Tryptophan-Containing Dual Neuroprotective Peptides: Prolyl Endopeptidase Inhibition and Caenorhabditis elegans Protection from β-Amyloid Peptide Toxicity
Neuroprotective peptides represent an attractive pharmacological strategy for the prevention or treatment of age-related diseases, for which there are currently few effective therapies. Lactoferrin (LF)-derived peptides (PKHs) and a set of six rationally-designed tryptophan (W)-containing heptapeptides (PACEIs) were characterized as prolyl endopeptidase (PEP) inhibitors, and their effect on β-amyloid peptide (Aβ) toxicity in a Caenorhabditis elegans model of Alzheimer’s disease (AD) was evaluated. Two LF-derived sequences, PKH8 and PKH11, sharing a W at the C-terminal end, and the six PACEI heptapeptides (PACEI48L to PACEI53L) exhibited significant in vitro PEP inhibition. The inhibitory pe…
Unraveling the mechanisms of action of lactoferrin-derived antihypertensive peptides: ACE inhibition and beyond
Hypertension is one of the most important causes of cardiovascular and renal morbidity and mortality, and it represents a serious health problem in Western countries. Over the last few decades scientific interest in food-derived antihypertensive peptides has grown as an alternative to drugs in the control of systemic blood pressure. Most of these peptides target the angiotensin I-converting enzyme (ACE) but emerging evidence points to other antihypertensive mechanisms beyond ACE inhibition. The milk protein lactoferrin (LF) is a good source of orally active antihypertensive peptides the characterization of which, including ex vivo functional assays and in vivo approaches, shows that they mi…
In vivo antihypertensive mechanism of lactoferrin-derived peptides: Reversion of angiotensin I- and angiotensin II-induced hypertension in Wistar rats
Novel peptides with antihypertensive effects in SHR rats have previously been identified in lactoferrin (LF) hydrolysates. To investigate their in vivo antihypertensive mechanism, we have assessed the blood pressure lowering effects of two of these LF-derived peptides (RPYL and DPYKLRP) in Wistar rats subjected to either angiotensin I- or angiotensin II-induced hypertension. Blood pressure was measured by the tail-cuff method, hypertension was induced by subcutaneous infusion of angiotensins, and then captopril, valsartan or LF-derived peptides orally administered. Angiotensin I- and angiotensin II-induced hypertension were reversed by captopril and valsartan, respectively. RPYL and DPYKLRP…
Requirement of the Lactobacillus casei MaeKR two-component system for L-malic acid utilization via a malic enzyme pathway.
ABSTRACTLactobacillus caseican metabolizel-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization ofl-malic acid via MLF does not support growth, the ME pathway enablesL. caseito grow onl-malic acid. In this work, we have identified in the genomes ofL. caseistrains BL23 and ATCC 334 a cluster consisting of two diverging operons,maePEandmaeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeKandmaeR). Homologous clusters were identified inEnterococcus faecalis,Streptococcus agalactiae,Streptococcus pyogenes, andStreptococcus uberis. Our results show that ME is …
Toxicological Assessment of Recombinant Xylanase X22 in Wine
Toxicological evaluation of xylanase X(22) from Aspergillus nidulans expressed in a wine yeast strain was carried out. The safety of the X(22) intake was assessed by digestibility, bioinformatic, and mouse short-term repeated dosing studies, although X(22) shows resistance to proteolytic degradation in the gastrointestinal system, is a minority protein component (<0.5 10(-)(6) %) of the produced wine, and shows no significant amino acid sequence homology to any known food allergens. The 4-week oral toxicity study was performed in Swiss mice at a dose level of 0.01, 0.1, or 1 mg/kg/day (these dosages correlate to 8, 80, and 800 times, respectively, the enzyme amount contained in 250 mL of wi…
An antihypertensive lactoferrin hydrolysate inhibits angiotensin I-converting enzyme, modifies expression of hypertension-related genes and enhances nitric oxide production in cultured human endothelial cells
This study was aimed to explore whether an antihypertensive lactoferrin hydrolysate (LFH) can inhibit angiotensin I-converting enzyme (ACE) activity and modify the expression of genes related to hypertension in human umbilical vein endothelial cells (HUVEC). LFH induced significant inhibition of ACE activity but it did not affect ACE mRNA levels after 24 h of exposure. LFH treatment significantly affected the expression of genes encoding for proteins involved in nitric oxide pathway such as soluble guanylate cyclase 1 α3 subunit (GUCY1A3; 4.42-fold increase) and nitric oxide synthase trafficking (NOSTRIN; 2.45-fold decrease). Furthermore, expression of the PTGS2/COX-2 gene encoding prostagl…
Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an -L-Rhamnosidase of enological interest
Monoterpenes such as geraniol, linalool, and -terpineol present in grapes determine the varietal flavor properties of young quality wines made from Muscat varieties (for reviews, see references 19 and 21). Geraniol and linalool are considered to be the most important of the monoterpene alcohols, as they are present in greater concentrations and have lower flavor thresholds than other major wine monoterpenes. In particular, linalool is thought to be responsible for the grapelike aroma of wines produced from the Muscat variety. A large proportion of
Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: From the in vitro ACE assay to the spontaneously hypertensive rat
Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural subst…
Antihypertensive effects of lactoferrin hydrolyzates: Inhibition of angiotensin- and endothelin-converting enzymes
The potential of bovine lactoferrin (LF) as a source of antihypertensive peptides acting on the renin-angiotensin system (RAS) and the endothelin (ET) system as dual vasopeptidase inhibitors has been examined. For this purpose enzymatic LF hydrolyzates (LFHs) were generated by trypsin and proteinase K digestions. Permeate fractions with molecular masses lower than 3 kDa (LFH <3 kDa) were orally administered to spontaneously hypertensive rats (SHRs). Although both LFHs <3 kDa showed in vitro angiotensin I-converting enzyme (ACE)-inhibitory activity, only proteinase K LFH <3 kDa exerted an in vivo antihypertensive effect. The proteinase K LFH <3 kDa and a previously characterized pepsin LFH <…
Bovine lactoferrin pepsin hydrolysate exerts inhibitory effect on angiotensin I-converting enzyme-dependent vasoconstriction
Abstract The inhibitory effect of a pepsin hydrolysate of bovine lactoferrin (LFH) on angiotensin I-converting enzyme (ACE) has been examined using in vitro and ex vivo functional assays. In vitro assays showed a LFH inhibitory effect on ACE activity with an IC 50 value of 0.95±0.06 mg mL −1 . Ex vivo functional assays using rabbit carotid artery segments showed a LFH inhibitory effect on ACE-dependent angiotensin I-induced contraction, but not on angiotensin II-induced contraction, suggesting that the effect of LFH is not due to antagonism of receptors for angiotensin II. LFH was shown to possess ACE inhibitory effect with potential to modulate hypertension, although the possible inhibitor…
Antihypertensive properties of lactoferricin B-derived peptides.
A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex viv…
Antihypertensive effect of a bovine lactoferrin pepsin hydrolysate: Identification of novel active peptides
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Potential of Antifungal Proteins (AFPs) to Control Penicillium Postharvest Fruit Decay
Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against …
Antihypertensive Mechanism of Lactoferrin-Derived Peptides: Angiotensin Receptor Blocking Effect
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Vasoactive properties of antihypertensive lactoferrin-derived peptides in resistance vessels: Effects in small mesenteric arteries from SHR rats
Aims: Bovine lactoferrin (LF) hydrolysates and peptides identified thereof have shown antihypertensive effects in rat models, mainly but not exclusively by angiotensin-converting enzyme inhibition. In this study we aimed to assess the vasoactive effects and mechanisms of an ultrafiltered (< 3 kDa) pepsin LF hydrolysate (LFH) and a heptapeptide identified in a LF hydrolysate produced by yeast proteolysis (DPYKLRP) in peripheral resistance arteries from spontaneously hypertensive rats (SHRs). Main methods: We used a myograph system for isometric tension recording in isolated small mesenteric arteries from SHRs. Direct vasoactive effects of LFH (30–100 μg/mL) and DPYKLRP (30–100 μM) were asses…
Dairy Debaryomyces hansenii strains produce the antihypertensive casein-derived peptides LHLPLP and HLPLP
The ability of dairy Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to release the casein-derived antihypertensive sequences RYLGY, AYFYPEL, LHLPLP, HLPLP, VPP and/or IPP was examined. Yeast strains were grown in medium with casein as sole nitrogen source and the yeast casein hydrolysates (CSHs) were analysed by HPLC-MS/MS to search for the six antihypertensive sequences. Only LHLPLP and HLPLP were identified in CSHs and exclusively in D. hansenii Dh1 and Dh14 hydrolysates in which both antihypertensive sequences represented approximately 6 (CSH Dh1) and 10% (CSH Dh14) of total peptide content. In addition, a complete analysis of selected CSHs by HPLC-MS/MS …
Bioavailability of antihypertensive lactoferricin B-derived peptides: Transepithelial transport and resistance to intestinal and plasma peptidases
The transepithelial transport of the angiotensin I-converting enzyme (ACE)-inhibitory and antihypertensive lactoferricin B (LfcinB)-derived hexapeptide LfcinB20-25 (RRWQWR) and of its two main fragments RWQ and WQ were investigated using a human intestinal cell (Caco-2) monolayer. The three peptides were susceptible to the action of brush-border peptidases. Intact LfcinB20-25 was not transported across Caco-2 whereas RWQ and WQ were both absorbed through the cell monolayer. Apparent permeability (Papp) values for absorptive transport across the monolayer were 0.7×10-8cms-1 (RWQ) and 3.9×10-8cms-1 (WQ). The effect of pathway-selective inhibitors on peptide absorption suggested paracellular d…
Increasing the levels of 2-phenylethyl acetate in wine through the use of a mixed culture of Hanseniaspora osmophila and Saccharomyces cerevisiae
The impact of mixed cultures of Hanseniaspora osmophila and Saccharomyces cerevisiae with different initial yeast ratios on wine composition has been examined. The mixed culture significantly affected sugar consumption, the main enological parameters and ester concentrations, with the exception of glycerol, isoamyl acetate and diethyl succinate levels. Remarkably, in wines obtained with mixed cultures the concentration of 2-phenylethyl acetate was approximately 3- to 9-fold greater than that produced by S. cerevisiae pure culture. Moreover sensory evaluation revealed a stronger fruity character in wines fermented with mixed cultures than in control wines. Independently of the mixed culture …
Differential susceptibility of mycotoxin-producing fungi to distinct antifungal proteins (AFPs).
Abstract The global challenge to prevent fungal spoilage and mycotoxin contamination on foods and feeds require the development of new antifungal strategies. Filamentous fungi encode diverse antifungal proteins (AFPs), which offer a great potential for the control of contaminant fungi. In this study, four AFPs from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC) belonging to classes A, B and C, were tested against a representative panel of mycotoxin-producing fungi. They included a total of 38 strains representing 32 different species belonging to the genera Alternaria, Aspergillus, Byssochlamys, Fusarium and Penicillium. PeAfpA exhibited a potent antifun…
α-L-rhamnosidases: Old and New Insights
L-Rhamnose is a component of plant cell wall pectic polysaccharides (Mutter et al., 1994; Ridley et al., 2001), glycoproteins (Haruko and Haruko, 1999) and secondary metabolites such as anthocyanins (Renault et al., 1997), flavonoids (Bar-Peled et al., 1991) and triterpenoids (Friedman and McDonald, 1997). It has also been found in bacterial heteropolysaccharides (Hashimoto and Murata, 1998), rhamnolipids (Ochsner et al., 1994) and in the repeating units of the O-antigen structure of the lipopolysaccharide component of bacterial outer membranes (Chua et al., 1999). Some rhamnosides are important bioactive compounds, e.g. cytotoxic saponins (Bader et al., 1998; Yu et al., 2002), antifungal p…
In vivo angiotensin I-converting enzyme inhibition by long-term intake of antihypertensive lactoferrin hydrolysate in spontaneously hypertensive rats
Abstract We evaluated the effect of the long-term intake of a bovine lactoferrin hydrolysate enriched in low molecular weight peptides (LFH in vivo mechanism of action. Male spontaneously hypertensive rats received tap water (negative control), captopril (positive control, 76 mg/kg/day), LFH in vivo mechanism for the antihypertensive effect of long-term oral administration of LFH
Novel Antihypertensive Lactoferrin-Derived Peptides Produced by Kluyveromyces marxianus: Gastrointestinal Stability Profile and In Vivo Angiotensin I-Converting Enzyme (ACE) Inhibition
Novel antihypertensive peptides released by Kluyveromyces marxianus from bovine lactoferrin (LF) have been identified. K. marxianus LF permeate was fractionated by semipreparative high performance liquid chromatography and 35 peptides contained in the angiotensin I-converting enzyme (ACE)-inhibitory fractions were identified by using an ion trap mass spectrometer. On the basis of peptide abundance and common structural features, six peptides were chemically synthesized. Four of them (DPYKLRP, PYKLRP, YKLRP, and GILRP) exerted in vitro inhibitory effects on ACE activity and effectively decreased systolic blood pressure after oral administration to spontaneously hypertensive rats (SHRs). Stab…
Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits
Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, wit…
Dairy yeasts produce milk protein-derived antihypertensive hydrolysates
Abstract The potential of 20 dairy yeast strains belonging to Debaryomyces hansenii , Kluyveromyces lactis and Kluyveromyces marxianus species was examined for the production of milk protein-derived antihypertensive hydrolysates. For this purpose yeast strains were grown in microbiological medium with casein or lactoferrin as sole nitrogen source, and the inhibitory effects of casein and lactoferrin hydrolysates (CSHs and LFHs) on angiotensin I-converting enzyme (ACE) activity were determined. Based on the ACE-inhibitory activity, four CSHs and five LFHs were selected, and permeate fractions with molecular masses lower than 3 kDa (pCSHs and pLFHs) were obtained. In vitro ACE-inhibitory pote…
Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories
Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatu…
Lactoferricin B-derived peptides with inhibitory effects on ECE-dependent vasoconstriction
Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an…