Characteristics of saltiness-enhancing peptides derived from yeast proteins and elucidation of their mechanism of action by molecular docking.

This study aimed to identify saltiness-enhancing peptides from yeast protein and elucidate their mechanisms by molecular docking. Yeast protein hydrolysates with optimal saltiness-enhancing effects were prepared under conditions determined using an orthogonal test. Ten saltiness-enhancing peptide candidates were screened using an integrated virtual screening strategy. Sensory evaluation demonstrated that these peptides exhibited diverse taste characteristics (detection thresholds: 0.13-0.50 mmol/L). Peptides NKF, LGLR, WDL, NMKF, FDSL and FDGK synergistically or additively enhanced the saltiness of a 0.30% NaCl solution. Molecular docking revealed that these peptides predominantly interacted with TMC4 by hydrogen bonding, with hydrophilic amino acids from both peptides and TMC4 playing a pivotal role in their binding. Furthermore, Leu217, Gln377, Glu378, Pro474 and Cys475 were postulated as the key binding sites of TMC4. These findings establish a robust theoretical foundation for salt reduction strategies in food and provide novel insights into the potential applications of yeast proteins.

Enhancing the AI-2/LuxS quorum sensing system in Lactiplantibacillus plantarum: Effect on the elimination of biofilms grown on seafoods.

The biofilm clustered with putrefying microorganisms and seafood pathogens could cover the surface of aquatic products that pose a risk to cross-contaminating food products or even human health. Fighting biofilms triggers synchronous communication associated with microbial consortia to regulate their developmental processes, and the enhancement of the quorum sensing system in Lactiplantibacillus plantarum can serve as an updated starting point for antibiofilm-forming strategies. Our results showed that the exogenous 25 mM L-cysteine induced a significant strengthening in the AI-2/LuxS system of Lactiplantibacillus plantarum SS-128 along with a stronger bacteriostatic ability, resulting in an effective inhibition of biofilms formed by the simplified microbial consortia constructed by Vibrio parahaemolyticus and Shewanella putrefaciens grown on shrimp and squid surfaces. The accumulation of AI-2 allowed the suppression of the expression of biofilm-related genes in V. parahaemolyticus under the premise of L. plantarum SS-128 treatment, contributing to the inhibition effect. In addition, strengthening the AI-2/LuxS system is also conducive to eliminating preexisting biofilms by L. plantarum SS-128. This study suggests that the enhancement of the AI-2/LuxS system of lactic acid bacteria enables the regulation of interspecific communication within biofilms to be a viable tool to efficiently reduce and eradicate potentially harmful biofilms from aquatic product sources, opening new horizons for combating biofilms.

Exploring the effect of Lactiplantibacillus plantarum Lac 9-3 with high adhesion on refrigerated shrimp: Adhesion modeling and biopreservation evaluation.

Lactic acid bacteria (LAB) have recently become ideal candidates for developing food biopreservatives. Adhesion is critical for LAB to perform biocontrol functions in food processing and preservation. In this study, we innovatively proposed an effective adhesion evaluation model related to the surface properties of LAB to excavate a LAB strain with high adhesion on the surface of shrimp. Then, the biocontrol potential regarding the quality of refrigerated shrimp was explored, especially for protein quality. The screening of highly adherent LAB was performed using 54 LAB strains tolerant to the low temperature (4 °C) and present antimicrobial activity. Based on surface hydrophobicity, autoaggregation, and biofilm formation, a new method for predicting LAB adhesion was established by stepwise multiple linear regression. The most relevant relationship between adhesion and biofilm formation was derived from the model. Lactiplantibacillus plantarum Lac 9-3 stood out for the strongest adhesion on the shrimp surface and the highest antimicrobial activity. The preservation results showed that Lac 9-3 significantly (p < 0.05) retarded the accumulation of total volatile basic nitrogen (TVB-N) and the growth of spoilage bacteria. The damage to the texture properties of shrimp was inhibited. Meanwhile, the degradation of myofibrillar protein was alleviated, including a significant delay (p < 0.05) in sulfhydryl (SH) group reduction, surface hydrophobicity increases, and protein conformation changes. This research optimized the evaluation of the bacteria adhesion potential, providing a new idea for developing biocontrol strategies to extend the commercial life of aquatic products.

The role of AI-2/LuxS system in biopreservation of fresh refrigerated shrimp: Enhancement in competitiveness of Lactiplantibacillus plantarum for nutrients.

Quorum sensing of lactic acid bacteria, mediated by the Autoinducer-2 (AI-2)/LuxS system, positively regulates antibacterial activity, which is an effective strategy for aquatic product preservation. This study revealed that AI-2/LuxS system regulates the nutritional competitiveness of LAB by facilitating membrane transport systems in preservation of vacuum-packaged refrigerated shrimp (VPRS), using Lactiplantibacillus plantarum SS-128 wild-type and the luxS mutant strain. In VPRS preservation, organisms that cause spoilage and total volatile basic nitrogen were significantly lower in the VPRS inoculated with L. plantarum SS-128 than those inoculated with the luxS mutant strain (L. plantarum ΔluxS/SS-128) (p < 0.05). Simulations in vitro using diluted shrimp juice showed the growth inhibitory effects of wild-type strain SS-128 on the main VPRS spoilage microorganism Shewanella baltica. This could potentially be attributed to more efficient nutrient utilization, presumably mediated by AI-2/LuxS system, as revealed by interaction analysis. In support of this, in vitro nutritional competition test showed that L. plantarum SS-128 was more competitive for nutrients when cocultured with S. baltica under conditions of limited nutrient availability. Subsequently, an integrated analysis of transcriptomic and metabolomic revealed that AI-2/LuxS enables a continuous expansion of L. plantarum SS-128 by balancing energy expenditure followed by enhancing membrane transport systems, which is the main driven forces for it to occupy a favourable niche quickly. Our results showed that the AI-2/LuxS system may regulate the nutritional competitiveness of lactic acid bacteria and may be a regulatory strategy for biological preservation of aquatic products.

Amelioration of dextran sulfate sodium-induced colitis by autoinducer-2-deficient Lactiplantibacillus plantarum is mediated by anti-inflammatory effects and alleviation of dysbiosis of the gut microbiota.

Lactic acid bacteria (LAB) attenuate dextran sulfate sodium (DSS)-induced colitis in mice by restoring gut flora homeostasis and modulating the immune response. Because synchronous behavior can be controlled by autoinducer-2 (AI-2)/LuxS-mediated quorum sensing, the Caco-2 cell model and DSS-induced model in C57BL/6 mice were used to explore the unknown effects of these communications involving AI-2 among various intestinal symbiotic species. The results of the cell viability and lactate dehydrogenase leakage assays indicated that the tested strains (the wild-type strains and AI-2-deficient mutants) were characterized by equal cytoprotection from hydrogen peroxide-induced injury independently of AI-2. The results of the assays of multiple indicators and proinflammatory cytokines characteristic for the symptoms of colitis in mice showed that oral administration of AI-2-deficient mutants for 7 days was more effective in ameliorating inflammation than the treatment with the wild-type strains. The treatment with AI-2-deficient mutants enriched potential probiotics (e.g., Lactobacillaceae) and controlled the proliferation of potentially harmful bacteria (e.g., Helicobacteraceae) to achieve the transformation of intestinal flora. These mutants regulated short-chain fatty acids and the intestinal epithelial barrier, thereby promoting the maintenance of relatively favorable intestinal homeostasis. These results demonstrated that the AI-2-deficient mutants provided a more pronounced ameliorative effect on colitis in a mouse model, suggesting that the background of the LAB effect is associated with the alterations in colonic flora induced by AI-2.

AI-2/Lux-S Quorum Sensing of Lactobacillus plantarum SS-128 Prolongs the Shelf Life of Shrimp (Litopenaeus vannamei): From Myofibril Simulation to Practical Application.

Retarding the protein deterioration of shrimp during storage is important for maintaining its quality. Lactobacillus plantarum SS-128 (L. plantarum SS-128) is a biocontrol bacterium that can effectively maintain the fresh quality of food. This research establishes a myofibril simulation system and refrigerated control system to explore the impact of L. plantarum SS-128 on the quality and shelf life of refrigerated shrimp (Litopenaeus vannamei). Through the bacterial growth assay and AI-2 signal molecule measurement, the effect of the AI-2/LuxS quorum sensing (QS) system of L. plantarum SS-128 and shrimp spoilage bacteria was established. In the myofibril simulation system, a study on protein degradation (dimer tyrosine content, protein solubility, sulfhydryl content, and carbonyl content) showed that adding L. plantarum SS-128 effectively slowed protein degradation by inhibiting the growth of food pathogens. The application to refrigerated shrimp indicated that the total volatile basic nitrogen (TVB-N) value increased more slowly in the group with added L. plantarum SS-128, representing better quality. The total viable count (TVC) and pH results exhibited similar trends. This study provides theoretical support for the application of L. plantarum SS-128 in storing aquatic products.

Sous-vide cooking endows a better microstructure for hairtail (Trichiurus lepturus) than traditional cooking: Mechanisms of moisture migration.

Sous-vide cooking is a highly praised method used to cook muscle foods because of its desired effect of providing better sensory properties by maintaining texture. In this study, we further explored the effect of water on texture by revealing the mechanisms of moisture migration. Low field nuclear magnetic resonance (LF-NMR) showed that the nonflowing water in sous-vide cooking hairtail was 2.36 ± 0.33% higher than that in traditional cooking. Magnetic resonance imaging (MRI) was used to clarify the law of moisture migration induced by temperature, and the moisture migration of the sous-vide cooking hairtail was slower during the holding heating stage. The microstructure explained the change rules of the texture. The degree of change was consistent with the moisture migration level. Digitalizing analysis quantitatively verified the effect of sous-vide cooking on the hairtail microstructure. The low moisture migration rate of sous-vide cooking resulted in a less damaged microstructure of the hairtail, manifesting as a desirable texture. PRACTICAL APPLICATION: LF-NMR and MRI showed that sous-vide hairtails exhibited a lower moisture migration rate. The holding heating stage only slightly changed the microstructure of the hairtail. The digitalizing analysis confirmed the moisture migration mechanisms. Heat-induced protein denaturation was closely related to the water state.

LuxS in Lactobacillus plantarum SS-128 Improves the Texture of Refrigerated Litopenaeus vannamei: Mechanism Exploration Using a Proteomics Approach.

This study illustrated the texture changes of Shewanella baltica-inoculated Litopenaeus vannamei during refrigerated storage with the exogenous addition of Lactobacillus plantarum SS-128. The group inoculated with SS-128 had an improved texture compared with that inoculated with the luxS-mutant group (ΔluxS). Proteomics were conducted to analyze the protein alterations in L. vannamei and supernatant, respectively. During storage, many texture-related proteins, including myosin heavy chain and beta-actin, were maintained due to luxS. Some endogenous enzymes related to the energy metabolism and hydrolysis of L. vannamei were downregulated. The luxS-induced interaction with S. baltica showed significant changes in the expression of some critical enzymes and pathways. The ATP-dependent zinc metalloprotease FtsH and protease subunit HslV were downregulated, and the oxidative phosphorylation and glycosaminoglycan degradation pathways in S. baltica were inhibited, resulting in the slow deterioration of L. vannamei. By exploring the mechanism underlying SS-128-led manipulation of the metabolism of spoilage bacteria, we clarified the texture maintenance mechanism of luxS in SS-128, providing theoretical evidence for SS-128 application in food preservation.

Combined Anti-Biofilm Enzymes Strengthen the Eradicate Effect of Vibrio parahaemolyticus Biofilm: Mechanism on cpsA-J Expression and Application on Different Carriers.

Vibrio parahaemolyticus is a human foodborne pathogen, and it can form a mature biofilm on food and food contact surfaces to enhance their resistance to antibacterial agents. In this study, the effect of anti-biofilm enzymes (combined lipase, cellulase and proteinase K) on the inhibition and eradication of pathogen biofilm was evaluated. The biofilm content of V. parahaemolyticus showed the highest level at the incubation time of 24 h, and the combined enzymes significantly inhibited the biofilm's development. The biofilm's inhibition and eradication rate at an incubation time of 24 h was 89.7% and 66.9%, respectively. The confocal laser scanning microscopic images confirmed that the microcolonies' aggregation and the adhesion of biofilm were inhibited with the combined enzyme treatment. Furthermore, combined enzymes also decreased the concentration of exopolysaccharide (EPS) and disrupted the EPS matrix network, wherein the expression of the EPS-related gene, cpsA-J, was likewise suppressed. The combined enzymes showed an excellent inhibition effect of V. parahaemolyticus biofilm on different carriers, with the highest inhibition rate of 59.35% on nonrust steel plate. This study demonstrates that the combined enzyme of lipase, cellulase and proteinase K could be a novel candidate to overcome biofilm's problem of foodborne pathogens in the food industry.

Dissecting of the AI-2/LuxS Mediated Growth Characteristics and Bacteriostatic Ability of Lactiplantibacillus plantarum SS-128 by Integration of Transcriptomics and Metabolomics.

Lactiplantibacillus plantarum could regulate certain physiological functions through the AI-2/LuxS-mediated quorum sensing (QS) system. To explore the regulation mechanism on the growth characteristics and bacteriostatic ability of L. plantarum SS-128, a luxS mutant was constructed by a two-step homologous recombination. Compared with ΔluxS/SS-128, the metabolites of SS-128 had stronger bacteriostatic ability. The combined analysis of transcriptomics and metabolomics data showed that SS-128 exhibited higher pyruvate metabolic efficiency and energy input, followed by higher LDH level and metabolite overflow compared to ΔluxS/SS-128, resulting in stronger bacteriostatic ability. The absence of luxS induces a regulatory pathway that burdens the cysteine cycle by quantitatively drawing off central metabolic intermediaries. To accommodate this mutations, ΔluxS/SS-128 exhibited lower metabolite overflow and abnormal proliferation. These results demonstrate that the growth characteristic and metabolism of L. plantarum SS-128 are mediated by the AI-2/LuxS QS system, which is a positive regulator involved in food safety. It would be helpful to investigate more bio-preservation control potential of L. plantarum, especially when applied in food industrial biotechnology.

Host-guest stoichiometry affects the physicochemical properties of beta-cyclodextrin/ferulic acid inclusion complexes and films.

An inclusion system of embedding ferulic acid into ß-cyclodextrin (FACD) with different host-guest stoichiometries was prepared by a co-precipitation method. Then, the physicochemical properties and release kinetics of the FACD were evaluated. The results of thermal analysis, X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy (FTIR) proved the successful embedding of FA into the ß-cyclodextrin matrix. Four mathematical models were applied to adjust the ferulic acid release profile and identify preferential kinetics. The results of physicochemical properties confirmed the successful formation of the complexes. The loading capacity (LC) and encapsulation efficiency (EE) of the inclusion complex (1 : 0.5) were 41.0 ± 3.28 mg g-1 and 52.1 ± 2.31%, respectively, which were significantly higher than other molar ratios. The release behaviour revealed that loaded FA molecules under various host-guest stoichiometries obey different release models. While lower host-guest stoichiometry (1 : 0.5) provided desirable EE, the moderate host-guest stoichiometry (1 : 1) exhibited faster release behaviour. The FACD inclusion complex could be a promising bioactive material for food preservation.

Effects of protein oxidation, cathepsins, and various freezing temperatures on the quality of superchilled sturgeon fillets.

Many aquatic products have been stored using superchilling technology, but rarely used for the storage of sturgeon fillets. In this study, we investigated the effects of protein oxidation, cathepsin, and various freezing temperatures on the quality of superchilled sturgeon fillets. Sensory evaluation results showed that the sensory attributes of superchilled (-3 °C) sturgeon fillets were acceptable three times longer (18 days) than samples stored at refrigeration temperatures (4 °C). The sarcoplasmic protein, carbonyl, myofibrillar protein, total sulfhydryl content and the surface hydrophobicity were determined using fluorescence spectrophotometry and SDS-PAGE. Results showed that superchilling might protect myofibrillar proteins from oxidation compared to refrigeration temperatures. The activity of the three cathepsins (B, L, and H) in terms of myofibrillar, mitochondria, lysosomes, and sarcoplasm demonstrated that superchilling can inhibit cathepsins activity in sturgeon and protect its muscle structure. Microscopic observations showed that as the temperature decreased, the gap area of the muscle fibers decreased, and the deformation of cross-sectional slices was gradually reduced. In addition, the freezing rate of ice crystals produced during the freezing process influenced the muscle structure, texture, and sensory attributes. Superchilled sturgeon fillets showed good hardness, chewiness, and water retention. In conclusion, superchilling technology shows promise for its ability to extend the shelf life while maintaining the texture and sensory attributes of fresh sturgeon fillets.

Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp.

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.

Chitosan/sodium tripolyphosphate nanoparticles as efficient vehicles for enhancing the cellular uptake of fish-derived peptide.

Methodology to enhance the intestinal absorption of peptides is an important challenge due to their easily degradation and poor permeability across the intestinal epithelium. In this study, the fish-derived peptide (DGDDGEAGKIG)-loaded chitosan (CS) nanoparticles (CS/PEP-NPs) were prepared and investigated in Caco-2 monolayer model. The results indicated zeta potential of CS/PEP-NPs increased with the increase in molecular weight of CS (10-50 kDa). Transmission electron microscopy images revealed the CS/PEP-NPs were uniform spherical-shaped nanoparticles with a diameter of 50-200 nm (150 kDa). Compared to other CS/PEP-NPs, 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29 × 10-5  cm s-1 ) and cumulative amount of peptide (120 min, 2,987 ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the intracellular fluorescence intensities of fluorescein isothiocyanate-labeled peptide. These findings suggest that chitosan nanoparticles are promising carriers to promote intestinal absorption of fish-derived peptide via paracellular pathway mediated by tight junctions. PRACTICAL APPLICATIONS: Chitosans are promising carriers to promote intestinal absorption of fish-derived peptide. The 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29 × 10-5  cm s-1 ) and cumulative amount of peptide (120 min, 2,987 ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the peptide uptake by paracellular pathway. Chitosan nanoparticles can be developed as vehicles for enhancing the cellular uptake of peptide in food industry.

Improving 3-phenyllactic acid production of Lactobacillus plantarum AB-1 by enhancing its quorum-sensing capacity.

3-Phenyllactic acid (PLA) is a novel and natural antimicrobial compound. However, the concentration of PLA produced by native microbes was rather low. To enhance the production of PLA of Lactobacillus plantarum AB-1, the microcapsules of L. plantarum AB-1 cells with a high quorum-sensing capacity was established and investigated. In addition, the relation between PLA production and quorum sensing was further investigated and confirmed by adding the exogenous 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor). The results indicated that the PLA production of L. plantarum AB-1 in microencapsulated cells (MC cells) was higher than that of the free cells, and the lactate dehydrogenase activity, autoinducer-2 (AI-2) levels and the relative expression of the luxS gene were also significantly increased in MC cells (P < 0.05). In addition, the cell growth, AI-2 levels and PLA production of L. plantarum AB-1 were also significantly promoted after adding 24 µM exogenous DPD. The results suggest that the PLA production of L. plantarum was partly regulated by the AI-2/LuxS system, and microencapsulation can increase the local AI-2 level and enhance QS capacity, which are beneficial to PLA production. The results may provide a new insight and experimental basis for the industrial production of PLA.

Cooperation of lactic acid bacteria regulated by the AI-2/LuxS system involve in the biopreservation of refrigerated shrimp.

Litopenaeus vannamei is an extremely perishable food because of rapid microbial growth and chemical degradation after harvesting. Biopreservation is a food preservation technology based on the addition of "positive" bacteria to kill or prevent the growth of undesirable microorganisms. In this study, the cooperation between lactic acid bacteria (LAB) strains (Lactobacillus plantarum AB-1 and Lactobacillus casei) regulated by the AI-2/LuxS was investigated in vitro and on shrimp. The antimicrobial activity of L. plantarum AB-1 was significantly increased in the co-culture compared with the mono-culture in vitro, and the transcription of the quorum sensing luxS gene and bacteriocin regulatory operons (plnB and plnC) in L. plantarum AB-1 were also significantly increased in co-culture (P < .05), indicating cooperation and that the production of bacteriocin in L. plantarum AB-1 might be related to the LuxS/AI-2 quorum sensing (QS) system. The results were confirmed by adding the exogenous AI-2 molecule signal to L. plantarum AB-1 in vitro. In the on shrimp experiments, the spoilage organisms (mainly Shewanella baltica) in shrimp samples were significantly inhibited after co-inoculation with L. plantarum AB-1 and L. casei, and the values of total volatile basic nitrogen (TVB-N) and pH in co-inoculated shrimp were also significantly decreased (P < .05). In addition, the AI-2 activities in co-inoculated shrimp were significantly higher during refrigerated storage. The results suggest that the cooperation and bacteriocin production of lactic acid bacteria might by regulated by the AI-2/LuxS system, and the co-inoculation of L. plantarum AB-1 and L. casei in shrimp is an effective strategy for biopreservation of shrimp.

AHLs-produced bacteria in refrigerated shrimp enhanced the growth and spoilage ability of Shewanella baltica.

Shewanella baltica is the predominant bacteria in spoiled shrimp (Litopenaeus vannamei), however, the spoilage ability and the mechanism of S. baltica is still unknown. S. baltica can't produce the signal molecule of acyl-homoserine-lactones (AHLs), so the aim of this study was to investigate how wild type S. baltica SA03 (WT SA03) eavesdrop exogenous AHLs to enhance its spoilage ability through LuxR receptor. The results indicated that Aeromonas spp. (Aer), Acinetobacter spp. (Aci) and Serratia spp. (Ser) isolated from refrigerated shrimp can produce different AHLs. WT SA03 can eavesdrop the AHLs of Aer (C4-HSL), Aci (O-C6-HSL) and Ser (C6-HSL, O-C6-HSL) to enhance its growth, especially Ser. Exogenous C4-HSL and C6-HSL enhanced biofilm formation of WT SA03, and C6-HSL and O-C6-HSL enhanced thioredoxin reductase trxB mRNA expression. However, the luxR mutant of WT SA03 (ΔluxR SA03) lost or weakened the role of using environmental AHLs. In vivo experiments, the lag time of WT SA03 was shortened by 6.4 h, 6.2 h and 14.4 h by co-inoculated with Aer, Aci and Ser, respectively. The total volatile basic nitrogen (TVB-N) were significantly enhanced in the samples co-inoculated with WT SA03 and Aer (or Aci, Ser) than those of ΔluxR SA03 and Aer (or Aci, Ser) (p < 0.05). The results showed that S. baltica SA03 can utilize AHLs produced by other bacteria to enhance its growth and spoilage ability through LuxR receptor system. Quorum sensing based on AHLs of bacteria might as the potential targets for food spoilage control.

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 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-1ß 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.

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.