The analysis of lipopolysaccharide (LPS) expression in gram negative bacteria currently relies on time-consuming and laborious extraction and detection steps. Herein, we report an extraction-free, in situ colorimetric assay based on silver deposition suitable for the detection of LPS. This method has been used to characterize LPS production in Vibrio parahaemolyticus (VP) under various environmental conditions, which was validated by SDS-PAGE silver staining and immunoblotting. Our results showed that LPS formed a brown deposit with a characteristic ag absorption peak at 430 nm after silver deposition, which was not observed for capsular polysaccharide or proteins. The silver absorption intensity exhibited a strong positive correlation with the LPS concentration (50-330 mu g/mL). Scanning electron microscopy and transmission electron microscopy confirmed the direct deposition of ag on both free LPS and the bacterial cell surface. The in situ silver staining results were consistent with those obtained using traditional methods to evaluate the LPS production of VP under different environmental conditions. This extraction-free approach facilitated the rapid, reliable assessment of LPS production levels in bacteria and LPS mutant strains, providing a practical alternative to conventional extraction-based methods

Studies exploring the potential of outer membrane protein C (OmpC) as a vaccine candidate and diagnostic antigen for Vibrio parahaemolyticus remain limited. In this study, native OmpC and a thioredoxin-fused form (TrxA-OmpC) were successfully expressed in a prokaryotic system and evaluated for their surface exposure and immunogenicity. Surface accessibility of OmpC in the presence of bacterial surface polysaccharides was investigated using antisera raised against polysaccharide-deficient mutant strains. Immune responses induced by both recombinant proteins were assessed through immunoassays, Western blotting, and serum bactericidal assays. The results demonstrated that although surface polysaccharides partially masked OmpC epitopes, OmpC remained accessible on the bacterial surface. Both OmpC and TrxA-OmpC induced strong antibody responses with high titers (27K-729K) and showed cross-reactivity with V. parahaemolyticus and two other Vibrio species. Moreover, antisera against TrxA-OmpC exhibited stronger antibody-dependent bactericidal activity at a serum dilution of 1:8, compared with 1:2 for OmpC antisera. These findings suggest that OmpC is an immunogenic surface antigen with promising in vitro bactericidal activity, although its in vivo protective efficacy requires further investigation.

Rapid detection of diverse Vibrio parahaemolyticus serotypes in seafood requires simple and reliable immunoassays. Our prior surface proteomic screening identified outer membrane protein A (OmpA) as a highly abundant and accessible surface antigen. Herein, monoclonal antibodies (mAbs) against OmpA (VPA1186) were generated and applied to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and a lateral flow immunoassay (LFIA). Among the 12 mAbs, 6D11 and 12B1 showed superior binding affinity (3.11-8.17 × 109) and higher positive-to-negative ratios (>20) when used as mAb pair. Notably, the capture mAb 6D11 showed high specificity for V. parahaemolyticus, whereas 12B1 exhibited broader cross-reactivity within the Vibrio Spp.; the Buckets effect of the mAb pair shapes the high specificity of the methods. The optimized DAS-ELISA achieved a lowest limit of detections (LODs) of 1.37 × 104 CFU/mL across multiple V. parahaemolyticus serotypes (6/6) and strains (9/9), while LFIA exhibited a visual LOD of approximately 105 CFU/g in spiked sample and 10 CFU/g after a 6-h enrichment. The ELISA and LFIA showed high specificity (93.10%, 27/29 strains) for V. parahaemolyticus. In real seafood samples analysis, this method showed high consistency (95.5%, 21/22 samples) with PCR and culture-based method. By targeting the surface-exposed, abundant, and conserved OmpA antigen, this study established an antigen-defined strategy for specific cross-serotype detection of V. parahaemolyticus in large aquatic products.

AIMS:Vibrio parahaemolyticus is a major zoonotic pathogen. Its outer membrane proteins (OMPs) are key targets for developing vaccines. This study aimed to evaluate the potential of OmpU and VP0802 as subunit vaccine candidates against Vibrio species. METHODS AND RESULTS:Unlike prior studies relying on qualitative survival endpoints and narrow strain panels, we employed serum bactericidal assays and an expanded strain panel to quantify cross-protective efficacy. The recombinant proteins OmpU and VP0802 were cloned, expressed, and purified. Their native abundance, surface exposure, immunogenicity, and protective efficacy were evaluated. Both recombinant OMPs exhibited strong binding to whole-cell murine antisera in an indirect enzyme-linked immunosorbent assay (titer 1:81 000). Confocal microscopy further confirmed high surface availability, with specific anti-OmpU and anti-VP0802 sera binding to bacterial cells. Immunization results showed that OmpU induced high-titer antisera (up to 1:243 000), accompanied by broad cross-reactivity and bactericidal activity against different V. parahaemolyticus serotypes and multiple Vibrio species, with a potent bactericidal antibody titer of 1:512. In contrast, VP0802 generated lower antibody titers (up to 1:9000), reduced immunoblot band, weaker immunofluorescence signals, and significantly reduced bactericidal activity (1:32). CONCLUSIONS:OmpU represents a promising cross-protective subunit vaccine candidate against multiple Vibrio species.

Although early-harvested Porphyra yezoensis has greater economic value, its nutritional variation from late-harvests remains unclear. Non-targeted UPLC-MS/MS and targeted LC-MS/MS analyses was used to characterize metabolic and nutritional differences between early (January) and late (April) harvests (n = 6 per group). The late-harvests exhibited significant enrichment (P < 0.05) in amino acid, lipid, organic acid, and carbohydrate metabolic pathways, accompanied by higher levels of crude protein, free amino acids, lipids, polyunsaturated fatty acids, vitamins, and crude fiber. Umami amino acids accounted for more than 64% of total free amino acids, while eicosapentaenoic acid constituted up to 60% of total fatty acids. Notably, ginsenoside Rg3 (0.94-0.97 μg/g) was detected for the first time in P. yezoensis. Although late-harvests exhibited reduced palatability due to higher fiber content and a firmer texture, it contained higher levels of several nutrients while maintaining comparable overall nutritional quality, indicating potential applications in functional foods or nutritional supplements.

The rising demand for sustainable, nutrient-rich foods has renewed interest in tempeh made from local Indonesian legumes. This study examines the quality and microbial characteristics of tempeh fermented from cowpea, jack bean, benguk, and bambara groundnut to assess their suitability for safe and standardized production. Fermentation with Rhizopus spp. increased protein digestibility, lowered antinutritional factors, and improved texture, though amino acid profiles and moisture retention varied by legume. Microbial analysis showed Rhizopus as the dominant fermenter, while background bacteria supported acidification and flavor formation. Low levels of spoilage organisms highlighted the need for hygiene, controlled fermentation, and proper packaging. Sensory results indicated that several legumes produced tempeh with aroma and texture comparable to soybean tempeh. Overall, these local legumes represent viable, sustainable soy alternatives, provided that fermentation processes are optimized and producer training and microbial monitoring are strengthened to ensure consistent quality and food system resilience.

High microbial counts present on dried laver can be of microbial quality and safety concerns. The current two-step drying, and post-drying decontamination methods had limited effects eliminating microorganisms. This work explored the efficacy of pre-drying decontamination methods for wet laver, including washing wet laver with slightly acidic electrolytic water (SAEW) or ozonated water (OW) before drying, or treating wet laver sheets with ultraviolet (UV) during early stage drying. The average APC and coliform in untreated wet laver were similar to 5.69 and 4.34 Log CFU/g. SAEW (100 ppm, 30 min), OW (1 mg/L, 30 min) and UV (5 cm, 30 min) decreased the APC and coliform of laver by 2.56 and 2.59, 2.95 and 2.84, and 1.03 and 1.22 Log CFU/g, respectively. While SAEW (100 ppm, 30 min) and OW wash (0.7 mg/L, 30 min) caused visible color changes to wet laver, the drying process (right after the treatment) mitigated the changes quite significantly. 16S sequencing results showed that OW significantly reduced Flavobacteriaceae in the Bacteroidota phylum, while the SAEW and UV significantly reduced the Bacteroidota and Proteobacteria phyla. Results showed that decontamination of wet laver before drying or during early drying stage was promising for control microbial loads on dried laver.

The color of fish during low-temperature preservation is one of the most important sensory indicators of their edible quality and shelf-life. This present review provides an in-depth look at color evaluation methods, including sensory, physical, chemical, and new color evaluation techniques. The key factors involved in discoloration of fish are elucidated in terms of major external factors such as temperature, time and processing methods, as well as internal factors such as myoglobin and lipid oxidation, ice crystal growth, and mitochondrial characteristics involved in the three major components of fish (moisture, proteins, and lipids). Meanwhile, the two most widely used methods reviewed in this paper: improved packaging methods and the addition of natural antioxidants and film coatings. This will contribute to provide a theoretical reference for analyzing the formation mechanism of fish color deterioration, and thus propose targeted regulatory strategies for fish during cryopreservation.

The microbial and physicochemical changes of hawthorn sticks in four packaging, including the traditional metallic twist tie packaging with biaxially oriented polypropylene and cast polypropylene, and vacuum packaging (VP), deoxygenated packaging (DP), and plastic packaging (PP) with polyamide/polyethylene (PA/PE) composite film, were compared during the storage at controlled temperature and relative humidity. After 60 days of storage, the control group showed an unacceptable increase in molds (0.65 log CFU/g) and maximum physicochemical losses (25

To understand better the high microbial load in dried laver (Porphyra yezoensis or nori), this study analyzed the aerobic plate count (APC), coliform count, temperature change, and microbiota of processing water, laver materials, and food contact surface (FCS) samples from three processing plants during the dried laver processing season from December 2023 to April 2024. The seawater used for the first washing had a low microbial load (APCs < 1–2.85 log CFU/g; coliform < 1 log CFU/g) and was dominated by Proteobacteria, Firmicutes, and Bacteroidota. The microbial load of fresh laver (4.21–4.76 log CFU/g) remained unchanged after seawater washing, but significantly increased after continuous shredding, sponge dehydration, first drying, and with the seasonal temperature rise. The microbiota of laver before drying was vulnerable between processing steps and seasons, but consistently shifted back to fresh laver microflora and was dominated by Flavobacteriaceae after drying. The FCSs (except for the curtain), which had a high microbial load (APCs 5.25–8.26 log CFU/g; coliform 1.52–4.84 log CFU/g) with similar microbiota to seawater, caused the secondary contamination of laver during processing. This study revealed the microbial proliferation of laver and seawater microflora in the continuous processing line with high nutrients and with the seasonal processing water temperature rise caused by the local weather, highlighting the need for routine cleaning and sanitizing, better washing of fresh laver, and low temperature control for future dried laver production.

This study investigated the production of antioxidant peptides from Porphyra yezoensis through fermentation with three strains of microorganisms: Lactiplantibacillus plantarum L13, Bacillus amyloliquefaciens MMB-02, and Saccharomyces cerevisiae A8. The crude peptides were extracted by aqueous acid precipitation and purified by Sephadex G-25 gel column to produce highly active antioxidant components with molecular weight of <4000 Da. The LC-MS/MS result revealed that the fermentation group contained more hydrophobic amino acids and oligopeptides, which were mainly originated from phycobiliproteins and algal blue proteins. Finally, the antioxidant activity of Porphyra yezoensis was determined with DPPH· and ABTS· scavenging rates of 54.87% and 57.39%, respectively. The ferric ion-reducing power (FRAP) and enzyme activities of SOD and CAT were significantly higher than those of the control group. This study provides a scientific foundation for the deep processing of striped seaweed and contributes to the theoretical understanding of synthetic antioxidant substitutes.

While dried laver serves as an important raw ingredient for seaweed snacks, the microbial quality and safety risks associated with the preparation and processing of dried laver have not been well studied. In this project, three commercial laver processing plants were sampled from 2020 to 2022. Results indicated that the current washing and two-step drying process increased the microbial loads in dried laver. The total aerobic plate count (APC), coliform, and mold counts in dried laver ranged from 7.34 to 7.91, 2.12 to 3.87, and 1.65 to 3.87 Log CFU/g respectively. The study further explored the decontamination efficacy of four post-drying treatments (three nonthermal and one thermal treatments). Dielectric barrier discharge (DBD) plasma (150 kV, 10 min), UV-C radiation (40 cm, 30 min), ozone (150 ppm, 40 min), and hot air (150 degrees C, 30 min) reduced the APC and coliform levels by 2.10 and -1.12, 0.98 and 1.03, 2.12 and -2.87, and 3.00 and 2.24 Log CFU/g respectively. Unfortunately, Ozone and hot air treatments led to significant color change and nutrient losses. Microorganisms become harder to control once dried on food, future study should explore the use of pre-drying treatments to better enhance the microbial quality and safety of dried laver.

Aeromonas hydrophila (Ah) is a zoonotic pathogen of great importance to aquaculture and human health. This study systematically evaluated the impact of salinity, sugar, ammonia nitrogen, and nitric nitrogen levels on the fitness of Ah by using Luria-Bertani (LB) broth supplemented with different concentrations of NaCl, sucrose, NH4Cl, urea, NaNO2 or NaNO3. Results showed that the static biofilm formation of Ah was higher at 28 °C compared to 37 °C (P < 0.05). At 28 °C, as the NaCl (>1 %) and sucrose levels increased, the Ah biofilm formation and the binding between Ah cells and monoclonal antibodies (mAbs, for immunodetection) decreased. Elevated ammonia nitrogen and nitric nitrogen levels generated no significant impact on Ah biofilm formation or immunodetection (P > 0.05). The expression of mAbs-targeted Omp remained unchanged under high NaCl or sucrose conditions. Further analysis showed that high sucrose conditions led to the over-expression of the extracellular polysaccharides (PS) and promoted the formation of capsule-like structures. These over-expressed PS and capsule structures might be one reason explaining the inhibited immunodetection efficacy. Results generated from this study provide crucial insights for the design of recovery and detection protocols for Ah present in food or environmental samples.

Roasted seaweed snacks are getting popular worldwide. To reveal the main origins of microorganisms present in the roasted seaweed snacks, samples of raw materials, finished products, and environment (both food-contact and nonfood-contact surfaces) were collected from four plants. The total aerobic plate (APC), coliform, and mold counts of these samples were analyzed and the selected samples were also used for 16S rRNA sequencing. Results showed that dried laver contained the highest levels of APC among all raw ingredients, ranging from 2.72 to 6.95 Log CFU/g. Crushed pumpkin seeds and almond flakes had the highest coliform (up to 5.57 Log CFU/g) and mold counts (up to 2.16 Log CFU/g). The addition of seasoning to seaweed sandwich snacks increased the total microbial loads. The roasting process decreased the APC, coliforms, and mold counts of seaweed snacks by 0.22–1.73 Log CFU/g, >0.18- >2.46 Log CFU/g, and >0.18- >0.88 Log CFU/g respectively. The 16S rRNA sequencing analysis showed that the microbiota of roasted seaweed snacks was closely correlated with that of dried laver and the microbial load of roasted sandwich seaweed was primarily from the dried laver while less from microorganisms present in the processing plant environment and line workers.

副溶血性弧菌外膜蛋白BamA是β-桶组装BAM复合物的核心成分,参与细胞外膜蛋白运送和安装过程,是潜在的新型靶标抗原,目前其在免疫检测抗原和疫苗中的潜在价值尚未有研究报道.本研究通过生物信息学软件SnapGene和Protean分析BamA的序列并筛选出多肽,采用PCR扩增出外膜蛋白BamA的基因片段,构建重组质粒pET-28a(+)-BamA;经大肠杆菌BL21诱导表达BamA重组蛋白(90 ku);多肽与BSA偶联,制备的抗原免疫BALB/c小鼠制备了血清.采用酶联免疫分析(ELISA)和蛋白质印迹法(Western blot)测定BamA蛋白及多肽免疫血清对24株弧菌的交叉反应.ELISA测定结果表明,免疫血清对BamA蛋白的效价在121 K以上,对副溶血性弧菌等弧菌的效价较弱(0.5 K);免疫印迹结果显示,BamA蛋白血清与副溶血性弧菌CICC 21617、CICC 21618、杀岩龙虾弧菌和非O1型霍乱弧菌等弧菌属细胞裂解物中90 ku左右的蛋白可发生特异性反应,而对费氏另类弧菌、嗜水气单胞菌和迟缓爱德华氏菌等非弧菌属无交叉反应.弧菌外膜蛋白BamA具有弧菌属保守性并可以诱导产生弧菌特异性抗体,多肽在菌体表面的暴露性可能受到其他抗原的影响,这给后续弧菌诊断抗原和疫苗抗原研究提供了依据.

为构建弧菌铁蛋白受体pvuA重组质粒,提高其在大肠杆菌BL21中的表达产量,优化表达条件,并为其免疫原性研究奠定基础,从副溶血弧菌基因组DNA扩增了弧菌铁蛋白受体pvuA基因,构建了重组质粒pET-28a(+)-ferric vibrioferrin receptor,转入大肠杆菌BL21并经异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达蛋白.在单因素试验的基础上,以菌体初始浓度、诱导时间、诱导温度、诱导剂浓度为自变量,菌体蛋白浓度为响应值,根据响应面法的Box-Benhnken中心设计原理,研究自变量及其交互作用对弧菌铁蛋白产量的影响,利用Design-Expert和响应面分析相结合的方法对诱导条件进行优化.IPTG诱导获得的重组蛋白以包涵体的形式存在,优化后最终确定重组弧菌铁蛋白受体pvuA最佳表达条件为菌体初始浓度OD600=0.6,诱导时间10 h,诱导温度37℃,IPTG浓度为1.0 mmol·L?1,此时包涵体沉淀中蛋白含量最高,为11.00 mg·mL?1.构建了弧菌铁蛋白受体pvuA的大肠杆菌重组表达质粒,通过优化表达条件提高了纯化蛋白产率,为研究弧菌铁蛋白受体蛋白的多克隆抗体的制备和应用奠定了基础.

The demand for roasted seaweed sandwich (Porphyra yezoensis) product has risen in recent years. The product slicing process has created a huge number of scraps that are not utilized effectively. Three lactic acid bacteria (LAB) strains were used to ferment P. yezoensis sauces in this study, including Lactobacillus fermentum, Lactobacillus casei, Streptococcus thermophilus, and the mixed strains (1:1:1, v/v). The fermentation characteristics, antioxidant capacity in vitro, sensory properties, and flavoring substances of fermented P. yezoensis sauces were analyzed. After 21 days of fermentation, all LAB strains grew well in the P. yezoensis sauces, with protease activity increased to 6.6, 9.24, 5.06, and 5.5 U/mL, respectively. Also, the flavors of P. yezoensis sauces fermented with L. casei and L. fermentum were satisfactory. On this premise, gas chromatography-mass spectrometry (GC-MS) was used to investigate the changes in gustatory compounds in P. yezoensis sauces fermented with L. casei and L. fermentum. In general, 42 and 41 volatile flavor chemicals were identified after the fermentation of L. casei and L. fermentum. Furthermore, the fermented P. yezoensis sauce possessed greater DPPH scavenging activity and ferric-reducing ability power than the unfermented P. yezoensis. Overall, the flavor and taste of P. yezoensis sauce fermented by L. casei was superior.

This study constructed the recombinant plasmid of a TonB-dependent receptor from V. parahaemolyticus and evaluated the immunogenicity of the recombinant protein in mice. The TonB-dependent receptor gene (GI: 28901321) was obtained by PCR amplification and cloned into plasmid pET-32a (+). The recombinant plasmids were transformed into Escherichia coli BL21, and the protein expression was induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The 6 × His-tagged TonB-dependent receptor inclusion bodies were purified by Ni-NTA Agarose column and renatured by gradient urea dialysis. The soluble and inclusion bodies of the TonB-dependent receptor were emulsified with Freund's adjuvant and subcutaneously injected into BALB/c mice. The serum titers with seven V. parahaemolyticus strains, eight Vibrio species, and nine other bacteria were studied by enzyme-linked immunosorbent assay and immunoblotting. The results showed that the serum homogenously bound the target protein in the V. parahaemolyticus cell lysates. The titers against the immunized protein were above 89K, while the titer against whole cells of seven V. parahaemolyticus strains ranged from 4.12K to 12.5K. However, the titers were higher for the soluble TonB-dependent receptor. The serums reacted with E. coli strains but did not cross-react with eight Vibrio species and Photobacterium damselae. These results showed that the TonB-dependent receptor proteins in this study were immunogenic, and the serums showed adequate specificity for V. parahaemolyticus. However, the availability of the TonB-dependent receptor on V. parahaemolyticus cells is probably limited.

This study aimed to investigate the antagonistic activity and mode of action of trypacidin from marine-derived Aspergillus fumigatus against Vibrio parahaemolyticus. Results indicated that the minimal inhibitory concentration and minimal bactericidal concentration of trypacidin against V. parahaemolyticus were 31.25 and 62.5 μg/mL, respectively, which was better than that of streptomycin sulfate. Trypacidin remarkably inhibited the growth of V. parahaemolyticus and had a strong destructive effect on cell wall permeability and integrity, cell membrane permeability, and morphological alterations. Its potential as an antibacterial agent for aquatic products must be further explored.

Lactic acid fermentation is a promising method for developing sandwich seaweed scraps. The objectives of this study were to investigate the effect of fermentation with Lactiplantibacillus plantarum MMB-05, Lactiplantibacillus casei FJAT-7928, mixed bacteria (1:1, v/v) and control on the physicochemical indexes, in vitro antioxidant activity, and volatile compounds of Porphyra yezoensis sauce. Sensory evaluation was also performed. The results indicated that all lactic acid bacteria strains grew well in P. yezoensis sauce after 72 h of fermentation, with the viable cell counts of L. plantarum MMB-05 exceeding 10.0 log CFU/mL, the total phenolic content increasing by 16.54%, and the lactic acid content increasing from 0 to 44.38 ± 0.11 mg/mL. Moreover, the metabolism of these strains significantly increased the content of umami, sweet and sour free amino acids in P. yezoensis sauce. The total antioxidant capacity of L. plantarum MMB-05, L. casei FJAT-7928, mix and control groups increased by 594.59%, 386.49%, 410.27%, and 287.62%, respectively. Gas chromatography-ion mobility spectrometry (GC-IMS) analysis suggested that aldehydes and ketones accounted for the largest proportion, and the relative contents of acids and alcohols in P. yezoensis sauce increased significantly after lactic acid bacteria fermentation. In addition, the analysis of dynamic principal component analysis (PCA) and fingerprinting showed that the volatile components of the four treatment methods could be significantly distinguished. Overall, the L. plantarum MMB-05 could be recommended as an appropriate starter for fermentation of sandwich seaweed scraps, which provides a fundamental knowledge for the utilization of sandwiched seaweed scraps.