Use of diacetyl to reduce the load of Vibrio vulnificus in the Eastern oyster,Crassostrea virginica

Vibrio vulnificus is a highly virulent human pathogen that occurs naturally among the microflora of oysters. This organism has two portals of entry into humans, one of which is ingestion. Oysters containing V. vulnificus consumed in a raw or undercooked state often serve as a vehicle for the transmission of this organism. Previous studies conducted in our laboratory have examined various generally recognized as safe compounds and have determined that diacetyl, a component of butter, is among the most effective of these compounds in reducing loads of V. vulnificus in oysters. The purpose of this study was to further examine the role of diacetyl, along with that of depuration, in reducing loads of V. vulnificus. Shellstock oysters were treated with various concentrations of diacetyl, and we found that many of the oysters ceased pumping when diacetyl was added. The data obtained in this study indicated that treatment with diacetyl is ineffective; however, any reduction in V. vulnificus numbers may be masked when groups of oysters, some of which may not have taken up diacetyl, are sampled. We then investigated the efficacy of diacetyl in lowering levels of V. vulnificus in shucked oysters. Diacetyl was found to significantly reduce the load of V. vulnificus in shucked oysters containing natural populations. Overall, it appears that treatment with diacetyl is ineffective for shellstock oysters, although it has potential for use in reducing loads of V. vulnificus in shucked oysters.     

Effectiveness of icing as a postharvest treatment for control of Vibrio vulnificus and Vibrio parahaemolyticus in the eastern oyster (Crassostrea virginica)

The focus of this research was to investigate the efficacy of icing as a postharvest treatment for reduction of the levels of Vibrio vulnificus and Vibrio parahaemolyticus in commercial quantities of shellstock oysters. The experiments were conducted in June and August of 2006 and consisted of the following treatments: (i) on-board icing immediately after harvest; (ii) dockside icing approximately 1 to 2 h prior to shipment; and (iii) no icing (control). Changes in the levels of pathogenic Vibrio spp. during wholesale and retail handling for 2 weeks postharvest were also monitored. On-board icing achieved temperature reductions in all sacks in accordance with the National Shellfish Sanitation Program standard, but dockside icing did not meet this standard. Based on one-way analysis of variance, the only statistically significant relationship between Vibrio levels and treatment occurred for samples harvested in August; in this case, the levels of V. vulnificus in the noniced oysters were significantly higher (P < 0.05) than were the levels in the samples iced on-board. When analyzing counts over the 14-day storage period, using factorial analysis, there were statistically significant differences in V. vulnificus and V. parahaemolyticus levels by sample date and/or treatment (P < 0.05), but these relationships were not consistent. Treated (iced) oysters had significantly higher gaping (approximately 20%) after 1 week in cold storage than did noniced oysters (approximately 10%) and gaping increased significantly by day 14 of commercial storage. On-board and dockside icing did not predictably reduce the levels of V. vulnificus or V. parahaemolyticus in oysters, and icing negatively impacted oyster survival during subsequent cold storage.     

Sodium Content of Oysters (Crassostrea virginica) and the Effect of Processing Method

The issue of the sodium content of foods and the labeling of food products as to their sodium content has become the target for legislative debate. Oysters, processed by three methods currently employed within the oyster processing industry, were analyzed for sodium content using atomic absorption spectrophotometry. Significant differences in the sodium levels were observed among the processes. Indications were that the variations observed were in part due to the viability of the mollusk during processing. The data indicate that it would be a significant problem for the oyster industry to declare in the label sodium content within a reasonable limit of certainty.     

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter‐feeding molluscan shellfish exposed to sewage‐contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male‐specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log₁₀ genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.     

Effects of a commercial heat-shock process on Vibrio vulnificus in the American oyster, Crassostrea virginica, harvested from the Gulf Coast

Oysters (Crassostrea virginica) harvested from the Gulf Coast, containing 10(2) to 10(4) most probable number (MPN) per gram of Vibrio vulnificus, were subjected to a commercial heat-shock process. After 1 to 4 min at internal oyster meat temperatures exceeding 50 degrees C, shellstock oysters were shucked, chilled, washed, and packed. V. vulnificus and total bacterial levels in Gulf Coast oysters were significantly reduced from 1 to 4 logs in the finished product. Similar reductions were not observed in shellstock oysters that were subject to conventional processing. Under the National Shellfish Sanitation Program, heat shocking is an acceptable process to use to assist in the shucking of shellstock. This research revealed that the heat-shock process may also serve to significantly reduce V. vulnificus in summer Gulf Coast oysters.     

Temperature Effects on the Depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American Oyster (Crassostrea virginica)

ABSTRACT:  This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster ( Crassostrea virginica ). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10⁴ to 10⁵ MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 °C. Depuration of oysters at 22 °C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 °C. Decreasing water temperature to 15 °C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 °C. However, depurations at 10 and 5 °C were less effective than at 15 °C in reducing the Vibrio spp. in oysters. Extended depuration at 15 °C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.     

Combined effect of ozonated water and chitosan on the shelf-life of Pacific oyster (Crassostrea gigas)

Effects of ozonated water and chitosan treatment on the shelf-life extension of Pacific oysters (Crassostrea gigas) stored at 5 ± 1 °C were studied. Results indicated that ozonated water treatment reduced the total microbial load of fresh oysters by about 10-fold (from 3.2 × 10³ CFU/g to 1.8 × 10² CFU/g) before storage and the microbial flora was different with that of raw samples. The wide-spectrum antibacterial property of chitosan against bacteria isolated from oysters was confirmed, and chitosan concentration of 5.0 g/l was eventually determined for application in oyster preservation. Based on microbiological analysis, biochemical indices determination and sensory evaluation, shelf-lives of 8–9 days for control, 10–12 days for ozonated water treated samples, 14–15 days for chitosan treated samples and 20–21 days for samples with combined treatment were observed, indicating that ozonated water and chitosan have a great potential for oyster preservation.Industrial relevanceAs seafood, Pacific oysters have a short shelf-life. Improvements in the shelf-life of oysters can have an important economic impact by reducing losses and by allowing the products to reach distant and new markets.In this work, Shelf-life of oysters with combined treatment of ozonated water and chitosan doubled, which has great practical meaning, and the process could be fully adopted by the food industry.We also did some research about the changes in microbial flora after ozonated water treatment. This work could help in preservation of oysters when ozone or ozonated water concerned.We discovered Wide-spectrum antibacterial property of chitosan against the strains isolated from raw oysters. The potential for using chitosan as a natural preservative in oysters was approved.     

超高压法提取牡蛎中牛磺酸的工艺研究

本文对超高压技术提取牡蛎中牛磺酸的工艺技术进行了研究,采用高效液相色谱法(HPLC)测定牛磺酸的提取量,并与传统的热水抽提法进行比较。单因素实验结果表明:压力、保压时间、温度以及料液比均对牛磺酸的提取量有较大影响。在此基础上通过正交实验得到最佳提取工艺条件为:压力300 MPa、保压时间25 min、温度45℃、料液比为1∶5,最高提取量22.96 mg/g,高于热水抽提法的牛磺酸提取量(17.07 mg/g)。本研究为发展高新技术应用于牡蛎中牛磺酸的提取提供理论依据。      

Amino acid changes in fermented oyster (Crassostrea gigas) sauce with different fermentation periods

Fermented oyster sauce (FOS) was prepared with 25% NaCl (w/w) at 20 °C with different fermentation periods. Biochemical changes during the fermentation were investigated. Results revealed that, during fermentation, protein contents were increased whereas carbohydrate contents were decreased. Amino acid composition showed that the contents of aspartic acid, lysine, glutamic acid, glycine and alanine were higher than other amino acids. Free amino acid contents of FOS fluctuated during the fermentation period but most of the free amino acids increased. Particularly, the contents of taurine, glutamic acid, lysine, glycine and alanine were high, which may contribute to the taste and flavour of FOS.     

Elimination of fecal coliforms and F-specific RNA coliphage from oysters (Crassostrea virginica) relaid in floating containers

Declining oyster (Crassostrea virginica) production in the Chesapeake Bay has stimulated aquaculture based on floats for off-bottom culture. While advantages of off-bottom culture are significant, the increased use of floating containers raises public health and microbiological concerns, because oysters in floats may be more susceptible to fecal contamination from storm runoff compared to those cultured on-bottom. We conducted four commercial-scale studies with market-size oysters naturally contaminated with fecal coliforms (FC) and a candidate viral indicator, F-specific RNA (FRNA) coliphage. To facilitate sampling and to test for location effects, 12 replicate subsamples, each consisting of 15 to 20 randomly selected oysters in plastic mesh bags, were placed at four characteristic locations within a 0.6- by 3.0-m "Taylor" float, and the remaining oysters were added to a depth not exceeding 15.2 cm. The float containing approximately 3,000 oysters was relaid in the York River, Virginia, for 14 days. During relay, increases in shellfish FC densities followed rain events such that final mean levels exceeded initial levels or did not meet an arbitrary product end point of 50 FC/100 ml. FRNA coliphage densities decreased to undetectable levels within 14 days (16 to 28 degrees C) in all but the last experiment, when temperatures fell between 12 and 16 degrees C. Friedman (nonparametric analysis of variance) tests performed on FC/Escherichia coli and FRNA densities indicated no differences in counts as a function of location within the float. The public health consequences of these observations are discussed, and future research and educational needs are identified.     

Effects of High Hydrostatic Pressure on the Physical,Microbial, and Chemical Attributes of Oysters (Crassostrea virginica)

The change in the quality attributes (physical, microbial, and chemical) of oysters (Crassostrea virginica) after high hydrostatic pressure (HHP) treatment at 300 MPa at room temperature (RT, 25 °C) 300, 450, and 500 MPa at 0 °C for 2 min and control oysters without treatment were evaluated over 3 wk. The texture and tissue yield percentages of oysters HHP treated at 300 MPa, RT increased significantly (P < 0.05) compared to control. Aerobic and psychrotrophic bacteria in control oysters reached the spoilage point of 7 log CFU/g after 15 d. Coliform counts (log MPN/g) were low during storage with total and fecal coliforms less than 3.5 and 1.0. High pressure treated oysters at 500 MPa at 0 °C were significantly higher (P < 0.05) than oysters HHP treated at 300 MPa at 0 °C in lipid oxidation values. The highest pressure (500 MPa) treatment in this study, significantly (P < 0.05) decreased unsaturated fatty acid percentage compared to control. The glycogen content of control oysters at 3 wk was significantly higher (P < 0.05) when compared to HHP treated oysters [300 MPa, (RT); 450 MPa (0 °C); and 500 MPa (0 °C)]. HHP treatments of oysters were not significantly different in pH, percent salt extractable protein (SEP), and total lipid values compared to control. Based on our results, HHP prolongs the physical, microbial, and chemical quality of oysters.     

Preparation and in vitro antioxidant activity of enzymatic hydrolysates from oyster (Crassostrea talienwhannensis) meat

Oyster is an abundant resource from ocean, which contains high content of protein. In the present study, oyster (Crassostrea talienwhannensis) meat was digested with three proteases including papain, neutrase and alcalase respectively and the derived hydrolysates were fractionated using a series of ultrafiltration membranes (molecular weight cut‐offs of 10, 5, 3 and 1 kDa). The resultant peptide fractions were evaluated for antioxidant activity using 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay and reducing power assay. Results indicated that oyster meat hydrolysates (OMHs) possessed DPPH radical scavenging capacity and reducing power in a dose‐dependent manner. The fractions below 1 kDa showed the strongest overall antioxidant activity. The present study demonstrated that the antioxidant potent of OMHs was strongly related to their concentration, size and amino acid composition.     

High salinity relay as a postharvest processing strategy to reduce vibrio vulnificus levels in Chesapeake Bay oysters (Crassostrea virginica)

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to <30 most probable number (MPN) per g and the impact on oyster mortality were assessed in the lower Chesapeake Bay. Two relay experiments were performed during the summer and fall of 2010. Oysters collected from three grow-out sites, a low salinity site (14 to 15 practical salinity units [psu]) and two moderate salinity sites (22 to 25 psu), were relayed directly to a high salinity site (≥30 psu) on Virginia's Eastern Shore. Oysters were assayed for V. vulnificus and Vibrio parahaemolyticus (another Vibrio species of concern) densities at time 0 prior to relay and after 7 and 14 days of relay, using the FDA MPN enrichment method combined with detection by real-time PCR. After 14 days, both V. vulnificus and V. parahaemolyticus densities were ≤0.8 MPN/g, and decreases of 2 to 3 log in V. vulnificus densities were observed. Oyster mortalities were low (≤4%) even for oysters from the low salinity harvest site, which experienced a salinity increase of approximately 15 psu. Results, although preliminary and requiring formal validation and economic analysis, suggest that high salinity relay could be an effective PHP method.     

High Salinity Relaying to Reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay Oysters (Crassostrea virginica)

Cases of Vibrio infections in the United States have tripled from 1996 to 2009 and these infections are most often associated with the consumption of seafood, particularly oysters (Crassostrea virginica). Information is needed on how to reduce numbers of Vibrio parahaemolyticus and Vibrio vulnificus in bi‐valve molluscan shellfish (for example, oysters). The purpose of this study was to evaluate the effectiveness of high salinity relaying or treatment in recirculating aquaculture systems (RASs) as methods to reduce the abundance of V. parahaemolyticus and V. vulnificus in oysters. For relaying field trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 h, and then transferred to high (29 to 33 ppt.) and moderate (12 to 19 ppt.) salinities. For RAS treatment trial, oysters were transferred to 32 to 34 ppt. salinity at 15 °C. After 7, 14, 21, and in some instances 28 d, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real‐time PCR. Initial levels of V. parahaemolyticus and V. vulnificus ranged from 3.70 to 5.64 log₁₀ MPN/g, and were reduced by 2 to 5 logs after 21 to 28 d in high salinity water (29 to 34 ppt.). Oyster mortalities averaged 4% or less, and did not exceed 7%. Relaying of oysters to high salinity field sites or transfer to high salinity RAS tanks was more effective in reducing V. vulnificus compared with V. parahaemolyticus. These results suggest that high salinity relaying of oysters is more effective in reducing V. vulnificus than V. parahaemolyticus in the oyster species used in this study.     

High‐pressure processing with hot sauce flavouring enhances sensory quality for raw oysters (Crassostrea virginica)

This study evaluated flavouring raw oysters by placing them under pressure in the presence of a commercially available hot sauce. Hand‐shucked raw oysters were processed at high pressure (600 MPa), in the presence or absence of hot sauce flavouring and evaluated by an experienced sensory panel 3 and 10 days after postharvest processing. The sensory panel evaluated high‐pressure‐processed oysters, with and without flavouring, for eleven flavours and three texture characteristics using an 11‐point intensity scale. Oysters were plump and characterised as moderately chewy and firm. Most oyster flavour characteristics were low in intensity with moderate intensity for briny and umami attributes. Flavoured oysters had a moderately intense tangy flavour and aftertaste. Flavouring a raw oyster by high‐pressure processing provides the potential to create a microbiologically safe product with unique sensory characteristics, which may influence consumer acceptance and marketability.     

Levels of Vibrio vulnificus and organoleptic quality of raw shellstock oysters (Crassostrea virginica) maintained at different storage temperatures.

Temperature abuse during raw oyster harvesting and storage may allow for the multiplication of natural spoilage flora as well as microbial pathogens, thus posing a potential health threat to susceptible consumers and compromising product quality. The objective of this study was to provide a scientific basis for determining whether different refrigeration and abuse temperatures for raw oysters would result in a spoiled product before it became unsafe. Raw shellstock oysters (Crassostrea virginica) purchased from a commercial Virginia processor were subjected to different temperature abuse conditions (7, 13, and 21 degrees C) over a 10-day storage period. Salinity, pH, halophilic plate count (HPC), total culturable Vibrio counts, and culturable Vibrio vulnificus counts were determined at each abuse condition. V. vulnificus isolates were confirmed by a specific enzyme-linked immunosorbent assay. Olfactory analysis was performed to determine consumer acceptability of the oysters at each abuse stage. The pH of the oysters decreased over time in each storage condition. The HPC increased 2 to 4 logs for all storage conditions, while olfactory acceptance decreased over time. V. vulnificus levels increased over time, reaching 10(5) to 10(6) CFU/g by day 6. The length of storage had a greater effect on the bacterial counts and olfactory acceptance of the oysters (P < 0.05) over time than did the storage temperature (P < 0.05).     

High hydrostatic pressure and UV light treatment of produce contaminated with Eimeria acervulina as a Cyclospora cayetanensis surrogate

The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (10(6) oocysts) and low (10(4) oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40 degrees C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (10(4) oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (10(4) oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.     

Coccidial contamination of raspberries: mock contamination with Eimeria acervulina as a model for decontamination treatment studies.

Numerous outbreaks have been reported since 1995 in the United States and Canada that were linked to the consumption of imported fresh raspberries contaminated with Cyclospora. Because Cyclospora has no laboratory animal hosts, Eimeria acervulina, a common chicken coccidium similar in characteristics to Cyclospora, was used as a surrogate to test decontamination treatments. Raspberries were mock contaminated with E. acervulina-sporulated oocysts in a water suspension, then exposed to washing, freezing, heat, or irradiation before they were fed to chicks. The presence of oocysts in the contaminated raspberries was confirmed either by duodenal lesions or oocysts in cecal contents 5 days postinoculation (PI) or in fecal contents 6 days PI, after 24 h of fecal collection. Washing of raspberries was generally not adequate in removing coccidial contamination, but freezing and heat treatment appeared effective. Gamma irradiation of E. acervulina-sporulated oocysts at a dose of 0.5 kGy was partially effective, but it was completely effective at 1.0 kGy and higher. We suggest that E. acervulina, for mock contamination of raspberries and subsequent decontamination treatments, is easy to handle, safe, and economical to study.