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In this study, the effect of the use of S. platensis, which is presented as an eco-friendly and alternative protein source, in the production of kefir, a probiotic dairy product, on various physicochemical properties as well as FAA, ACE inhibitory activity, proteolysis, TPC, DPPH, ABTS + , and mineral values was investigated. It was observed that the addition of S. platensis at different ratios to the kefir samples had a statistically very significant (p < 0.01) effect on all physicochemical analyses; L. mesenteroides count; all amino acids except isoleucine, aspartic acid, and glutamic acid; ACE inhibitory activity, TN, TCAN, TCAN/TN, mM Gly, TPC, DPPH, ABTS + , Na, Mg, K, and Fe. In plain kefir samples, mineral contents were determined by order of K > P > Na > Ca > Mg > Zn >> Fe > Cr > Cr > Mn. Furthermore, a general increase was observed in FAA, ACE inhibitory activity, TPC, DPPH, ABTS + , and mineral values, as well as in the counts of Lactococcus spp. and L. mesenteroides in the samples, depending on the proportion of S. platensis added, compared to plain kefir samples. In this context, it was concluded that the addition of S. platensis to kefir at different rates could meet various components required by the body on a daily basis and result in a nutraceutical product., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Biofilters are the important source and sink of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB) in the drinking water. Current studies generally ascribed the prevalence of BAR in biofilter from the perspective of gene behavior, i.e. horizontal gene transfer (HGT), little attentions have been paid on the ARGs carrier- ARB. In this study, we proposed the hypothesis that ARB participating in pollutant metabolism processes and becoming dominant is an important way for the enrichment of ARGs. To verify this, the antibiotic resistome and bacterial functional metabolic pathways of a sand filter was profiled using heterotrophic bacterial plate counting method (HPC), high-throughput qPCR, Illumina Hiseq sequencing and PICRUSt2 functional prediction. The results illustrated a significant leakage of ARB in the effluent of the sand filter with an average absolute abundance of approximately 10 2 -10 3  CFU/mL. Further contribution analysis revealed that the dominant genera, such as Acinetobacter spp., Aeromonas spp., Elizabethkingia spp., and Bacillus spp., were primary ARGs hosts, conferring resistance to multiple antibiotics including sulfamethoxazole, tetracycline and β-lactams. Notably, these ARGs hosts were involved in nitrogen metabolism, including extracellular nitrate/nitrite transport and nitrite reduction, which are crucial in nitrification and denitrification in biofilters. For example, Acinetobacter spp., the dominant bacteria in the filter (relative abundance 69.97 %), contributed the majority of ARGs and 53.79 % of nitrite reduction function. That is, ARB can predominate by participating in the nitrogen metabolism pathways, facilitating the enrichment of ARGs. These findings provide insights into the stable presence of ARGs in biofilters from a functional metabolism perspective, offering a significant supplementary to the mechanisms of the emergence, maintenance, and transmission of BARin drinking water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 10 3  CFU/mL) and ampicillin (≥ 8.128 × 10 3  CFU/mL). The real-time PCR-based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6')-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Antimicrobial resistance (AMR) is now recognized as a leading global threat to human health. Nevertheless, there currently is a limited understanding of the environment's role in the spread of AMR and antibiotic resistance genes (ARGs). In 2019, the U.S. Geological Survey conducted the first statewide assessment of antibiotic resistant bacteria (ARB) and ARGs in surface water and bed sediment collected from 34 stream locations across Iowa. Environmental samples were analyzed for a suite of 29 antibiotics and plated on selective media for 15 types of bacteria growth; DNA was extracted from culture growth and used in downstream polymerase chain reaction (PCR) assays for the detection of 24 ARGs. ARGs encoding resistance to antibiotics of clinical importance to human health and disease prevention were prioritized as their presence in stream systems has the potential for environmental significance. Total coliforms, Escherichia coli (E. coli), and staphylococci were nearly ubiquitous in both stream water and stream bed sediment samples, with enterococci present in 97 % of water samples, and Salmonella spp. growth present in 94 % and 67 % of water and bed sediment samples. Bacteria enumerations indicate that high bacteria loads are common in Iowa's streams, with 23 (68 %) streams exceeding state guidelines for primary contact for E. coli in recreational waters and 6 (18 %) streams exceeding the secondary contact advisory level. Although antibiotic-resistant E. coli growth was detected from 40 % of water samples, vancomycin-resistant enterococci (VRE) and penicillinase-resistant Staphylococcus aureus (MRSA) colony growth was detected from nearly all water samples. A total of 14 different ARGs were detected from viable bacteria cells from 30 Iowa streams (88 %, n = 34). Study results provide the first baseline understanding of the prevalence of ARB and ARGs throughout Iowa's waterways and health risk potential for humans, wildlife, and livestock using these waterways for drinking, irrigating, or recreating., Competing Interests: Declaration of competing interest David M. Cwiertny reports financial support was provided by The Chicago Community Foundation., (Published by Elsevier B.V.)

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study is to explore differences in the peptidomics of Saccharomyces pastorianus protein hydrolysates treated with different enzymes. Briefly, differences in the peptide fingerprints and active peptides of neutral protease/papain-hydrolyzed S. pastorianus were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) combined with PEAKS Online 1.7 analysis software, Peptide Ranker, and the BIOPEP database. Compared to traditional databases, the PEAKS Online uses de novo sequencing for analysis to obtain oligopeptides smaller than pentapeptides. It provides more comprehensive data of the peptide sample. In this study, enzymatic hydrolysates of S. pastorianus protein were prepared under the optimum conditions of neutral protease and papain respectively. In total, 7221 and 7062 polypeptides were identified in the hydrolysates of neutral protease and papain, respectively; among these polypeptides, 980 were common to the two enzymes. The 6241 and 6082 unique peptides found in the hydrolysates of neutral protease and papain, respectively, indicated that the peptide fingerprints of the two hydrolysates are quite different. Peptide Ranker predicted that 3013 (41.73%) and 3095 (43.83%) peptides were potentially bioactive in the hydrolysates of neutral protease and papain, respectively. According to the BIOPEP database, neutral protease and papain contained 295 and 357 active peptides, respectively; these peptides were mainly composed of angiotensin converting enzyme (ACE) inhibitors and dipeptidyl peptidase IV inhibitors and antioxidant peptides. The number of active peptides in the hydrolysate of papain was higher than that in the hydrolysate of neutral protease, but the total ion intensity of active peptides in the former was lower than that in the latter. This study revealed the influence of protease type on the composition of enzymatic hydrolysates from S. pastorianus protein. The above results provide a reference for the development of functional products of S. pastorianus protein peptides and the high-value utilization of yeast resources.

Integrated and monoculture freshwater aquaculture systems are often regarded as important reservoirs for antimicrobial resistance genes (ARGs) and antimicrobial resistance bacteria (ARBs), yet only a few studies have assessed differences in the antimicrobial resistome and antibiotic residues between aquaculture modes. In this study, a metagenomic approach was used to comprehensively explore the dynamic patterns and potential transmission mechanisms of ARGs in ducks, human workers, fish, water and sediments during the transition from an integrated to a monoculture freshwater aquaculture mode and to investigate the associations of ARGs with potential hosts in microbial communities using network analysis and a binning approach. The results showed that the abundance and diversity of ARGs were higher under integrated fish-duck farming than in single fish ponds. During the transition from an integrated to a monoculture aquaculture farm, ARGs in workers and sediments were not easily removed. However, ARGs in the aquatic environment underwent regular changes. In addition, duck manure was probably the most dominant source of ARGs in the duck farm environment. Network analysis indicated that Escherichia spp. were the most dominant hosts of ARGs. Variation partitioning analysis (VPA) showed that in water samples, the bacterial community played an important role in the ARG profile. In addition, we identified a potential risk of the presence of highly virulent and antimicrobial-resistant Klebsiella pneumoniae in workers. These results help assess the risk of ARG transmission in integrated and monoculture aquaculture farms and suggest that we should strengthen the monitoring of long-term resistance in integrated aquaculture environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL -1 , despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Francesc Corbera-Rubio reports financial support was provided by Dunea Duin & Water. Brent Pieterse reports a relationship with Dunea Duin & Water that includes: employment. David de Ridder reports a relationship with Evides NV that includes: employment., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Dissolved organic carbon (DOC) is a significant component of regional and global carbon cycles and an important surface water quality indicator. DOC affects the processes of solubility, bioavailability and transport for a number of contaminants, such as heavy metals. Therefore, it is crucial to understand DOC fate and transport in the watershed and the transport pathways of DOC load. We modified a previously developed watershed-scale organic carbon model by incorporating the DOC load from glacier melt runoff and used the modified model to simulate periodic daily DOC load in the upper Athabasca River Basin (ARB) in the cold region of western Canada. The calibrated model achieved an overall acceptable performance for simulating daily DOC load with model uncertainties mainly from the underestimation of peak loads. Parameter sensitivity analysis indicates that the fate and transport of DOC load in upper ARB are mainly controlled by DOC production in the soil layers, DOC transport at the soil surface, and reactions in the stream system. The modeling results indicated that the DOC load is mainly from the terrestrial sources and the stream system was a negligible sink in the upper ARB. It also indicated that rainfall-induced surface runoff was the major transport pathway of DOC load in the upper ARB. However, the DOC loads transported by glacier melt runoff were negligible and only accounted for 0.02% of the total DOC loads. In addition, snowmelt-induced surface runoff and lateral flow contributed 18.7% of total DOC load, which is comparable to the contribution from the groundwater flow. Our study investigated the DOC dynamics and sources in the cold region watershed in western Canada and quantified the contribution of different hydrological pathways to DOC load, which could provide a useful reference and insight for understanding watershed-scale carbon cycle processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Background: Mahkota Dewa [Phaleria macrocarpa (Scheff) Boerl.] fruit in vitro and in- vivo can decrease and prevent elevation of the blood pressure, lower plasma glucose levels, possess an antioxidant effect, and recover liver and kidney damage in rats. This study aimed to determine the structure and inhibitory activity of angiotensin-converting enzyme inhibitors (ACE) from the Mahkota Dewa fruit., Methods: The fruit powder was macerated using methanol and then partitioned by hexane, ethyl acetate, n-butanol, and water. The fractions were chromatographed on the column chromatography and incorporated with TLC and recrystallization to give pure compounds. The structures of isolated compounds were determined by UV-Visible, FT-IR, MS, proton ( 1 H-NMR), carbon ( 13 C-NMR), and 2D-NMR techniques encompassing HMQC and HMBC spectra. The compounds were evaluated for their ACE inhibitory activity, and the strongest compound was determined by the kinetics enzyme inhibition., Results: Based on the spectral data, the isolated compounds were determined as 6,4-dihydroxy-4-methoxybenzophenone-2-O-β-D-glucopyranoside (1), 4,4'-dihydroxy-6-methoxybenzophenone-2-O-β-D-glucopyranoside (2) and mangiferin (3). IC 50 values of the isolated compounds 1, 2 and 3 were 0.055, 0.07, and 0.025 mM, respectively., Conclusion: The three compounds have ACE inhibitor and mangiferin demonstrated the best ACE inhibitory activity with competitive inhibition on ACE with the type of inhibition kinetics is competitive inhibition., (© 2023. The Author(s).)

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) has become an increasingly serious global public health issue. This study investigated the distribution characteristics and influencing factors of ARB and ARGs in greenhouse vegetable soils with long-term application of manure. Five typical ARGs, four heavy metal resistance genes (MRGs), and two mobile genetic elements (MGEs) were quantified by real-time quantitative polymerase chain reaction (qPCR). The amount of ARB in manure-improved soil greatly exceeded that in control soil, and the bacterial resistance rate decreased significantly with increases in antibiotic concentrations. In addition, the resistance rate of ARB to enrofloxacin (ENR) was lower than that of tylosin (TYL). Real-time qPCR results showed that long-term application of manure enhanced the relative abundance of ARGs in vegetable soils, and the content and proportion of quinolone resistance genes were higher than those of macrolide resistance genes. Redundancy analysis (RDA) showed that qepA and qnrS significantly correlated with total and available amounts of Cu and Zn, highlighting that certain heavy metals can influence persistence of ARGs. Integrase gene intI1 correlated significantly with the relative abundance of qepA, qnrS, and ermF, suggesting that intI1 played an important role in the horizontal transfer of ARGs. Furthermore, there was a weakly but not significantly positive correlation between specific detected MRGs and ARGs and MGEs. The results of this study enhance understanding the potential for increasing ARGs in manure-applied soil, assessing ecological risk and reducing the spread of ARGs., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Some natural products are important sources of treatments for hypertension based on their potential inhibitory effects on angiotensin-converting enzyme (ACE); however, it is difficult to identify natural ACE inhibitors (ACEIs) due to the complex secondary metabolite environment of natural products. Enzyme immobilization is an important method for screening active constituents in natural products, but this method can sometimes return false-positive and false-negative results. To improve the accuracy and reliability of ligand-fishing methods, we established a novel strategy based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment technologies. We first synthesized ACE-immobilized mesoporous magnetic beads and then verified the screened compounds by molecular docking and in vitro activity detection. We then used active-site blocking to exclude non-specific binding constituents and applied directional enrichment to enrich the low-content constituents for ligand fishing. The screening identified six potential ACEIs from Scutellariae Radix and eight potential ACEIs from Lonicerae japonicae flos, and their inhibitory activity was confirmed by molecular docking simulations and in vitro activity detection. This process screened six additional compounds and excluded two false-positive results as compared with results exclusively using enzyme immobilization. This strategy provides a feasible method for screening active compounds in natural products., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Angiotensin-converting enzyme (ACE) inhibitory peptides are potential alternatives to the synthetic ACE inhibitory drugs, but the in vivo antihypertensive effects of most of them have not been confirmed. The tripeptide Leu-Pro-Pro (LPP) is one of the few peptides that have been proved clinically effective in reducing the blood pressure of hypertensive patients and casein is currently its major source. LPP is contained in multiple fractions of zein, and corn gluten meal (CGM) is hence a potential new source of LPP. For this purpose, CGM was fermented with a Lactobacillus helveticus strain and the medium composition was optimized; the decoloration of the resultant hydrolysate was investigated as well., Results: LPP could be successfully released from CGM by fermentation with the strain Lactobacillus helveticus CICC 22536. The highest LPP content and protein recovery of 561 mg kg -1 and 14.92% occurred in the medium containing 20 g L -1 glucose, 15 g L -1 beef extract, 60 g L -1 CGM, 10 g L -1 CaCO 3 , 0.5 g L -1 NaCl, and inoculation amount 6%. The supplementation of Flavourzyme® further improved the two parameters to 662 mg kg -1 and 36.94%, respectively. The permeate of the hydrolysate after ultrafiltration through a 5 kDa membrane could be effectively decolored by the macroporous resin XAD-16 without notable protein loss, and its LPP content was further boosted to 743 mg kg -1 ., Conclusion: CGM is a potential new source of LPP and its ultrafiltered and decolored hydrolysate could be used to develop new antihypertensive functional foods. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

A simple sensor was developed for the colorimetric determination of captopril (CPT). Herein, hierarchical hollow MnO 2 microspheres (HH-MnO 2 ) were applied as nanozymes with peroxidase-mimetic activity. Free cation radicals with a strong absorption signal (λ max at 653 nm) were generated via a redox reaction between 3, 3', 5, 5'-tetramethylbenzidine (TMB) and HH-MnO 2 . Captopril could successfully prevent the generation of blue-colored free cation radicals. The influence of CPT concentration on the absorption of the generated radicals was monitored by UV-Vis spectroscopy. The corresponding linear concentration range was from 1.0 to 30.0 μg mL -1 (4.6-138.1 μmol L -1 ), and the detection limit was found to be 0.26 μg mL -1 (1.2 μmol L -1 ). As a practical usage, the developed sensor was effectively utilized to measure the content of CPT in pharmaceutical formulations., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Channa striatus is high-protein food with many health functions. This study aimed to prepare, screen and identify the angiotensin-converting enzyme inhibition peptides (ACEIPs) from C. striatus hydrolysates by response surface methodology and bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking. The optimal conditions for preparing ACEIPs were hydrolysis temperature 55 °C, hydrolysis time 3 h, pH 9, solid-liquid ratio 1:20 g/mL, and enzyme addition 5%, the ACE inhibition and molecular weight distribution of obtained hydrolysate was 54.35% and 8770-160 Da, respectively. Seven novel ACEIPs were screened through the established high-throughput screening approach, among which, EYFR and LPGPGP showed the strongest ACE inhibition with the IC 50 value of 179.2 and 186.3 μM, respectively (P > 0.05). Molecular docking revealed that three and ten hydrogen bonds were formed between ACE and LPGPGP and EYFR, respectively, S1 and S2 were the major active pockets, but the major driving forces varied., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Distilled spent grain (DSG) is the biggest by-product in baijiu (Chinese liquor) production, releasing approximately 23.44 million tons every year. Aiming at comprehensive identification of more bioactive peptides, in this work, the new bioassay-guided proteomics and Biolynx peptide sequencer based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) were developed. Moreover, 22 peptides with angiotensin converting enzyme (ACE) inhibitory activities were identified. Seven peptides were successfully quantified using electrospray ionization with triple-quadrupole mass spectrometry (ESI-QQQ-MS) in the multiple reaction monitoring (MRM). Of these identified peptides, Pro-Arg was the most abundant (92.14 μg g -1 dry weight (DW)) and acted as a competitive inhibitor of ACE by molecular docking. Therefore, peptides from DSG can be considered as promising candidates for ACE inhibition; in addition, the new strategy for peptide sequencing can be extended to any food matrices containing peptide mixture or protein hydrolysate., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The survival kinetics of Listeria monocytogenes (9 log CFU/mL) as a post-fermentation contaminant in probiotic fermented milk (Lactobacillus acidophilus La-5, 8-9 log CFU/mL) processed with milk subjected to ohmic heating (0, 4, 6, and 8 V/cm; CONV, OH 4 , OH 6 , OH 8 , 90-95 °C/5 min) was investigated using Weibull predictive model. Additionally, the presence of bioactive compounds (antioxidant activity, inhibition of the enzymes α-glucosidase, α-amylase, and angiotensin-converting) and sensory analysis (consumer test) of probiotic fermented milks were evaluated. Overall, OH provided a decrease in the viability of Listeria monocytogenes, suitable Lactobacillus acidophilus counts, and satisfactory results in the gastrointestinal tract survival. The Weibull model presented an excellent fit to the data of all conditions. Furthermore, lower δ values (217-298 against 665 h, CONV), and increased R 2 values (0.99 against 0.98, CONV) were obtained for the OH-treated samples, emphasizing the best performance of OH data. In addition, OH improved the generation of bioactive compounds as well as the sensory acceptance. Indeed, considering functional and safety purposes, OH presented as an interesting technology to be used in milk for manufacturing probiotic fermented milk., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8-76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu 2+ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe 2+ chelating activity. In what concerns the α-amylase inhibitory activity, the IC 50 values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.

This study attempts to investigate natural angiotensin-I converting enzyme (ACE) inhibitors. Soybean protein isolated (SPI) hydrolysate (SPIH) was prepared by Alcalase from inexpensive SPI, and their ACE inhibitory peptides were obtained via membrane separation, ethanol precipitation and adsorption chromatography enrichment. Activated carbon was more suitable for peptide enrichment than eight macroporous resins. The peptide fraction yielded under optimal conditions (protein-active carbon mass ratio 2:1; adsorption pH 3.0 and time 2 h; desorption time 2 h) exhibited a 10.4 times higher ACE-inhibitory activity than SPIH. Novel peptides IY, YVVF, LVF, WMY, LVLL and FF (hydrophobicity values 10.51-12.87; activity scores 0.2373-0.999) might be the main contributors to SPIH's ACE inhibition. IY had the lowest IC50 (0.53 ± 0.02 μM). YVVF had the greatest affinity (-9.8 kcal/mol) for 2OC2 (ACE's C-domain receptor) via H-bonds. IY and WMY could be potent ACE inhibitors, and their ACE-inhibitory activities unaltered and increased after in vitro gastrointestinal digestion., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Evidence-based pharmacotherapy with carvedilol and enalapril in children suffering from heart failure is insufficient owing to limited pharmacokinetic data. Although a few data sets regarding enalapril, its metabolite enalaprilat and carvedilol in children have been published, pharmacokinetic data on carvedilol metabolites are missing. However, for both drug substances, their active metabolites contribute substantially to drug efficacy. As data can hardly be derived from adults owing to the unknown impacts of enzymatic maturation and ontogeny during childhood, customised assays are important to facilitate paediatric evidence-based pharmacotherapy. Considering ethical paediatric constraints, a low-volume liquid chromatography coupled to mass spectrometry (LC-MS/MS) assay was developed using whole blood or plasma for the quantification of enalapril, enalaprilat, carvedilol, O-desmethyl carvedilol, 4- and 5-hydroxyphenyl carvedilol as well as 3- and 8-hydroxy carvedilol. To facilitate broader applications in adults, the elderly and children, a wide calibration range-between 0.024/0.049 and 50.000 ng/ml-was achieved with good linearity (r ≥ 0.995 for all analytes). In compliance with international bioanalytical guidelines, accuracy, precision, sensitivity and internal standard normalised matrix effects were further successfully validated with the exception of those for 3-hydroxy carvedilol, which was therefore assessed semi-quantitatively. Distinct haematocrits did not impact matrix effects or recoveries when analysing whole blood. Blood-to-plasma ratios were determined for all analytes to form the basis for pharmacokinetic modelling. Finally, incurred sample reanalysis of paediatric samples confirmed the reproducibility of the developed low-volume LC-MS/MS method during study sample analysis. The assay facilitates the reliable generation of important data and contributes towards a safe drug therapy in children., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

We investigated the effects of supplementation of yogurt with elderberry juice (Sambucus nigra L.), in both natural and restructured forms, on certain technological, physicochemical, sensory, and health-promoting properties of yogurt, including antioxidant activity and inhibition of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase activity. Consumer acceptance of the yogurt-juice products was assessed. Gel-strength restructured elderberry juice retained a spherical shape and most of the juice, despite decreasing in mass from 57.2 to 50.9 g during storage. As a result, yogurt supplemented with 10 and 25% restructured elderberry juice appeared to be more desirable from a sensory and technological perspective than yogurt with natural juice. Yogurt supplemented with restructured elderberry juice had a high water-holding capacity (94.4-96.4%), exhibited no spontaneous whey syneresis, and maintained a dense consistency (up to 5,626 g). Consumer penalty analysis of the just-about-right diagnostic attributes indicated that the flavor of these yogurts may not be sufficiently refreshing. High correlation was demonstrated between ACE inhibition, ABTS, α-amylase, and α-glucosidase in yogurt supplemented with restructured juice. An in vitro gastrointestinal simulation estimated bioaccessibility of antioxidants to be in the range of 62 to 66%. This model fermented yogurt supplemented with restructured elderberry juice is a novel dairy-juice beverage that represents a new approach for the development of functional fruit yogurt beverages., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In order to evaluate differences in the peptide profile and bioactive potential in dairy products, by increasing the protein content and using proteolytic bacteria strain to enable the release of bioactive peptides, a high-protein yogurt with adjunct culture was developed. The effect of protein content, the addition of Lactobacillus helveticus LH-B02, and storage time were evaluated. The qualitative analysis of peptide profile was performed using a mass spectrometry approach (MALDI-ToF-MS), and the potential bioactivity evaluated by ACE inhibition activity. Protein content did not affect the peptide profile in yogurts, and the addition of Lactobacillus helveticus LH-B02 favored the formation of peptides recognized as bioactive, such as α S1 -CN f(24-32) and β-CN f(193-209). Increased protein content and adjunct culture addition increased the ACE inhibitory activity. The combination of both factors had no additional effect on the bioactive potential of yogurts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Jasminum is an important genus in the olive family (Oleaceae), comprising about 200 species distributed all over the world. In the current study, the methanolic extract of Jasminum grandiflorum subsp. floribundum aerial parts and its respective fractions; dichloromethane and n-butanol fractions were analyzed using ultra-performance liquid chromatography coupled to high resolution mass (UPLC-HRMS) for profiling and characterization of the plant metabolites. More than seventy metabolites were identified belonging to different classes including phenolic acids, flavonoids, secoiridoids, iridoids, lignans, fatty acids, and triterpenes. The samples were also assessed for their angiotensin-I-converting enzyme (ACE) and renin inhibitory activity along with their antioxidant potential using five complementary assays: TAC (total antioxidant capacity), DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid), FRAP (ferric reducing antioxidant power) and iron reducing power. The results revealed that the n-butanol fraction showed a potent ACE and renin inhibition as compared to Lisinopril and Aliskiren standard drugs (24.66 ± 2.41 ng/mL vs. 18.37 ± 1.21 ng/mL and 141.14 ± 5.28 ng/mL vs. 447.87 ± 3.2 ng/mL, respectively) and also a strong antioxidant activity. Interestingly, the secoiridoids, dominated metabolites detected in the n-butanol fraction, revealed the potential of them for management of the hypertension diseases. The total extract and fractions were also standardized using HPLC analysis of the major secoiridoid glycoside; oleuropein. Finally, J. grandiflorum standardized extract could be considered as a target for further studies to discover a new therapeutic anti-hypertensive drug., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To promote the nutritional and pharmacological values of four sea cucumber species ( Holothuria poli , H. tubulosa , H. arguinensis , and H. sanctori ), harvested from the Algerian coast, we aimed to study their proximate composition, fatty acid profile and angiotensin-converting enzyme (ACE) inhibitory activity. Their phospholipids were also used to elaborate nanoliposomes and to encapsulate peptides obtained from the same source. After the physico-chemical characterization of nanoliposomes and peptides, in vitro analyses were realized. The four holothurian species showed a high amount of protein (49.26-69.34%), and an impressive lipid profile of 27 fatty acids, mainly composed of polar fatty acids (91.16-93.85%), with a high polyunsaturated fatty acids (PUFA) content (50.90-71.80%), particularly eicosapentaenoic acid (EPA) (5.07-8.76%) and docosahexaenoic acid (DHA) (4.86-7.25%). A high phospholipids amount was also found (55.20-69.85%), mainly composed of phosphatidylcholine (PC) (51.48-58.56%). Their peptide fractions exhibited a high ACE inhibitory activity (IC 50 0.30 to 0.51 mg/mL). The results also showed that the nanoliposomes do not induce cytotoxicity and cell death in human MSCs and no perturbation of proliferation for all the times and the tested concentrations, as well as the combined nanoliposomes and hydrolysates (HTS) at a concentration of 0.1 mg/mL. All four sea cucumbers show potential as a new source for omega-3, omega-6, and bioactive peptides., Competing Interests: The authors declare no conflict of interest.

Addition of probiotics to yogurt with desired health benefits is gaining increasing attention. To further understand the effect of probiotic Lactobacillus plantarum on the quality and function of fermented milk, probiotic fermented milk (PFM) made with probiotic L. plantarum K25 and yogurt starter ( L. delbrueckii ssp. bulgaricus and Streptococcus thermophilus ) was compared with the control fermented milk (FM) made with only the yogurt starter. The probiotic strain was shown to survive well with a viable count of 7.1 ± 0.1 log CFU/g in the PFM sample after 21 days of storage at 4°C. The strain was shown to promote formation of volatiles such as acetoin and 2,3-butanediol with milk fragrance, and it did not cause post-acidification during refrigerated storage. Metabolomics analysis by GC-MS datasets coupled with multivariate statistical analysis showed that addition of L. plantarum K25 increased formation of over 20 metabolites detected in fermented milk, among which γ-aminobutyric acid was the most prominent. Together with several other metabolites with relatively high levels in fermented milk such as glyceric acid, malic acid, succinic acid, glycine, alanine, ribose, and 1,3-dihydroxyacetone, they might play important roles in the probiotic function of L. plantarum K25. Further assay of the bioactivity of the PFM sample showed significant ( p < 0.05) increase of ACE inhibitory activity from 22.3% at day 1 to 49.3% at day 21 of the refrigerated storage. Therefore, probiotic L. plantarum K25 could be explored for potential application in functional dairy products.

Radix Rehmanniae (RR) is the root tuber of Rehmannia glutinosa Libosch. Herein, the methanol extracts of dried RR (DRR) and processed RR (PRR) were partitioned to obtain ethyl acetate, aqueous, and n -butanol layers. The angiotensin-I converting enzyme (ACE) inhibition test indicated that the ethyl acetate extracts of DRR (DRRE) and PRR (PRRE) show better inhibitory activity. Therefore, changes in blood pressure were tested over 24 h in spontaneously hypertensive rats, with DRR showing good anti-hypertensive activity. DRRE was further subjected to column chromatography; 28 fractions were separated and tested for ACE inhibition. Ultimately, six compounds were identified by spectral analysis, and literature comparison. Specifically, ursolic acid and oleanolic acid showed better ACE inhibition than the other compounds. This study confirmed that DRR has anti-hypertensive activity. In future, DRR's potential as a health food should be further assessed.

Purification of small bioactive peptides from complex biological samples is a difficult task due to the interference of concentrated large biomolecules. In this study, a magnetic immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether (IMACM@mPEG) was prepared and applied for the rapid purification of angiotensin I-converting enzyme (ACE) inhibitory peptides from casein hydrolysate. The proposed IMACM@mPEG considerably reduced the non-specific adsorption of large proteins and exhibited improved purification efficiency towards ACE inhibitory peptides. A novel peptide with moderate ACE inhibitory activity (IC 50 value of 274 ± 5 μM) was identified as LLYQEPVLGPVR. Lineweaver-Burk plot confirmed the non-competitive inhibition pattern of LLYQEPVLGPVR. The purified peptide was digested after simulated gastrointestinal digestion and produced shorter peptides which contributed to enhanced ACE inhibitory activity. These results indicated that the IMACM@mPEG is an effective method for the prepurification of ACE inhibitory peptide and the purified peptide LLYQEPVLGPVR may have potential as nutraceutical ingredient in functional foods for hypertension treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Siraitia grosvenorii fruit (SGF) has been used as a natural sweetener and traditional medicine in China for more than two centuries. This study evaluated the effect of SGF extract supplementation (0.5%, 1%, and 2%) on the chemical, microbial and sensory properties of probiotic yogurt. The antioxidant, angiotensin-converting-enzyme inhibitory (ACE-I) and antibacterial bioactivities were determined. SGF extract supplementation improved some of the chemical and physicochemical characteristics. Probiotic yogurt with the fruit extract had significantly more Lactobacillus casei and Lactobacillus bulgaricus, whereas there was no significant effect on the number of Streptococcus thermophiles. The bioactivities were significantly increased by SGF extract supplementation. Probiotic yogurt with 2% SGF extract showed the highest antioxidant, ACE-I, and antibacterial activities, whereas the one with 1% SGF extract conferred the highest sensory attributes score. Overall, SGF extract offers a promising option as a dietary supplement to produce novel dairy products that have high nutritional and bioactivity values., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A N-doped hollow copolymer tube (NHCT) was fabricated via template-free one-pot asynchronous polymerization strategy. Discrepancies of monomer polymerization speed and their hydrophilic-hydrophobic interaction resulted in the assembly of a hollow tube having inner diameter and double wall thickness of ∼230 and 40 nm, respectively. The formation and growth mechanism of NHCT analyzed via advanced characterization revealed that the unique growth processes tuned a demarcating surface layer between inner (hydrophilic) and outer (hydrophobic) layers. The screening and recognition ability of NHCT were determined for two specific dipeptides (WW and RR) possessing great discrepancies in hydrophilicity and angiotensin converting enzyme inhibitory (ACE-I) activity. NHCT realized high adsorption capacity (1.57 mmol/g) and selectivity (∼1274) for hydrophilic dipeptide RR (low ACE-I activity) from the mixture of RR/WW. As a result, ACE-I activity for residual solution were enhanced about 4.1 times as compared to original solution from natural silkworm pupae protein hydrolysate. Awarding to these results and its facile and discerning ability, NHCT can be envisioned to be of great value for the separation of small functional peptides from a natural edible source.

This research paper addresses the hypothesis that the fortification of goat milk base with whey protein concentrate (WPC) could affect both the textural and the biofunctional properties of set-style yoghurt. The effect of fortification of goat milk base with two different WPCs on thermophilic bacteria counts, proteolysis, physical and biofunctional properties of set-style yoghurts was studied at specific sampling points throughout a 4-week storage period. Fortification and storage did not influence thermophilic counts. Physical properties were affected significantly (P < 0.05) by the composition of the protein and the mineral fraction of the WPC but not by the storage. ACE-inhibitory activity was moderate in accordance to low lactobacilli counts and lack of proteolysis. DPPH-radical scavenging activity, Fe2+-chelating activity and superoxide scavenging activity were high. At 28 d an anti-inflammatory effect was observed, which was not affected by WPC addition.

The development of botanical materials as therapeutic agents involves the meticulous assessment of safety, efficacy, and quality. Compared with small-molecule drugs, quality control of botanical drugs confronts with more significant challenges due to their inherent complexity. Current quality control methods for botanical drugs, either prevailing chemical tests or emerging biological assays, are not able to meet recent demands of multiplexing, sensitivity, and speed. Here, we propose an on-demand strategy based on a direct analysis in real time-mass spectrometry (DART-MS) platform, which is capable of simultaneously analyzing multiple constituents and bioactivities of botanical drugs. Notably, the bioactivities are assessed by a multiple-enzyme assay that adopts cleavable mass spectrometry probes as enzymatic substrates: these probes labeled with a piperazine tag make possible sensitive, multiplexed, and quantitative enzyme activity measurements. The concept is successfully demonstrated via a case study of Danshen ( Salvia miltiorrhiza ) Injection where simultaneous detection of 34 constituents and inhibitory activities on two target enzymes can be achieved in just minutes. This proof-of-concept application also gives evidence that combining MS-sensitive probes with DART-MS can provide an environmentally friendly, highly sensitive analytical approach for botanical quality control.

Angiotensin-converting enzyme inhibitory peptides were isolated and identified from milk fermented using Lactobacillus delbrueckii QS306. The peptide with the highest angiotensin-converting enzyme inhibitory activity (C5) was purified using ultrafiltration with 10 and 3 kDa molecular mass cut-off membranes, Sephadex G-15 (Sigma-Aldrich, St. Louis, MO) gel filtration chromatography, reversed-phase HPLC, and Orbitrap Elite (Thermo Fisher Scientific Inc., Waltham, MA) liquid chromatography-tandem mass spectrometry. We obtained peptide LPYPY by microbial fermentation, which was derived from κ-casein f (AA 77-81). We synthesized LPYPY using an Fmoc solid-phase synthesis method and explored the secondary structure of the pentapeptide. The half maximal inhibitory concentration for the angiotensin-converting enzyme inhibitory activity of LPYPY was 12.87 μg/mL. The results provide additional information for ongoing research and the development of functional foods having antihypertensive effects., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors., (Copyright © 2018. Published by Elsevier B.V.)

YA is an angiotensin-I-converting enzyme- (ACE-) inhibitory peptide from oyster hydrolysate with antihypertensive activity. Its antioxidant and anti-inflammatory activity were investigated in this study. YA can dose-dependently quench DPPH and ABTS radical and inhibit lipopolysaccharide-induced nitric oxide in RAW 264.7 cells. YA is a multifunctional peptide and was selected as an indicator for quality control and efficacy evaluation of oyster hydrolysate. A practical HPLC/UV assay for YA quantification was developed and validated. It was proved to be accurate and reliable, according to parameters such as specificity, linearity, precision, and accuracy. The quantity results of YA showed that the stage of enzymatic hydrolysis was a critical control point for quality control; the efficacy of oyster hydrolysate can be enhanced after digested in the gastrointestinal tract due to the release of YA by brush border peptidases. Therefore, YA from oyster hydrolysate is a potential bioactive ingredient for functional foods to combat hypertension.

The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us, being the activity ascribed to polyphenols and cyclitols like l-(+)-bornesitol. We herein evaluated the stability of the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, employing both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and showed adequate linearity (r 2  > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was categorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first report on bornesitol stability under different stress conditions and the obtained data are relevant for the development and quality control of standardized products from H. speciosa leaves., (Copyright © 2018. Published by Elsevier B.V.)

A simple, specific, and rapid kinetic study of benazepril (BNZ) hydrolysis was developed and validated using HPLC. BNZ was degraded using 0.1 N sodium hydroxide at room temperature to produce benazeprilat, which is an active metabolite of BNZ and acts as an angiotensin-converting enzyme inhibitor. Analysis was carried out using an Athena C18 column (4.6 × 250 mm, 5 µm particle size). The mobile phase consists of a mixture of phosphate buffer (pH 4.5) and acetonitrile (53 + 47, v/v) at a flow rate of 1 mL/min. UV detection was accomplished at 242 nm using moexipril as the internal standard. The method was validated according to International Conference on Harmonization guidelines, and the calibration curve was linear over the range 10-100 µg/mL, with acceptable accuracy and precision. Kinetic profiling of the hydrolysis was shown to follow pseudo-first-order kinetics. The method was applied to the assay of BNZ in combined dosage form with no interference from other ingredients. The obtained results were statistically compared with those of the official method, showing no significant difference.

Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity. Graphical abstract Workflow showing the entire peptidomic approach developed for identification of bioactive peptides in microalgae.

Cheese is a suitable matrix to deliver probiotic strains but it contains a high amount of sodium. The effect of partial substitution of NaCl by KCl and the addition of flavor enhancers (l-arginine, yeast and oregano extract) on probiotic Prato cheese was investigated after 1, 30, and 60 d of refrigerated storage (immediately after manufacturing, and during ripening and storage). Microbiological (lactic acid bacteria and probiotic Lactobacillus casei 01 counts and survival under gastrointestinal conditions), physicochemical (pH, proteolysis, fatty acids), bioactivity (antioxidant effect and angiotensin I-converting enzyme inhibitory activity), rheological, and water mobility by means of time domain low-field nuclear magnetic resonance were investigated. Significant changes in probiotic survival were observed; however, the sodium reduction and the addition of flavor enhancers did not constitute an obstacle to L. casei 01 (>10 8  CFU/g) during storage. Slight changes were observed in proteolysis, bioactivity, water mobility, texture profile, and fatty acids of the cheeses as a function of the flavor enhancer added. The sodium reduction and the supplementation of Prato cheese with probiotic cultures may be an effective alternative to the production of a potentially functional cheese., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study explored a novel strategy to develop solid-state whole-grain oats as a novel carrier of lactic acid bacteria and a nutraceutical supplement rich in ACE inhibitory peptides. Oats were fermented by Lactobacillus plantarum B1-6, Rhizopus oryzae, or a combination of L. plantarum B1-6 and R. oryzae. L. plantarum showed a much better growth performance in oats when it was combined with R. oryzae than when it was cultured alone, as evidenced by an increase in viable cell count to 9.70 log cfu g-1 after 72 h of fermentation. The coinoculated fermented oats (CFO) and the R. oryzae-fermented oats (RFO) were then selected for investigations on protein hydrolysis and on the functional properties of the released bioactive peptides. The results showed that the soluble protein contents changed from 7.05 mg g-1 to 14.43 and 10.21 mg g-1 for CFO and RFO, respectively. However, the degree of hydrolysis and the content of peptides with molecular masses less than 10 000 Da indicated that the CFO proteins can be degraded to a greater degree. As analyzed by electrophoresis and reversed-phase high-performance liquid chromatography, the protein and peptide profiles of CFO and RFO demonstrated that the proteins from CFO were more obviously hydrolyzed and more small peptides were obtained. In addition, both CFO and RFO presented higher ACE inhibitory activities than unfermented oats, whereas the protein extracts from CFO exerted a lower IC50 value of 0.42 mg protein per mL compared with the protein extracts from the other samples. This research has broadened our knowledge on the development of whole-grain oat products as a probiotic carrier and on the difference between mixed solid-state fermentation (SSF) and fungi SSF in terms of protein degradation and the capacity to release ACE inhibitory peptides. Our approach could be used to obtain probiotic food products and probably to develop oats as a potential therapeutic ingredient targeting hypertension.

Sodium caseinate hydrolysates (NaCaH) contain biologically active peptides that can positively influence human health. However, their intense bitterness hinders their inclusion in food products. To our knowledge, no studies have investigated whether a correlation between bitterness and bioactivity exists in NaCaH, so it is not yet known what effect selective removal of bitterness has on NaCaH bioactivity. A deeper understanding of the physicochemical characteristics affecting both bitterness and bioactivity is therefore needed. The aim of this study was to use in silico analysis to elucidate the relationship between bitterness and bioactivity of the insulinogenic NaCaH. The NaCaH fractions were generated by membrane filtration and flash chromatography and were subsequently evaluated for bitterness by a sensory panel. In this present study, peptidomic and bioinformatic processing of these NaCaH fractions allowed for the identification of insulinogenic peptides as well as other literature-identified peptides in each of the fractions. The results showed that the most bitter fraction contained the highest abundance of insulinogenic peptides, whereas another bitter fraction contained the highest abundance of other literature-identified bioactive peptides exhibiting angiotensin-converting enzyme-inhibition activity. Although some bioactive peptides were identified in the least bitter fractions, the abundance of these peptides was very low. These observations show a correlation between bitter taste and bioactivity, highlighting potential complications in removing bitterness while maintaining bioactivity. However, as the most bitter fraction contained the highest abundance of insulinogenic peptides, there is potential for using a lower dose of this enriched bioactive fraction to exert health benefits. The second most bitter fraction contained a very low abundance of insulinogenic peptides and other bioactive peptides. Therefore, removal of this fraction could reduce the NaCaH product's bitterness without significantly altering overall bioactive potential., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Prunus genus fruit seeds are sources of highly angiotensin-I-converting enzyme (ACE)-inhibitory peptides. The presence of peptides IYSPH, IYTPH, IFSPR, and VAIP seems to be related to this activity but no previous work has demonstrated the direct relationship between the concentration of these peptides and the antihypertensive activity of hydrolysates. This work describes the development of a method for the quantification of these peptides in Prunus seeds hydrolysates based on capillary liquid chromatography-IT-MS/MS. The analytical characteristics of the method were evaluated through the study of the linearity, LOD, LOQ, presence of matrix interferences, precision, and recovery. The developed methodology was applied to the determination of the four peptides in seed hydrolysates from different Prunus genus fruits: peaches (7 varieties), plums (2 varieties), nectarines (3 varieties), apricots (2 varieties), cherry, and paraguayo. Peaches and plums seed hydrolysates yielded the highest concentrations of these peptides while paraguayo one showed the lowest concentrations. A high correlation between peptides concentrations was demonstrated suggesting that the four peptides could be released from the same seed proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography-mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Our study assayed angiotensin-converting enzyme (ACE) inhibitory activity and fermentation characteristics of 41 food-originated Lactobacillus casei strains in fermented milk production. Twenty-two of the tested strains produced fermented milks with a high ACE inhibitory activity of over 60%. Two strains (IMAU10408 and IMAU20411) expressing the highest ACE inhibitory activity were selected for further characterization. The heat stability (pasteurization at 63°C for 30 min, 75°C for 25 s, and 85°C for 20 s) and resistance to gastrointestinal proteases (pepsin, trypsinase, and sequential pepsin/trypsinase treatments) of the ACE inhibitory activity in the fermented milks produced with IMAU10408 and IMAU20411 were determined. Interestingly, such activity increased significantly after the heat or protease treatment. Because of the shorter milk coagulation time of L. casei IMAU20411 (vs. IMAU10408), it was selected for optimization experiments for ACE inhibitory activity production. Our results show that fermentation temperature of 37°C, inoculum density of 1 × 10 6 cfu/g, and fermentation time of 12 h were optimal for maximizing ACE inhibitory activity. Finally, the metabolite profiles of L. casei IMAU20411 after 2 and 42 h of milk fermentation were analyzed by ultra-HPLC electron spray ionization coupled with time-of-flight mass spectrometry. Nine differential abundant metabolites were identified, and 2 of them showed a strong and positive correlation with fermented milk ACE inhibitory activity. To conclude, we have identified a novel ACE inhibitory L. casei strain, which has potential for use as a probiotic in fermented milk production., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

A first of its kind, simple, rapid, and sensitive liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for quantification of perindopril and perindoprilat in both human plasma and breast milk. The analytes and internal standards (phenazone and acetyl salicylic acid) were extracted from biological matrices by protein precipitation. A Phenomenex® C-18 column was used to provide an appropriate chromatographic separation of the analytes, followed by detection with tandem mass spectrometry. Gradient chromatographic and mass spectrometric detection conditions with mobile phases (A: 5% methanol + 0.1% formic acid in water v/v, and B: 95% methanol + 0.1% formic acid in water v/v) were developed to achieve a LOQ of 0.5 ng/mL in both human plasma and milk. The method was suitable of evaluating clinical samples. The mass transition was followed as m/z 369.10/172.00 for perindopril, m/z 339.00/168.10 for perindoprilat, m/z 188.90/55.95 for phenazone, and m/z 179.04/137.02 for acetyl salicylic acid. The developed method was optimized and validated with a linear range of 0.1-200 ng/mL (r 2  = better than 0.99 for both perindopril and perindoprilat). The precision and accuracy values were within 15% CV. The overall recovery of the analytes was 80-110%. The method has good specificity and repeatability. Stability studies were conducted in both human plasma and bovine milk for up to 3 months, at the storage conditions of 25, 4, and -80 °C.

A capillary zone electrophoresis (CZE) method for the quantitation of captopril (CPT) using UV detection was developed. Influence of electrolyte concentration and system variables on electrophoretic separation was evaluated and a central composite design (CCD) was used to optimize the method. Variables investigated were pH, molarity, applied voltage and capillary length. The influence of sodium metabisulphite on the stability of test solutions was also investigated. The use of sodium metabisulphite prevented degradation of CPT over 24 hours. A fused uncoated silica capillary of 67.5cm total and 57.5 cm effective length was used for analysis. The applied voltage and capillary length affected the migration time of CPT significantly. A 20 mM phosphate buffer adjusted to pH 7.0 was used as running buffer and an applied voltage of 23.90 kV was suitable to effect a separation. The optimized electrophoretic conditions produced sharp, well-resolved peaks for CPT and sodium metabisulphite. Linear regression analysis of the response for CPT standards revealed the method was linear (R2 = 0.9995) over the range 5-70 μg/mL. The limits of quantitation and detection were 5 and 1.5 μg/mL. A simple, rapid and reliable CZE method has been developed and successfully applied to the analysis of commercially available CPT products.

The effect of partial substitution of NaCl with KCl and the flavor enhancers addition (arginine, yeast extract and oregano extract) on Probiotic Prato cheese processing with (L. casei 01, 7logCFU/mL) was investigated. Microbiological (lactic acid bacteria and probiotic counts), physicochemical (proximate composition, pH, proteolysis), bioactivity (antioxidant and angiotensin I-converting enzyme inhibitory activity), rheological (uniaxial compression and creep tests), water mobility (time domain low field magnetic resonance), microstructure (scanning electron microscopy) and sensory evaluation (consumer test) were performed. Sodium reduction and flavor enhancers addition did not constitute an obstacle to the survival of lactic and probiotic bacteria. Proximate composition, antioxidant and angiotensin I-converting enzyme inhibitory activity, and the rheological parameters were affected by the addition of flavor enhancer. No change in the fatty acid profile of cheeses was observed while good performance in the consumer test was obtained by the addition of yeast extract and oregano extract. Prato cheese can be an adequate carrier of probiotics and the addition of different flavor enhancers can contribute developing this functional product in the cheese industry., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We present an environmentally friendly method for the analysis of three angiotensin-converting enzyme inhibitors and hydrochlorothiazide simultaneously using a green micellar eluent for the first time. The chromatographic separation of enalapril maleate, lisinopril dihydrate, benazepril hydrochloride, and hydrochlorothiazide was implemented on an octadecyl silica column with a solution containing sodium dodecyl sulfate (0.12 M), 1-propyl alcohol (10% v/v), triethylamine (0.3% v/v), and H 3 PO 4 (0.02 M) at pH 3.6 as the mobile phase and UV detection at 210 nm. Validity of the method was confirmed and it exhibited good linearity within the ranges of 5.0-50.0 μg/mL for hydrochlorothiazide and 10.0-60.0 μg/mL for the three angiotensin-converting enzyme inhibitors with a limit of detection of 0.39 to 1.15 μg/mL for all the studied drugs. The developed micellar high-performance liquid chromatography method enables the quantification of the targeted angiotensin-converting enzyme inhibitors in combined tablets with hydrochlorothiazide by isocratic elution. There is no need for special precautions to prevent broadening and splitting of their chromatographic peaks. The method fulfills the society rights for safe and green analytical methods. The retention behavior of the four studied drugs was fitted to Foley's model and their association equilibria to the micelles (K AM ) and to the surface-modified stationary phase (K AS ) were calculated., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study presents information on the biochemical composition (dry weight basis), nutritional aspects, and angiotensin I-converting enzyme (ACE) inhibitory properties of selected macroalgae (Ulva compressa, Ulva rigida, Gelidium microdon, and Pterocladiella capillacea) from Azores. Moisture content was very high (83.2% to 90.0% of fresh weight). Total dietary fiber (33.7% to 41.0%) that presented a good balance of both soluble and insoluble fibers (15.5% to 19.2% and 18.2% to 21.8%, respectively) was the most abundant component in these macroalgae. Protein and ash (ranged from 15.7% to 23.4% and 10.7% to 20.7%, respectively) were the 2nd most abundant components in red and green macroalgae, respectively. Moderate soluble carbohydrate contents (14.5% to 19.8%) were found in all species. Lipid contents were low (1.0% to 4.3%), particularly in Ulva species (1.0% to 1.7%), but contained higher unsaturated fatty acids (FAs) (7.5% to 32.9% and 29.6% to 69.2% of total FA for monounsaturated fatty acid and polyunsaturated fatty acid groups, respectively) than saturated fatty acid (23.3% to 46.8% of total FA) contents. All the macroalgal proteins had high digestibility in vitro (82.2% to 89.4%, relatively to sodium caseinate), contained high quantity of essential amino acids (45.3% to 58.1% of total amino acids), but in different proportions, and were rich in aspartic and glutamic acids that together account for 17.2% to 36.2% of the total amino acids. These results suggested that regular consumption of the selected macroalgae may improve human health and revealed that they can be used for producing food supplements for human and animal nutrition and/or pharmaceuticals with potential effect on the regional economy. Furthermore, the ACE-inhibitory IC 50 values of 0.095 to 0.695 mg/mL for the <1 kDa protein hydrolysate fraction revealed a potential impact on hypertension disorder., (© 2017 Institute of Food Technologists®.)