International Journal of Biosciences IJB ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 8, p. 43-49, 2015 RESEARCH PAPER OPEN ACCESS Comparative analysis of microbiological status between raw and ready-to-eat product of black tiger shrimp (Penaeus monodon) Md. Shakir Hossain 1, Mehedi Mahmudul Hasan 1, Md. Golam Sarwer 1, Shuva Bhowmik 2* 1 Department of Fisheries and Marine Science, Noakhali Science and Technology University, Sonapur 3814, Bangladesh 2 Department of Fisheries Technology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh Key words: Microbiological status, raw products, ready-to-eat product, Salmonella spp. and Vibrio cholerae. http://dx.doi.org/10.12692/ijb/6.8.43-49 Article published on April 25, 2015 Abstract The investigation was carried out to detect the health hazard microbiological status in raw (head on and shell on) and ready-to-eat product of black tiger shrimp (Penaeus monodon). The abundance of total bacterial load, total coliform, faecal coliform, Salmonella spp., Vibrio cholerae were determined with three different samples of raw and ready-to-eat product of black tiger shrimp. The study represents that raw (head on and shell on) shrimp contains more microorganisms, because in raw shrimp elimination of head and shell was not occurred which contains maximum number of microorganisms of the total body. Total bacterial load (Aerobic Plate Count) of raw black tiger shrimp (head on and shell on) was 1.8967 10 5 ±0.187 10 5 CFU/g whereas in ready-to-eat product of shrimp was 0.17 10 5 CFU/g ± 0.0088 10 5 CFU/g respectively. However total coliform of raw black tiger shrimp and ready-to-eat product of shrimp were 66.67±14.50 MPN/g and <3 MPN/g respectively, and faecal coliform of raw black tiger shrimp (head on and shell on) and ready-to-eat product of black tiger shrimp were 2.20±1.11 MPN/g and <3MPN/g, respectively. Furthermore Salmonella spp. and Vibrio cholerae were totally absent in raw and ready-to-eat product of shrimp. The above mentioned data were under the limit of international standard. This study implies that the quality of ready-to-eat product of black tiger shrimp was excellent for exporting and presented no harm to human health. * Corresponding Author: Shuva Bhowmik shuva_bhowmik@yahoo.com 43 Hossain et al.
Introduction Shrimp and prawn have long been playing a major role in the diet and nutrition of Bangladeshi people and a key contributor in country s export earnings. Major export items from Bangladesh are raw shrimp block frozen, cooked IQF (tail-on/tail-off), IQF Shrimp and prawn, consumer pack of raw frozen shrimp, dry, salted and dehydrated fish and a little quantity of value added shrimp products. 63% of frozen shrimp exported to the European countries and 34% to USA from Bangladesh (DoF, 2011). Black tiger shrimps become contaminated by microorganism and enter seafood processing chain because of inadequate process control, poor standards of hygiene and sanitation in processing plants and post-production contamination during incorrect handling or storage. Most of the publications concluded that bacterial pathogens such as Salmonella spp. and Vibrio spp. are responsible for the majority of 80% disease outbreaks including death (Hamdan et al., 2008). Due to insufficient attention toward standard of hygiene and quality of the products such as shrimp products has suffered considerable losses in rejection from 1975 to 1978 and the country was placed under automatic detention by United State Food and Drug Authority USFDA (Limpus, 1978). Bangladesh was black listed along with other countries by USFDA for having the evidence of Vibrio, Salmonella, filth, flies, cockroach and other insects in frozen marine fishes. It faced heavy loss in the form of rejection and of relatively low price offered by the foreign buyers for fish products from Bangladesh. The processing industry was worried about the quality deterioration of fish fillets. Thus the microbial analysis should be done for maintaining own quality and to gain satisfaction of buyers of European countries. Isolation and identification of microbial food contaminants help to understand how infectious agents enter and spread through the food chain and therefore come up with procedures to prevent or minimize exposure of the consumer to such agents. A study carried out an experiment on microbiological quality of processed frozen black tiger shrimps in fish processing plant (Hossain et al., 2010). But the processed shrimp and fish are sometimes rejected by the importing countries because of high bacterial load, decomposition, filth, unexpected foreign matters as well as pathogenic microbes (E coli, Salmonella, V cholerae etc). Although frozen and ready-to-eat product are being exported in a large scale from our country, there is a lot of information available from the Export promotion Bureau (EPB) that the exported fisheries items earned less price competing with neighboring Countries (Ahmed, 1999). This is undoubtedly a great economic loss for our country. These activities occur occasionally due to improper implementation of HACCP system as well as poor establishment of GMPs (Good Manufacturing Practices) and inadequate sanitation procedures. GMPs are the foundation of HACCP and a pre-requisite program before starting HACCP. HACCP systems must be built-upon a firm foundation of compliance with GMPs and acceptable sanitation procedures. Nowadays, Bangladesh government has been taking serious action against the shrimp and fish processing plants which are not following importers conditions and not implementation HACCP system for ensuring quality products. Therefore, the study was undertaken to asses microbial status between raw and ready-toeat product for exporting to different countries of the world. Materials and methods Sample selection for Microbial Analysis Three samples of black tiger shrimp (head on and shell on) and three samples of ready-to-eat products were collected randomly from three different lots in receiving hall at the beginning of processing. Microbial Analysis Microbial analysis was performed according to the standard procedure for the enumeration and identification of microorganisms. Enumeration of Total Bacterial Load (APC) 20g of the raw shrimp sample was blended for 1 min 44 Hossain et al.
with 180 ml of sterile dilute 0.1% peptone in an automatic blender. That provided a dilution of 10-1. This process was repeated, using the progressively increasing dilution to prepare dilution of 10-2, 10-3, 10-4, and 10-5. The same procedure was followed for the ready-to-eat product samples.1ml 10-1 solution was added with 9ml, 0.1% peptone water (10-2 ) and 1ml with 9ml LTB (Lauryl Tryptose Broth) in the Durham s tube (10-1 ). Then 10-2 solution was converted to 10-3, 10-4, 10-5 solution with the 0.1% peptone solution (Haq et al., 2009). Poured 1ml of solution from each test tube was the sterile Petri dish. Approximately 15 ml agar which has been melted and brought to 45 C was poured into the plates. Plates were rotated by hand 5 times in the clockwise direction, 5 times in the counter clockwise direction and several times crosswise for equal distribution of the media. Fewer than 15 minutes were elapsed between making the dilution and pouring the agar. After solidification of the media, the plates were inverted and placed in incubator to incubate at 37 C for 18 hrs (USFDA, 1984). After 48 hours, the number of colonies which were developed in the Petri dishes was properly counted by colony counter machine. The total number of bacteria per gram of sample was obtained by multiplying the average number of colonies on Petri dishes by the respective dilution factor. The total number of bacteria found from each Petri dish for each dilution was averaged to find a reliable aerobic Plate Count (APC). Enumeration of Total Coliform 20g of the sample was blended for 1 min with 180 ml of sterile dilute 0.1% peptone in an automatic blender. That provided a dilution of 10-1. 1ml 10-1 solution was added with 9ml. 0.1% peptone water (10-2 ) and 1ml with 9ml LTB (Lauryl Tryptose Broth) in the Durham s tube (10-1 ). Then 10-2 solution was converted to 10-3, 10-4, 10-5 solution with the 0.1% peptone solution. Solution of 10-1, 10-2, and 10-3 were incubated Durham s tubes at 37 c for 48 hours. The formation of gas after 48 hours was considered sufficient evidence of the presence of coliform. Gas formation was recorded and the result was computed by using MPN chart (USFDA, 1984). Detection of Faecal Coliform Tubes of lauryl tryptose broth that was positive for gas production were selected and a loopful of Broth from each positive culture were inoculated into a tube of Brilliant Green Bile (2%) Broth and a tube of tryptone water tube were tasted with Kovac s reagent to determine the presence of indole. A positive indole reaction in a broth that has produced gas at 44 0 C indicates the presence of E. coli. The positive gas production tubes were recorded and the results were computed using MPN chart (USFDA, 1984). Detection of Salmonella The presence of Salmonella spp. was detected by homogenizing a 25 g portion of the composite sample in 225 ml (p H 7.5) sterile buffered peptone water aseptically and incubating for 24-48 hr. at 37 c in an incubator(figure 3.20). After incubation 1 ml sample was transferred to duplicate tubes of Tetrathionate (9 ml) and Selenite Cysteine Broth (9 ml), incubated for 24 hours at 37 c and sub-cultured into Xylose Lysine Deoxycholate (XLD) and Brilliant Green Agar (BGA). After incubation for 24 hr at 37 c, characteristics colonies (on XLD- black centered, colony, convex, entries, glossy and on BGA-pink, red, convex, entree glossy colonies surrounded by brilliant red zones in the agar) were streaked with sterile platinum wise loop incubated at 37 c for 6 hrs. After incubation characteristics change may help about presence or absence of Salmonella. H2S gas was not found which indicate that Salmonella was absent in the sample (USFDA, 1984). Detection of V. cholerae 25g portion of the composite sample was added in 225 ml. sterile alkaline peptone water aseptically and incubated at 37 c for 24 hrs. Loopful alkaline peptone water was streaked on the surface of separate plates of Thiosulfate Citrate Bile Salts (TCBS) agar in such a manner to obtain individual colony and incubated at 37 c for 24 hrs. After 24 hours, V. cholerae colony was tested. The colony of V. cholerae was plain, 45 Hossain et al.
yellow color and very big size (generally 2-3 mm). From TCBS the selected colony was transferred to the butt of Triple Sugar Iron Agar (TSIA) Slant with streaking. Then, TSIA tubes are incubated at 37 C for 24 hrs. Black color gas was observed in TSIA indicated that V. cholerae was absent (USFDA, 1984). Data Analysis All data were analyzed with Microsoft Excel 2007. Data were presented as mean ± SEM. Results Aerobic Plate Count (APC), total coliform, faecal coliform, Solmonella spp., Vibrio cholerae of raw (head on and shell on) and ready-to-eat product of black tiger shrimp were measured. The comparison with microbiological quality between raw (head on, shell on) and ready-to-eat product of black tiger shrimp also were measured. Total Bacterial Load (APC) The APC of raw black tiger shrimp among three samples were 2.14 10 5 CFU/g, 1.45 10 5 CFU/g, 2.02 10 5 CFU/g of sample 1, 2 and 3, respectively (Table 1). Furthermore, SPC of ready-to-eat product of black tiger shrimp in three samples were 0.17 10 5 CFU/g, 0.15 10 5 CFU/g, 0.18 10 5 CFU/g of sample 1, 2 and 3, respectively (Table 2). Total bacterial load detected in ready-to-eat product of black tiger shrimp was significantly lower than raw samples of black tiger shrimp (Figure 1). Total Coliform and Faecal Coliform Total coliform of raw (head on and shell on) black tiger shrimp among three samples were 93 MPN/g, 43 MPN/g and 64 MPN/g (Table 1). Furthermore, total coliform in ready-to-eat product of black tiger shrimp among three samples were <3MPN/g, <3 MPN/g, <3 MPN/g (Table 2). Faecal coliform of raw (head on and shell on) black tiger shrimp of three samples were 3.6MPN/g, 3.0 MPN/g, <3 MPN/g (Table 1). The faecal coliform of ready-to-eat product of black tiger shrimp was <3 MPN/g, <3 MPN/g, <3 MPN/g (Table 2). MPN count (Mean ± SEM) of total coliform per gram of sample observed in different samples of raw (head on and shell on) shrimp was 66.67±14.50 MPN/gm, while in ready-to-eat shrimp product, the MPN count of total coliform per gram of all samples was <3 MPN/gm. In raw samples of shrimp, the MPN count of total coliform per gram of sample ranges from 43 to 93, the values varied too much. The mean MPN count per gram of sample of faecal coliform in raw (head on and shell on) shrimp and ready-to-eat shrimp product were 2.20±1.11MPN/gm, 2.20 ± 1.11 MPN/gm respectively (Table 1 and 2). Table 1. Density (CFU/g) of total aerobic bacteria, MPN Count of Total Coliform, Faecal Coliform and presence of Salmonella spp., Vibrio cholerae detected in three different samples of raw (head on and shell on) black tiger shrimp. Sample No. APC (CFU/g) Total Coliform (MPN/g) Faecal Coliform (MPN/g) Salmonella spp. Vibrio cholerae Sample 1 2.14 10 5 93 3.6 Absent Absent Sample 2 1.43 10 5 43 3 Absent Absent Sample 3 2.02 10 5 64 <3 Absent Absent Mean ± SEM 1.8967 10 5 ± 0.187 10 5 66.67±14.50 2.20±1.11 Table 2. Density (CFU/g) of total aerobic bacteria, MPN Count of Total Coliform, Faecal Coliform and presence of Salmonella spp., Vibrio cholerae detected in three different samples of ready-to-eat product of black tiger shrimp. Sample No. APC (CFU/g) Total Coliform (MPN/g) Faecal Coliform (MPN/g) Salmonella spp. Vibrio cholerae Sample 1 0.17 10 5 <3 <3 Absent Absent Sample 2 0.15 10 5 <3 <3 Absent Absent Sample 3 o.18 10 5 <3 <3 Absent Absent Mean ± SEM 0.17 10 5 ± 0.0088 10 5 - - CFU: Colony Forming Unit; MPN: Most Probable Number and APC: Aerobic Plate Count. 46 Hossain et al.
Salmonella and Vibrio cholerae Salmonella spp. and Vibrio cholerae both were absent in raw (head on and shell on) and ready-to-eat product of black tiger shrimp in selected samples (Table 1 and 2). Discussion The standard cooking time and temperature were maintained for ready-to-eat black tiger shrimp product in this study. According to ICMSF (1982), the acceptable upper limit of total bacterial load, total coliform and faecal coliform is cfu/g, 100 MPN/g and <3 MPN/g respectively, while salmonella and V. cholerae should not present. In the study, APC, total coliform and faecal coliform in the ready-to-eat shrimp was 0.17 10 5 CFU/g, <3 MPN/g and <3MPN/g respectively, which is under the limit of ICSMF. Besides, Salmonella spp. and Vibrio cholerae were absent in both the selected samples. Thus, the sample of Black Tiger shrimp ready-to-eat product was under the acceptable limit according of ICMSF and FDA guidelines (ICMSF, 1982 and FDA, 2001). The mean value of total coliform observed in Cooked IQF shrimp was <3 ±0.00 MPN/g, while it was 23.50 ± 13.72 MPN/g in raw block frozen shrimp (Yusuf et al., 2012). Total coliform in Cooked IQF shrimp <3 MPN and it was 21.00 ± 0.25 and 4.20 ± 1.20 MPN in Raw Block Frozen Shrimp and Raw IQF shrimp respectively (Hossain et al., 2010). Faecal coliform, Salmonella spp. and Vibrio cholerae were absent in the selected samples. The present study also observed total coliform and faecal coliform in ready-to-eat product of shrimp <3 MPN, and <3 MPN. In raw shrimp total coliform and faecal coliform were 66.67±14.50 MPN/g, and 2.20±1.11 MPN/g. In readyto-eat shrimp product, elimination of bacteria occurs in two steps first during cooking and then freezing. On the other hand, in raw block frozen and raw IQF shrimp, elimination occurs only during freezing. The lowest count in ready-to-eat shrimp product might be of this reason. From the present study, it has been revealed that ready-to-eat product of shrimps is highly qualified for export purpose. Ahmed and Anwar (2007) performed a bacteriological assessment of value-added ready-to-cook shrimps processed for export from Bangladesh Escherichia coli counts of all the samples were within the acceptable limit of ICMSF standard and coagulate-positive Staphylococcus aureus, Salmonella and Listeria monocytogenes were absent from any specimen. In the present study the absence of Salmonella spp. and Vibrio cholera in ready-to-eat black tiger shrimp was due to proper handling, cooking, freezing and storing at -18 0 C. Sreenivassam (1958) worked on freshly caught whole shrimp. He found that 1.8 10 6 CFU/g bacteria per gram in freshly caught whole shrimp. The result of present study indicate that the raw (head on and shell on) black tiger shrimp was found to contain APC 1.8967 10 5 CFU/g due to geographical variation and management of aquaculture. Alam et al., (2003) performed a study on the prevalence of bacteria in the muscle of shrimp in processing plant were bacteria levels in the muscle decreased over time in shrimp in five suppliers and varied between 8 10 5 CFU/g and 6 10 5 CFU/g. This study indicated that the raw (head on and shell on) black tiger shrimp was found to contain APC 1.8967 10 5 ±0.187 10 5 CFU/g due to the variation in processing methods. Detection of coliform bacteria is used as an indicator of water sanitation or as a general indicator of sanitary condition in the food-processing environment. The presence of faecal coliform is not permitted in the shrimp samples in Japan, USA and European countries. This research indicates that faecal coliform was absent due to proper processing in the fish processing plant where the study was conducted. Shewan (1970) studied the frequency of occurrence of pathogens Salmonella and Clostridium botulinum and frequency of occurrence of Vibrio parahaemolyticus in fish and fishery products. He suggested bacteriological standards of fish and fishery products and presented bacteriological data on some fishery products. Salmonella has been isolated from fresh, frozen, canned and sun dried marine fish products (Natarajan et al., 1985). This study assumes that the absence of Salmonella spp. and Vibrio cholerae in ready-to-eat product of black tiger shrimp was because of proper handling, cooking, freezing and storing at -18 0 C in the fish processing plant. It is possible to reduce pathogenic bacteria load in raw 47 Hossain et al.
shrimp by proper management of farming and handling. Bangladesh exports the frozen sea foods mainly in the EU, USA and other developed countries of the world. After 17 December, 1997 it was mandatory to prepare all the seafood products under the HACCP regulations (USFDA, I997). But the implementation of HACCP in a processing plant is not only narrative task. Good Manufacturing Practices (GMPs) and sanitation are the prerequisite for the implementation of HACCP system. The plant or factory layout, facilities, Standard Operation Procedure (SOPs) and Sanitation Standard Operating Procedures (SSOPs) are the principal points of GMPs and sanitation procedures which are obviously needed to implement HACCP system. acknowledged. References Ahmed MK. 1999. Solving of systematic sea food quality problems cross functional team in the fish processing plants of Bangladesh. Paper Read to the 4 th Annual Quality Convention Proceedings, Dhaka, Bangladesh. Ahmed S, Anwar MN. 2007. Bacteriological assessment of value added ready to cook /eat shrimps processed for export from Bangladesh following the guidelines of international standards. Bangladesh Journal of Microbial 24, 81-84. http://dx.doi.org/10.3329/bjm.v24i2.1247 Alam SMN, Mostafa G, Bhuiyan MDH. 2003. Prevalence of bacteria in the muscle of shrimp in processing plant. Internet Journal of Food Safety 5, 21-23. Fig. 1. Aerobic Plate Count (APC) in three sample of both raw and ready to eat shrimp product. Conclusion Good quality of sea food depends on the careful manipulation of every stage from initial production to processing, storage, marketing and consumption. As it ensured the reduce amount of microbial load so it may be said that is ready for eat as well as export. The GMPs and sanitation procedures of the selected fish processing plant were excellent; hence their product performance (quality) earned a better place in the foreign markets. It is revealed that ready-to-eat product of black tiger shrimp a reduced amount of microbes product than raw (head on and shell on) tiger shrimp for export purpose. Acknowledgement The facilities provided to us in the microbiological laboratory of Ark Sea Foods Ltd., Chittagong, Bangladesh to carry out this study gratefully Do F. 2011. National fish week compendium (in Bengali). Department of Fisheries, Ministry of Fisheries and Livestock, Bangladesh. 144 P. FDA. (Food and Drug Administration), 2001. Fish and fisheries products hazards and controls guidance, (3rd edition). US Food and Drug Administration, Center for Food Safety and Applied Nutrition. Hamdan RH, Musa N, Wei LS, Sarman A. 2008. Isolation and enumeration of coliform bacteria and salmonella spp. from short necked clam orbicularia at east coast, Malaysia. Internet Journal of Food Safety 10, 58-64. Hossain A, Mandal SC, Rahman MS, Rahman MM, Hasan M. 2010. Microbiological quality of processed frozen black tiger shrimps in fish processing plant. World Journal of Fish and Marine Sciences 2, 124-128. Huq KA, Rahman SMB, Islam MB, Shaha BM, Ghosh AK, Sayeed MA. 2009. Quality aspects of frozen shrimp product in processing industry: a case 48 Hossain et al.
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