Comparative 1H NMR-Based Metabolomics of Traditional Landrace and Disease-Resistant Chili Peppers (Capsicum annuum L.).

Chili peppers (Capsicum annuum L.) are economically valuable crops belonging to the Solanaceae family and are popular worldwide because of their unique spiciness and flavor. In this study, differences in the metabolomes of landrace (Subicho) and disease-resistant pepper cultivars (Bulkala and Kaltanbaksa) widely grown in Korea are investigated using a 1H NMR-based metabolomics approach. Specific metabolites were abundant in the pericarp (GABA, fructose, and glutamine) and placenta (glucose, asparagine, arginine, and capsaicin), highlighting the distinct physiological and functional roles of these components. Both the pericarp and placenta of disease-resistant pepper cultivars contained higher levels of sucrose and hexoses and lower levels of alanine, proline, and threonine than the traditional landrace cultivar. These metabolic differences are linked to enhanced stress tolerance and the activation of defense pathways, imbuing these cultivars with improved resistance characteristics. The present study provides fundamental insights into the metabolic basis of disease resistance in chili peppers, emphasizing the importance of multi-resistant varieties to ensure sustainable agriculture and food security. These resistant varieties ensure a stable supply of high-quality peppers, contributing to safer and more sustainable food production systems.

Profile change of the volatile and non-volatile compounds in dried or baked laver by photooxidation.

Effects of light or dark storage condition on the profile changes of volatile and non-volatile compounds were evaluated in dried and baked laver for 60 days. Volatile and non-volatile compounds were analyzed using gas chromatography-mass selective detection and high-performance liquid chromatography-quadrupole-time of flight-mass spectrometry, respectively. Baked laver stored in light conditions for 60 days produced the most volatile compounds, whereas dried laver stored in the dark produced the least volatile compounds. Total 11 classes of volatile compounds were detected, including alkanes, alkenes, and ketones, with aldehydes being most abundant in dried laver stored under light. Metabolite analysis of non-volatile compounds led to the selection of 12 compounds with a higher variable importance projection (VIP) value of >1.0: 6 fatty acids (VIP 1.2-2.0), 2 flavanols (VIP 1.3-1.8), hydroxybenzoic acid (VIP 1.5), hydroxycinnamic acid (VIP 2.3), a phenolic acid ester (VIP 1.9), and phloroglucinol (VIP 1.2). Generally, levels of these compounds decreased more following storage in the light than under dark, irrespective of laver preparation. The content of linolenic acid was particularly affected by storage conditions, with light conditions causing a fourfold reduction in linolenic acid level compared with dark conditions, which could result in an increased formation of aldehydes. Gallic acid and sinapinic acid were detected in dried but not baked laver, as they are destroyed by heat treatment. Therefore, laver should be baked and stored in dark conditions to prevent the development of rancidity. PRACTICAL APPLICATION: Laver is one of the representative seaweeds, and the popularity among consumers increases. Although commercially available laver is prepared in dried or baked condition, scientific studies on the changes of metabolites, including volatile and non-volatile compounds during storage, are scarce. The results of this study can be applied to improve proper storage methods to maintain the quality of laver, which can be beneficial for consumers and food industry.

Detection of Red Pepper Powder Adulteration with Allura Red and Red Pepper Seeds Using Hyperspectral Imaging.

This study used shortwave infrared (SWIR) technology to determine whether red pepper powder was artificially adulterated with Allura Red and red pepper seeds. First, the ratio of red pepper pericarp to seed was adjusted to 100:0 (P100), 75:25 (P75), 50:50 (P50), 25:75 (P25), or 0:100 (P0), and Allura Red was added to the red pepper pericarp/seed mixture at 0.05% (A), 0.1% (B), and 0.15% (C). The results of principal component analysis (PCA) using the L, a, and b values; hue angle; and chroma showed that the pure pericarp powder (P100) was not easily distinguished from some adulterated samples (P50A-C, P75A-C, and P100B,C). Adulterated red pepper powder was detected by applying machine learning techniques, including linear discriminant analysis (LDA), linear support vector machine (LSVM), and k-nearest neighbor (KNN), based on spectra obtained from SWIR (1,000-1,700 nm). Linear discriminant analysis determined adulteration with 100% accuracy when the samples were divided into four categories (acceptable, adulterated by Allura Red, adulterated by seeds, and adulterated by seeds and Allura Red). The application of SWIR technology and machine learning detects adulteration with Allura Red and seeds in red pepper powder.

Susceptibility of emetic and enterotoxigenic Bacillus cereus grown at high temperature to disinfectants.

The prevalence of emetic strains in food products is rare; however, infection with these may be fatal to the vulnerable population. Bacterial control of the emetic Bacillus cereus strains is still unclear. This study aimed to assess the influence of high temperature on the disinfection of emetic and enterotoxigenic B. cereus. Emetic B. cereus strains survived up to 50 °C; the lag time and maximum growth rate were higher at 42 °C than those at 30 °C. Compared to enterotoxigenic B. cereus, all emetic food strains showed higher minimum inhibitory concentrations and minimum bactericidal concentrations for sodium hypochlorite and citric acid. The disinfectant susceptibility of the emetic B. cereus OS-05 strain incubated at a higher temperature did not increase and was maintained at the highest MBC value. In all emetic B. cereus strains, enterotoxin gene expression was upregulated at 42 °C and 45 °C. Increased ces gene expression was also found in emetic B. cereus strains GP-15 and OS-05, with upregulation of 128- and 820-fold at 42 °C. Thus, emetic B. cereus grown at high temperatures may resist common disinfectants of the food industry. The findings may help control B. cereus in food or the food processing industry.

Determination of Freshness of Mackerel (Scomber japonicus) Using Shortwave Infrared Hyperspectral Imaging.

Shortwave infrared (SWIR) hyperspectral imaging was applied to classify the freshness of mackerels. Total volatile basic nitrogen (TVB-N) and acid values, as chemical compounds related to the freshness of mackerels, were also analyzed to develop a prediction model of freshness by combining them with hyperspectral data. Fresh mackerels were divided into three groups according to storage periods (0, 24, and 48 h), and hyperspectral data were collected from the eyes and whole body, separately. The optimized classification accuracies were 81.68% using raw data from eyes and 90.14% using body data by multiple scatter correction (MSC) pretreatment. The prediction accuracy of TVB-N was 90.76%, and the acid value was 83.76%. These results indicate that hyperspectral imaging, as a nondestructive method, can be used to verify the freshness of mackerels and predict the chemical compounds related to the freshness.

Seaweed metabolomics: A review on its nutrients, bioactive compounds and changes in climate change.

Seaweed, an important food resource in several Asian countries, contains various metabolites, including sugars, organic acids, and amino acids; however, their content is affected by prevailing environmental conditions. This review discusses seaweed metabolomics, especially the distribution of primary and functional secondary metabolites (e.g., carotenoids, polyphenols) in seaweed. Additionally, the effects of global warming on seaweed metabolite profile changes are discussed. For example, high temperatures can increase amino acid levels in seaweeds. Overall, understanding the effects of global warming on seaweed metabolite profiles can be useful for evaluating the nutritional composition of seaweeds as food. This review provides an overview of recent applications of metabolomics in seaweed research as well as a perspective on the nutrient content and cultivation of seaweeds under climate change scenarios.

Effect of Moisture Content Difference on the Analysis of Quality Attributes of Red Pepper (Capsicum annuum L.) Powder Using a Hyperspectral System.

The variety of characteristics of red pepper makes it difficult to analyze at the production field through hyperspectral imaging. The importance of pretreatment to adjust the moisture content (MC) in the process of predicting the quality attributes of red pepper powder through hyperspectral imaging was investigated. Hyperspectral images of four types of red pepper powder with different pungency levels and MC were acquired in the visible near-infrared (VIS-NIR) and short-wave infrared (SWIR) regions. Principal component analysis revealed that the powders were grouped according to their pungency level, color value, and MC (VIS-NIR, Principal Component 1 = 95%; SWIR, Principal Component 1 = 91%). The loading plot indicated that 580-610, 675-760, 870-975, 1020-1130, and 1430-1520 nm are the key wavelengths affected by the presence of O-H and C-H bonds present in red pigments, capsaicinoids, and water molecules. The R2 of the partial least squares model for predicting capsaicinoid and free sugar in samples with a data MC difference of 0-2% was 0.9 or higher, and a difference of more than 2% in MC had a negative effect on prediction accuracy. The color value prediction accuracy was barely affected by the difference in MC. It was demonstrated that adjusting the MC is essential for capsaicinoid and free sugar analysis of red pepper.

Changes in Volatile Compounds in Short-Term High CO2-Treated 'Seolhyang' Strawberry (Fragaria × ananassa) Fruit during Cold Storage.

'Seolhyang' strawberry is harvested before it is fully ripened and treated with CO2 to extend the shelf-life. However, the volatile changes in the 'Seolhyang' strawberry after short-term CO2 treatment have not been investigated, although the volatile profile is an important quality attribute. Herein, we investigated the effect of short-term high CO2 treatment on the changes in the composition of volatile compounds in 'Seolhyang' strawberries at two ripening stages (i.e., half-red and bright-red) during cold storage using headspace solid-phase microextraction and gas chromatography-mass spectrometry. Furthermore, the effect of CO2 treatment on fruit quality with respect to the aroma was investigated. A total of 30 volatile compounds were identified. Storage increased the volatile compound concentrations, and the total concentration of volatiles in the CO2-treated strawberries was lower than that of the untreated strawberries during storage. The production of some characteristic strawberry volatiles (e.g., 4-methoxy-2,5-dimethyl-3(2H)-furanone) was inhibited in CO2-treated strawberries. However, CO2 treatment helped maintain the concentrations of hexanal and 2-hexenal, which are responsible for the fresh odor in strawberries. Interestingly, CO2 treatment suppressed the production of off-odor volatiles, acetaldehyde, and hexanoic acid during strawberry storage. Thus, short-term CO2 treatment may help maintain the fresh aroma of strawberries during cold storage.

Toxigenic Potential of Mesophilic and Psychrotolerant Bacillus cereus Isolates from Chilled Tofu.

The prevalence, toxin gene profile, antibiogram, and biofilm formation to determine the virulence potential of mesophilic and psychrotolerant Bacillus cereus (B. cereus) isolated from chilled tofu were investigated. Among 58 isolates, 21 isolates were capable of growth at 7 °C, and these isolates shared a potential hazard for food poisoning with mesophilic isolates. B. cereus harboring enterotoxin genes was more frequently found in psychrotolerant isolates than in mesophilic isolates. Thirty-seven (62.2%) mesophilic isolates and all psychrotolerant isolates carried four or more enterotoxin genes. The hemolysin BL (42.9%) and nonhemolytic enterotoxin complexes (90.5%) were found at a higher frequency in psychrotolerant isolates than in mesophilic isolates. Some B. cereus isolates showed resistance to rifampicin or clindamycin, regardless of mesophilic and psychrotolerant isolates. A total of 56% and 40% mesophilic isolates displayed the strongest biofilm formation at 40 and 42 °C, respectively. However, the biofilm formation of psychrotolerant isolates was not significantly affected by temperature. The results of this study provide new strategies for the development of bacterial control, which allows us to optimize technologies to inhibit B. cereus, including psychrotolerant isolates, in the food industry.

Transcriptomic analysis of effects of 1-methylcyclopropene (1-MCP) and ethylene treatment on kiwifruit (Actinidia chinensis) ripening.

Ethylene (ET) is a gaseous phytohormone with a crucial role in the ripening of many fruits, including kiwifruit (Actinidia spp.). Meanwhile, treatment with 1-methylcyclopropene (1-MCP), an artificial ET inhibitor delays the ripening of kiwifruit. The objective of this study was to determine the effect of ET and 1-MCP application during time-course storage of kiwifruit. In addition, we aimed to elucidate the molecular details underlying ET-mediated ripening process in kiwifruit. For this purpose, we conducted a time-course transcriptomic analysis to determine target genes of the ET-mediated maturation process in kiwifruit during storage. Thousands of genes were identified to be dynamically changed during storage and clustered into 20 groups based on the similarity of their expression patterns. Gene ontology analysis using the list of differentially expressed genes (DEGs) in 1-MCP-treated kiwifruit revealed that the identified DEGs were significantly enriched in the processes of photosynthesis metabolism and cell wall composition throughout the ripening process. Meanwhile, ET treatment rapidly triggered secondary metabolisms related to the ripening process, phenylpropanoid (e.g. lignin) metabolism, and the biosynthesis of amino acids (e.g. Phe, Cys) in kiwifruit. It was demonstrated that ET biosynthesis and signaling genes were oppositely affected by ET and 1-MCP treatment during ripening. Furthermore, we identified a ET transcription factor, AcEIL (Acc32482) which is oppositely responsive by ET and 1-MCP treatment during early ripening, potentially one of key signaling factor of ET- or 1-MCP-mediated physiological changes. Therefore, this transcriptomic study unveiled the molecular targets of ET and its antagonist, 1-MCP, in kiwifruit during ripening. Our results provide a useful foundation for understanding the molecular details underlying the ripening process in kiwifruit.

140 GHz Ultra-Long Bessel-Like Beam with Near-Wavelength Beamwidth.

The Bessel-Gauss beam has outstanding features, such as long depth of focus (DOF) and super resolution for nondestructive imaging inspection. However, most approaches for generating a nondiffractive beam have mainly focused on extending the DOF. In this study, the ultra-long high-resolution Bessel-like beam was first demonstrated in a sub-THz wave range (140 GHz). An axicon lens having an apex angle of 110° was used to generate the highly focused Bessel-like beam. To extend the depth of focus, we varied the incident beam angle on the axicon by moving the first lens distance. With the newly developed beam profiler, 3D beam profiles were acquired for characterizing in detail the beam propagation. As a result, even if the depth of focus was 72 times (154 mm) the source wavelength (2.143 mm), the focusing beamwidth was simultaneously maintained at 1.4 times (3.0 mm) the wavelength (i.e., the near-wavelength beamwidth). An ultra-long needle beam of near-wavelength size can promote the applicability of the sub-THz imaging technique in noninvasive sensing applications, such as computer tomography, materials inspection, and through-the-wall-imaging.

The Bactericidal Effect of a Combination of Food-Grade Compounds and their Application as Alternative Antibacterial Agents for Food Contact Surfaces.

Chemical antibacterials are widely used to control microbial growth but have raised concerns about health risks. It is necessary to find alternative, non-toxic antibacterial agents for the inhibition of pathogens in foods or food contact surfaces. To develop a non-toxic and "green" food-grade alternative to chemical sanitizers, we formulated a multicomponent antibacterial mixture containing Rosmarinus officinalis L., Camellia sinensis L., citric acid, and ε-polylysine and evaluated its bactericidal efficacy against Staphylococcus aureus, Escherichia coli, Bacillus cereus, Salmonella Enteritidis, and Listeria monocytogenes on food contact surfaces. A combination of the agents allowed their use at levels lower than were effective when tested individually. At a concentration of 0.25%, the multicomponent mixture reduced viable cell count by more than 5 log CFU/area, with complete inactivation 24 h after treatment. The inhibitory efficacy of the chemical antibacterial agent (sodium hypochlorite, 200 ppm) and the multicomponent antibacterial mixture (0.25%) on utensil surfaces against S. aureus, E. coli, S. Enteritidis, and L. monocytogenes were similar, but the multicomponent system was more effective against B. cereus than sodium hypochlorite, with an immediate 99.999% reduction on knife and plastic basket surfaces, respectively, and within 2 h on cutting board surfaces after treatment. A combination of these food-grade antibacterials could be a useful strategy for inhibition of bacteria on food contact surfaces while allowing use of lower concentrations of its components than are effective individually. This multicomponent food-grade antibacterial mixture may be a suitable "green" alternative to chemical sanitizers.

Effects of different storage conditions on the metabolite and microbial profiles of white rice (Oryza sativa L.).

Microbial populations in white rice (Oryza sativa L.) samples stored for 6 months in open or closed conditions were studied and their metabolite profiles analyzed using GC/MS to elucidate the relationship between storage and rice quality. Rice samples stored in open conditions at 25 °C were contaminated by Aspergillus tritici, Cladosporium cladosporioides, and Penicillium sp., whereas the control stored in closed conditions at 5 °C was mainly contaminated by Hyphopichia burtonii and A. tritici. These differences resulted in significantly different metabolite profiles. Increased mold population decreased the levels of fresh rice flavor-associated volatile metabolites and primary energy sources, but increased the levels of metabolites associated with lipid oxidation, polyols, and energy production. Thus, rice quality, especially flavor, could be significantly influenced by the increased mold population caused by open storage at 25 °C; volatile metabolites and polyols are potential indicators of rice quality.

Prevalence, Enterotoxin Genes, and Antibiotic Resistance of Bacillus cereus Isolated from Raw Vegetables in Korea.

Bacillus cereus has long been recognized as an important pathogen in foodborne poisoning worldwide. Fresh vegetables are often contaminated with enterotoxigenic B. cereus and have been implicated as a vehicle for the transmission of this bacterium. This study reports on the occurrence, virulence gene profile, and antibiotic resistance of B. cereus in fresh vegetables. Of 102 examined samples, 48 (47%) of the samples were contaminated with B. cereus (>1 log CFU/g) and 7 (6.8%) of the samples showed more than 3 log CFU/g. In total, 118 B. cereus isolates were examined for the virulence genes nheA, nheB, nheC, hblA, hblC, hblD, cytK, and entFM and for resistance to antibiotics. Of these B. cereus isolates, 70% harbored nheA, nheB, nheC, and cytK. Eighteen (80%) of 21 isolates from bell peppers possessed eight enterotoxin genes. B. cereus isolates were susceptible to imipenem, vancomycin, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, whereas 22.4% of isolates from garlic chives, 48.7% from perilla leaf, and 40.5% from romaine lettuce showed antibiotic resistance to rifampin and 6% of isolates from garlic chives exhibited resistance to tetracycline. Three isolates from garlic chives were resistant to both tetracycline and rifampin. Raw vegetables were revealed to be major sources of B. cereus containing multiple toxin genes and exhibiting antibiotic resistance. Therefore, the potential health risks of consuming these vegetables raw or undercooked should not be underestimated. This study provides basic information for monitoring the antibiotic resistance and toxigenicity of B. cereus in the food chain during vegetable distribution and for developing food safety management to reduce the contamination with and transmission of B. cereus.

Chemical and sensory profiles of makgeolli, Korean commercial rice wine, from descriptive, chemical, and volatile compound analyses.

The chemical and sensory profiles of 12 commercial samples of makgeolli, a Korean rice wine, were determined using descriptive sensory, chemical, and volatile components analyses. The sample wines were analysed for their titratable acidity, ethanol content, pH, Hunter colour value and total reducing sugars. The chemical compositions of the makgeolli samples were found to be significantly different. The volatile compounds were extracted with solid-phase microextraction and analysed by gas chromatography time-of-flight mass spectrometry. In all, 45 major volatile compounds, consisting of 33 esters, 8 alcohols, 1 aldehyde, 1 acid, 1 phenol and 1 terpene, were identified; each makgeolli sample included 28-35 volatile compounds. Based on principal component analysis of the sensory data, samples RW1, RW2, RW5, RW8 and RW12 were associated with roasted cereal, mouldy, bubbles, sweet and sour attributes; the other samples were associated with sensory attributes of yellowness, yeast, full body, turbidity, continuation, swallow, alcohol, fruit aroma and whiteness.

Buckwheat (Fagopyrum esculentum M.) sprout treated with methyl jasmonate (MeJA) improved anti-adipogenic activity associated with the oxidative stress system in 3T3-L1 adipocytes.

Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE) treated with methyl jasmonate (MeJA) significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties.

Effect of salinity stress on phenolic compounds and carotenoids in buckwheat (Fagopyrum esculentum M.) sprout.

The effect of salinity stress on the nutritional quality of buckwheat sprouts cultivated for 1, 3, 5, and 7d was investigated by analysis of the antioxidant activity and levels of phenolic compounds and carotenoids. Treatment with various concentrations of NaCl (10, 50, 100, and 200mM) resulted in an increase in the amount of phenolic compounds and carotenoids in the sprouts compared with the control (0mM). The phenolic contents of sprouts treated with 10, 50, and 100mM after 7d of cultivation were 57%, 121%, and 153%, respectively, higher than that of the control (0mM NaCl). Moreover, the accumulation of phenolic compounds was primarily caused by an increase in the levels of 4 compounds: isoorientin, orientin, rutin, and vitexin. The carotenoid content of sprouts treated with 50 and 100mM NaCl was twice higher than that of the control. In addition, the antioxidant activity of ethanol extracts of the sprouts was increased by NaCl treatment. Although the growth rate of sprouts decreased with >50mM NaCl, these results suggest that treatment of an appropriate concentration of NaCl improves the nutritional quality of sprouts, including the level of phenolic compounds, carotenoids, and antioxidant activity.

Metabolomic analysis of phenolic compounds in buckwheat (Fagopyrum esculentum M.) sprouts treated with methyl jasmonate.

The effects of exogenous methyl jasmonate (MeJA) on phytochemical production in buckwheat sprouts cultivated under dark conditions (0, 1, 3, 5, and 7 d) were investigated by metabolomic analysis, using ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF) mass spectroscopy (MS) and partial least-squares-discriminant analysis (PLS-DA). MeJA-treated and control groups showed no differences in growth but were clearly discriminated from each other on PLS-DA score plots. The metabolites contributing to the discrimination were assigned as chlorogenic acid, catechin, isoorientin, orientin, rutin, vitexin, and quercitrin, which have various health effects. Moreover, isoorientin, orientin, rutin, and vitexin were assigned as the main phytochemicals of sprouts cultivated under dark conditions. The accumulation of these metabolites caused the phenolic compound content and antioxidant activity of the sprouts to increase. Further, this study revealed that their accumulation resulted from the stimulation of the phenylpropanoid pathway by MeJA treatment. Therefore, these metabolites may be useful for better understanding the effects of MeJA on buckwheat sprout phytochemicals and contribute to improving the functional quality of the sprouts.

Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR.

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately 84 degrees for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or 103 V. vulnificus cells from pure cultured broth and 103 cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

Identification of an iron-regulated hemin-binding outer membrane protein, HupO, in Vibrio fluvialis: effects on hemolytic activity and the oxidative stress response.

In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.