Microbial Indicators and Possible Focal Points of Contamination during Production and Processing of Catfish.

The catfish industry is important to the United States economy. The present study determined the levels of microbial indicators and the prevalence of Listeria spp. and Listeria monocytogenes at catfish farms and catfish processing plants. Live fish, water, and sediment samples were analyzed in farms. Fish skin, fillets, chiller water, and environmental surfaces were assessed at the processing plants both during operation and after sanitation. Live fish had 2% prevalence of Listeria monocytogenes, while sediment and water were negative for Listeria. Live fish skin counts averaged 4.2, 1.9, and 1.3 log CFU/cm2 aerobic (APC), total coliform (TCC) and generic Escherichia coli counts, respectively. Water and sediment samples averaged 4.8 and 5.8 log CFU/g APC, 1.9 and 2.3 log CFU/g TCC, and 1.0 and 1.6 log CFU/g generic E. coli counts, respectively. During operation, Listeria prevalence was higher in fillets before (57%) and after (97%) chilling than on fish skin (10%). Process chiller water had higher (p ≤ 0.05) APC, TCC, and Listeria prevalence than clean chiller water. After sanitation, most sampling points in which Listeria spp. were present had high levels of APC (>2.4 log CFU/100 cm2). APC combined with Listeria spp. could be a good approach to understand microbial contamination in catfish plants.

A New Approach for Quantifying Purpurogallin in Brewed Beverages Using LC-MS in Combination with Solid Phase Extraction.

Purpurogallin (PPG) is a phenolic compound known for its high antioxidant properties in plant-based food materials. However, there is no easy and reliable method for direct determination of PPG in brewed beverages owing to its hydrophobicity, which makes it hard to separate from the background hydrophobic components. Therefore, a method employing solid-phase extraction (SPE) and liquid chromatography-mass spectrometry (LC-MS) was developed for detection and quantification of PPG in brewed beverages, and PPG content was quantified in commercial coffee, cocoa, and tea samples. The limits of detection and quantification were 71.8 and 155.6 ng/g dry weight (dw), respectively. The recovery with SPE was 26.6%. When combined with acetonitrile extraction (ANE), the recovery was 6.8%, higher than 2.6% with water extraction (WTE). Test tube extractions were better than moka pot brewing (MPB) for PPG quantification. Total PPG content of ground coffees prepared by ANE, WTE, and MPB ranged between 635 and 770, 455 and 630, and 85 and 135 ng/g dw, respectively. PPG was detected in two English breakfast tea samples (335−360 ng/g dw) using WTE, but not in cocoa samples. ANE showed higher (p < 0.05) PPG levels, but WTE (r = 0.55, p < 0.01) correlated better with MPB than ANE (r = 0.43, p < 0.01). The result indicated that WTE is the best method to determine PPG in brewed beverages. This work demonstrated that PPG was significant in brewed coffee, and our pioneer study in developing the method for beverage sample preparation and LC-MS analysis has made possible industrial applications and provided new perspectives for future research.

Research Note: Identification and characterization of Salmonella spp. in mechanically deboned chickens using pulsed-field gel electrophoresis.

Salmonella is one of the common foodborne bacteria, causing 80.3 million illnesses every year worldwide. This study was conducted to isolate and identify Salmonella enterica serovars from poultry samples responsible for causing foodborne poisoning in the Mississippi area, United States. A total of 55 S. enterica serovars-Enteritidis (6), Oranienburg (1), Schwarzengrund (8), Heidelberg (4), Kentucky (22), 4, [5], 12:i:- (1), Montevideo (2), Infantis (9), and multi serotypes (2)-were isolated from approximately 110 poultry samples. Through pulsed-field gel electrophoresis (PFGE) analysis, 8 to 13 bands were obtained. The profiles showed >90% similarity in strains within the same type. Consequently, PFGE could be a useful tool to determine chromosomal similarity (clonality of strains) that can be used to trace down epidemiologic sources and geographical origins of Salmonella.

Effect of Chlorine-Induced Sublethal Oxidative Stress on the Biofilm-Forming Ability of Salmonella at Different Temperatures, Nutrient Conditions, and Substrates.

The present study was conducted to evaluate the effect of chlorine-induced oxidative stress on biofilm formation by various Salmonella strains on polystyrene and stainless steel (SS) surfaces at three temperatures (30, 25 [room temperature], and 4°C) in tryptic soy broth (TSB) and 1/10 TSB. Fifteen Salmonella strains (six serotypes) were exposed to a sublethal chlorine concentration (150 ppm of total chlorine) in TSB for 2 h at the predetermined temperatures. The biofilm-forming ability of the Salmonella strains was determined in 96-well polystyrene microtiter plates by using a crystal violet staining method and on SS coupons in 24-well tissue culture plates. All tested strains of Salmonella produced biofilms on both surfaces tested at room temperature and at 30°C. Of the 15 strains tested, none (chlorine stressed and nonstressed) formed biofilm at 4°C. At 30°C, Salmonella Heidelberg (ID 72), Salmonella Newport (ID 107), and Salmonella Typhimurium (ATCC 14028) formed more biofilm than did their respective nonstressed controls on polystyrene ( P ≤ 0.05). At room temperature, only stressed Salmonella Reading (ID 115) in 1/10 TSB had significantly more biofilm formation than did the nonstressed control cells ( P ≤ 0.05). Salmonella strains formed more biofilm in nutrient-deficient medium (1/10 TSB) than in full-strength TSB. At 25°C, chlorine-stressed Salmonella Heidelberg (ATCC 8326) and Salmonella Enteritidis (ATCC 4931) formed stronger biofilms on SS coupons ( P ≤ 0.05) than did the nonstressed cells. These findings suggest that certain strains of Salmonella can produce significantly stronger biofilms on plastic and SS upon exposure to sublethal chlorine.

Effect of deboning time on the growth of Salmonella, E. coli, aerobic, and lactic acid bacteria during beef sausage processing and storage.

The objective of the current study was to determine the effects of deboning time, three steps of sausage processing (grinding, salting, and batter formulation), and storage time (of raw materials and cooked sausage) on the growth (log CFU/g) of aerobic bacteria, lactic acid bacteria, and inoculated Salmonella and E. coli. Beef deboning time did not influence bacterial counts (P≥0.138). However, salting of raw ground beef resulted in a 0.4-log reduction in both aerobic plate count (APC) and Salmonella (P≤0.001). Lactic acid bacteria were increased from non-detectable concentration (0.54 log) on d 0 to 3.8 log on d 120 of vacuum storage (P≤0.019). Salmonella counts were increased (P<0.001) over storage time (3.2 to 3.3 log CFU/g from d 0 to 10). Results indicated that salting and batter formulation had a greater impact on bacterial counts than rigor state of raw beef.

Molecular characteristics, biofilm-forming abilities, and quorum sensing molecules in Vibrio parahaemolyticus strains isolated from marine and clinical environments in Korea.

Vibrio parahaemolyticus is an inhabitant of marine and estuarine environments and causes seafood-borne gastroenteritis in humans. In this study, an UltraFast LabChip Real-Time PCR assay was evaluated for rapid detection and quantification of pathogenic V. parahaemolyticus isolates. Escherichia coli and Vibrio harveyi were used as negative controls. Twenty-six tdh-positive, biofilm-producing V. parahaemolyticus isolates were analyzed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). REP-PCR analysis showed that the majority of the V. parahaemolyticus isolates originated from seafood and that clinical specimens formed two major clusters at 92.8% and 32% similarity levels. The presence and quantification of Autoinducer-2 was carried out using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after derivatization of Autoinducer-2 with 2, 3-diaminonaphthalene. The presence of tdh-positive V. parahaemolyticus in marine samples highlights the need for constant environmental monitoring to protect public health.

The Effects of Sequential Environmental and Harvest Stressors on the Sensory Characteristics of Cultured Channel Catfish (Ictalurus Punctatus) Fillets.

Stress during fish culture alters physiological homeostasis and affects fillet quality. Maintenance of high-quality seafood is important to ensure the production of a marketable product. This study assessed how sequential stressors affect the sensory and quality characteristics of catfish (Ictalurus punctatus) fillets. Three stress trials were conducted where temperature (25 or 33 °C) and dissolved oxygen (DO, approximately 2.5 or >5 mg/L) were manipulated followed by socking and transport stress. After each stage of harvest (environmental stress, socking, and transport), fillet yield, consumer acceptability, descriptive evaluation, cook loss, tenderness, and pH were evaluated. Fillet yield decreased with increasing severity of environmental stress. Fillets from the severe stress treatment (33 °C, approximately 2.5 mg/L) received the highest acceptability scores (P < 0.05). Control fillets (25 °C, >5 mg/L) were the least acceptable (P < 0.05). Increased intensity of less favorable flavor attributes commonly associated with catfish resulted in the differences in acceptability among treatments. As fish progressed through the harvest event, cook loss decreased, tenderness increased, and pH increased, indicating that stress induced textural changes. The data suggest that although environmental stress results in slight changes in flavor attributes, its effects on acceptability are minor with fillets from all treatments still liked (>6 on a 9 point scale). Socking and transport were identified to positively affect textural characteristics of catfish fillets. Although the effects observed were not likely to negatively impact consumer acceptance, a strict management plan should be followed to maintain consistency in the product and avoid changes in stressors that might alter quality more drastically.

Potassium acetate and potassium lactate enhance the microbiological and physical properties of marinated catfish fillets.

Sodium or potassium salts such as lactate and acetate can be used to inhibit the growth of spoilage bacteria and food-borne pathogens, and thereby prolong the shelf-life of refrigerated seafood. However, minimal information is available regarding the combined effects of potassium salts (acetate and lactate) with an agglomerated phosphate blend on the quality and safety of refrigerated catfish fillets. The objective of this study was to determine the microbiological and quality characteristics of marinated catfish fillets treated with organic acid salts. Catfish fillets were vacuum-tumbled with a brine solution with and without the added organic acid salts, at 10% over initial, raw weight prior to tray-packing and storage at 4 °C for 14 d. Fillets were evaluated for yields, color, pH, tenderness, consumer acceptability, and shelf-life. No differences (P > 0.05) existed among the treated and untreated fillets with regards to solution pick-up and pH, but all treated fillets increased (P < 0.05) cooking yields and Intl. Commission on Illumination (CIE) a* values, and decreased (P < 0.05) CIE L* and b* values in the catfish fillets when compared to the untreated fillets. The fillets treated with a combination of potassium acetate and potassium lactate had lower (P < 0.05) psychrotrophic plate counts and lower spoilage scores than the control treatments on days 7, 10, and 14. In addition, consumers preferred (P < 0.05) treated catfish fillets (fried) with respect to appearance, flavor, and overall acceptability over the negative control. In conclusion, the combination of potassium acetate and potassium lactate enhanced sensory quality and extended the shelf-life of refrigerated catfish fillets.

Incidence and persistence of Listeria monocytogenes in the catfish processing environment and fresh fillets.

Incidence of Listeria spp. in whole raw catfish, catfish fillets, and processing environments from two catfish processing facilities was determined in August 2008 and August 2009. Thirty-nine (18.4%) of 212 samples collected in August 2008 were positive for Listeria monocytogenes. Prevalences of Listeria species L. innocua and L. seeligeri-L. welshimeri-L. ivanovii were 11.3 and 23.6%, respectively. Of 209 samples collected in August 2009, 12.4% were positive for L. monocytogenes, 11% for L. innocua, and 19.6% for L. seeligeri-L. welshimeri-L. ivanovii. No Listeria grayi was detected in any of the samples. L. monocytogenes was not found in catfish skins and intestines, but was detected in catfish fillets, on food contact surfaces, and on non-food contact surfaces with frequencies of 45.0, 12.0, and 11.1%, respectively. In August 2008 isolates, serotypes 1/2b (62.2%) and 3b (15.6%) were frequently isolated, whereas the majority of the August 2009 isolates (92.3%) were serotype 1/2b. Genotyping analyses revealed that some genotypes of L. monocytogenes isolates were detected in one facility even after a year, but no persistence of L. monocytogenes was observed in the other facility. In addition, some L. monocytogenes isolates from fresh fillets showed genotypes that were either identical, or more than 90% similar, to those of L. monocytogenes isolates from food contact surfaces in the processing lines. The results of this study suggest that processing environment rather than whole raw catfish is an important source of L. monocytogenes contamination in the catfish fillets. These results should assist the catfish industry to develop better control and prevention strategies for L. monocytogenes.

Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-ß-hydroxybutyrate polymer.

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii ∆arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-ß-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E(1) component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.

Prevalence and contamination patterns of Listeria monocytogenes in catfish processing environment and fresh fillets.

Catfish skins, intestines, fresh fillets, processing surfaces at different production stages, chiller water and non-food contact surfaces were sampled for Listeria monocytogenes and other Listeria species. Among 315 samples, prevalence of L. monocytogenes, Listeria innocua and a group of Listeria seeligeri-Listeria welshimeri-Listeria ivanovii was 21.6, 13.0 and 29.5%, respectively. No Listeria grayi was detected in this survey. While no L. monocytogenes strains were isolated from catfish skins and intestines, the strains were found with a frequency of 76.7% in chilled fresh catfish fillets and 43.3% in unchilled fillets. L. monocytogenes and Listeria spp. were also detected in fish contact surfaces such as deheading machine, trimming board, chiller water, conveyor belts at different stages, and fillet weighing table. Among L. monocytogenes, 1/2b (47.0%), 3b (16.0%) and 4c (14%) were the predominant serotypes isolated, whereas 4b, 4e, 1/2c and 1/2a were detected at much lower frequencies. Genotype analyses of L. monocytogenes isolates using serotyping, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed that chiller water represented an important contamination source of L. monocytogenes in the chilled catfish fillets of two processing facilities, whereas fillet weighing table significantly contributed to the catfish fillet contamination of the third facility. This study suggests that L. monocytogenes contamination in the processed catfish fillets originates from the processing environment, rather than directly from catfish. Results from this study can aid the catfish industry to develop a plant-specific proper cleaning and sanitation procedure for equipment and the processing environment designed to specifically target L. monocytogenes contamination.

Enhanced antimicrobial activity of starch-based film impregnated with thermally processed tannic acid, a strong antioxidant.

Starch-based films impregnated with fresh tannic acid (FTA/starch film) and thermally processed tannic acid (PTA/starch film) were assessed for inhibition of Escherichia coli O157:H7 and Listeria monocytogenes. Disc-diffusion assay revealed that the PTA/starch film showed larger clear zone around the film on the bacterial lawn than the FTA/starch film at the same tannic acid concentrations (0.45 to 4.5mg per disc). Viable cell count assays in tryptic soy broth showed that the PTA/starch film also had a stronger antimicrobial activity on these foodborne pathogens than the FTA/starch film. L. monocytogenes did not replicate in trypic soy broth containing the FTA/starch film for the first 8h but multiplied up to 9.22 log CFU/ml at 48 h of incubation. The PTA/starch film caused a 2.72-log decrease in L. monocytogenes cells over the same time period. While 5-log E. coli O157:H7 cells were inactivated by the FTA/starch film within 48 h, more than 7-log E. coli O157:H7 cells were killed by the PTA/starch film over the same period. The antimicrobial activity of FTA/starch and PTA/starch film was primarily pH independent. HPLC measurement of the FTA or PTA release from starch film in water revealed that their release kinetic curves were in well match with their inactivation curves for E. coli O157:H7 and L. monocytogenes in 0.1% peptone water. In addition to antimicrobial activity, FTA showed antioxidant activity on soybean oil by doubling the induction time of oil oxidation. PTA further enhanced the oxidative stability of the oil by 17%. These results suggested that the use of processed tannic acid in starch films could improve the safety and quality of foods.

Overproduction of poly-beta-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF.

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-beta-hydroxybutyrate (PHB), a polymer of industrial importance. This arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated approximately 61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also approximately 11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine-Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.