Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cutoff percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do so.
Omega-3 fatty acids have been shown to reduce the risk of chronic diseases like cardiovascular disease and cancer as well as promote brain development among infants and children. This study was carried out to compare total protein, fat and omega-3 fatty acids content of raw and pressurized fish of P. pangasius (yellowtail catfish) and H. macrura (long tail shad). The fish was cooked using pressure cooker for six minute to be pressurized. The protein content was determined by using Kjedahl method while total fat was determined using solvent extraction using chloroform and methanol. Fatty acid methyl esters (FAME) were prepared by a direct transesterification method, and quantified by gas chromatography using external standard. Results showed that marine fish H. macrura (long tail shad) had higher content (p < 0.05) of protein (18.30 ± 0.040 g/100 g), fat (10.965 ± 1.610 g/100 g), EPA (11.83 ± 0.02 g/100 g) and DHA (5.96 ± 0.31 g/100 g) compared to freshwater fish P. pangasius (yellowtail catfish). The protein content of pressurized fish was higher compare to raw fish, but there was no difference in total fat and omega-3 fatty acids content between raw and pressurized of freshwater fish P. pangasius and marine fish, H. macrura. In conclusion, marine fish are better source of protein, fat and omega-3 content, while pressurized fish shown to have comparable amount of protein, fat and omega-3 fatty acids content with raw fish. The result obtained assist the consumers to prepare a healthy menu in order to retain the protein and omega-3 fatty acids content of fish through healthy way of cooking.
Vibrio parahaemolyticus is a foodborne pathogen and their human infection is regularly associated with the consumption of raw or undercooked seafood and contaminated water supplies. Many conventional biochemical identification and confirmation procedures are performed to detect the presence of this pathogen, both from seafood or environmental samples. However, these procedures not only require two or more days to complete, they do not have the capabilities to determine the number of V. parahaemolyticus cells in any given samples. Thus, in this study we describe the development of a rapid SYBR green based real-time PCR assay, targeting the thermo labile (tl) gene of V. parahaemolyticus for the detection and enumeration of this bacterium from seafood and environmental samples. We report that the real-time PCR assay and the primers designed are highly specific, and only generated the desired amplicons with V. parahaemolyticus DNA samples against other bacteria and fungi species. Our assay is also highly sensitive, and, is able to detect V. parahaemolyticus with high coefficient values in concentrations as low as 1.0 pg/μl DNA for pure genomic DNA solutions and 10 cells/ml in serially diluted cell suspension and spiked samples. This assay can be completed in less than 3 hours and may be used as a tool for rapid determination of V. parahaemolyticus densities in the food industries, environmental risk assessment and for clinical diagnostics purposes.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
Hepatitis A is a liver infection caused by the hepatitis A virus (HAV). Outbreaks of hepatitis A have been linked to the consumption of both raw and cooked shellfish. These outbreaks could induce a public confidence problem over shellfish safety and may result in important economic losses for the seafood industry. The work presented in this study investigated the presence of HAV in shellfish from Peninsular Malaysia. A total of 365 of cultured and wild shellfish from 36 sampling locations located throughout Peninsular Malaysia were examined using a commercial nucleic acid extraction and reverse transcription -polymerase chain reaction (RT-PCR) kit. HAV was not detected in almost all of the shellfish samples xamined. Only one cockle sample from Changkat, Seberang Perai was positive for HAV. The results suggest the absence of HAV or very low amount of HAV viral particles in most of the shellfish examined.
The status ofhve l1eavy metals: cadmium, lead copper, zinc and mercury were determined in seafood and its products imported_ti·om Thailand via Bukit Kayu Hiram, Kedah, Flame Atomic Absorption Spectrophotometer was used to determine the level of these heary metals except for mercury, where the cold vapor technique was used, Randomized sampling was done according to a predetermined sampling plan based on the previous years consignments. Data collected were compared with the maximum permitted level of -metal contaminants in fish and fish products- ofthe Fourteenth Schedule (Regulation 38) of Food Act I983 and Food Regulations I 985 to ascertain compliance. lt was noted form this study, that the level of metals detected in seafood and its products had a very wide range, The levels detected for cadmium was at a range of 0. 00] - 3.9/2, 0.07 — 0.29, 0,04 - 4,4 mg/kg in fish, shellfish and cuttlefish respectivelv. In general, cadmium level in some samples was notably higher particularly in shellfish. All samples had lead level less than the permitted value except for fish where the highest value detected was 3.28 mg/kg. The level of copper and zinc was higher than the permitted value in octopus, prawn and crab, Mercury level in all samples analvzed was found to be below the detection level. As for fish samples, zinc level was found to be higher whereas copper was within the limit. However, it was also noted that the level of all the heavy metals in jiozen jish was within the permitted limit.
Biological amines are nitrogenous compounds that occur naturally in wide variety of food. Histamine, putrescine, cadavarine, tyramine, spermine, spermidine, tryptamine and β-phenylethylamine are the biogenic amines that are normally present in foods. Although the biogenic amines play some important physiological functions but high level of amines can cause toxicological effects. High amount of amines can be produced by bacteria during amino acids decarboxylation and have been identified as one of the important agent causing seafood intoxication. Temperature is the major factor for controlling the biogenic amines formation in food. The effects of other alternatives are also discussed including salting, packaging, irradiation, high pressure processing and the use of starter culture. A variety of techniques can be combined together to control the microbial growth and enzyme activity during processing and storage for better shelf life extension and food safety.
Antibiotic susceptibility and genetic diversity of E. coli isolated from cultured catfish and their surrounding environment were determined. The levels of resistance of the E. coli isolates towards six different antibiotics tested differed considerably. Though the isolates displayed resistance towards some of the antibiotics tested, none of the isolates showed resistant towards norfloxacin, sulphametoxazole/trimethoprim and chloramphenicol. RAPD-PCR analysis using single primer and primers combination clustered the E. coli isolates into 3 and 5 groups, respectively. The results of this study suggest that the E. coli isolates from the catfish and their surrounding environment derived from a mixture of sensitive and resistant strains with diverse genetic contents. The use of the RAPD analysis is sufficiently discriminatory for the typing of the E. coli isolates.
Gelatin from fish skin is known to be an alternative source for mammalian gelatin. However, it has weaker properties compared to bovine and porcine gelatin, which limits its use in the industry. The conventional method for fish gelatin extraction requires long production time and could cause serious water pollution and chemical treatments are often being used to enhance the yield of fish gelatin and its properties but it may affect the amino acid content of the gelatin. In this regard, High-Pressure Processing (HPP) is a novel method suggested for fish gelatin extraction. The HPP method is classified as green technology as it requires low electricity throughout the process. This study will discuss the impact of HPP the technique gelatin extracted from fish skin. Skins from four types of fish, namely red tilapia (Oreochromis niloticus), black tilapia (Oreochromis mossambicus), grouper (Epinephelus areolatus) and threadfin bream (Nemipterus tambuloides), were used. High pressure was applied at either pretreatment in citric acid solution or during thermal extraction; and the pressure was maintained at 250 MPa with pressure holding time of 10 minutes and 18 hours of water extraction. Gelatin extract from traditional acid-base method was prepared as a standard for comparison. The study found that there was an increment in the yield of gelatin and the concentration of gelatin extract, and the pre-treatment time was also reduced.
The current study was conducted to evaluate the nutritional characteristics (moisture, protein, lipids, ash and fatty acid composition) of the flesh of oil sardine (Sardinella longiceps) and Indian mackerel (Rastrelliger kanagurta) caught from Hadhramout coast of the Arabian Sea. The protein content was 21.6% and 18.1% (wet weight basis) for mackerel and sardine, respectively. The lipid content was much higher in sardine (10.1%) compared with mackerel (1.7%). The fatty acid composition showed that total saturated fatty acids had the highest relative value (37.5%) among other fatty acid groups in the flesh lipids of sardine, followed by polyunsaturated fatty acids (29.9%) and monounsaturated fatty acids (23.4%). In mackerel, polyunsaturated fatty acids was present at 37.4%, followed by saturated fatty acids (36.7%) and then monounsaturated fatty acids (14.3%). The majority of polyunsaturated fatty acids in both fish were deposited as omega-3 (89.8% in sardine and 87.9% in mackerel), of which docosahexaenoic acid and eicosapentaenoic acid were the most abundant. In conclusion, oil sardine and Indian mackerel which are locally available and affordable fish in Yemen can be considered valuable sources of nutrients particularly protein and health-beneficial omega-3 long chain polyunsaturated fatty acids.
This article contains data on the bacterial communities and its diversity associated with Anadara granosa. The A. granosa samples were obtained from two major estuaries in Penang, Malaysia using a culture dependent and 16S rRNA Illumina sequencing approaches. A. granosa, a commercial blood cockles and popular seafoods, is fragile to the surrounding environments. Thus, our research focused to better understand the bacterial communities and it diversity in the A. granosa, as well as on the generation of a metagenomic library from A. granosa to further understanding on it diversity. The bacteria Vibrionaceae (34.1%) was predominant in the A. granosa from both environments followed by Enterobacteriaceae (33.3%) and Bacillaceae (16.75%). Vibrio sp., Klebsiella sp., and Bacillus subtilis were the most abundant species present. The data generated in this research is the first metagenomic examination of A. granosa and will provide as a baseline to understand the bacterial communities associated with A. granosa and its surrounding natural environments.
This study was conducted to determine the cholesterol and alpha-tocopherol contents of 20 marine fish and four other seafood from the Straits of Malacca. Cholesterol and alphatocopherol contents of the fish and other seafood were determined using high-performance liquid chromatography. The results showed that most of the fish contained low amounts of cholesterol, except sixbar grouper (Epinephelus fasciatus), long-tailed butterfly ray (Gymnura sp.), yellowstripe scad (Selaroides leptolepis), cuttlefish (Sepia officinalis), large-scale tongue sole (Cynoglossus arel), and longtail shad (Hilsa macrura) that contained high amounts of cholesterol (119.39-353.97 mg/100 g wet samples). Indian mackerel (Rastrelliger kanagurta), giant seaperch (Lates calcarifer), prawn (Metapenaeus affinis), and moonfish (Trachinotus blochii) had high alpha-tocopherol contents (462-989 μg/100 g wet sample). Regular consumption of fish and other seafood is highly recommended partly due to the high alphatocopherol content. Due to the high cholesterol in certain types of fish, consumption of the fish fillets of sixbar grouper, long-tailed butterfly ray, yellowstripe scad, cuttlefish, and large scale tongue sole should be < 100 g per day and < 50 per day for longtail shad. Validation of the analytical method also showed a high accuracy and reproducibility of the HPLC method.
Aquaculture production of the Pacific white shrimp is the largest in the world for crustacean species. Crucial to the sustainable global production of this important seafood species is a fundamental understanding of the shrimp gut microbiota and its relationship to the microbial ecology of shrimp pond. This is especially true, given the recently recognized role of beneficial microbes in promoting shrimp nutrient intake and in conferring resistance against pathogens. Unfortunately, aquaculture-related microbiome studies are scarce in Southeast Asia countries despite the severe impact of early mortality syndrome outbreaks on shrimp production in the region. In this study, we employed the 16S rRNA amplicon (V3-V4 region) sequencing and amplicon sequence variants (ASV) method to investigate the microbial diversity of shrimp guts and pond water samples collected from aquaculture farms located in Malaysia and Vietnam. Substantial differences in the pond microbiota were observed between countries with the presence and absence of several taxa extending to the family level. Microbial diversity of the shrimp gut was found to be generally lower than that of the pond environments with a few ubiquitous genera representing a majority of the shrimp gut microbial diversity such as Vibrio and Photobacterium, indicating host-specific selection of microbial species. Given the high sequence conservation of the 16S rRNA gene, we assessed its veracity at distinguishing Vibrio species based on nucleotide alignment against type strain reference sequences and demonstrated the utility of ASV approach in uncovering a wider diversity of Vibrio species compared to the conventional OTU clustering approach.
Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.
This study was conducted to evaluate umami taste in protein hydrolysate produced from green mussel (Perna viridis) by hydrolysing with flavorzyme at pH 8, enzyme substrate ratio (E/S) 3% with or without the presence of 0.4% sodium tripolyphosphate (STPP) and 1.5% NaCI. Degree of hydrolysis (DH), yield, amino acid compositions, molecular weight distribution and sensory evaluation were determined. The highest DH (23.18%), darkest color and highest yield (8.34%) were recorded for hydrolysate produced in the presence of both STPP and NaCI. Electrophoresis analysis showed the presence of protein bands between 10 to 70 kDa where hydrolysate with addition of STPP and NaCI had bands with lower intensities. Amino acids which contribute to the umami taste such as glutamic acid, glycine and aspartic acid were higher in hydrolysate produced with STPP and NaCI addition. The hydrolysate has lesser fishy odor and flavor than those produced with only in the presence of flavorzyme and was also rated with highest score for all the five basic tastes including bitterness. However, the score for bitterness was still lower than the reference solutions. Therefore, green mussel hydrolysate produced in this study has a good potential as a food flavorant.
Metallic contamination in seafood, especially fish, has been of increasing concern to human health. Moreover, with increasing dependency on farmed fish for fish resources, the metallic contamination in them is still questionable. This study aimed to investigate the effects of cooking (steaming) on heavy metal concentration in farmed fish and to estimate its potential human health risk. Farmed sea basses (Lates calcarifer) from Setiu Lagoon were used to study the difference in metal uptake through human consumption of raw and cooked (steamed) fish samples. Selected heavy metals, namely copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), mercury (Hg) and lead (Pb), were measured using ICP-MS following Teflon bomb closed digestion of the fish samples. Cooking of the fish muscle by steaming was applied to investigate if cooking changes the concentration of heavy metals. Mercury and As were found accumulated more notably in the fish muscle, though only Hg was found to show significant (p > 0.05) increase when L. calcarifer is cooked. The amount of As in the fish muscle throughout its growth can be potentially harmful to humans, with the highest averaged concentration at 3.29 ± 0.65 mg/kg dw. above the standard set by the Malaysian Food Regulation (1985) of >1 mg/kg. All the other heavy metals were at relatively safe concentrations well below the standard set by both national and international guidelines. The PTWI per individual of L. calcarifer for As was at 0.84 mg/kg bw., which indicated that the amount of this fish safe for consumption without any adverse effect is 170 g/week. Therefore, long-term intake of these fish may pose a risk to human health due to the relatively higher Hg and As concentration found in these fishes.
Vibrio parahaemolyticus is a foodborne bacterial pathogen that may cause gastroenteritis in humans through the consumption of seafood contaminated with this microorganism. The emergence of antimicrobial and multidrug-resistant bacteria is another serious public health threat worldwide. In this study, the prevalence and antibiotic susceptibility test of V. parahaemolyticus in blood clams, shrimps, surf clams, and squids were determined. The overall prevalence of V. parahaemolyticus in seafood was 85.71% (120/140), consisting of 91.43% (32/35) in blood clam, 88.57% (31/35) in shrimps, 82.86% (29/35) in surf clams, and 80% (28/35) in squids. The majority of V. parahaemolyticus isolates from the seafood samples were found to be susceptible to most antibiotics except ampicillin, cefazolin, and penicillin. The MAR indices of V. parahaemolyticus isolates ranged from 0.04 to 0.71 and about 90.83% of isolates were found resistant to more than one antibiotic. The high prevalence of V. parahaemolyticus in seafood and multidrug-resistant isolates detected in this study could pose a potential risk to human health and hence appropriate control methods should be in place to minimize the potential contamination and prevent the emergence of antibiotic resistance.
Gelatin from salmon (Salmo salar) skin with high molecular weight protein chains (α-chains) was extracted using trypsin-aided process. Response surface methodology was used to optimise the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gelatin were used as responses in the optimization study. The optimum conditions were determined as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and extraction time at 6 h 16 min. This response surface optimized model was significant and produced an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%). Twofold higher yields of gelatin with high molecular weight protein chains were achieved in the optimized process with trypsin treatment when compared to the process without trypsin.
Human anisakiasis is a zoonosis acquired by eating raw or undercooked infected seafood. Herein, we report a case of acute dysentery caused by anisakiasis in a 64-year-old man in Malaysia. A colonoscopy was performed and a nematode larva was found penetrating the mucosa of the ascending colon. Bleeding was observed at the site of penetration. Y-shaped lateral epidermal cords were seen from the cross section of the worm, which is a prominent feature of Anisakis larva. Molecular analysis using polymerase chain reaction of cytochrome oxidase 2 (cox2) gene confirmed the specimen to be larva of Anisakis simplex.
The trace metal concentrations in edible muscle of red tilapia (Oreochromis spp.) sampled from a former tin mining pool, concrete tank and earthen pond in Jelebu were analysed with microwave assisted digestion-inductively coupled plasma-mass spectrometry. Results were compared with established legal limits and the daily ingestion exposures simulated using the Monte Carlo algorithm for potential health risks. Among the metals investigated, arsenic was found to be the key contaminant, which may have arisen from the use of formulated feeding pellets. Although the risks of toxicity associated with consumption of red tilapia from the sites investigated were found to be within the tolerable range, the preliminary probabilistic estimation of As cancer risk shows that the 95th percentile risk level surpassed the benchmark level of 10(-5). In general, the probabilistic health risks associated with ingestion of red tilapia can be ranked as follows: former tin mining pool > concrete tank > earthen pond.