. A better understanding of these associations may support the development of therapeutic strategies to mitigate the reproductive consequences of T1D and improve male fertility outcomes. Full article

Myocardial ischemia–reperfusion injury (MI/R) is a negative and adverse cardiovascular outcome following myocardial ischemia, cardiac surgery, or circulatory arrest. Prolyl endopeptidase (PREP) appears to be involved in inflammatory responses, so it could be a possible therapeutic target for counteracting ischemia injury. This study aimed to investigate the role of PREP inhibitor, KYP-2047 (4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrolidine), in the modulation of molecular and biochemical processes involved in MI/R. MI/R was induced through coronary artery occlusion (15 min), followed by reperfusion (2 h). KYP-2047 was intraperitoneally administrated at doses of 2.5 mg/kg and 5 mg/kg 24 h before the surgical procedures. The hearts were removed and processed for analysis. KYP-2047 treatment limited ischemic myocardial-induced histological damage and neutrophil accumulation, limiting inflammation, fibrosis, and apoptosis processes. Additionally, KYP-2047 was able to modulate p-38 and p-ERK expression, suggesting an improving role in recovering cardiac function. These findings highlighted the protective effects of KYP-2047 pretreatment in MI/R injury, suggesting PREP as a potential target therapy for the pathogenesis of MI/R. Although the molecular mechanisms underlying the action of KYP-2047 are still to be explored, these results suggested that the regulation of NF-κB, apoptosis, and MAPK pathways by KYP-2047 treatment could preventatively limit the damage caused by MI/R. Full article

This current study aims to analyze the potential bioactivities possessed by the enzymatic hydrolysates of commercial bovine, porcine, and tilapia gelatins using bioinformatics in combination with in vitro and in vivo studies. The hydrolysate with superior inhibition of angiotensin converting enzyme (ACE) activity was used to treat the D-galactose (DG)-induced amnesic mice. In silico digestion of the gelatins led to the identification of peptide sequences with potential antioxidant, ACE-inhibitory, and anti-amnestic properties. The results of in vitro digestion revealed that the <1 kDa peptide fraction of porcine gelatin hydrolysate obtained after 1 h digestion with papain (PP) (PP1, <1 kDa) potently inhibited ACE, acetylcholinesterase, and prolyl endopeptidase activities at 87.42%, 21.24%, and 48.07%, respectively. Administering the PP1 to DG-induced amnesic mice ameliorated the spatial cognitive impairment and Morris water maze learning abilities. The dentate area morphology in the PP1-treated mice was relatively similar to the control group. In addition, PP1 enhanced the antioxidant capacity in the DG-induced amnesic mice. This study suggests that PP1 could serve as a potential treatment tool against oxidative stress, hypertension, and neurodegenerative diseases. Full article

The peptidomes from the literature of 24 prolyl-endopeptidase-treated beers during fermentation, declared gluten-free, and 13 untreated beers have been characterised and subjected to an extensive study to investigate their safety for celiac patients. The analysis contains 1996 gluten peptides, ascribed to the treated beers, and 1804 to the untreated beers. The prolyl-endopeptidase-untreated malt beers are hazardous for celiac patients. Peptides of most of these beers showed matches with complete celiac immunogenic motifs, and an additional 28% of the peptides have partial matches with complete immunogenic motifs. On the other hand, after the enzyme treatment during fermentation no celiac hazardous gluten peptides are identified in the treated beers. Due to partial matches with complete celiac immunogenic motifs, 11% potentially hazardous gluten peptides are still identified in the treated beers. Only a maximum of 17% of these peptides can be detected by ELISA analysis. A mass spectrometry analysis or the recently developed method based on G12/A1 monoclonal antibody lateral flow immunochromatographic assay seems necessary to thoroughly reveal the potential risk of the treated beers. The actual immune response of treated beer, described in the literature by the response of the serum antibodies of celiac disease (CD)-active patients and by in vitro immune response, could not be related to the presence of known (partial) CD-immunogenic motifs in the gluten peptides. Full article

Euphorbia species are important sources of polycyclic and macrocyclic diterpenes, which have been the focus of natural-product-based drug research due to their relevant biological properties, including anticancer, multidrug resistance reversal, antiviral, and anti-inflammatory activities. Premyrsinane, cyclomyrsinane, and myrsinane diterpenes are generally and collectively designated as myrsinane-type diterpenes. These compounds are derived from the macrocyclic lathyrane structure and are characterized by having highly oxygenated rearranged polycyclic systems. This review aims to describe and summarize the distribution and diversity of 220 myrsinane-type diterpenes isolated in the last four decades from about 20 Euphorbia species. Some myrsinane diterpenes obtained from Jatropha curcas are also described. Discussion on their plausible biosynthetic pathways is presented, as well as isolation procedures and structural elucidation using nuclear magnetic resonance spectroscopy. Furthermore, the most important biological activities are highlighted, which include cytotoxic and immunomodulatory activities, the modulation of efflux pumps, the neuroprotective effects, and the inhibition of enzymes such as urease, HIV-1 reverse transcriptase, and prolyl endopeptidase, among other biological effects. Full article

Sea fennel and seaside arrowgrass are two abundant but underutilized halophytes along the Atlantic and Mediterranean coasts. This study investigated the antioxidant capacity and the potential antihypertensive (Angiotensin Converting Enzyme I, ACE-I inhibition), hypoglycaemic (Dipeptidyl Peptidase IV, DPP-IV inhibition), and nootropic (Prolyl Endopeptidase, PEP inhibition) activity of their polyphenol extracts. They had a high phenol content (21–24 mEq GA/g), antioxidant capacity evaluated using the ABTS (17–2 mg ascorbic acid/g) and FRAP (170–270 mM Mohr’s salt/g) assays, and effective ACE-inhibiting properties (80–90% inhibiting activity at final concentration of 0.5 mg/mL). Additionally, the sea fennel extract displayed high DPP-IV inhibitory capacity (73% at 1 mg/mL), while the seaside arrowgrass extract exhibited potent Prolyl endopeptidase inhibitory capacity (75% at 1 mg/mL). Fractionation by HPLC concentrated the bioactive molecules in two fractions, for which the composition was analyzed by LC-MS/MS. Different chlorogenic acids seemed to play an important role in the bioactivity of sea fennel extract, and different flavonoids, mainly apigenin, luteolin and chrysoeriol, in the bioactivity of the seaside arrowgrass extract. Given their potential health benefits, these extracts could serve as valuable bioactive ingredients and could potentially encourage the cultivation of these species in regions where traditional crops face challenges in growth. Full article

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17β-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction. Full article

This study provides a comprehensive comparison among the most common gluten-free (GF) brewing practices, with a focus on the impact of each treatment on physicochemical parameters and consumer acceptability of the final beer. In addition, the influence of a longer cold maturation on the natural reduction of the gluten content was investigated. Prolyl endopeptidase addition was found to be the most effective treatment in reducing gluten levels (−75.93%), followed by silica gel (−53.09%), longer cold maturation (−4.32%), and tannins (−1.85%). Nonetheless, none of the treated beer samples was gluten-free (gluten content > 20 ppm) due to the high nitrogen content of the original wort. The silica gel application treatment affected the physicochemical and sensory characteristics of the final beer the least. According to the difference from control test results, no significant difference in terms of overall liking, appearance, odor/aroma, or taste was observed between the silica gel-treated sample and control beer (p > 0.05). On the other hand, the application of enzymes and tannins significantly affected the appearance and the beer odor/aroma. Nevertheless, all beer samples received positive sensory acceptance scores. Full article

Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD. Full article

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48–64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity. Full article

The present work shows a procedure to valorize non-commercial boiled shrimp to produce functional ingredients, using a combined treatment based on enzymatic hydrolysis and subsequent glycation under mild conditions. Antioxidant and prolyl endopeptidase-inhibiting activities were determined as a function of hydrolysis and glycation times (0–120 min and 0–180 min, respectively). The reaction products were characterized by determining the degree of hydrolysis, browning, fluorescent compounds, free amino acids, phenol content, Fourier transform infrared spectroscopy (FTIR), and molecular weight of the different fractions obtained. Enzymatic hydrolysis generated hydrolysates with significant antioxidant and prolyl endopeptidase-inhibiting activities. Glycation under mild conditions was used as a strategy to improve the antioxidant and potential nootropic properties of the hydrolysates. During glycation, the free amino acid content decreased, total phenols and fluorescent compounds increased significantly, and low molecular weight melanoidins were formed. The presence of peptide-glucose conjugates was also confirmed by FTIR. Glycation increased the antioxidant activities of the hydrolysates; however, their prolyl-endopeptidase-inhibiting activity was lost. Results showed that compounds with promising antioxidant (hydrolysis and glycation) and potential nootropic (hydrolysis) activities and applications in food systems were obtained from the biotechnological strategy used. Full article

Background: Intestinal ischemia/reperfusion injury (IRI) remains a clinical event that contributes to high morbidity and mortality rates. Intestinal epithelium is exposed to histological and vascular changes following tissue ischemia. Prolyl endopeptidase (PREP), involved in inflammatory responses, could be targeted for recovery from the permanent consequences following intestinal ischemia. Our aim was to investigate the role of PREP inhibitor KYP-2047 in tissue damage, angiogenesis, and endothelial barrier permeability after intestinal IRI in mice. Methods: KYP-2047 treatments were performed 5 min prior to intestinal damage. Intestinal IRI was induced in mice by clamping the superior mesenteric artery and the celiac trunk for 30 min, followed by 1 h of reperfusion. Results: PREP inhibition by KYP-2047 treatment reduced intestinal IR-induced histological damage and neutrophil accumulation, limiting inflammation through decrease of NF-ĸB nuclear translocation and fibrotic processes. KYP-2047 treatment restored barrier permeability and structural alteration following intestinal IRI, attenuating neovascular processes compromised by ischemia/reperfusion. Additionally, loss of epithelial cells during intestinal ischemia occurring by apoptosis was limited by KYP-2047 treatment, which showed strong effects counteracting apoptosis and DNA damage. Conclusions: These findings provide the first evidence that PREP inhibition through KYP-2047 inhibitor use could be a validate strategy for resolving alterations of intestinal epithelium the pathophysiology of intestinal disease. Full article

Tetraponera rufonigra (Arboreal Bicoloured Ant) venom induces pain, inflammation, and anaphylaxis in people and has an increased incident in Southeast Asia regions. The bioactive components and mechanism of action of the ant venom are still limited. The aim of this research was to identify the protein composition and inflammatory process of the ant venom by using RAW 264.7 macrophage cells. The major venom proteins are composed of 5’ nucleotidase, prolyl endopeptidase-like, aminopeptidase N, trypsin-3, venom protein, and phospholipase A₂ (PLA₂). The venom showed PLA₂ activity and represented 0.46 μg of PLA₂ bee venom equivalent/μg crude venom protein. The venom induced cytotoxic in a dose- and time-dependent manner with IC₂₀ approximately at 4.01 µg/mL. The increased levels of COX-2 and PGE₂ were observed after 1 h of treatment correlating with an upregulation of COX-2 expression. Moreover, the level of mPGES-1 expression was obviously increased after 12 h of venom induction. Hence, our results suggested that the induction of COX-2/mPGEs-1 pathway could be a direct pathway for the ant venom-induced inflammation. Full article

Wheat storage proteins and products of their hydrolysis may cause coeliac sprue in genetically predisposed individuals with high expression of main histocompatibility complex HLA-DQ2 or DQ8, since by consuming wheat, they become exposed to proline- (P) and glutamine (Q)-rich gluten. In bread-making, the hydrolysis of gliadins and coeliac-toxic peptides occurs with varied efficiency depending on the fermentation pH and temperature. Degradation of gliadins catalysed by Lactobacillus acidophilus 5e2 peptidases and a commercial prolyl endopeptidase synthesised by A. niger, carried out at pH 4.0 and 37 °C, reduces the gliadin concentration over 110-fold and decreases the relative immunoreactivity of the hydrolysate to 0.9% of its initial value. Hydrolysis of coeliac-toxic peptides: LGQQQPFPPQQPY (P1) and PQPQLPYPQPQLP (P2) under the same conditions occurs with the highest efficiency, reaching 99.8 ± 0.0% and 97.5 ± 0.1%, respectively. The relative immunoreactivity of peptides P1 and P2 was 0.8 ± 0.0% and 3.2 ± 0.0%, respectively. A mixture of peptidases from L. acidophilus 5e2 and A. niger may be used in wheat sourdough fermentation to reduce the time needed for degradation of proteins and products of their hydrolysis. Full article