Haohao Wu

Formation of ferric oxyhydroxide nanoparticles mediated by peptides in anchovy (Engraulis japonicus) muscle protein hydrolysate.

Nanosized iron fortificants appear to be promising and can be synthesized in a greener way using peptides as biotemplates. Anchovy is a huge underdeveloped source of muscle protein that enhances human nonheme iron absorption. This paper shows that peptides in anchovy ( Engraulis japonicus ) muscle protein hydrolysate (AMPH) mediate the formation of monodispersed ferric oxyhydroxide nanoparticles (FeONPs) with diameters of 20-40 nm above pH 3.0. Peptides in AMPH nucleate iron through carboxyl groups and crystal growth then occur as a result of condensation of carboxylate-ligated hydroxide iron centers, yielding Fe-O-Fe cross-link bonds. Monomers of FeONPs are formed after steric obstruction of further crystal growth by peptide backbones with certain lengths and further stabilized by surface-adsorbed peptides. The iron-loading capacity of peptides in AMPH is up to 27.5 mg iron/g peptide. Overall, the present study provides a greener alternative route to the synthesis of FeONPs.

Enhancement of non-heme iron absorption by anchovy (Engraulis japonicus) muscle protein hydrolysate involves a nanoparticle-mediated mechanism.

The mechanisms by which meat enhances human absorption of non-heme iron remain unknown. Recently, anchovy (Engraulis japonicus) muscle protein hydrolysate (AMPH) was found to mediate the formation of nanosized ferric hydrolysis products in vitro. The current paper evaluates the effects of AMPH on the bioavailability and the intestinal speciation of non-heme iron in rats, followed by an investigation of cellular uptake pathways of in vitro-formed AMPH-stabilized nanosized ferric hydrolysis products (ANPs) by polarized human intestinal epithelial (Caco-2) cells. The hemoglobin regeneration efficiencies in anemic rats followed the order ferric citrate (9.79 ± 2.02%) < commercial bare α-Fe2O3 nanoparticles (16.37 ± 6.65%) < mixture of ferric citrate and AMPH (40.33 ± 6.36%) ≈ ferrous sulfate (40.88 ± 7.67%) < ANPs (56.25 ± 11.35%). Percentage contents of intestinal low-molecular-weight iron in the groups of FC+AMPH, FeSO4, and ANPs were significantly lower than the corresponding hemoglobin regeneration efficiencies (P < 0.05), providing strong evidence for the involvement of nanosized iron in intestinal iron absorption from FC+AMPH, FeSO4, and ANPs. Calcein-fluorescence measurements of the labile iron pool of polarized Caco-2 cells revealed the involvement of both divalent transporter 1 and endocytosis in apical uptake of ANPs, with endocytosis dominating at acidic extracellular pH. Overall, AMPH enhancement of non-heme iron absorption involves a nanoparticle-mediated mechanism.

Quorum Sensing Involved in the Spoilage Process of the Skin and Flesh of Vacuum-Packaged Farmed Turbot (Scophthalmus maximus) Stored at 4 °C

Fish skin has both positive and negative effects on the shelf-life of the fish. This study aimed to investigate the spoilage process of the skin and flesh of refrigerated farmed turbot (Scophthalmus maximus) with vacuum packaging. Microbial community changes were analyzed by combining culture-dependent and culture-independent methods. The results indicated that the shelf-life of vacuum-packaged refrigerated turbot was 16 d; skin mucus was the interference factor of turbot quality. The culture-dependent analysis demonstrated that the total viable counts and the population of H2 S-producing bacteria, Pseudomonas spp., Enterobacteriaceae spp., and lactic acid bacteria in skin had a range of 0.45 to 1.40 log (CFU/g) higher than the microbial numbers in flesh after 16 d in storage. 16S high throughout sequencing results demonstrated that the compositions of spoilage microbes were similar in skin and flesh. Shewanella spp., followed by Carnobacterium spp., was the dominant spoilage organism at day 16. Quorum sensing (QS) signaling activity increased during the storage. Exogenous N-butanoyl-L-homoserinelactone(C4-HSL) and N-hexanoyl-Lhomoserine lactone (C6-HSL) significantly accelerated the spoilage process of refrigerated turbot, while the addition of 4, 5-Dihydroxypentane-2, 3-dione (DPD) prolonged the lag phase duration. Therefore, QS may be involved in the spoilage process of refrigerated turbot.

Spoilage of refrigerated (4 °C) Litopenaeus vannamei: cooperation between Shewanella species and contribution of cyclo‐(L‐Pro‐L‐Leu)‐dependent quorum sensing

Summary nThe spoilage potential of Shewanella putrefaciens and S.xa0baltica isolated from spoiled refrigerated Litopenaeus vannamei was evaluated by inxa0vitro assays for trimethylamine oxide reduction, extracellular hydrolytic enzymes and biofilm formation, and inxa0vivo inoculation into surface-sterilised shrimp followed by microbial, biochemical and sensory analyses during storage for 5xa0days at 4xa0°C. S.xa0baltica displayed higher spoilage potential than S.xa0putrefaciens both inxa0vitro and inxa0vivo. Shrimp co-inoculated with them had one-day shorter shelf life than those mono-inoculated, based on the results of bacterial density, volatile base nitrogen, trimethylamine, volatile organic compounds and sensory analysis, which strongly suggests cooperation of Shewanella species in shrimp spoilage. Exogenous cyclo-(L-Pro-L-Leu) boosted bacterial growth, extracellular protease and collagenase activities, and biofilm formation of S.xa0putrefaciens and S.xa0baltica at least before they entered the stationary phase, indicating that cyclo-(L-Pro-L-Leu)-dependent quorum sensing, a recently suggested communication mechanism between them, contributes to the cooperation.

Novel Natural Angiotensin Converting Enzyme (ACE)-Inhibitory Peptides Derived from Sea Cucumber-Modified Hydrolysates by Adding Exogenous Proline and a Study of Their Structure–Activity Relationship

Natural angiotensin converting enzyme (ACE)-inhibitory peptides, which are derived from marine products, are useful as antihypertensive drugs. Nevertheless, the activities of these natural peptides are relatively low, which limits their applications. The aim of this study was to prepare efficient ACE-inhibitory peptides from sea cucumber-modified hydrolysates by adding exogenous proline according to a facile plastein reaction. When 40% proline (w/w, proline/free amino groups) was added, the modified hydrolysates exhibited higher ACE-inhibitory activity than the original hydrolysates. Among the modified hydrolysates, two novel efficient ACE-inhibitory peptides, which are namely PNVA and PNLG, were purified and identified by a sequential approach combining a sephadex G-15 gel column, reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), before we conducted confirmatory studies with synthetic peptides. The ACE-inhibitory activity assay showed that PNVA and PNLG exhibited lower IC50 values of 8.18 ± 0.24 and 13.16 ± 0.39 μM than their corresponding truncated analogs (NVA and NLG), respectively. Molecular docking showed that PNVA and PNLG formed a larger number of hydrogen bonds with ACE than NVA and NLG, while the proline at the N-terminal of peptides can affect the orientation of the binding site of ACE. The method developed in this study may potentially be applied to prepare efficient ACE-inhibitory peptides, which may play a key role in hypertension management.

Spoilage of refrigerated Litopenaeus vannamei : eavesdropping on Acinetobacter acyl-homoserine lactones promotes the spoilage potential of Shewanella baltica

Shewanella baltica and Acinetobacter are among the predominant spoilage bacteria in refrigerated shrimp (Litopenaeus vannamei). S. baltica are incapable of producing acyl-homoserine lactone (AHL) quorum sensing signals, but can respond to environmental AHLs. In this paper, Acinetobacter was found to produce three AHLs, i.e. N-butanoyl-l-homoserine lactone (C4-HSL), N-(3-oxohexanoyl)-l-homoserine lactone (O-C6-HSL) and N-(3-oxooctanoyl)-l-homoserine lactone (O-C8-HSL), according to thin-layer chromatography using the bioreporter Agrobacterium tumefaciens A136. The agar diffusion and β-galactosidase assays revealed that S. baltica could eavesdrop on these three AHLs from Acinetobacter. Eavesdropping on Acinetobacter AHLs especially C4-HSL was found to boost the growth of S. baltica particularly under nutrient limiting conditions (up to 40-fold increase) in the co-culture experiments. The azocasein assay revealed that S. baltica produced fourfold more extracellular proteases in response to Acinetobacter AHLs. As demonstrated by the biofilm crystal violet staining assay and confocal laser scanning microscopy, eavesdropping also decreased the biofilm-forming capacity of Acinetobacter. By inoculation of S. baltica and Acinetobacter onto surface-sterilized shrimp, eavesdropping was found to endow a growth advantage to S. baltica in vivo, resulting in a 0.5xa0day shortened shelf life of shrimp according to total volatile basic nitrogen levels and sensory analysis. Overall, the AHL-dependent eavesdropping increased the spoilage potential of S. baltica, providing a fresh perspective on the spoilage process of refrigerated L. vannamei, and this may inspire the development of novel preservation techniques in the future to further reduce post-harvest loss of shrimp.

Non-Heme Iron Loading Capacities of Anchovy (Engraulis japonicus) Meat Fractions under Simulated Gastrointestinal Digestion

A ferric oxyhydroxide nanoparticle (FeONP)-mediated mechanism has been suggested recently for anchovy (Engraulis japonicus) meat (AM) enhancement of non-heme iron absorption. The current paper fractionates AM biomass into protein (70.67%), lipid (20.98%), and carbohydrate (i.e., glycogen and mucopolysaccharide, 1.07%) and evaluates their capacities in templating the formation of FeONPs under simulated gastrointestinal digestion. Results show that their iron-loading capacities (mg/g) follow the ascending order glycogen (2.43 ± 0.65), protein (20.16 ± 0.56), AM (28.19 ± 0.86), lipid (33.60 ± 1.12), and mucopolysaccharide (541.33 ± 32.33). Protein and lipid act in synergy to contribute the overwhelming majority (about 90%) of AMs iron-loading capacity. l-α-Phosphatidylcholine and l-α-lysophosphatidylcholine are the predominant iron-loading fractions in the lipid digest. Dynamic light scattering and transmission electron microscopy exhibit coating of inorganic cores of the formed FeONPs with peptides or phospholipid-based mixed micelles. Overall, protein and phospholipid are key players in the nanoparticle-mediated meat factor mechanism.

Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro

Probiotic-derived polyphosphates have attracted interest as potential therapeutic agents to improve intestinal health. The current study discovered the intracellular accumulation of polyphosphates in a marine cyanobacterium Synechococcus sp. PCC 7002 as nano-sized granules. The maximum accumulation of polyphosphates in Synechococcus sp. PCC 7002 was found at the late logarithmic growth phase when the medium contained 0.74 mM of KH2PO4, 11.76 mM of NaNO3, and 30.42 mM of Na2SO4. Biogenic polyphosphate nanoparticles (BPNPs) were obtained intact from the algae cells by hot water extraction, and were purified to remove the organic impurities by Sephadex G-100 gel filtration. By using 100 kDa ultrafiltration, BPNPs were fractionated into the larger and smaller populations with diameters ranging between 30–70 nm and 10–30 nm, respectively. 4′,6-diamidino-2-phenylindole fluorescence and orthophosphate production revealed that a minor portion of BPNPs (about 14–18%) were degraded during simulated gastrointestinal digestion. In vitro studies using lipopolysaccharide-activated RAW264.7 cells showed that BPNPs inhibited cyclooxygenase-2, inducible nitric oxide (NO) synthase expression, and the production of proinflammatory mediators, including NO, tumor necrosis factor-α, interleukin-6, and interleukin-1β through suppressing the Toll-like receptor 4/NF-κB signaling pathway. Overall, there is promise in the use of the marine cyanobacterium Synechococcus sp. PCC 7002 to produce BPNPs, an anti-inflammatory postbiotic.

A Novel Natural Influenza A H1N1 Virus Neuraminidase Inhibitory Peptide Derived from Cod Skin Hydrolysates and Its Antiviral Mechanism

In this paper, a novel natural influenza A H1N1 virus neuraminidase (NA) inhibitory peptide derived from cod skin hydrolysates was purified and its antiviral mechanism was explored. From the hydrolysates, novel efficient NA-inhibitory peptides were purified by a sequential approach utilizing an ultrafiltration membrane (5000 Da), sephadex G-15 gel column and reverse-phase high-performance liquid chromatography (RP-HPLC). The amino acid sequence of the pure peptide was determined by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was PGEKGPSGEAGTAGPPGTPGPQGL, with a molecular weight of 2163 Da. The analysis of the Lineweacer–Burk model indicated that the peptide was a competitive NA inhibitor with Ki of 0.29 mM and could directly bind free enzymes. In addition, docking studies suggested that hydrogen binding might be the driving force for the binding affinity of PGEKGPSGEAGTAGPPGTPGPQGL to NA. The cytopathic effect reduction assay showed that the peptide PGEKGPSGEAGTAGPPGTPGPQGL protected Madin–Darby canine kidney (MDCK) cells from viral infection and reduced the viral production in a dose-dependent manner. The EC50 value was 471 ± 12 μg/mL against H1N1. Time-course analysis showed that PGEKGPSGEAGTAGPPGTPGPQGL inhibited influenza virus in the early stage of the infectious cycle. The virus titers assay indicated that the NA-inhibitory peptide PGEKGPSGEAGTAGPPGTPGPQGL could directly affect the virus toxicity and adsorption by host cells, further proving that the peptide had an anti-viral effect with multiple target sites. The activity of NA-inhibitory peptide was almost inactivated during the simulated in vitro gastrointestinal digestion, suggesting that oral administration is not recommended. The peptide PGEKGPSGEAGTAGPPGTPGPQGL acts as a neuraminidase blocker to inhibit influenza A virus in MDCK cells. Thus, the peptide PGEKGPSGEAGTAGPPGTPGPQGL has potential utility in the treatment of the influenza virus infection.

Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC 7002 Exhibit Intestinal Protective Potential in Human Intestinal Epithelial Cells In Vitro and Murine Small Intestine Ex Vivo

Polyphosphates are one of the active compounds from probiotics to maintain gut health. The current research extracted and purified intact biogenic polyphosphate nanoparticles (BPNPs) from Synechococcus sp. PCC 7002 cells. BPNPs were near-spherical anionic particles (56.9 ± 15.1 nm) mainly composed of calcium and magnesium salt of polyphosphate and were colloidally stable at near-neutral and alkaline pH. BPNPs survived gastrointestinal digestion in mice and could be absorbed and transported by polarized Caco-2 cell monolayers. They dose-dependently increased the tightness of intercellular tight junction and the expression of claudin-4, occludin, zonula occludens-1, and heat shock protein 27 in Caco-2 cell monolayers. BPNPs also effectively attenuated H2O2-induced cell death, plasma membrane impairment, and intracellular superoxide production in NCM460 cells. In addition, they conferred resistance to H2O2-induced barrier disruption in freshly excised mouse small intestine. Our results suggest that BPNPs are a promising postbiotic nanomaterial with potential applications in gut health maintenance.