Journal of Fisheries science and Technology – No 3/2016 present content ace-inhibitory activity of protein hydrolysate from the skin of striped catfish (Pangasius hypophthalmus); potential impacts of climate change on fisheries and aquaculture in Sri Lanka (Roaming through the climate change management stories of Sri Lanka); effect of cation concentrations (K+, Ca2+) and hormonal stimulation on sperm motility of common carp cyprinus carpio...
Journal of Fisheries science and Technology No.3 - 2016 ACE-INHIBITORY ACTIVITY OF PROTEIN HYDROLYSATE FROM THE SKIN OF STRIPED CATFISH (Pangasius hypophthalmus) Hue Quoc Hoa1,2, Nguyen Xuan Duy3 Received: 21/7/2016; Revised: 09/8/2016; Accepted: 26/9/2016 ABSTRACT There has recently been an increasing demand to produce protein hydrolysates containing peptides with specific biological properties, which could be marketed as functional food ingredients The objective of this study was to evaluate the in vitro angiotensin converting enzyme inhibitory activity of striped catfish skin hydrolysates and its corresponding fractionates The striped catfish skin from fillet processing was extracted in an autoclave at 1210C for 30 minutes to obtain an extracted protein Then it was further hydrolysed with Alcalase with the enzyme to substrate ratio of 20 units/gram protein at 50oC, pH for 7h to obtain protein hydrolysate The degree of hydrolysis (DH) increased with the increase of hydrolysis time and reached the highest DH of 91.9% after 7h hydrolysis The 5-h hydrolysate (DH= 60.8%) exhibited the highest ACE-inhibitory activity (IC50 = 831 µg/ml) Therefore, the 5-h hydrolysate sample was used as material for studying enrichment of ACE-inhibitory peptides by ultrafiltration using three different molecular weight cut-off membranes (10, 5, and kDa) Six sample fractions obtained during ultrafiltration process (permeate and retentate) were tested for angiotensin converting enzyme inhibition activity Permeate of kDa membrane showed the highest activity The obtained hydrolysates were fractioned using SephadexM G-15 Based on gel filtration chromatography results, angiotensin converting enzyme inhibitory peptides had molecular weight ranging of 307 Da to 429 Da Our findings revealed the potential of using catfish skin as a promising material for retrieving angiotensin converting enzyme inhibitory substances Keywords: Alcalase, ACE-inhibitory activity, hydrolysate, ultrafiltration, Pangasius hypophthalmus I INTRODUCTION High blood pressure is a major risk factor associated with cardiovascular disease, the biggest cause of casualty Hypertension is commonly treated with antihypertensive or blood pressure lowering drugs, such as captopril, benazepril, enalapril These drugs are angiotensin I converting enzyme (ACE) inhibitors ACE (EC 3.4.15.1) is a zinc-metallopeptidase that needs zinc and chloride ions for its activity In the renin-angiotensin system (RAS), ACE plays a crucial role in the regulation of blood pressure as well as cardiovascular function (Li et al., 2004) Within the enzyme cascade of the RAS, ACE converts the inactive angiotensin I by cleaving dipeptide from the C-terminus into the potent vasoconstricting angiotensin II This potent vasoconstrictor is also involved in the release of a sodium-retaining steroid, aldosterone, from the adrenal cortex, which has a tendency to increase blood pressure As many synthetic drugs like ACE inhibitors have side effects, peptides from food sources provide an attractive alternative (Howell and Kasase, 2010) Recent researches have reported discoveries of peptides, which are isolated and characterized from a number of fish skin by-products such as Nile tilapia skin (Vo et al., 2011), Pacific cod skin (Ngo et al., 2011), Atlantic salmon skin (Gu et al., 2011), Skate skin (Lee et al., 2011), Pangasius catfish (Mahmoodani et al., 2014) Nutraceutical and Functional Food R&D Center, Prince of Songkla University, Thailand Department of Technology, Dong Thap Community College, Vietnam Faculty of Food Technology, Nha Trang University, Vietnam * Correcponding email: hqhoa@dtcc.edu.vn NHA TRANG UNIVERSITY • Journal of Fisheries science and Technology that inhibited ACE and can be used as nutraceuticals and functional food ingredients A group of peptides from sardine (Fujita, 2001) could decrease blood pressure and approved products containing these components can claim that the product is suitable for individuals with slightly elevated blood pressure A commercial product from sardine peptides that lowers blood pressure was approved by food for specified health uses (FOSHU), an official functional food approved by the consumer affairs agency of Japan (Shimizu, T, 2003) Striped catfish (Pangasius hypophthalmus) is a large freshwater fish It is an important species in freshwater aquaculture in Vietnam, Thailand, Malaysia, Indonesia and China The fillet processing generates considerable quantities of by-products, including abdominal organs, head, bone and skin, that in total represent about 65% of the fish by weight (Thuy et al., 2007) The objective of this study was to investigate ACE inhibitory activity of protein hydrolysate from striped catfish skin by-products by enzymatic hydrolysis using Alcalase II MATERIALS AND METHODS Materials Catfish skins were obtained from a striped catfish processing plant (Dong Thap, Vietnam), the skins were frozen and stored at -20oC before use Alcalase from Bacillus licheniformis 2.4 L, o-phthalaldehyde, DL-dithiothreitol, ACE from rabbit lung and other chemicals were purchased from Sigma-Aldrich Chemical Company Polysulphone hollow fiber membranes with 10, 5, and kDa MWCOs (diameter = 1, 1, and 0.5 mm; area = 0.01, 0.01, and 0.014 m2) were purchased from GE Healthcare Bio-Science Ltd (Bangkok, Thailand) Methods 2.1 Extraction of protein from striped catfish skin The clean skins were added with distilled water (1:2, w/v) and the protein was extracted • NHA TRANG UNIVERSITY No.3 - 2016 using an autoclave at 121oC for 30 After extraction, the extracted protein solution was filtered through a metal sieve to remove skin residues Extracted protein solution was centrifuged at 3,000g for 20 at 25oC to remove insoluble residues and used as a substrate for enzyme hydrolysis Protein content in the skin and the extracted protein solution were determined by Kjeldahl method (AOAC, 1999) 2.2 Enzymatic hydrolysis of extracted protein solution The extracted protein solution was diluted to obtain a protein concentration of 1% (w/v) by 0.1 M sodium phosphate buffer, pH 8.0 The protein solution was hydrolysed by 20 units/g protein of Alcalase 2.4 L at pH 8.0 and 50oC in a 4-L reactor for 6h The pH of the mixture was measured by a pH meter (Eutech, Cyber Scan pH 110, Singapore) and manually adjusted to pH 8.0 during the hydrolysis by 6N NaOH and 6N HCl Aliquots of hydrolysate were collected every 60 mins during the hydrolysis The sample aliquots were heated in boiling water (950C) for 10 mins to inactivate Alcalase They were kept in plastic bottles at - 20oC for analyses The degree of hydrolysis (DH) of the sample was determined by measure the available cleaved peptides bonds upon hydrolysis, using the o-phthalaldehyde (OPA) method as described by Hue et al (2013) 2.3 Enrichment of ACE-inhibitory peptides derived from hydrolysate of striped catfish skin by ultrafiltration The protein hydrolysate was separated using three different MWCO membranes (10, 5, and kDa) The operating condition in batch mode was transmembrane pressure (TMP) of 1.5 bars, and cross flow velocity (CFV) of 1.5 m/s The ACE-inhibitory activity of the feed and permeate were analyzed Journal of Fisheries science and Technology No.3 - 2016 2.4 Angiotensin-I converting enzyme inhibitory fractionation It was dried using freeze dryer activity of protein hydrolysates from striped (Flexi Dry, Dura Dry, NY, USA) The hydrolysate catfish skin was The inhibition of ACE activity was determined by the method of Cushman and Cheung (1971) described by Lee et al (2010) with some modifications The reaction mixture contained 8.3 mM Hippuryl-L-HistidylL-Leucine (Hip-His-Leu) in 0.5M NaCl and mU ACE in 50 mM sodium borate buffer (pH 8.3) A sample (50 μl) was added to above reaction mixture (50 μl) and mixed with 8.3 mM HHL (150 μl) containing 0.5 M NaCl After incubation at 37oC for h, the further reaction was stopped by the addition of 0.1M HCl (250 μl) The resulting hippuric acid was extracted by fractioned using SephadexM G-15 The elution was carried out with 50 mM sodium phosphate buffer pH 7.0 at a flow rate of 0.3 ml/min The ml fractions were collected and their absorbance was read at 220 and 280 nm A standard distribution was determined by chromatographing independently using the following standards: Reduced glutathione (429 Da), Hip-His-Leu (307 Da), and Tyrosine (181.91 Da) The fractions of SephadexM G-15 column were determined for their ACE inhibitory activity All fractions were determined soluble protein content by Lowry method the addition of 1.5 ml of ethyl acetate After (Lowry et al., 1951) centrifugation (800 x g, 15 mins), ml of the 2.6 Statistical analysis upper layer was transferred into a glass tube All experiments were carried out in and evaporated at room temperature for h in triplicate Analysis of variance was performed a vacuum The hippuric acid was redissolved Mean comparisons were run by Duncan’s in ml of distilled water, and absorbance was multiple range tests Analysis was performed measured at 228 nm using a spectrophotometer using an SPSS package (GENESYS 10S UV-VIS Thermo Scientific, Tokyo, Japan) The control and blank were III RESULTS AND DISCUSSION prepared in the same manner, except that 50 μl Effect of hydrolysis time on degree of of buffer was used instead of the sample The hydrolysis (DH) ACE inhibitory activity was expressed as IC50 value (μg/ml) The IC50 value was defined as The DH is generally used as a proteolysis monitoring parameter, and it is the most widely the concentration of inhibitor required to inhibit used indicator for comparison among different 50% of the ACE activity The percentage of protein hydrolysates (Guérard et al., 2002) inhibition level was calculated as follows: Inhibition level (%) = AControl - ASample AControl - ABlank x 100 Where AControl is the absorbance of control There was a sharp increase of DH in the first 30 (DH = 28%) and it increased slightly during 30 to 180 hydrolysis stage From 180 onwards, the DH rose dramatically ASample is the absorbance of the sample and reached a peak of 91.9% at the end of the 2.5 Fractionation of ACE-inhibitory peptides from the increase of short peptides These from hydrolysate results indicated that rapid cleavage of ABlank is the absorbance of the blank The obtained hydrolysate from UF with the highest ACE-inhibitory activity was used for period (Figure 1) High value of DH resulted peptides from the extracted protein solution by Alcalase occurred after h NHA TRANG UNIVERSITY • Journal of Fisheries science and Technology No.3 - 2016 Figure Degree of hydrolysis of protein hydrolysate during hydrolysis with Alcalase Effect of hydrolysis time on ACE inhibitory activity of hydrolysate ACE inhibitory activity of hydrolysate with different hydrolysis time expressed as IC50 is shown in Figure IC50 value of hydrolysate decreased as hydrolysis time increased (p < 0.05) ACE inhibitory activity of extracted protein (IC50 value of 1,556 ± 16.61 µg/ml) increased after hydrolysis (IC50 value ranging from 1,233 ± 29.31 µg/ml to 831 ± 33.39 µg/ml) It was suggested that peptides with ACE inhibitory activity could be generated during hydrolysis The ACE inhibitory activity appeared to increase as hydrolysis time increase because the lengths of peptides were shortened and increased ACE inhibitory activity (Je et al., 2004) The highest ACE inhibitory activity of striped catfish skin protein hydrolysate (IC50 value of 831 ± 33.39 µg/ml) was found at hydrolysis time of h The highest ACE inhibitory activity of skin hydrolysate in the present study was almost similar with that of blacktip shark gelatin (0.94 1.77mg/ml) (Kittiphattanabawon et al., 2013), salmon skin gelatin (1.17 mg/ml) (Gu et al., 2011), and skate skin gelatin (1.89 mg/ml) (Lee et al., 2011) Enzyme hydrolysis was performed in order to achieve the desired degree of hydrolysis to obtain biologically active peptides From previous studies, ACE inhibitory activity of peptides increased with prolonged incubation with enzyme However, longer hydrolysis time led to the peptides lost their ability to inhibit ACE (Wu et al., 2008; Xu et al., 2014) The structure of amino acid for interactions between the substrate and the active site of ACE affected ACE inhibitory activity (Ondetti et al., 1977) Cushman and Cheung (1971) reported that peptides containing aromatic at the C-terminal end and the branch-chain aliphatic amino acid at the N-terminal were effective for high ACE inhibitory activity because of the interaction between these amino acids at the active site of ACE Figure ACE inhibitory activity of striped catfish hydrolysate at various hydrolysis times Different letters on the bars indicate significant differences (p < 0.05) The lower IC50 value represents the higher ACE inhibitory activity • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology Effect of different MWCO membranes on ACE-inhibitory activity of peptides Permeate of MWCO kDa membrane showed the highest ACE inhibitory activity The results indicated that molecular weight of most ACE inhibitory peptides, which was No.3 - 2016 produced and separated from the hydrolysate, was smaller than kDa This result was in accordance with Je et al (2004), who reported that Alaska pollack frame protein hydrolysate that having a molecular mass below kDa showed the highest ACE inhibitory activity Figure ACE inhibitory activity of peptides in permeate and retentate during ultrafiltration of 5-h hydrolysate in batch mode (TMP = 1.5 bars, CFV = 1.5 m/s, temperature = 50oC) 10 kDa MWCO (A), kDa MWCO (B), and kDa MWCO (C) membranes The lower IC50 value represents the higher ACE inhibitory activity Figure shows filtration time versus ACE inhibitory activity of peptides in permeation and retentiveness during ultrafiltration of protein hydrolysate In general, the ACE inhibitory activity of peptides in permeance and retention fell steadily when the operating time increase (IC50 value increased steadily) The ACE inhibitory activity of peptides in permeates was always higher than that in the retentate because low molecular weight of peptides in permeates exhibited high ACE inhibitory activity The ACE inhibitory activity (IC50 average value) of permeates of MWCO 10, 5, and kDa membranes were 159.7, 125.0, and 8.3 àg/ml, respectively NHA TRANG UNIVERSITY Journal of Fisheries science and Technology No.3 - 2016 Fractionation of ACE-inhibitory peptides activity was obtained at fractions 15 to 18 that from hydrolysate having molecular weights 307 Da to 429 Da The chromatogram of hydrolysate subjected Similar findings were also observed from to Sephadex G-15 column is shown in Figure previous works by Je et al (2004); Mahmoodani Amarowicz and Shahidi (1997) reported that et al (2014); Raghavan and Kristinsson (2009), the optical density at 220 nm (A220) indicates who reported that peptides with molecular the peptide bonds and the optical density at masses below kDa showed the highest ACE 280 nm (A280) represents peptides, proteins inhibitory activity The peaked fractions showed or amino acids with aromatic rings Figure the highest ACE inhibitory activity (IC50 value shows the chromatogram of the hydrolysate ranging from 1.22 to 5.88 µg/ml) (Table 1), from permeates of UF kDa MWCO membrane which ranged from 141.45 to 681.72 fold which was fractionated using Sephadex G-15 gel higher than hydrolysate (IC50 value 831.7 µg/ml) filtration chromatography A peak of A220 Fractions 15-18 showing the highest ACE was observed in fraction 4, reflecting the inhibitory activity The result suggests that presence of peptides bonds and a distinct peak peptides without or low ACE inhibitory activity of A280 was found in the same fraction indicated was the presence of peptides containing aromatic peptides with high ACE inhibitory activity were amino acids The highest ACE inhibitory concentrated M M removed during fractionation Figure Elution profile of striped catfish skin hydrolysate (from UF kDa MWCO membrane) separated by size exclusion chromatography on SephadexM G-15 Reduced glutathione (MW = 429 Da), Hip-His-Leu (MW = 307 Da), Tyrosine (MW = 181.91 Da), were used to calibrate the standard molecular weights Table ACE inhibitory activity of peaked fractions from SephadexM G-15 column Fraction No ACE inhibitory activity (IC50) 15 4.98 ± 0.03 µg/ml 16 1.22 ± 0.01 àg/ml 22 5.88 0.06 àg/ml NHA TRANG UNIVERSITY while Journal of Fisheries science and Technology IV CONCLUSION This study found that the protein hydrolysate from striped catfish skin exhibited strong ACE-inhibitory activity The ultrafiltration usage of kDa was successful for separation ACE inhibitory activity peptides since ultrafiltration of the hydrolysate resulted in a significant increase its ACE inhibitory activity in the permeate fractions (IC50 = 8.3 µg/ml) It was concluded that peptides receiving from alcalase hydrolysis of striped catfish skin No.3 - 2016 could be utilized as a part of functional food or ingredients of a formulated drug in order to control high blood pressure ACKNOWLEDGMENTS The authors would like to express their sincere thanks to the Mekong 1,000 Project The People’s Committee of Dong Thap Province - Vietnam, and the Faculty of Agro-Industry, Prince of Songkla University Thailand REFERENCES Amarowicz, R and Shahidi, F., 1997 Antioxidant activity of peptide fractions of capelin protein hydrolysates Food Chem, 58 (4): 355-359 AOAC, 1999 Official Methods of Analysis, 16th ed Arlington, VA: Association of Official Analytical Chemists Cushman, D W and Cheung, H S., 1971 Spectrophotometric assay and properties of the angiotensin I-converting enzyme of rabbit lung Biochem Pharmacol, 20: 1637-1648 Fujita, H 2001 Human study of sardine peptides on blood pressure Nutrition Reseach, 21: 1149 Gu, R Z., Li, C Y., Liu, W Y., Yi, W X and Cai, M Y., 2011 Angiotensin I converting enzyme inhibitory activity of low molecular weight peptides from Atlantic salmon (Salmo salar L.) skin Food Res Int, 44: 1536-1540 Guérard, F., Guimas, L., Binet, A., 2002 Production of tuna waste hydrolysates by a commercial neutral protease preparation Journal of Molecular Catalysis B: Enzymology, 19: 489-498 Howell, N K and Kasase, C., 2010 Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Blackwell Publishing Ltd John Wiley & Sons, Inc Iowa: 203-219 Hue, Q H., Youravong, W., Sirinupong, N., 2013 Antioxidant activities of protein hydrolysate from the skin of striped catfish (Pangasius hypophthalmus) fillet processing waste J Fish Sci Technol Special issue: 70-77 Je, J Y., Park, P J., Kwon, J Y and Kim, S K., 2004 A novel angiotensin I converting enzyme inhibitory peptide from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate J Agric Food Chem, 52 (26): 7842-7845 10 Kim, S K and Byun, H G., 2001 Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin Process Biochem, 36: 1155-1162 11 Kittiphattanabawon, P., Benjakul, S., Visassanguan, W and Shahidi, F., 2013 Inhibition of angiotensin converting enzyme, human LDL cholesterol and DNA oxidation by hydrolysates from blacktip shark gelatin LWT Food Sci Technol, 51: 177-182 12 Li, G H., Le, G W., Shi, Y H and Shrestha, S 2004 Angiotensin-converting enzyme inhibitory peptides from food proteins and their physiological and pharmacological effects Nutr Res 24: 469-486 13 Lee, J K., Jeon, J K and Byun, H G., 2011 Effect of angiotensin-I converting enzyme inhibitory peptide purified from skate skin hydrolysate Food Chem, 125: 495-499 14 Lee, S H., Qian, Z J and Kim, S K., 2010 A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats Food Chem, 118: 96-102 NHA TRANG UNIVERSITY • Journal of Fisheries science and Technology No.3 - 2016 15 Lowry, O H., Rosebrough, N J., Farr, A L and Randall, R J., 1951 Protein measurement with the folin phenol reagent J Bio Chem, 193: 265-275 16 Mahmmdani, M., Ghassem, M., Babji, A S., Yusop, S M., 2014 ACE inhibitory activity of pangasius catfish (Pangasius sutchi) skin and bone gelatin hydrolysate/ J Food Sci Technol 51 (9): 1847-1856 17 Ngo, D H., Ryu, B., Vo, T S., Himaya, S W A., Wijesekara, I and Kim, S K., 2011 Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin Int J Biol Macromol, 49: 1110-1116 18 Ondetti, M.A., Rubin, B and Cushman, D W., 1977 Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents Science, 196: 441–444 19 Raghavan, S and Kristinsson, H G., 2009 ACE inhibitory activity of tilapia protein hydrolysate Food Chem, 117: 582-588 20 Shimizu, T 2003 Health claims on functional foods: the Japanese regulations and an international comparison Nutrition Research Reviews, 16: 241-252 21 Thuy, N T., N T Loc, J E Lindberg., Ogle B., 2007 Survey of the production, processing and nutritive value of catfish by-product meals in the Mekong Delta of Vietnam Livestock Research for Rural Development, 19: 124-103 22 Vo, T S., Ngo, D H., Kim, J A., Ryu, B and Kim, S K., 2011 An antihypertensive peptide from Tilapia gelatin diminishes free radical formation in murine microglial cells J Agric Food Chem, 59: 12193-12197 23 Wu, H., He, H L., Chen, X L., Sun, C Y., Zhang, Y Z and Zhou, B C., 2008 Purification and identification of novel angiotensin-I converting enzyme inhibitory peptides from shark meat hydrolysate Process Biochem, 43: 457-461 24 Xu, W., Kong, B H and Zhao, X H., 2014 Optimization of some conditions of Neutrase-catalyzed plastein reaction to mediate ACE-inhibitory activity in vitro of casein hydrolysate prepared by Neutrase J Food Sci Technol, 51 (2): 276-284 10 • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology No.3 - 2016 POTENTIAL IMPACTS OF CLIMATE CHANGE ON FISHERIES AND AQUACULTURE IN SRI LANKA (ROAMING THROUGH THE CLIMATE CHANGE MANAGEMENT STORIES OF SRI LANKA) Jayasinghe A.D1, Niroshana K.H.H2 Received: 07/6/2016; Revised: 29/7/2016; Accepted: 26/9/2016 ABSTRACT There is an increasing concern over the effects of climate change on aquaculture worldwide Given a mounting evidence of the impacts of climate variability and change on aquatic ecosystems, the resulting impacts on fisheries and aquaculture are likely to be substantial in Sri Lanka This paper reviews potential impacts of climate change on Sri Lankan Fisheries and Aquaculture together with certain possible measures that the nation can adapt to cope with the impacts The sea level rise has been identified as the mostly affecting factor on the Sri Lankan Fisheries and Aquaculture Through ArcGIS techniques, the study has found that in Puttalam district 32.8%, 33.7%, 35.8% and 37.6% of aquaculture farm areas will be irreversibly affected by the future sea level rising in 2025, 2050, 2075 and 2100, respectively Insights to the possible coping strategies were also provided in the study for the government, NGOs and the private sector to cooperate collectively in search of most suitable solutions to deal with climate change Keywords: Climate change, impacts, aquaculture I INTRODUCTION It is a well-known fact that Sri Lanka earns a high amount of income from its fisheries and moreover the sector provides about 540,000 direct and indirect employment opportunities people island wide (The Ministry of Fisheries & Aquatic Resources Development, 2016) Fishery constitutes the major economic activity in the coastal region which contains 25% of the population The fishery sector has received much attention in the national development agenda as shown by its recognition (Climate Change Secretariat and Ministry of Environment Sri Lanka, 2010) Although Sri Lanka is endowed with large fresh and brackish water resources it does not have a tradition of aquaculture and only shrimp aquaculture and ornamental fish culture have been developed to any extent (FAO, 2016) Capture fishery of Sri Lanka was given more weight in the history of the climate change impacts on Sri Lanka Being touched thoroughly the SL stories of climate change and the fisheries this paper provides insights on one of the most critical issue of climate change impact on Sri Lanka: the sea level rise Literature is available in supporting to get predictions on the sea level rise in the Puttalam District of Sri Lanka Yet, no evidence is available in favor of predicting the possible consequences of such impacts This paper consists of two main parts: (i) the holistic nature of the CC and then the CC impact on local level specifically Sri Lanka and (ii) the ArcGIS technique was used to make predictions about scenarios of sea level rise in Puttalam District This paper gives more weight to the impact of sea level rise on shrimp farming in the Puttalam district of Sri Lanka as it was the blood vein of the Sri Lankan export fishery sector Least developed countries in tropical regions have already been identified as particularly Institute of Aquaculture, Nha Trang University, Vietnam Department of Oceanography and Marine Geology, University of Ruhuna NHA TRANG UNIVERSITY • 11 Journal of Fisheries science and Technology vulnerable to climate change because of their greater economic and nutritional dependence on fish and fewer available resources to invest in climate adaptation (Barange et al., 2014) Human population growth is faster in least developed countries where fish provides a larger contribution to non-grain protein needs South Asian region stands out as a place that is not only projected to face decreasing catches, but also has a high dependency on fisheries and a sizeable, rapidly growing population whose consumption of fish is likely to increase with its rapid economic development (Delgado et al., 2003) Climate change affects people and nature in countless ways, and it often increases the prevailing threats that have already put pressure on the environment According to the statistics presented by the National Aquaculture Development Authority of Sri Lanka the fish production by inland and aquaculture sector is much lower than the production by the marine fish catch For example, in the year 2014 total fish catch from fisheries counted as 459300 million tons (MT) while the fish catch from aquaculture accounted as 75750 MT Therefore, studies should prioritize to investigate the impacts of climate change on aquaculture in terms Nevertheless it is also necessary to highlight that we should revise the aquaculture impacts on accelerating the climate change II METHODOLOGY Study area Though Sri Lanka is bestowed with a great potential of aquaculture there was no virtually history on aquaculture until the beginning of 1980s along the coastal border of the Puttalam district of the North Western region of Sri Lanka Afterwards there was a rapid expansion of the shrimp production along the coastal boarder of the Puttalam district mainly due to three reasons Firstly, Puttalam is endowed with number of abundant natural resources such as mangrove swamps, coastal lagoons, 12 • NHA TRANG UNIVERSITY No.3 - 2016 Figure Distribution of aquaculture farms in Puttalam district, Sri Lanka Inserted figure is the map of Sri Lanka with the location of Puttalam district This map was constructed by using ArcGIS® software by Esri tidal flats and estuaries well-suited to shrimp farming Secondly, this region is close to the Katunayake International airport and Colombo harbor together with good road facilities which allowed for swift access to infrastructure needs for the export of special fresh products And the third reason is that “industry development in the Puttalam district coincide with a heavy demand for shrimp in international markets” (Munasignhe et al, 2010) Depending on the aforementioned facts, this study was carried out in Puttalam District based on mainly IPCC reports and other secondary data on Aquaculture industry in Sri Sri Lanka and Climate Change Assessing sea level rise impacts on aquaculture farming industry in Puttalam district In order to assess the sea level rise impacts on aquaculture industry in Puttalam district, information was required on the distribution of aquaculture farms in Puttalam district and the areas of inundation due to predicted sea level rise Hence, the distribution of the aquaculture farms in the Puttalam district were extracted from Google Satellite Images Journal of Fisheries science and Technology Risks of L monocytogenes & Salmonella in seafood products 2.1 Bacterial contamination in seafood products The bacteria can be introduced through many routes to food processing plants, and may establish on food processing equipment Subsequently, food products may become contaminated during processing The soil on shoes and clothing of workers, transport equipment, raw materials or even possibly healthy human carriers are the main sources that lead to the settlement of these organisms in food processing facilities The contamination may occur from the initial raw material source, at each processing stage such as slaughtering, cutting, filleting, smoking, packaging lines, etc During processing, cross contamination may occur (Gravani, 1999) The hygiene condition of the factory and the factory personnel may contribute to the transmission of these bacteria into finished products (Miettinen et al., 2001) The high humidity and the presence of nutrients create an advantageous environment for bacterial growth L monocytogenes and Salmonella are most often detected in moist areas such as floors, drains, condensed and stagnant water, product residues, and processing equipment (Swaminathan, 2001) 2.2 The occurrence of foodborne pathogens in seafood products L monocytogenes has been described as a bacterium with a wide distribution and it can be detected regularly in many foods, including seafood The reported incidences in seafood products vary from to 75% in the literature Unprocessed or frozen fish reported to harbour this bacterium is from to 50% of examined samples The reported figures for processed seafood products are 0-26%, shrimps 0-20%, shellfish 0-7.5% and smoked salmon or trout 0-75% (Lunestad, 2005) There have been many studies on the incidences of the organism in seafood processing factories A smoked-salmon processing plant, including a smokehouse and a slaughterhouse was 114 • NHA TRANG UNIVERSITY No.3 - 2016 examined for the occurrence of L monocytogenes and other Listeria spp The results reflected that L monocytogens was often detected in samples from the smokehouse, but in lower frequencies in the slaughterhouse, indicating that the smokehouse may be considered a reservoir for the bacterium (Rørvik et al., 1995) Other authors also showed that the contamination by L monocytogenes of seafood varied with the product category The highest prevalence was found in cold-smoked fish (34-60%) while the lowest was found in heat treated and cured seafood (4-12%) (Jørgensen and Huss, 1998) Huss et al (2000) have classified four groups of seafood as potential high risk foods for listeriosis They comprise molluscs including fresh and frozen mussels, clams, and oysters; raw fish; lightly preserved fish products including salted, marinated, fermented, cold-smoked, and “ravid” fish; and mildly heat processed fish products and crustaceans Among these seafood, ravid rainbow trout and cold smoked rainbow trout were found the sources of infection in two outbreaks in Sweden and Finland (Miettinen et al., 1999) The original amount in products is usually below 1CFU/g but it may increase in the preservation process, and sometimes exceeds a certain level of 100 CFU/g at the end of the shelf-life of products Although, there are few reports of cases of listeriosis from eating fish and seafood but controlling growth and its hazards are very important (Jinneman et al., 1999) The Food and Drug Administration (FDA) of the US showed that Salmonella was the most common contaminant of fish and fishery products (Allshouse et al., 2004) The presence of Salmonella in seafood may derive from contamination occurring in the natural aquatic environment and in aquaculture The study of Huss and Gram (2004) showed that up to 10-15% of fish samples from India and Mexico were positive of Salmonella Crustacean and molluscan products imported from India Journal of Fisheries science and Technology and Malaysia were also contaminated with Salmonella Salmonella contamination was also reported from other countries like Thailand, Hong Kong, Spain and Turkey The highest Salmonella incidence in fishery products was determined in Central Pacific and African countries while it was lower in Europe and North America (Heinitz et al., 2000) Salmonella has also been detected in US market oysters and in other US imported seafood from different countries For the nine-year period of 1990 - 1999, the FDA examined imported and domestic fish and seafood for Salmonella The results showed that 7.2% of the 11.312 imported samples were positive and only 1.3% of the 768 domestic samples were positive The most common serovar found in the world was S Weltvreden In seafood the commonest serotype encountered was S Worthington followed by S Weltevreden The diversity of Salmonella serovars associated with fish and fishery product was highest in Southeast Asia and next highest in South America (Jay et al., 2005) Bacterial biofilm and measures for bacterial biofilm control 3.1 Bacterial biofilm Biofilm is a community of microbial cells that are attached to biotic or abiotic surfaces and surrounded by a self produced extracellular polymeric substances (EPS) Genetic diversity of this community has shown that biofilm is an ancient ubiquitous life form of microorganisms Virulence and pathogenicity of microorganisms are often enhanced when growing as biofilm Pathogenic bacteria in biofilm protect themselves against the host immune system and increases tolerance to antibiotics treatment, resulting in chronic infections Besides, biofilm associated bacteria are also protected from environmental influences due to the EPS (Chmielewski and Frank, 2003) They are considered highly resistant to cleaning and disinfection Biofilm may consist of spoilage No.3 - 2016 bacteria and a mixture of pathogenic microorganisms such as L monocytogenes, Salmonella spp., Shigella, and become a main concern for the food industry Many studies have reported that 60-80% of microbial infections in the body caused by bacterial biofilm as opposed to planktonic bacteria (Andrews and Bäumler, 2005; Svjetlana and Jasmina, 2007) Biofilm formation is regulated by different genetic and environmental factors Genetic studies have shown that bacterial motility, cell membrane proteins, extracellular polysaccharide and signaling-molecules play significant roles in biofilm formation (Chmielewski and Frank, 2003) Bacterial motility is enabled by two types of protein on the cell surface including flagella and filmbrie Cell membrane proteins help to stimulate the process of adhesion activity EPS are high-molecular weight compounds secreted by microorganisms into their environment They include polysaccharide, proteins, DNA, and lipids that establish the function and structure of biofilm and cell attachment on surfaces (Dufour et al., 2010) Different signals from the environment such as availability of nutrients, presence of oxygen, temperature and pH take part in the regulation of a biofilm formation Growing as biofilm, the bacteria are protected from adverse environmental factors, increasing availability of nutrients for growth, increasing binding of water molecules, reducing the possibility of dehydration, and facilitating higher rates of DNA transfer As a result, inactivation of these bacteria by conventional methods such as use of antibiotics and disinfectants is often ineffective (Dufour et al., 2010) The process of formation consists of steps including attachment or colonization, growth of colonies and periodic detachment of planktonic cells (Svjetlana and Jasmina, 2007) Microcolony in biofilm is the basic structural unit of biofilm Colonies are in most cases mushroom-shaped or rod-like, and consist of one or more types of bacteria, microfungi, NHA TRANG UNIVERSITY • 115 Journal of Fisheries science and Technology algae and protozoa The proportion consists of 10-25% of bacterial cells and 80-90% of EPS matrix depending on bacteria type Water channels flow between microcolonies distributing nutrients to them and receiving the harmful metabolites (Svjetlana and Jasmina, 2007) 3.2 Persistence of L monocytogenes and Salmonella in food processing environments L monocytogenes can adhere and grow as biofilm to different surface materials such as plastic, metal, rubber, polymer, glass, stainless steel, etc that are commonly used in food industry Studies have shown that L monocytogenes can form multi-layer biofilms with three-dimensional structure The cells are not only embedded in extracellular material but also by thread-like extracellular one (Jay et al., 2005; Møretrø and Langsrud, 2004) In biofilm L monocytogenes becomes more resistance to disinfection than free living form and the removal of complex biofilm is also more difficult than adherent single cell of L monocutogenes (Møretrø and Langsrud, 2004) Molecular typing techniques have shown that certain clones of L monocytogenes can be persistent in food processing environments for months or years (Møretrø and Langsrud, 2004) Persistent L monocytogenes strains can be isolated from food processing surfaces after cleaning and disinfections Besides, food processing equipment including conveyor belts, slicing, dicing, packaging machines are extremely difficult to sanitize, and thus become a reservoir of L monocytogenes (Møretrø and Langsrud, 2004) Salmonella was demonstrated that biofilm may form on a variety of surfaces including living tissues, medical devices, oil drilling, paper production, waste water treatment and food processing The bacteria in biofilm are more tolerant to antibiotics than those in planktonic form (Giaouris et al., 2012) In food processing plants, many reports have shown that this organism can attach to food-contact surfaces and a variety of abiotic ones such as 116 • NHA TRANG UNIVERSITY No.3 - 2016 stainless steel, plastic, rubber, glass, cement, marble and granite, etc (Andrews and Bäumler, 2005; Giaouris et al., 2012) Thus, Salmonella biofilm in this environment shows its survival and as a persistent source of microbial contamination that may lead to food spoilage and transmission of diseases pH, temperature, osmolarity, oxygen level, nutrient composition, and presence of mixed bacteria play important roles in biofilm formation (Huss et al., 2000) 3.3 Measures for bacterial biofilm control As discussed above, microorganisms will enhance virulence and pathogenicity when growing as biofilm as well as resistance to antimicrobial agents and environmental influences Biofilm growth in food processing environments may increase the opportunity of contamination of processed products and the transmission of spoilage and pathogenic bacteria in biofilm into the factory environments Therefore it is truly difficult to completely eradicate bacterial biofilm from food processing facilities, thus becoming a great concern and a hazard for human health and food industry Some of the following methods to control biofilm formation and development in food processing industry nowadays may include cleaning, sanitizing and bacteriophages a Cleaning It is very necessary to remove all factors that support bacterial biofilm growth such as water, soil, nutrients, humidity, or dirty clothes and equipment in food processing environment Due to the adhesion of bacteria to surfaces and biofilm protected by extracellular polymeric substances, it is very difficult to clean completely all the surfaces, equipment and machines in food processing plants The common chemical cleaning agents used in food processing industry are surfactants or alkali agents that can act as detergents for fat and protein They can be used in combination with sequestrant or chelator and nonionic wetting agents (Chmielewski and Frank, 2003) These substances may emulsify fats and denature Journal of Fisheries science and Technology protein thus they can dissolve food residues Additionally, acid cleaners are also occasionally used to clean surfaces in case of having high food debris content or hard soiled stains with precipitated minerals Chmielewski and Frank, (2003) suggested that surfaces of glass, stainless steel or ceramic should be cleaned with alkali or nonionic detergents These agents are also used for plastics All detergent solutions should be applied at temperatures between 40 and 90oC depending on soil type and ability of redeposition The cleaning procedure is effective when it removes all food residues and others that may promote the growth of bacterial biofilm However, bacterial biofilm may be formed and grow very quickly at any locations in processing environment when living conditions are favorable after cleaning Therefore, the application of cleaning and sanitation or disinfection should be implemented at the same time (Chmielewski and Frank, 2003, Simões et al., 2010) b Sanitation The aim of the sanitizing process is to reduce the viable bacterial cells left after cleaning and prevent regrowth on surfaces by using chemical antimicrobial agents In addition, all these substances must be safe, non toxic, easy to rinse off from surfaces and not No.3 - 2016 harm human health and final products The conventional antimicrobial products applied in food industry are iodophor, chlorine, hypochlorite, acid anionic, peroxyacetic acid, fatty acid and quaternary ammonium compound sanitizers (Simões et al., 2010) Chlorine is the agent used very common nowadays In seafood processing plants, chlorine is used to disinfect water for production such as thawing frozen products, washing raw materials and in making ice for chilling fishery products (Lunestad, 2005) Other sanitizers such as chlorine dioxide (ClO2) and chloramines are also used in food industry ClO2 is considered as a bactericidal and fungicidal agent and it is widely used in Europe and US as an alternative to chlorine and hypochlorite Monochloramine is evaluated better than chlorine but it requires contact time longer in order to get effectiveness (Chmielewski and Frank, 2003) Besides, EO water is also a strong agent against various foodborne pathogens The gaseous and dissolved forms of ozone are also used as antimicrobial agents in seafood industry (Lunestad, 2005) The following table shows commonly used disinfectants to control of bacterial biofilm of various types in dairy processing plants (Simões et al., 2010) Table Antimicrobial agents are commonly used to control bacterial biofilm in dairy processing plants (Simões et al., 2010) NHA TRANG UNIVERSITY • 117 Journal of Fisheries science and Technology c Bacteriophages Bacteriophage or phage is known as a virus that infects bacteria Nowadays, bacteriophages are more studied and applied widely for decontamination of bacterial biofilm in food industry and medical treatment Their effectiveness is highly evaluated in reducing biofilm bacteria (Fu et al., 2010) The study of Woolston et al (2013) showed that using lytic bacteriophages, SalmoFresh™ can reduce Salmonella contamination on the stainless steel and glass surfaces by 4.3 log CFU/surface (99.995%) and 3.0 log CFU/surface (99.90%), respectively This reduction is reported to be similar to the study for Yersinia pestis-specific bacteriophages (>99%) and Escherichia coli O157:H7-specific bacteriophages (94%) For L monocytogenes, the finding of the study of Soni and Nannapaneni, (2010) found that bacteriophage LISTEX P100 is considered a listericidal for reducing L monocytogeneson raw salmon fillets Phage P100 was justified that it can be used against L monocytogenes serotypes 1/2a and 4b on the surface of raw salmon fillet tissues (Soni and Nannapaneni, 2010) The study found that there is a significantly higher reduction in L monocytogenes count with an increase in phage density However, since phage P100 is highly specific to only Listeria spp., it requires further testing and more caution for eradication of other harmful microflora (Soni and Nannapaneni, 2010) The application of phage is also effective in reducing pathogenic bacterial biofilm on indwelling medical devices (Fu et al., 2010) The study of Fu et al (2010) found that the surfaces of indwelling medical devices are frequently colonized with bacterial biofilm, using phage M4 can reduce viable biofilm count of Staphylococcus aeroginosa on indwelling catheters Measures for prevention of L monocytogenes and Salmonella contamination in seafood products Nowadays, a large number of seafood processing enterprises has increased in Vietnam 118 • NHA TRANG UNIVERSITY No.3 - 2016 Control of food quality and food safety becomes a leading concern with respect to consumer health for manufacturers The Hazard Analysis and Critical Control Points (HACCP) is an effective food safety management system for seafood processing and exporting enterprises This control system helps manufacturers control hazards for prevention of food contamination from stages such as seafood farming, fishing, processing and final products In order to ensure food safety, all these plants should comply strictly with all the regulations of HACCP as well as implement adequately prerequisite programs including GMP - Good Manufacturing Practices, GHP - Good Hygiene Practices, and SSOP - Sanitation Standard Operating Procedures Generally, preventive measures for bacterial contamination in seafood processing plants focus on three main points including the control of factory infrastructure, plant sanitation and sources of raw materials Factory infrastructure: The factory infrastructures should be periodically monitored The wet ceilings, floors, drains, pipe lines, water tank, production equipment, product packaging systems, product transportation and delivery systems may be degraded and damaged by time, thus it may create a favorable environment for the growth of microorganisms Factory management operators should have suitable measures and precaution for the upgrade and maintenance their factory infrastructure Plant sanitation: The development of seafood industry leads to huge waste material from seafood processing plants These pollutants may cause a lot of harm to human health as well as the surrounding environment They include liquid wastes, solid wastes, waste odors or other hazardous wastes Solid waste materials such as heads, bones, skins, scales or fins of seafood are organic products that are easily rotten, fermented and decomposed in hot and humid weather condition Although some Journal of Fisheries science and Technology plants have set up plans to resolve all these waste materials, for example, solid wastes are provided to other manufacturers for producing by-products such as fish oil, chitozan or animal feed, the small amount left is also enough to affect the surrounding environment within the processing plants (Thủy sản Việt Nam, 2012) If the cleaning and disinfection are not implemented regularly at processing areas, it may lead to the formation and development of bacterial biofilm at these sites Other places such as floors or drains are also the potential reservoirs of microorganisms Processing and finished products are easily contaminated by not only Listeria monocytogenes or Salmonella spp., but also by other foodborne pathogens such as Clostridium perfringens or Yershinia, etc existed in a mixture of bacteria community Therefore, all processing enterprises should have long strategies for suitable treatments of waste materials after processing, and they should implement good hygiene practices that may prevent the regrowth and spread of these foodborne pathogens from the environment to finished products Additionally, cross contamination during processing and packaging are also a great concern for manufacturers Some suggested preventive measures for limitating cross contamination in seafood products such as: using water source for production according to the national technical regulation on drinking water quality (QCVN 01: 2009/ BYT) by the Ministry of Health; equipment used for production must be made from specific materials that are easy to clean, disinfect, or sterile and must be regularly maintained; the health of working personnel should be periodically examined for production In case of occurring infectious disease, they should be cared and cured completely until they recover their health On the other hand, all the staff and working personnel should also be trained for knowledge on food safety in manufacturing and environmental protection in order to help them No.3 - 2016 raising awareness to comply with all restrictive regulations for cross contamination prevention Raw materials: Seafood products are also easily contaminated at the early stage of the processing such as filleting, cutting, trimming due to untreated raw materials L monocytogenes and Salmonella are usually present in raw seafood so that raw materials need to be tested for microbiological quality safety before they come into the plants Raw material sources contaminated with pathogenic bacteria should not be allowed for production Testing raw materials may be an effective way to monitor how often products from different suppliers are contaminated with these bacteria The testing also ensures that the imported raw materials from specific sources are not a significant source of pathogenic bacteria The type and frequency of testing will depend on the type of products and the suppliers Besides, raw materials also need suitable treatments before they are used for processing The aim of this procedure is aimed to kill these pathogenic organisms to the extent possible The common treatments to eliminate or reduce the amount of L monocytogenes or Salmonella on raw fish are usage of water containing chlorine or chlorine dioxide for washing seafood (Huss et al., 2000) Other treatments can be used such as ozone, acidified sodium chlorite (ASC) solution in reducing a variety of different foodborne pathogens including Listeria in many different food products (Lunestad, 2005) In reality, some seafood processing and exporting enterprises usually not manage and comply with all the regulations for food safety, so the problem of bacterial recontamination in seafood products frequently occurs in plants III CONCLUSIONS AND RECOMMENDATIONS Seafood products are susceptibly contaminated with pathogenic bacteria such as L monocytogenes or Salmonella by many routes in seafood processing plants The formation of bacterial biofilm in food processing NHA TRANG UNIVERSITY • 119 Journal of Fisheries science and Technology facilities makes the bacteria more persistent and resistant to cleaning and disinfection, thus it enhances the risks of bacterial contamination for food including seafood The contamination of L monocytogenes and Salmonella in seafood products should be resolved by the control strategies of bacterial biofilm formation and development in food processing facilities The application of good manufacturing procedures, sanitary and hygiene practices, No.3 - 2016 and the hazard analysis and critical control point (HACCP) systems should be more strictly implemented in food processing plants Additionally, all seafood processing sectors in Vietnam should comply strictly with all the regulations of the National technical regulations on fisheries food business operators - HACCP based program for quality and safety assurance of the Ministry of Agriculture and Rural Development (QCVN 02-02: 2009/BNNPTNT) REFERENCES 10 11 12 13 14 Allshouse, J., Buzby, J., Harvey,D., and Zorn, D., 2004 United states department of Agriculture Economic Research Service, Seafood Safety and Trade Agriculture Information Bulletin, Number 789-7 Andrews, H L and Bäumler, A J., 2005 Salmonella species In Fratamico P M., Bhunia A K and Smith J L (eds) Foodborne pathogens Caister Academic Press, UK: 327-339 Chmielewski, R A N and Frank, J F., 2003 Biofilm formation and control in food processing facilities Comprehensive Reviews In Food Science And Food Safety 2(1): 22-32 Dufour, D., Leung, V., and Lévesque, C.M., 2010 Bacterial biofilm: structure, function, and antimicrobial resistance Endodontic Topics 22(1): 2-16 Farber, J M and Peterkin, P I., 1991 Listeria monocytogenes, a Foodborne Pathogen Microbiological Review 55(3): 476- 511 Fishnet, 2010 Tổng cục Thủy sản Thống kê (http://www.fistenet.gov.vn/thong-tin-huu-ich/thong-tin-thong-ke Fu, W., Forster, T., Mayer, O., Curtin, J.J., Lehman, S M., and Donlan, R M., 2010 Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system Antimicrobial Agents and Chemotherapy 54(1): 397-404 Giaouris, E., Chorianopoulos, N., Skandamis, P and Nychas, G J 2012 Attachment and biofilm formation by Salmonella in food processing environments Salmonella - A Dangerous Foodborne Pathogen, Dr Dr Barakat S M Mahmoud (Ed.), ISBN: 978-953-307-782-6, InTech, Available from: http://www.intechopen.com/books/ salmonella-a-dangerous-foodborne-pathogen/attachment-and-biofilmformation-by-salmonella-in-foodprocessing-environments Gravani, R., 1999 Incidence and control of Listeria in food processing facilities In Ryser, E T., and Marth, E H.(eds) Listeria, Listeriosis and Food Safety, Second edition, Revised and Expanded, Marcel Dekker, New York, USA: 75-95 Heinitz, M L., Ruble, R D., Wagner, D E and Tatini, S.R., 2000 Incidence of Salmonella in fish and seafood Journal of Food Protection 63(5): 579-592 Huss, H H., Reilly, A and Embarek, P K B., 2000 Prevention and control of hazard in seafood Food Control 11: 149-156 Ivanek, R Gröhn, Y T, and Wiedmann, M 2006 Listeria monocytogenes in multiple habitats and host populations: Review of available data for mathematical modeling Foodborne Pathogens and Disease3(4):319-336 Jay, J M., Loessner, M J and Golden, D.A., 2005 Modern Food Microbiology Seventh Edition Food Science Text Series,751p Jemmi, T and Stephan, R., 2006 Listeria monocytogenes: Foodborne pathogen and hygiene indicator Scientific and Technical Review of the Office International des Epizooties 25(2): 571-580 120 • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology No.3 - 2016 15 Jinneman, K.C, Wekell, M.M and Eklund, M.W., 1999 Incidence and behavior of Listeria monocytogenes in fish and seafood In Ryser, E T., and Marth, E H (eds) Listeria, Listeriosis and Food Safety, Second edition, Revised and Expanded, Marcel Dekker, New York, USA: 601-630 16 Jørgensen, L.V and Huss, H.H 1998 Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood International Journal of Food Microbiology 42: 127-131 17 Liu, D 2006 Review: Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen Journal of Medical Microbiology 55: 645-659 18 Lunestad, B T., 2005 Compendium in Seafood Microbiology, NIFES, Bergen, Norway 19 Miettinen, M K., Siitonen, A., Heiskanen, P., Haajanen, H., Bjorkroth, K J., and Korkeala, H J., 1999 Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria monocytogenes in cold smoked rainbow trout Journal of Clinical Microbiology37: 2358-2360 20 Miettinen, H., Aarnisalo, K., Salo, S., and Sjöberg, A M., 2001 Evaluation of surface contamination and the presence of Listeria monocytogenes in fish processing factories Journal of Food Protection 64(5): 635-639 21 Møretrø, T and Langsrud, S., 2004 Listeria monocytogenes: Biofilm formation and persistence in food-processing environments Biofilms 1: 107-121 22 QCVN 01: 2009 / BYT National technical regulation on drinking water quality, 2009 The Ministry of Health 23 QCVN 02-02: 2009 / BNNPTNT National technical regulation Fisheries Food Business Operators –HACCP Based Program for Quality and Safety Assurance, 2009 Ministry of Agriculture and Rural Development 24 Rocourt, J., 1999 The Genus Listeria and Listeria monocytogenes: Phylogenetic position, taxonomy, and identification In: Ryser, E T., and Marth, E H.(eds) Listeria, Listeriosis and Food Safety, 2nd Ed Marcel Dekker, New York, USA: 1-20 25 Rørvik, L M., Dominique, A C., and Magne, Y., 1995 Contamination pattern of Listeria monocytogenes and other Listeria spp in a salmon slaughterhouse and smoked salmon processing plant International Journal of Food Microbiology 25:19-27 26 Simões, M., Simões, L C and Vieira, M J., 2010 A review of current and emergent biofilm control strategies LWT Food Science and Technology 43: 573-583 27 Soni, K.A and Nannapaneni, R., 2010 Bacteriophage significantly reduces Listeria monocytogenes on raw Salmon fillet tissue Journal of Food Pretection 73 (1):32-38 28 Svjetlana, M and Jasmina, V., 2007 Characteristics and significance of microbial biofilm formation Periodicum Biologorum 109(2): 1-7 29 Slutsker, L and Schuchat, A., 1999 Listeriosis in humans In Ryser, E T., and Marth, E H.(eds) Listeria, Listeriosis and Food Safety, Second edition, Revised and Expanded, Marcel Dekker, New York, USA: 75-95 30 Swaminathan, B., 2001 Listeria monocytogenes In: Doyle, M P., Beuchat, L R., and Montville, T J (eds) Food Microbiology: Fundamentals and Frontiers, 2nd ed, ASM Press, Washington, D C: 383-409 31 Swaminathan, B and Smidt, P G., 2007 The epidemiology of human listeriosis Microbes and Infection 9: 1236-1243 32 Thủy sản Việt Nam, 2012 Thực trạng môi trường sở chế biến thủy sản (http://thuysanvietnam.com.vn/ thuc-trang-moi-truong-o-cac-co-so-che-bien-thuy-san-article-2943.tsvn) 33 Walker, S J., Archer, P and Banks, J G., 1990 Growth of Listeria monocytogenes at refrigeration temperatures Journal of Applied Bacteriology 68: 157-162 34 VASEP, 2013 Sản xuất xuất thủy sản Việt Nam (http://www.vasep.com.vn/Bao-cao-xuat-khau-thuysan/253_32243/Xuat-khau-thuy-san-9-thang-dau-nam-dat-48-ty-USD.htm) 35 WHO, 2004 Risk Assessment of Listeria monocytogenes in Ready-to-Eat Foods In: Microbiological Risk Assessment Series, No Technical Report ISBN 92 1562625 (http://www.who.int/foodsafety) 36 Woolston, J., Parks, A R., Abuladze, T., Anderson B., Li, M., Carter, C., Hanna, L F., Heyse, S., Charbonneau, D and Sulakvelidze, A., 2013 Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces Bacteriophage 3(3): 1-7 NHA TRANG UNIVERSITY • 121 Journal of Fisheries science and Technology No.3 - 2016 CHARACTERISTICS OF AGE DISTRIBUTION AND GROWTH PARAMETERS OF LOBSTER (Nephrops norvegicus LINNAEUS, 1758) IN ICELAND Vu Trong Dai1, Hreidar Thor Valtysson2 Received: 28/7/2016; Revised: 20/9/2016; Accepted 26/9/2016 ABSTRACT The characteristics of age distribution and growth parameters of lobster population (N norvegicus) in Icelandic waters were analysed based on length frequency distribution and cohort slicing method The lobster population had twenty five age groups in which the mean carapace length (CL) was ranged from 7.22 mm at the first age to 76.08 mm at the twentieth five ages In the period from 1970 to 2011, the age groups of to 10 year olds were in high abundant with 81% of catch prorduction as well as the mean carapace length ranged from 32.41 to 52.42 mm The mean carapace length of lobster catched in the Southwest fishing area (47.94 mm) was signifficantly higher when compared to those in the Southeast fishing area (44,84 mm) The growth parameters of this species estimated according to von-Bertalanffy equation were: CL∞ = 82.5 (mm), k = 0.1 (yr-1), t0 = - 0.1 and the growth performance index (Φ’) is 3.87 Keywords: Age distribution, carapace length, growth parameter, Nephrops I INTRODUCTION Norway lobster or Nephrops (N norvegicus) is a high value benthic decapod crustacean species and is a commercially important target in fisheries of many European countries due to its wide distribution [17] The global capture production of this species is 66.5 thousand tons [6] They are caught mainly by trawling during their periodic emergence [2, 3] In Icelandic waters, this species is distributed in the warmer waters off the southern coast, being most abundant at depths of 110-270 m and temperatures of 6-9°C Stock assessment of Nephrops seems to be complicated and difficult because of the fact that this species is only caught during the periodic emergence of the individual from them the burrows However, its emergence varies with time of day, season, animal size, sex, and reproductive status Moreover, this species lacks hard structures that can be used for ageing, so the standard age-based Institute of Aquaculture, Nha Trang University, Vietnam University of Akureyri, Iceland 122 • NHA TRANG UNIVERSITY methodologies applied in fishery-dependent stock assessment cannot be applied [2, 8] Therefore, length frequency distribution and cohort slicing method have typically been used in age distribution and growth parameters of this species to attend to its stock assessment as well as informed a fisheries management policies advice II MATERIALS AND METHODS Data collection Data were collected from the Marine Research Institute database of Iceland including both biomass indices from survey and commercial fisheries data in the period from 1970 to 2011 In the standard survey, the research vessel was rigged with a conventional lobster-trawl of 45m headline and mesh size 80 mm (headline length 150 feet) Two 200 mm window-panels were on the upper deck of the trawl, the one towards the opening was × m and the other × m located m Journal of Fisheries science and Technology behind that As the research trawl has the same design as commercial ones all tows from the research vessels were usable as catch-samples for estimation of stock-size Tow-speed was kept around 2.5 nm and tow-length was usually nm but a minimum of one hour was towed to be valid The research areas included all nephrops fishing grounds from the Southeast to the Southwest of the Icelandic water The total catch of nephrops was recorded (numbers and weight) and number of by-catch species on each station From the catch, one basket (approximately 20kg or 200 300 nephrops) was taken aside for length measurements Each nephrops, carapace length was recorded and sex was identified as well as females assigned to maturity stage and noted if the lobster has recently molted its shell The same parameters and numbers were measured from the commercial catches A total of 200 samples of about 20kg have been processed or roughly sample for each 10 tons landed in recent years Logbooks were mandatory in the fishery where information on catch, effort (towed hours) and locations were also given Data analyses The CL of nephrops was measured as a distance between the back of the eye socket to the rear edge of body carapace [12] In length distributions and cohort slicing analysis, the data were used to compute for only males The total length was calculated based on the relationship equation with CL and then the asymptotic length (L∞) was estimated based on total length according to Sarda et al., (1998) [13] The length-frequency distributions were plotted by year and analysed as a combination of cohort length distributions, each of which was assumed to be in the form of a Gaussian distribution [15] The mean length at No.3 - 2016 age of each cohort and the standard deviations was estimated and then the average growth rate can be modelled The von-Bertalanffy (VB) growth equation was applied to the estimated mean length-at-age and the standard deviation data according to the equation: Lt = L∞ (1− e−k(t−t0) ) (where Lt as the average length-at-age t, L∞ as the asymptotic length, k as the growth rate parameter and t0 as hypothetical age at length equal to 0) The VB growth model assumes that growth was faster when the species was young and get slow onwards after they become mature In order to run this equation in R, some initial values as L∞, k and t0 were set and the least sums of squared error (SSE) was minimised using an iterative process [15] The parameter t0 was fixed at 0.1 as the first two age groups were completely missing from the data series and t0 was always estimated really low To compare the estimated growth parameters of nephrops in this study with other studies, the growth performance index (Φ’) were used which calculated according to equation: Φ = log10k + * log10 L∞ (where k as the growth rate parameter and L∞ as the asymptotic length) These data were analysed by models in the R software package according to Stefansson (2012) [14] III RESULTS AND DISCUSSION Characteristics of age distribution 1.1 Age distribution at different years The age distribution of lobster population from 1970 to 2011 had twenty five age groups corresponding several peaks of carapace length (CL) frequency distribution (Figure 1) The mean CL of N norvegicus at age estimated was ranged from 7.22 mm at age to 76.08 mm at age 25 NHA TRANG UNIVERSITY • 123 Journal of Fisheries science and Technology No.3 - 2016 Figure The age distribution of N norvegicus based on CL distribution in catches from 1970 to 2011 The growth rate of nephrops was high in the first four years with mean increment of about 7.0 mm per year After this age, they reached a maturity size, therefore, the growth rate gradually decreased The proportions corresponding to relative ages revealed a difference between decades In the period 1970 - 1979, the age groups of to year olds were in high abundant as the mean CL ranged 32.41 - 49.19 mm The age groups of to 10 year olds of Nephrops showed a dominant proportion in the catches in the periods from 1980 - 1989 corresponding to mean CL of 32.41 - 52.42 mm On the other hand, in recent years (1990 - 2009) and the whole data from 1970 to 2011, the landings showed high abundant of age groups from to 10 year olds or mean CL of 37.26 - 52.42 mm For the other groups, the age proportions were small (Table 1) Table Mean CL and age proportion of N norvegicus in catches from 1970 to 2011 Proportion (%) Age Mean CL (mm) 1970-1979 1980-1989 1990-1999 2000-2009 1970-2011 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 7.22 14.51 21.09 27.03 32.41 37.26 41.64 45.61 49.19 52.42 55.34 57.98 60.36 62.52 64.46 66.22 67.81 69.25 70.54 71.71 72.77 73.73 74.59 75.37 76.08 3.904e-04 6.690e-04 0.00333 0.04052 0.12999 0.23606 0.15564 0.16582 0.10511 0.04347 0.06881 0.02776 6.564e-07 0.01122 0.00688 5.898e-07 2.331e-07 5.413e-04 9.469e-04 3.102e-06 3.665e-07 3.959e-07 6.670e-08 9.341e-08 0.00285 4.026e-04 7.699e-04 0.00135 0.02019 0.10445 0.14914 0.21745 0.16544 0.11656 0.12218 0.04140 0.03125 0.01685 4.244e-07 0.00959 3.531e-07 8.992e-08 5.351e-08 7.151e-08 3.660e-08 1.911e-08 8.855e-08 1.921e-07 3.232e-07 0.00296 9.454e-05 1.606e-04 6.967e-04 0.02611 0.09711 0.20350 0.19869 0.14899 0.11814 0.10038 0.06161 0.03545 1.367e-06 0.02809 2.685e-07 4.401e-07 7.086e-10 4.058e-07 1.810e-07 9.089e-05 2.404e-04 1.732e-04 3.134e-05 5.930e-06 4.472e-04 3.805e-06 1.172e-04 4.670e-04 0.00776 0.06046 0.12269 0.15398 0.17378 0.11922 0.13062 0.06519 0.08298 3.115e-06 0.05616 0.02014 2.007e-06 6.164e-06 6.843e-04 0.00549 2.262e-04 3.802e-06 2.856e-06 1.450e-06 1.744e-06 1.065e-06 1.471e-04 2.719e-04 0.00155 0.02083 0.09547 0.16539 0.18045 0.15539 0.11871 0.10176 0.05339 0.06087 8.279e-08 0.03708 1.075e-04 9.292e-07 0.00543 0.00211 2.967e-07 3.539e-07 2.488e-07 7.566e-07 1.331e-07 1.397e-06 0.00103 124 • NHA TRANG UNIVERSITY SD 0.37146 0.74646 1.08544 1.39185 1.66882 1.91919 2.14551 2.35008 2.35008 2.70215 2.85326 2.98984 3.11330 3.22490 3.32578 3.41697 3.49939 3.57391 3.64126 3.70214 3.75713 3.80691 3.85189 3.89253 3.92928 Journal of Fisheries science and Technology No.3 - 2016 In general, the mean CL of N norvegicus in catches increased from 1970 to 2011 (Figure 2) Figure The mean CL of N norvegicus in catches from 1970 to 2011 The mean CL of this species caught in 1970 1.2 Age distribution at different fishing areas was 45.10 mm and presented a fluctuation The age distributions of N norvegicus onwards with minimum value of 42.60 mm in were compared the Southeast with Southwest 1973 Until 1995, the CL increased rapidly and fishing grounds based on the mean of CL reached a peak of 49.66 mm in 2010 frequency distributions Figure The age distribution of N norvegicus based on CL distribution in catches from 1970 to 2011 at different fishing areas (A: Southeast area and B: the Southwest area) Figure showed that the plotted of age distribution was wider than in the Southeast distributions as CL frequency distributions areas, which revealed a difference in the were similar, but in the Southwest areas, the proportion of age groups (Table 2) NHA TRANG UNIVERSITY • 125 Journal of Fisheries science and Technology No.3 - 2016 Table Age proportion of N norvegicus in catches at different fishing areas from 1970 to 2010 Age Proportion (%) Southeast areas Southwest areas 2.106e-04 2.023e-04 3.733e-04 Age Proportion (%) Southeast areas Southwest areas 14 0.01436 0.06853 3.489e-04 15 0.00406 0.01124 0.00159 0.00144 16 4.538e-04 2.992e-05 0.02245 0.02029 17 7.999e-04 0.01403 0.11301 0.08017 18 8.431e-04 0.01562 0.19184 0.13409 19 6.784e-06 5.318e-08 0.19557 0.15062 20 3.183e-07 3.339e-07 0.16140 0.13479 21 1.625e-07 2.269e-07 0.10207 0.12198 22 1.087e-07 4.741e-07 10 0.10534 0.09338 23 4.469e-07 1.386e-09 11 0.02944 0.07735 24 1.988e-07 7.967e-06 12 0.05462 0.07474 25 0.00155 0.00142 13 5.323e-07 3.542e-07 In the Southeast fishing areas, age groups with the highest proportions ranged between and 10 corresponding to mean CL of 32.41 - 52.42 mm, while the proportion of age groups to 12 with mean CL ranged from 32.44 mm to 57.98 mm were most abundant in the Southwest fishing areas Figure The mean CL of N norvegicus in catches at different fishing areas from 1970 to 2011 Figure showed that the mean CL of nephrops caught in the Southeast areas fluctuated but did not show any consistent trend with the mean CL was 44.84 mm from 1970 to 2011 Meanwhile, in the Southwest areas, the mean CL of this species revealed an increase from a minimum value of 40.27 mm in 1970 to maximum value of 61.42 mm in 2003 and then gradually decreased to 50.0 126 • NHA TRANG UNIVERSITY mm in 2011 The mean carapace length of N norvegicus in this fishing areas was 47.94 which is significantly higher when compared to Southeast areas (p