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      The Effects of Storage Temperature on the Growth of Vibrio parahaemolyticus and Organoleptic Properties in Oysters

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          Abstract

          During harvesting and storage, microbial pathogens and natural spoilage flora may grow, negatively affecting the composition and texture of oysters and posing a potential health threat to susceptible consumers. A solution to these problems would mitigate associated damaging effects on the seafood industry. The purpose of this study was to investigate the effects of storage temperature on growth of vibrios as well as other microbial, sensory, and textural characteristics of post-harvest shellstock Eastern oysters ( Crassostrea virginica). Oysters harvested from the Chesapeake Bay, Maryland, during summer months (June, July, and August, 2010) were subjected to three storage temperatures (5, 10, and 20°C) over a 10-day period. At selected time intervals (0, 1, 3, 7, and 10 days), two separate samples of six oysters each were homogenated and analyzed for pH, halophilic plate counts (HPC), total vibrios, and Vibrio parahaemolyticus ( Vp). Oyster meats shucked after storage were also organoleptically evaluated (acceptability, appearance, and odor). Texture analysis was performed using a texture analyzer on meats shucked from oysters held under the same conditions. The pH of the oyster homogenates showed no consistent pattern with storage time and temperature. The HPC (4.5–9.4 log CFU/g) were highest on day 7 at 20°C while olfactory acceptance reduced with time and increasing storage temperatures. The Vp counts increased over time from 3.5 to 7.5 log MPN/g by day 10. Loss of freshness as judged by appearance and odor was significant over time ( p < 0.05). Toughness of oysters increased with storage time at 5 and 10°C from days 1 to 3 but was inconsistent after day 7. The results indicate that the length of storage and temperature had a significant effect on bacterial counts and olfactory acceptance of oysters but had an inconsistent effect on texture.

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          Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.

          Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected 10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.
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            Relationships between environmental factors and pathogenic Vibrios in the Northern Gulf of Mexico.

            Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.
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              Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters.

              The objectives of this study were to investigate the seasonal distribution of total and pathogenic Vibrio parahaemolyticus in the Chesapeake Bay oysters and waters, and to determine the degree of association between V. parahaemolyticus densities and selected environmental parameters. Oyster and water samples were collected monthly from three sites in Chesapeake Bay, Maryland from November 2004 through October 2005. During collection of samples, water temperature, salinity, turbidity, dissolved oxygen, pH, chlorophyll a, and fecal coliform levels in oysters were also determined. V. parahaemolyticus levels were enumerated by a quantitative direct-plating method followed by DNA colony hybridization; presence/absence was further determined by overnight broth enrichment followed by either standard colony isolation or real-time PCR. The thermolabile hemolysin (tlh) gene and thermostable direct hemolysin (tdh) gene were targeted for detection of total and pathogenic V. parahaemolyticus, respectively, for both direct plating and enrichment. The thermostable related hemolysin (trh) gene, which is a presumptive pathogenicity marker, was targeted only for the enrichment approach. By direct plating, colonies producing tlh signals were detected in 79% of oyster samples at densities ranging from 1.5x10(1) to 6.0x10(2) CFU/g. Pathogenic V. parahaemolyticus (tdh+) was detected in 3% (level was 10 CFU/g) of oyster samples while no V. parahaemolyticus was detected in water samples. By the enrichment approach with standard colony isolation, 67% of oyster and 55% of water samples (n=33) were positive for total V. parahaemolyticus, and all samples were negative for pathogenic V. parahaemolyticus. In contrast, enrichment followed by real-time PCR detected tlh, tdh and trh in 100%, 20% and 40% of oyster and 100%, 13% and 40% of water enrichments collected from June to October 2005, respectively. V. parahaemolyticus densities in oysters varied seasonally and were found to be positively correlated with water temperature, turbidity, and dissolved oxygen.
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                Author and article information

                Contributors
                Journal
                Front Public Health
                Front Public Health
                Front. Public Health
                Frontiers in Public Health
                Frontiers Media S.A.
                2296-2565
                25 March 2014
                16 May 2014
                2014
                : 2
                : 45
                Affiliations
                [1] 1Food Science and Technology Program, Department of Agriculture, Food and Resource Sciences, University of Maryland Eastern Shore , Princess Anne, MD, USA
                Author notes

                Edited by: Khwaja M. Sultanul Aziz, Bangladesh Academy of Sciences, Bangladesh

                Reviewed by: Nur A. Hasan, University of Maryland College Park, USA; Sangita Ahmed, University of Dhaka, Bangladesh

                *Correspondence: Salina Parveen, University of Maryland Eastern Shore, Center for Food Science and Technology, 1 Backbone Road, Princess Anne, MD 21853, USA e-mail: sparveen@ 123456umes.edu

                This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health.

                Article
                10.3389/fpubh.2014.00045
                4032938
                24904911
                5fc8a017-9bc5-43a0-8ec6-6233493f17f1
                Copyright © 2014 Mudoh, Parveen, Schwarz, Rippen and Chaudhuri.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 19 February 2014
                : 01 May 2014
                Page count
                Figures: 8, Tables: 1, Equations: 0, References: 26, Pages: 7, Words: 5788
                Categories
                Public Health
                Original Research

                vibrio parahaemolyticus,halophilic plate count,oysters,sensory,texture

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