Zunying Liu

A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate

Body wall protein from the sea cucumber (Acaudina molpadioidea) was hydrolyzed sequentially with bromelain and alcalase. The hydrolysate was fractionated into two ranges of molecular weight (PH-I, >2 kDa; PH-II, <2kDa) using an ultrafiltration membrane bioreactor system. The PH-II brought about a high angiotensin I-converting enzyme (ACE) inhibitory activity. An ACE inhibitory peptide was isolated from the PH-II, using the chromatographic methods including gel filtration, ion-exchange chromatography and reversed phase high-performance liquid chromatography. The purified ACE inhibitory peptide was a novel peptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as MEGAQEAQGD. It was found that the inhibitory activity of the peptide was intensified by 3.5 times from IC(50) 15.9 to IC(50) 4.5 microM after incubation with gastrointestinal proteases. The ACE inhibitory peptide from A. molpadioidea showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 3 microM/kg.

The involvement of bacterial quorum sensing in the spoilage of refrigerated Litopenaeus vannamei

Quorum-sensing signals in refrigerated shrimp (Litopenaeus vannamei) undergoing spoilage were examined using bioreporter assays, thin-layer chromatography and gas chromatography-mass spectrometry, and the results revealed the presence of three types of autoinducers including acetylated homoserine lactones (AHLs) (i.e., N-hexanoyl-homoserine lactone, N-oxohexanoyl-homoserine lactone and N-octanoyl-homoserine lactone), autoinducer-2, and cyclic dipeptides (i.e., cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe)). Autoinducer-2, rather than any AHL, was detected in extracts from pure cultures of the specific spoilage organisms (SSO), i.e., Shewanella putrefaciens (SS01) and Shewanella baltica (SA02). As for the cyclic peptides, only SA02 was determined to produce cyclo-(L-Pro-L-Leu). According to the transcription levels of LuxR (the master quorum-sensing regulator) in the SSO in response to exogenous autoinducers, the SSO could sense AHLs and cyclo-(L-Leu-L-Leu), rather than autoinducer-2, cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). In accordance with the results of LuxR expression, the production of biofilm matrixes and extracellular proteases in the SSO was regulated by exogenous AHLs and cyclo-(L-Pro-L-Leu), rather than 4,5-dihydroxy-2,3-pentanedione (the autoinducer-2 precursor), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). Exogenous N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) increased the growth rates and population percentages of the SSO in shrimp samples under refrigerated storage, and interestingly, exogenous 4,5-dihydroxy-2,3-pentanedione also increased the population percentages of the SSO in vivo by inhibiting the growth of the competing bacteria. However, according to the levels of TVB-N and the volatile organic components in the shrimp samples, exogenous 4,5-dihydroxy-2,3-pentanedione did not accelerate the shrimp spoilage process as N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) did. In summary, our results suggest that quorum sensing involves the spoilage of refrigerated Litopenaeus vannamei.

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.

Antioxidant balance and accumulation of advanced glycation end products after the consumption of standard diets including Maillard reaction products from silver carp peptides

The aim of this research was to investigate the accumulation and excretion of AGEs including fluorescent compounds and N-ε-carboxymethyllysine (CML) in streptozotocin-induced diabetes C57BL/6J mice fed on a diet containing Maillard reaction products (MRPs) from a silver carp peptide-glucose model system at different heating temperatures for the first time, and analyze the relationship between their in vitro antioxidant activity based on DPPH and ORAC assays and in vivo oxidative stress. The in vitro antioxidant activity of MRPs positively correlated with their AGEs level and molecular weight distribution. However, the opposite results were found by malondialdehyde (MDA) and super oxide dismutase (SOD) assays that MRPs from a higher heating temperature could aggravate the oxidative stress of diabetic mice. CML accumulation in serum was aggravated, but changes of MRPs in diets hardly affected CML accumulation in the kidney and liver. Therefore, the high AGEs levels from a diet containing peptide MRPs have negative effects on the oxidative stress of diabetic mice.

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.

Assessing the Antioxidant Activity of the Ultrafiltration Fractions From Silver Carp Protein Hydrolysate by Different Antioxidant Methods

Silver carp protein hydrolysate (SH) was fractionated by 5- and 1-kDa cutoff ultrafiltration (UF) membrane, and three ultrafiltrates were obtained: SHI (>5 kDa), SHII (1–5 kDa), and SHIII (<1 kDa) which were screened for antioxidant activity by five in vitro assays. The SHIII showed higher 2, 2-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) values, inhibiting effects for liposome oxidation induced by Fe3+/VC. The SHII showed higher ferric reducing power (FRAP) and inhibiting effects for liposome oxidation induced by 2, 2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH). However, the SHI showed the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity. Therefore, the antioxidant activity of silver carp protein hydrolysates was mainly associated with their molecular weight distribution, and the antioxidant assays used to test different samples gave comparable results.

Effects of Calcium-Binding Peptide from Tilapia Scale Protein Hydrolysates on Calcium Absorption in Caco-2 Cells

ABSTRACT Fish scales are usually discarded as an industrial solid waste. In this study the fish scale protein was recovered and employed to prepare protein hydrolysates using commercial proteases. The hydrolysates were further separated into four peptide fractions (F1, F2, F3, and F4) by Sephadex G-15. Among the fractions, the F3 fraction, with the highest calcium-binding activity, was further investigated to enhance calcium uptake in Caco-2 cells. The results showed calcium uptake from F3-Ca by Caco-2 cells after 2 h of incubation was significantly higher than those of CaCl2 and tilapia scale protein hydrolysates (TSPH), respectively, and was similar to that of casein phosphopeptide (CPP). Therefore, the F3 fraction improved Ca uptake versus the CPP samples and can be employed as an alternative to CPP in the manufacture of functional foods.

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.

Development, Characterization, and Antimicrobial Activity of Gelatin/Chitosan/ZnO Nanoparticle Composite Films

ABSTRACT In order to enhance antimicrobial activity of the film, zinc oxide (ZnO) nanoparticles were incorporated in gelatin/chitosan composite film, and the physical-mechanical properties were investigated. The film-forming solution of gelatin/chitosan/ZnO films showed increased gel strength (GS), elastic modulus (G’), and viscous modulus (G’’) compared to that of the Control. Fish gelatin/chitosan/ZnO films showed lower water vapor permeability (WVP), higher glass transition temperatures (Tg), and antibacterial activities against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Vibrio parahemolyticus. Fourier transform infrared spectra (FTIR) and microstructure studies indicated gelatin, chitosan, and ZnO interactions determined the properties of the composite film.

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.