Search Results (741)

In the context of the valorization of agri-food by-products, tomato (Solanum lycopersicum L.) seeds represent a protein-rich matrix containing potential bioactives. The aim of the present work is to develop a biochemical pipeline for (i) achieving high protein recovery from tomato seed, (ii) optimizing the hydrolysis with different proteases, and (iii) characterizing the resulting peptides. This approach was instrumental for obtaining and selecting the most promising peptide mixture to test for antifungal activity. To this purpose, proteins from an alkaline extraction were treated with bromelain, papain, and pancreatin, and the resulting hydrolysates were assessed for their protein/peptide profiles via SDS-PAGE, SEC-HPLC, and RP-HPLC. Bromelain hydrolysate was selected for antifungal tests due to its greater quantity of peptides, in a broader spectrum of molecular weights and polarity/hydrophobicity profiles, and higher DPPH radical scavenging activity, although all hydrolysates exhibited antioxidant properties. In vitro assays demonstrated that the bromelain-digested proteins inhibited the growth of Fusarium graminearum and F. oxysporum f.sp. lycopersici in a dose-dependent manner, with a greater effect at a concentration of 0.1 mg/mL. The findings highlight that the enzymatic hydrolysis of tomato seed protein represents a promising strategy for converting food by-products into bioactive agents with agronomic applications, supporting sustainable biotechnology and circular economy strategies. Full article

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value. Full article

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu²⁺ ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu²⁺ and Zn²⁺ that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu²⁺ or Zn²⁺ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu²⁺ and those treated with Zn²⁺ formed distinct groups based on DNA sequence and methylation markers. Zn²⁺ treated and control samples formed separate groups. Also, Cu²⁺ discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu²⁺ or Zn²⁺ ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye. Full article

The quality of Crocus sativus L. flowers, beyond their stigmas, is influenced by the presence of bioactive metabolites also in their floral bio-residues. Given the effect of climatic and soil variables on these bioactive compounds, the aim of this research was to develop an agroecological zoning of saffron crop areas within the Protected Designation of Origin (PDO) La Mancha region (Castilla-La Mancha, Spain) by integrating the floral metabolite content with climatic and soil variables. To achieve this, a total of 173 samples were collected during the 2022 and 2023 harvests and analyzed via RP-HPLC-DAD to determine crocins, picrocrocin, kaempferols, and anthocyanins. Two new indices, Cropi (crocins + picrocrocin) and Kaeman (kaempferols + anthocyanins), were defined to classify flowers into four quality categories (A–D). High-quality classifications (A and B) were consistently associated with plots grouped in the meteorological stations of Ontur, El Sanchón, and Bolaños, indicating favorable edaphoclimatic conditions and climatic parameters, such as moderate temperatures and reduced humidity, for metabolite biosynthesis. In contrast, plots included in the meteorological stations of Tarazona and Pedernoso were mostly assigned to lower categories (C and D). Spatial analysis using thematic maps revealed that areas with an intermediate carbonate content, less calcareous soils, and higher organic matter levels were linked to higher flower quality. These findings highlight the influence of soil characteristics and climate, with distinct seasonal contrasts, that positively influence metabolite synthesis and flower quality. Full article

Nowadays, there is special interest in promoting the consumption of ancestral crops and minimally processed foods with high nutritional value. However, besides nutritional issues, safety assessments must be addressed. This study aimed to evaluate mycotoxin contamination in five minimally processed traditional Ecuadorian foods: ochratoxin A (OTA), fumonisin B₁ (FB₁), and aflatoxins (AFs) in brown rice, lupin, and quinoa; OTA, FB₁, and deoxynivalenol (DON) in whole-wheat flour; and OTA and AFs in peanuts. Samples (45 samples of peanuts and whole-wheat flour, 47 of brown rice, 46 of quinoa, and 36 of lupin) were collected from local markets and supermarkets in the three most populated cities in Ecuador. Mycotoxins were determined by RP-HPLC with fluorescence and detection. Results were compared with the maximum permitted levels (MPLs) of European Regulation 2023/915/EC. Overall contamination reached up to 59.8% of the analyzed samples (38.4% with one mycotoxin and 21.5% with co-occurrence). OTA was the most prevalent mycotoxin (in 82.6% of quinoa, 76.7% of whole-wheat flour, 53.3% of peanuts, 48.6% of lupin, and 25.5% of brown rice), and a modest number of quinoa (17%) and lupin (5.7%) samples surpassed the MPLs. DON was found in 82.2% of whole-wheat flour (28.9% > MPL). FB₁ was detected in above 25% of brown rice and whole-wheat flour and in 9% of the quinoa samples. FB₁ levels were above the MPLs only for whole-wheat flour (17.8%). AFB₁ and AFG₁ showed similar prevalence (about 6.5 and 8.5%, respectively) in quinoa and rice and about 27% in peanuts. Overall, these findings underscore the importance of enhancing fungal control in the pre- and post-harvest stages of these foods, which are recognized for their high nutritional value and ancestral worth; consequently, the results present key issues related to healthy diet promotion and food sovereignty. This study provides compelling insights into mycotoxin occurrence in minimally processed Ecuadorian foods and highlights the need for further exposure assessments by combining population consumption data. Full article

In order to innovatively develop high-activity ACE inhibitory peptides from edible fungi, the conditions for a double-enzymatic hydrolysis preparation of ACE inhibitory peptides from Flammulina velutipes were optimized by response surface methodology. After purification by macroporous resin, gel chromatography, and RP-HPLC, a crude peptide fraction was obtained; its ACE inhibition rate was 85.73 ± 0.95% (IC₅₀ = 0.83 ± 0.09 mg/mL). Based on LC-MS/MS sequencing, the four novel peptides, namely, FAGGP, FDGY, FHPGY, and WADP, were screened by computer analysis and molecular docking technology. The four peptides exhibited a binding energy between −9.4 and −10.3 kcal/mol, and formed hydrogen bonds with Tyr523, Ala354, and Glu384 in the S1 pocket, Tyr520 and His353 in the S2 pocket, and His383 in the HEXXH zinc-coordinating motif of ACE, indicating their good affinity with the ACE active site. The IC₅₀ values of the four ACE inhibitory peptides were 29.17, 91.55, 14.79, and 41.27 μM, respectively, suggesting that these peptides could potentially contribute to the development of new antihypertensive products. Full article

The selection of plant-based substrates for mushroom cultivation is a key factor influencing their growth and metabolism. The aim of this study was to demonstrate, in an innovative approach, differences in the content of biologically active compounds, bioelements, and antioxidant properties of Hericium erinaceus (Bull.) Pers. cultivated on various plant-based substrates derived from waste materials, specifically hemp straw and beech sawdust. Another objective was to compare various extraction methods in terms of their impact on the concentration of these compounds. Elemental analysis was performed using the TXRF method, while bioactive constituents were determined using the DAD/UV RP-HPLC technique. The plant-based substrate and extraction method influenced the levels of obtained metabolites. Dual extraction with moderate ethanol concentrations was most effective for isolating key bioactive compounds from H. erinaceus—notably ergothioneine, lovastatin, L-phenylalanine, and ergosterol—while antioxidant activity did not correlate with the concentration of the solvent used. Although dual extracts enhanced certain antioxidants and metabolites, whole fruiting bodies contained higher levels of bioelements. Overall, fruiting bodies grown on beech sawdust had greater amounts of most bioactive compounds compared to those cultivated on hemp straw, emphasizing that both substrate choice and extraction method critically influence the mushroom’s bioactive profile and its potential health benefits. Full article

This study aimed to ferment protein-rich amaranth flour with different strains of lactic acid bacteria (LAB) and to analyse the fermented dough’s functional properties. The fermented dough analysis was conducted using titrimetric, spectrophotometric, and chromatographic methods. The antioxidant activity of the fermented doughs was evaluated using the DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) methods, finding ABTS radical scavenging values ranging from 26.00 ± 1.05% to 58.92 ± 6.05%, while the DPPH values ranged from 21.29 ± 0.83% to 28.24 ± 5.48%. By RP-HPLC (Reversed Phase-High Performance Liquid Chromatography) characterisation, several phenolic acids and flavonoids were identified and quantified. Among these compounds, epigallocatechin was the most abundant, with the highest concentration recorded at 7789.88 ± 17.0 ng/µL in the control sample. This was followed by a 6942.47 ± 5.632 ng/µL concentration in the dough fermented with Lacticaseibacillus rhamnosus MIUG BL38 strain and 4983.16 ± 7.29 ng/µL in the dough fermented with Lactiplantibacillus pentosus MIUG BL24 strain. These two LAB strains (Lc. rhamnosus MIUG BL38 and Lp. pentosus MIUG BL24), with probiotic properties previously demonstrated, were selected based on their acidification potential, antioxidant activity, and bioactivity for future optimisation studies. Lactic acid fermentation significantly enhances bioactive characteristics of the amaranth flour, enabling the design of diverse gluten-free products with increased functional properties based on the attributes induced by the prebiotic, probiotic and postbiotic contents (tribiotics). Full article

The application of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) in the analysis of peptide therapeutics demonstrates its capacity to achieve high sensitivity and selectivity, which are essential qualities for the expanding peptide therapeutic industry. Given the challenges posed by hydrophilic peptides in reversed-phase chromatography, we investigated the necessity of a derivatization procedure to improve chromatographic separation and quasimolecular ion fragmentation during MS/MS detection. We investigated how eight different derivatizing agents react with a hydrophilic lysine- and arginine-containing human ezrin peptide-1 (HEP-1) to identify the most suitable one. The results showed that the reaction of HEP-1 with propionic anhydride proceeds most rapidly and completely, providing a high and reproducible yield of the product, which has sufficient retention on the RP column. The 4-propionylated derivative of HEP-1, compared to the other derivatives considered, demonstrates the most pronounced MS/MS fragmentation. The retention time of 2.42 min allows the separation of the substance from the interfering components of the blood plasma matrix and provides a limit of quantification of 5.00 ng/mL, which allows the use of this derivatizing agent for subsequent applications in pharmacokinetic studies, and this approach can improve the analytical parameters of similar peptides in other HPLC-MS/MS studies. Full article

In this study, camel milk (CM) and Gir cow milk (GCM) were fermented through cofermentation via yeast–lactic cultures, i.e., Lacticaseibacillus rhamnosus (M9, MTCC 25516) and Saccharomyces cerevisiae (WBS2A, MG101828), and their antioxidant and antidiabetic effectiveness were studied. To optimize the growth conditions, the level of proteolysis was evaluated by exploring various inoculation levels (1.5, 2.0 and 2.5%) as well as incubation durations (0, 12, 24, 36 and 48 h). Peptides were extracted and purified through 2D gel electrophoresis as well as SDS–PAGE. Water-soluble extracts (WSEs) of ultrafiltered (UF) peptide fractions were evaluated via reversed-phase high-performance liquid chromatography (RP-HPLC) to identify the peptide segments. By applying the Peakview tool, peptide sequences obtained from liquid chromatography–mass spectrometry (LC/MS) were reviewed by comparison with those in the BIOPEP database. Furthermore, the elevated levels of TNF-α, IL-6, IL-1β and nitric oxide (NO) in RAW 267.4 cells treated with lipopolysaccharide (LPS) are considerably lower than those in cultured CM and GCM. Protein macromolecules in CMs and GCMs have been captured via confocal laser scanning microscopy (CLSM) and Fourier transform infrared (FTIR) spectroscopy both before and after fermentation. Full article

Edaravone is used to treat motor neurone disease (MND) by slowing disease progression and prolonging survival time. Currently, it is available as an IV infusion (Radicava^®, Jersey City, NJ, USA) and an oral liquid suspension (Radicava ORS^®, Jersey City, NJ, USA). Development of novel edaravone formulations is still an active field of research that requires a validated stability-indicating assay capable of providing specific, precise, and accurate quantification of edaravone content. In this study, we developed and validated a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for edaravone quantification. Ten RP-HPLC methods based on the previously published literature were evaluated during method development. The optimal method employed a gradient method on an Agilent ZORBAX Extend-C18 column (150 × 4.6 mm, 5 µm) and produced a sharp and symmetrical drug peak. The method was further validated according to ICH Q2(R2) guidelines for specificity, linearity, sensitivity, accuracy, and precision. Successful separation of edaravone from void signals and degradant products was achieved. The method was precise and accurate at the concentration range of 6.8–68.6 µg/mL and was recommended to use without methyl hydroxybenzoate (MHB) as an internal standard. Full article

Background: Xerophilous scrubland is a semi-desert ecosystem characterized by a wide diversity of shrubs, which have secondary compounds with nutraceutical potential that could be used as feed for livestock, specifically by goats, since this species has developed behavioral and physiological adaptations that allow it to take advantage of the plant resources of said scrubland. Objective: To evaluate the nutraceutical potential of Havardia pallens and Vachellia rigidula, native species of the xerophilous scrubland, when incorporated as ingredients in goat diets. Methods: Integral diets for male goats were prepared, formulated with 35% inclusion of Havardia pallens, Vachellia rigidula, and Medicago sativa, the latter used as a plant control species. The content of flavonoids and total phenols was compared using colorimetric methods, and the antioxidant capacity was measured using the FRAP method. RP-HPLC-ESI-MS characterized the bioactive compounds in the different extracts. Statistical analysis was performed by ANOVA. Results: The aqueous extraction of Vachellia rigidula showed the highest concentration of total phenols ($\overline{\mathrm{x}}$ = 18.22 mg GAE/g⁻¹), followed by the ethanolic extract in the same species ($\overline{\mathrm{x}}$ = 17.045 mg GAE/g⁻¹). Similarly, Vachellia rigidula presented the highest antioxidant capacity ($\overline{\mathrm{x}}$ = 144,711.53 µmol TE/g⁻¹), while Medicago sativa presented the lowest ($\overline{\mathrm{x}}$ = 11,701.92 µmol TE/g). The RP-HPLC-ESI-MS analysis revealed that Vachellia rigidula presented a higher abundance of flavones, catechins, flavonols, methoxyflavones, and tyrosols. However, Harvardia pallens presented higher levels of methoxycinnamic and hydroxycinnamic acids. One-way ANOVA results showed that diets containing 35% Vachellia rigidula and Havardia pallens significantly contrasted (p < 0.05), increased the content of secondary compounds and antioxidant capacity compared to the control species. Furthermore, including Vachellia rigidula led to a significantly higher antioxidant capacity (p < 0.05) than diets with Havardia pallens or Medicago sativa. Conclusions: Incorporating the leguminous shrubs Vachellia rigidula and Havardia pallens into the formulation of comprehensive diets for buck goats improves the content and availability of phenols, flavonoids, and antioxidants. However, in vivo evaluation of these diets is important to determine their physiological and productive effects on the animals. Full article

Microalgae-based bioremediation is increasingly recognized as a sustainable, efficient, and straightforward technology. Despite this growing interest, the potential of Parachlorella hussii for metal biosorption remains underexplored. This study is the first report evaluating the metal biosorption activity in Parachlorella hussii ACOI 1508 (N9), highlighting the impact of the culture age on the monosaccharide composition and its correlation to the metal binding capacity. The capsular strain (N9) was isolated from the hypersaline ecosystem—Lake Chott Aïn El-Beida—in southeastern Algeria. Cultivated in Bold’s Basal medium, the strain produced 0.807 ± 0.059 g L⁻¹ of RPSs and 1.975 ± 0.120 g L⁻¹ of CPSs. Biochemical analysis of the extracts revealed a high total sugar content (% w/w) that ranged from 62.98 ± 4.87% to 95.60 ± 87% and a low protein content (% w/w) that ranged from 0.49 ± 0.08% to 1.35 ± 0.69%, with RPS-D7 and RPS-D14 having high molecular weight (≥2 MDa). HPLC-based monosaccharide characterization demonstrated compositional differences between the exponential and stationary phases, with rhamnose dominating (~55%) in RPS-D14 and with the presence of uronic acids comprising 7–11.3%. Metal removal efficiency was evaluated using the whole biomass in two growth phases. Copper uptake exhibited the highest capacity, reaching 18.55 ± 0.61 mg Cu g⁻¹ DW at D14, followed by zinc removal with 6.52 ± 0.61 mg Zn g⁻¹ DW. Interestingly, removal efficiencies increased to about twofold during the stationary phase, reaching 51.15 ± 1.14% for Cu, 51.08 ± 3.35% for Zn, and 36.55 ± 3.09% for Ni. The positive results obtained for copper/zinc removal highlight the biosorption potential of P. hussii, and notably, we found that the metal removal capacity significantly improved with culture age—a parameter that has been poorly investigated in prior studies. Furthermore, we observed a growth phase-dependent modulation in monosaccharide composition, which correlated with enhanced functional properties of the excreted biomolecules involved in biosorption. This metabolic adjustment suggests an adaptive response that may contribute to the species’ effectiveness in heavy metal uptake, underscoring its novelty and biotechnological relevance. Full article

Bioactive extracts from living plants (LR) and beach deposits (NR) of the submerged brackish water angiosperm Ruppia maritima were examined for their antioxidant activity and anticancer potential. LR extract scavenged effectively free radicals with IC₅₀ values of 38.00 μg/mL (DPPH^•), 12.00 μg/mL (ABTS^•+), 281.00 μg/mL (OH^•), and 53.00 μg/mL (O₂^•), and exhibited reducing activity with an RP_0.5AU value of 37.00 μg/mL. NR extract retained a significant part of LR extract’s antioxidant activity by scavenging free radicals with IC₅₀ values of 180.00 μg/mL (DPPH^•), 60.00 μg/mL (ABTS^•+), and 164.00 μg/mL (O₂^•), and exhibited reducing activity with an RP_0.5AU value of 107.00 μg/mL. Importantly, NR extract (IC₅₀ value: 60.00 μg/mL) exhibited much higher inhibitory activity than LR extract (IC₅₀ value: 1100.00 μg/mL) in XTT assay. HPLC analysis revealed that both R. maritima extracts contained phenolics, such as chicoric acid, quercetin-3-O-glucopyranoside, p-coumaric acid, 3,5-dimethoxy-4-hydroxicinnanic acid, trans-ferulic acid, and rutin hydrate, possessing antioxidant and/or anticancer activity. Thus, the present study showed for the first time that R. maritima extracts from either LR or NR are a promising source of bioactive compounds having beneficial properties for human health. Full article

Background: A key challenge in modern pharmaceutical research is developing predictive models for drug formulation behavior. Since permeability is closely linked to molecular properties, considering a broad of characteristics is essential for building reliable predictive tools. Near-infrared spectroscopy (NIR), a non-destructive, non-invasive, and chemically specific method, offers a powerful alternative to current gold-standard methods approved by regulatory agencies. Objectives: This study aims to apply a partial analytical quality by design (AQbD) approach to enhance the understanding and development of NIR and RP-HPLC methodologies. Methods: The employment of NIR with multivariate data analysis enabled the establishment of chemometric models for the classification and quantification of bifonazole (BFZ) in cream formulations. Results: An analytical target profile (ATP) was defined to guide the selection of critical method variables and support method design and development activities. Risk assessment was carried out using an Ishikawa diagram. For the RP-HPLC method, key performance parameters such as peak area, theoretical plates, tailing factor, and assay were evaluated, while NIR spectra and BFZ concentration were considered for method performance. The quantification models enabled the accurate determination of BFZ content, yielding results of 8.48 mg via NIR and 8.34 mg via RP-HPLC, with an RSD of 1.25%. Conclusions: These findings demonstrate the robustness and reliability of the models, making them suitable for routine quality control of BFZ formulations. Future research should aim to explore its use for monitoring permeation dynamics in real time and integrating it into regulatory frameworks to standardize its application in pharmaceutical quality control and formulation development. Full article