Investigation of hygienic quality and freshness of marketed fresh seafood in Northern Taiwan

To survey the hygienic quality and freshness of fresh fish and shellfish, 533 specimens of fish and shellfish were collected from fishing piers, traditional markets and supermarkets (including warehouses) in northern Taiwan during the winter (January-February) and summer (July-August) seasons for hygienic quality and freshness examinations. The indicators included total bacterial count, fecal coliform, Escherichia coli, volatile basic nitrogen (VBN) and K value judged from ATP-related compounds. It was found that the hygienic quality and freshness of fish and shellfish decreased in summer, especially for clam and mussel. The ratio of unacceptable hygienic quality or freshness of fish and shellfish was the highest in fecal coliform, followed by E. coli and others. The fecal coliform count in products from the traditional markets was the highest, followed by those from supermarkets and fishing piers. Secondary contamination seems to be the most important problem for hygienic quality and freshness of fish and shellfish.     

Occurrence of histamine and histamine-forming bacteria in kimchi products in Taiwan

Twenty kimchi products sold in supermarkets and 17 products sold in retail markets were purchased from southern Taiwan and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH and salt content in all samples ranged from 3.6 to 5.1 and 1.5–16.0%, respectively. The supermarket kimchi products had 1–7.2 log CFU/g of APC and <3–600 MPN/g of total coliform (TC), and the retail market kimchi products had 4–8.03 log CFU/g of APC and <3 to >2400 MPN/g of TC. Only one of the retail market kimchi products contained 20 MPN/g Escherichia coli. Although, supermarket kimchi products had an average histamine content of 49.8 mg/100 g, 15 of them had histamine content greater than 5 mg/100 g, the allowable level set by the US Food and Drug Administration (FDA) for scombroid fish and/or product. In contrast, only eight retail market kimchi products had histamine levels greater than 5 mg/100 g. Among the supermarket samples, three contained histamine at 50.2, 273 and 535 mg/100 g, that are more than the 50 mg/100 g hazard action level. Four histamine-producing bacteria capable of producing 13.6–43.1 ppm of histamine in MRS broth supplemented with 0.25% l-histidine were identified as Lactobacillus para. paracasei (one strain), Lb. brevis (one strain), and Brevibacillus brevis (two strains). To our knowledge, this is the first report to demonstrate the occurrence of high contents of histamine and histamine-forming bacteria in kimchi products.     

Change of Hygienic Quality and Freshness in Tuna Treated with Electrolyzed Water and Carbon Monoxide Gas during Refrigerated and Frozen Storage

ABSTRACT:  Among different fish slices used for sashimi preparation, tuna is the most popular and preferable fish type for Taiwanese people. To improve the hygienic quality of fish slices, electrolyzed (EO) water containing 10, 50, and 100 mg/L chlorine, was used in combination with CO gas treatment. Effect of different treatment on aerobic plate count (APC), volatile basic nitrogen (VBN), K value, and Hunter L*, a*, b* values of yellow-fin tuna steak during storage (4 °C and −20 °C) were evaluated. It was found that APC, VBN, and K values increased with storage time for all treatment. Except for K value, APC and VBN of tuna steak treated with the combination of more than 50 mg/L chlorine EO water and CO gas had the lowest value after 8 d of refrigerated storage. Hunter a* value of tuna steak treated with only CO gas was the highest, followed by those treated with EO water and CO gas. These results demonstrated that EO water containing 50 mg/L chlorine combined with CO gas treatment in tuna fish steak would be an effective method for enhancing the hygienic quality and freshness for tuna meat and extending refrigerated storage time. Tuna treated with EO water containing 100 mg/L chlorine and CO gas combination had the lowest APC immediately after treatment and reduced further to below detection limit after 1 mo frozen storage at −20 °C.     

Species identification of snapper: A food poisoning incident in Taiwan

A snapper (Lutjanidae fish) is a carnivorous coral reef fish that is distributed in sea areas around Taiwan. In December 2008 in southern Taiwan, a food poisoning incident occurred due to the ingestion of snapper, and the causative residue of ciguatera was investigated using a toxicity assay. To identify the species of the causative sample, six suspected species of Lutjanidae fish commonly found in Taiwan were analyzed using both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. According to the low molecular weight region (<30.0 kD) of species-specific patterns extracted from myofibrillar and sarcoplasmic proteins, the 6 snapper species could be clearly differentiated by the SDS-PAGE method. Furthermore, a consistent 465 bp sequence of the mitochondrial cytochrome b gene from the 6 snapper species was amplified by the PCR method and was rapidly distinguished by the analysis of restriction enzymes. According to both SDS-PAGE and PCR-RFLP methods, the poisonous sample was identified as Lutjanus bohar, which is also a notorious Lutjanidae species containing ciguateric toxins.     

A potential methodology for differentiation of ciguatgoxin-carrying species of moray eel

A food poisoning incident due to ingestion of an unknown moray eel occurred in Taiwan. To identify the species of causative moray eel implicated in food poisoning, a 376-bp fragment sequence of cytochrome b gene was successfully obtained from four species of moray eel by using a pair of primers (L newest-1/H 15149). This fragment could be amplified using fish meat treated with different heating processes (100 °C for 30 min, 100 °C for 60 min, 121 °C for 15 min, and 121 °C for 30 min). After sequencing, it was found that no variation in sequence was detected among individuals within each species. The species of causative moray eel implicated in food poisoning was judged as Gymnothorax javanicus based on sequence analysis. In addition, using restriction enzyme analysis, including Taq I and Sau96 I, could distinguish these four species of moray eel and identify the fish species of poisoning sample as G. javanicus. The toxicity of viscera in 24 specimens of four moray eel species was not detected, but the food poisoning sample was found to be toxic. Overall, this study proved that DNA sequence and restriction enzyme analyses are useful in identifying that the causative moray eel species was G. javanicus.     

Application of electrolyzed oxidizing water on the reduction of bacterial contamination for seafood

For reducing bacterial contamination, electrolyzed oxidizing water (EO water) has been used to reduce microbial population on seafood and platform of fish retailer. The specimens of tilapia were inoculated with Escherichia coli and Vibrio parahaemolyticus, and then soaked into EO water for up to 10 min. EO water achieved additional 0.7 log CFU/cm² reduction than tap water on E. coli after 1 min treatment and additional treatment time did not achieved additional reduction. EO water treatment also reduced V. parahaemolyticus, by 1.5 log CFU/cm² after 5 min treatment and achieved 2.6 log CFU/cm² reduction after 10 min. The pathogenic bacteria were not detected in EO water after soaking treatment. In addition, EO water could effectively disinfect the platform of fish retailer in traditional markets and fish markets.     

Identification of tetrodotoxin in a marine gastropod (Nassarius glans) responsible for human morbidity and mortality in Taiwan

The toxicity of the gastropod Nassarius glans was investigated. This gastropod was implicated in an incident of food paralytic poisoning on Tungsa Island, Taiwan, in April 2004. Six victims consumed both digestive glands and muscle. These tissues contained high concentrations of toxin; their highest toxicity scores were 2,048 and 2,992 MU/g, respectively, based on the tetrodotoxin (TTX) bioassay. The toxin was purified from these gastropods and analyzed by high-performance liquid chromatography, which revealed TTX and related compounds 4-epi TTX and anhydro-TTX; paralytic shellfish poisons were not found. The urine and blood samples from patients were cleansed using a C18 Sep-Pak cartridge column and 3,000 molecular weight cutoff Ultrafree microcentrifuge filters, and the eluate was filtered and analyzed by liquid chromatography and mass spectrometry. The detection limit for TTX was 1 ng/ml. The standard curves were linear in the range 30 to 600 ng/ml for urine and 1 to 30 ng/ml for blood. TTX was detected in all urine samples but in only three of four blood samples tested. Thus, the causative agent of gastropod food poisoning was identified as TTX.     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.     

Histamine level and species identification of billfish meats implicated in two food-borne poisonings

Two incidents of food-borne poisonings, causing illness in 59 and 43 victims due to ingestion of billfish meats, occurred in May 2004, in Pingtung, southern Taiwan and in December 2004, Taichung, central Taiwan, respectively. One fried billfish fillet and five frozen billfish fillet samples collected, respectively, from the suspected restaurants in Pingtung and Taichung, respectively, were tested to determine the histamine levels and identify fish species. Analyses of histamine showed that the suspected billfish samples in two food poisonings contained more than 150 mg/100 g of histamine, which is higher than the hazard action level of 50 mg/100 g. Judging from the allergy-like symptoms of the victims and the high histamine levels in the suspected billfish samples, both food-borne poisonings were strongly suspected to be caused by histamine intoxication. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the species of the suspected billfish samples in both food poisonings. The 348 bp amplified fragment of the mitochondrial cytochrome b gene by PCR was digested with BsaJI, Cac8I and HpaII enzymes to distinguish the species of the suspected billfish samples. Consequently, the species of Pingtung and Taichung billfish samples implicated in food poisonings were identified as Makaira nigricans and Xiphias gladius, respectively.     

Bacteriological quality and histamine-forming bacteria associated with fish meats and environments in HACCP and non-HACCP fish processing factories

Twenty fish meats and 21 environmental surface samples obtained from a Hazard Analysis Critical Control Points (HACCP) fish processing factory and two non-HACCP fish processing factories in Pingtung, southern Taiwan were tested to determine the hygienic quality and histamine-forming bacteria. The levels of aerobic plate count (APC), total volatile basic nitrogen and total coliform in fish samples obtained from the HACCP factory were significantly lower than those of fish samples obtained from the two non-HACCP factories. The average content of the eight biogenic amines in HACCP fish samples was less than 1.0 mg/100 g, while that in non-HACCP fish samples was less than 1.5 mg/100 g. In environmental surface samples, the average levels of ATP bioluminescence and APC (swabbing method) of HACCP processing factory were significantly (P < 0.05) lower than those of non-HACCP processing factories. Fifteen histamine-forming bacterial strains isolated from fish meats and environmental samples producing 2.3–561.5 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH), belonged to Enterobacteriaceae (thirteen strains) and Staphylococcus spp. (two strains).