Food and Nutrition Sciences, 2013, 4, 1201-1208 Published Online December 2013 (http://www.scirp.org/journal/fns) http://dx.doi.org/10.4236/fns.2013.412154 Open Access FNS Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials Olusegun Ayodele Oyelese, Jacob Oyeleye Oyedokun Department of Aquaculture and Fisheries Management, Faculty of Agriculture and Forestry, University of Ibadan, Ibadan, Nigeria. Email: segunoyelese@yahoo.co.uk Received July 13th, 2013; revised August 13th, 2013; accepted August 20th, 2013 Copyright © 2013 Olusegun Ayodele Oyelese, Jacob Oyeleye Oyedokun. This is an open access article distributed under the Crea- tive Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accordance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual property Olusegun Ayodele Oyelese, Jacob Oyeleye Oyedokun. All Copyright © 2013 are guarded by law and by SCIRP as a guardian. ABSTRACT Three different packaging materials of (37 cm × 25 cm) size (Sealed Transparent Polythene Bag (STPB) Sealed Paper Bag (SPB) (Brown envelope), Open Mouth Polythene Bag (OMPB) (Black incolour)) were used for Oreochromis- niloticus (O), Clariasgariepinus (C) and Mormyrusrume (M). Twenty fish samples per species (averaging 250 gm) were hot smoked dried whole for 36 hours at an average temperature of 100˚C. Packaged hot at the rate of 6 fishes per pack- age for each species (three packs for each packaging treatment i.e. 18 pieces were packed while the remaining 2 pieces were used for initial bacteria load and microbial load). Microbial load (Total Viable Count (TVC), Total Coliform Count (TCC) and Total Fungi Count (TFC)) for the fresh fish was initial hot smoked and finally at the end of 12 weeks was monitored. The TVC (bacterial load) of O. niloticus dropped from (10.6 - 8.4) × 104 (fresh state-hot smoked) and M. rume (9.8 - 7.0) × 104, while C. gariepinus slightly in creased from (12.4 - 12.6 ) × 104. After hot smoking, highest TVC of 8.6 × 104 (OMPBC), 8.3 × 104 (SPBC) and 8.2 × 104 (STPBC) was recorded in C. gariepinus among the 9 packag- ing at 12 weeks. However highest tendency for heavy TVC is in all OMPB with highest bacteria load in the OMPBC (8.6 × 104), 7.6 × 104 (OMPBO) and 6.6 × 104 (OMPBM). After 12 weeks highest ranged TFC of (0.6 - 0.7) × 10 4 was recorded in M. rume as against 0.2 × 104 recorded in the initial smoked for all. TCC was high est in C. gariepinus (4.0 - 4.3) × 104. Packaging did not limit the existence of micro-organisms. Six bacteria species (Micrococcus (acidiophilus, luteus), Bacillus (subtilis, cereus, aureus), Staphylococcus aureus, Streptococcus lactis, Proteus (vulgaricus, morganii), Pseudomonas aureginosa) and three fungi species (Aspergillus (niger, tamari), Rhizopusnigricans, fusariumoxysporum) were repr esen ted in all th e pack ag es. On the av er age f ive bacteria and two fungi species were represented, excepting for OMPBM and OMPBO with six bacteria species. Keywords: Bacteria; Coliform; Mould Count/Flora; Freshwater Fish Species; Packaging Material 1. Introduction Bacteria are unicellular microscopic organisms which occur almost everywhere in nature. Up to 1500 species of bacteria have been isolated since bacteria are living things; they acquire a source of food, moisture and suit- able temperature to grow, when thes e condition s are ade- quate. Bacteria cause spoilage of improperly dried fish by multiplying inside the fish flesh thereby causing pu- trefaction. Once bacteria spoilage sets in there it is hard to remedy. The result of bacteria attack is off odour and flavor and when pathogenic bacteria are involved, it could result in illness to consumer [1]. The bacteria that most often involved in the spoilage of fish are part of the natural flora of the external slime of fishes and their intestinal content [2]. They lamented that the predominant kinds of bacteria causing spoilage vary with the temperatures at which the fish are held as follows: Chilling temperature Species of pseudomonas Achromebacter and Flavobacterium Higher temperature Genera micrococcus and
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials 1202 Bacillus Atmospheric temperature Escherichia Proteus Serratia Sarcina and Clostridium Bacteria are unicellular microscopic organisms which occur almost everywhere in nature [1]. Up to 1500 spe- cies of bacteria have been isolated since bacteria are liv- ing things. They acquire a source of food, moisture and suitable temperature to grow [3], when these conditions are adequate. Bacteria will grow by a process known as Binary Fission in which the cell divides into two new cells. Some bacteria causing fish spoilage might have a generation time of 20 minutes at 30˚C [1]. In such a case, a single bacterium may give billions in 10 hours [4]. Whilst increase in the population of micro-organisms by geometric progression is theoretically possible, its practical application is limited by environment factors prevailing. These factors are: 1) Temperature Table 1 below shows the ranges of temperature for the growth of micro-organism. 2) Water Content Table 2 shows the minimum water activity for the growth of micro-organism. 3) Acidity or Alkalinity (pH) Bacteria grow well over a wide range of hydrogen ion concentration pH ranging from 4.0 - 9.0. The optimum pH growth for most bacteria lies between pH 6.5 and 7.5 although some bacteria are capable of growing at the Table 1. Temperature ranges for growth of micro-organ- ism. Types of micro-organism Minimum (˚C) Optimum (˚C) Maximum (˚C) Psychrophiles 0 15 - 25 30 Mesophiles 10 37 43 Thermopliles 25 50 - 65.5 85 Table 2. Minimum water activity for growth of micro-or- ganism [5]. Micro-organism Minimum water activity Normal bacteria 0.90 Normal yeast 0.88 Normal moulds 0.80 Halophilic bacteria 0.75 Dryness resistant moulds 0.05 Osmotic pressure resistant yeast 0.61 extremes of the pH ranges. Bacteria growth and toxin production are inhibited if the conditions are more lethal to micro-organisms than alkaline [6]. 4) Nutrient Composition Bacteria are living organisms and like other living things such as plants and animals, they require a source of energy to survive. Such energy can be obtained from sunlight or by breakdown of nutrients which are mainly carbohydrates, proteins, fats and oil, vitamins and other growth factors. The breakdown of each of these nutrients requires the possession of the appropriate enzymes by bacteria [1,6]. 1.1. Bacterial in Smoked Fish Smoked fish and shellfish products can be a source of microbial hazards including listeria monocytogenes, Sal- monella species and Clostridium botulinium, L. mnono- cytogens has been identified in several food borne out- breaks, in which pasteurized milk, coleslaw and soft cheese were implicated [7]. These organisms have also been isolated from a variety of fish and shellfish products [8]. 1.2. Fungal Attacks in Smoked Fish Insufficient dried fish (still containing approximately 40% moisture) especially at the processing location are prone to fungal infection, principally from the non-spe- cific Penicillium spp., Aspergillus spp. Substantial quali- ties of fish are usually discarded during drying due to fungal growth. Fungal spp. also associated with smoked fish include: Aspergillus fumigates, Absidia spp., Rhizo- pus spp., Mucor spp., Cladosporium spp. [9-12]. It was observed that though smoking fish provides longer shelf life than other preservative methods, smoking will be effective if properly done (especially to reduce packag- ing). Adebayo-Tayo et al. [9] identified 12 different fungi and aflatoxin B1 and G1 in three main markets in Nigeria on smoked dried fish with moisture content ranging from 22.7% - 27.6%. He said the level of infestation might be due to high percentage of moisture content of the smoked fish. 2. Materials and Methods 2.1. Collection of Samples\Packaging Twenty pieces (sample) of each fish species of average weight 250 grams were collected for Oreochromisniloti- cus (O), Clariasgariepinus (C) and Mormyrusrume (M). Also fresh samples were collected for the initial proxi- mate analysis while the remaining fresh fishes were transported to the processing unit for smoking. After Open Access FNS
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials 1203 which the initial proximate analysis of the hot smoked fish was also taken before packaging in the 37 cm × 25 cm packaging materials for each of the smoked fish spe- cies (using each of the three different packaging material for each fish species) at the rate of six (6) fish species per package and labeled e.g. for Oreochromis (STPBO— Sealed Transparent Polythene Bag Oreochromis, SPBO— Sealed paper Bag Oreochromis, OMPBO—Open Month Polythe ne Bag Oreochromis. 2.2. Hot Smoking of the Fish Species The smoking kiln was locally improvised. Three broken blocks each of 0.3 m height was used to raise the wire gauze (on which the fish were laid) to avoid direct con- tact with fire. Big wire gauze of mesh size 2 cm was set on the fire when the fire was fully lit. The three species of the fish to be smoked were placed on the gauze. Big aluminum basin with a opening at the centre was used to cover the fish species in order to conserve the fire. It was through the opening that the temperature of the smoking kiln (chimney) was taken daily, until the three fish spe- cies were hot smoked dried. Hot smoking was done for 36 hours (this was achieved in three days at an average of 12 hours smoking per day) at an average temperature of 100˚C. Hot smoking was done with an exotic hard wood (Eucalyptus species), collected from the Forestry De- partment of the University of Ib adan. Turning of the fish species were done at the same time to maintain uniform drying\smoking at an interval of one hour (1.5 hr) thirty minutes for 3 days. 2.3. Packaging and Shelfing After three days of intensive smoking, each species of the three freshwater fish species were packaged under three different packaging materials (Sealed Transparent Polythene Bag (STPB), Sealed Paper Bag (SPB) (Brown envelope), Open Mouth Polythene Bag (OMPB) (Black in colour)) under room ambient temperature range of 25˚C - 32˚C for 12 weeks. Mould growth: insect infesta- tion was checked daily during this period for each of the fish species. The three different materials used were: A. Sealed Transparent Polythene Bag (STPB) 1. Tilapia (Oreochromisniloticus) (STPBO) 2. Clariasgariepinus (STPBC) 3. Mormyrusrume (STPBM) B. Sealed Paper Bag (SPB) 1. Tilapia (Oreochromisniloticus) (SPBO) 2. Clariasgariepinus (STBC) 3. Mormyrusrume (STBM) C. Open Mouth Polythene Bag (OMPB) 1. Tilapia (Oreochromisniloticus) (OMPBO) 2. Clariasgariepinus (OMPBC) 3. Mormyrusrume (OMPBM) The fishes were packaged hot in the packaging bags and stored in the laborator y for 12 weeks. 2.4. Preparation of Media All analytical procedures in this study are according to the A.O.A.C [13]. 2.4.1. Nutrie nt Agar Twenty eight (28) grams of powdered commercially prepared of nutrient agar was weighed on Analytical metller balance into a clean dry 1 litre conical flask and 1000 ml of distilled water placed inside a water bath set about 90˚C, allow the agar to dissolve. Distribute them into MacCantney bottles and placed them inside auto- clave and set the autoclave at 121˚C for 15 mins. 2.4.2. Macconkey Agar (Mcca) Fifty five (55) grams of macConkey Agar was weighted into a 1 litre capacity of conical flask and brings to boil to dissolve the agar. Distribute them into Mac Cartney bottles and autoclave as for Nutrient Agar. 2.4.3. Potato Dextrose Aga r (PDA) Thirty nine (39) grams of PDA was weighted into a 1 litre capacity of conical flask bring to boil an d distributed them into Mac Cartney bottles and placed them inside an autoclave as for Nutrient Agar. 2.5. Pouring of Plates After autoclaving the media were placed inside a water bath set at 45˚C to maintain the media in a molten state. 1 g each of the sample was weighed into a test-tube containing 9 ml of sterile distilled water and serially di- lute them until you reach your dilution factor (10-5) and plate out 1 ml of the last dilution factor into a sterile plates (sterilized by placing them in an over set at 160˚C for an hour). Pour the media individually i.e. Nutrient Agar, Mac Conkey Agar and Potato Dextrose Agar into a separate plate i.e. each sample will have 3 plates and they were duplicated. After solidifying the plates were incubated in an incu- bator set at 370˚C for Nutrient Agar and Mac Conkey Agar while the potato Dextrose Agar was incubated at 280˚C - 30˚C. All the plates were incubated invertedly. 2.6. Microbial Count The plate was counted at 48 hours for Nutrient Agar and Mac Conkey Agar while it was read for potato Detrose Agar t 72 hours. Open Access FNS
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials Open Access FNS 1204 2.7. Lactic Acid Bacterial Count est TCC of 0.8 × 104, was recorded for O. niloticus, fol- lowed by C. gariepinus (0.4 × 104) and lastly M. rume with TCC of 0.3 × 104. Fifty five (55) grams of Man De Rogsa and shape me- dium (MRS) was weighed as for the above nutrient agar preparation procedures. Table 4 and Figure 1 show that the TVC of O. niloti- cus dropped from 10.6 × 104 (in the fresh state) to 8.4 × 104 in the initial hot smoked and M. rume dropped from 9.8 × 104 (fresh state) to 7.0 × 104 after hot smoking, while the TVC of C. gariepinus slightly increased from 12.4 × 104 in the fresh state to 12.6 × 104 after hot smok- ing. While TFC increased from zero to 0.2 × 104 for the three fish species; highest TCC of 9.8 × 104 was recorded in C. gariepinus, followed by 4.2 × 104 in O. niloticus and lastly 3.0 × 104 in M. rume. 2.8. Statistical Analysis Analysis of Variance (ANOVA) in completely random- ized design was performed on the data obtained using SPSS (2006). Significant means were compared at 5% probability level using Duncan’s New Multiple Range Test (DMRT) as provided in the same SPSS (2006). Table 5 shows that the least bacteria load (TVC) was recorded in the SPBM and STPBM, both recording TVC 6.4 × 104 in each case. Generally highest TVC of 8.6 × 104 (OMPBC), 8.3 × 104 (SPBC) and 8.2 × 104 (STPBC) were recorded in all C. gariepinus among the nine pack- ages at the end of 12 weeks storage/packaging. Next is O. niloticus packaging 7.6 × 104 (OMPBO), 7.4 × 104 (STPBO) and 7.2 × 10 4 (SPBO) and lastly M. rume 6.6 × 3. Result As shown in Table 3 the microbial load varied signifi- cantly (P < 0.05) among the three species. In the fresh fish the highest TVC of 12.4 × 104 was recorded in C. gariepinus, this is followed by O. niloticus with 10.6 × 104 and lastly M. rume 9.8 × 104. However while TFC was zero in the fresh fish for the three fish species, high- Table 3. Microbial load of fresh fish samples. Fish species Total Viable Count (TVC) Total Coliform Count (TCC) Total Fungi Count (TFC) C. gariepinus 12.4 × 104 0.4 × 104 NIL O. niloticus 10.6 × 104 0.8 × 104 NIL M. rume 9.8 × 104 0.3 × 104 NIL Table 4. Microbial load of initial hot smoked fish. Fish species Total Viable Count (TVC) Total Coliform Count (TCC) Total Fungi Count (TFC) C. gariepinus 12.6 × 104 9.8 × 104 0.2 × 104 O. niloticus 8.4 × 104 4.2 × 104 0.2 × 104 M. rume 7.0 × 104 3.0 × 104 0.2 × 104 Table 5. Final microbial load at the end of twelve weeks storage/packaging of the three hot smoked freshwater fish species. Fish species Total Viable Count (TVC) Total Coliform Count (TCC) Total Fungi Count (TFC) SPBC 8.3 × 104 4.0 × 104 0.6 × 104 OMPBC 8.6 × 104 4.3 × 104 0.6 × 104 STPBC 8.2 × 104 4.2 × 104 0.5 × 104 SPBO 7.2 × 104 3.8 × 104 0.4 × 104 OMPBO 7.6 × 104 3.4 × 104 0.3 × 104 STPBO 7.4 × 104 3.3 × 104 0.5 × 104 SPBM 6.4 × 104 3.2 × 104 0.6 × 104 OMPBM 6.6 × 104 3.1 × 104 0.7 × 104 STPBM 6.4 × 104 3.2 × 104 0.7 × 104
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials 1205 104 (OMPBO), 6.4 × 104 (SPBM) and 6.4 × 104 (STPBM) respectively. However all the OMPB packages (Open Mouth Polythene Bag)—OMPBC (8.6 × 104), OMPBO (7.6 × 104) and OMPBM (6.6 × 104) had the highest bac- teria load in each of the 3 fish species. However the high- est ranged fungi (TFC) of 0.6 × 104 - 0.7 × 104 was re- corded in M. rume. This is followed by C. gariepinus with 0.5 × 104 - 0.6 × 104 while least TFC range of 0.3 × 104 - 0.5 × 104 was recorded in O. niloticus. TCC was highest in C. gariepinus ranging from 4.0 × 104 - 4.3 × 104 followed by O. niloticus (3.3 × 104 - 3.8 × 104) and lastly M. rume (3.1 × 104 - 3.2 × 104) respectively. Generally M. rume was the best packaged in terms of bacteria load (TVC) with the least range of (6.4×104 - 6.6×104) followed by O. niloticus (7.2×104 -7.6×104) and lastly C. gariepinus (8.2×104 - 8.6×104) which is the poorest in terms of bacteria loads. There were significant (P < 0.05) differences between and within the TVC (i.e bacteria load), TCC and TFC for the three species in this study. 0 2 4 6 8 10 12 14 TOTALVIABLECOUNT(TVC) C.gariepinus O.niloticus M.rume Figure 1. Total Viable Count (TVC) (Bacteria load) for the three fish species for the fresh fish, initial hot smoked and inal/smoked packaged at the end of 12 weeks. f Table 6 shows that 6 bacteria species were identi- fied in the fresh O. niloticus, while 5 species each were identified for C. gariepinus and M. rume in their fresh state. Also Micrococcus acidiophilus and Proteus vulga- ricus were identified in the three fresh fish species under study, while Streptococcus lactis and Staphylococcus au- reus were absent in C. gariepinus. However Serra- ticmacescenes was only present in the fresh C. garie- pinus. Table 7 shows that only C. gariepinus had only one fungi species (Rhizopusnigrica) represented in the ini- tially hot smoked three (3) fish species. While 6 bacteria species were each represented in C. gariepinus and M. rume; O. niloticus had 5 bacteria species; also only Staphylococcus aureus was present throughout in the 3 initially hot smoked fish species. Table 8 shows th at only the OMPB for M. rume had 6 bacteria species and 2 fungi species, while the remaining 8 packages had 5 bacteria species and 2 fungi species. The prominent fungi species represented all the 9 pack- ages are Aspergillu ssp (niger, tamari), Rhizopusni- gricans (in SPBO, OMPBO, OMPBC, SPBM AND STPBM), WHILE Fusari umoxysporum is only repre- sented in STPBO. The prominent bacteria species represented in all the 9 packages are Micrococcus sp (acidiophilus and luteus), Bacillus sp (aureus, cereus andluteus ). Staphylococcus aureus is present in 8 packages with the exception of OMPBO, Streptococcus lactis is also present in 8 pack- ages excepting SPBO. Proteus sp (vulgaricus and mor- gani) were presented in 7 packages, excepting OMPBC and STPBM. Lastly, Pseudomonas aureginosa is present in only 3 packages (SPBO, OMPBO and OMPBC). Since micro-organisms are ubiquitous the type of pack aging (as shown in the study) will not limit their exis- tence. Tables 6. Bacteria species identified from the fresh three fish species. Fish species Micro organism C. gariepinus Micrococcus acidiophilus, Bacillus cereus, Serraticmacescenes, Bacillus subtilis, Proteus vulgaricus (5 b a c t e ri a specie s ) O. niloticus Pseudomonas auregionosa, Streptococcus lactis, Micrococcus acidiophilus, Micrococcus luteus, Staphylococcus aureus, Proteusvulgaricus (6 bacteria species) M. rume Staphylococcus aureus, Bacillus subtilis, Micrococcus acidiophilus, Proteusvulgaricus, Streptococcus lactis (5 bacteria species) Table 7. Bacteria and fungi species identified from the initial hotsmoked three fish species. Fish species Micro organism C. gariepinus Micrococcus luteus, Bacillus cereus, Staphylococcus aureus, Streptococcus lactis, Pseudomonas aureginosa, Proteus vulgaricus, 6 bacteria species + 1 fungi (Rhizopusnigrica) O. niloticus Streptococcus lactis, Micrococcus acidiophilus, Staphylococcus aureus, Bacillus subtilis, Micrococcus acidiophilus, Bacillus macerans. 5 bacteria species + Nil (0) fungi species M. rume Staphylococcus aureus, Bacillus subtilis, Micrococcus acidiophilus, Micrococcus luteus, Proteus morganii, Pseudomonas aureginosa (6 bacteria species + Nil (0) fungi species) Open Access FNS
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials 1206 Table 8. Bacteria and fungi species identified from the smoked three fish species at 12 weeks of storage/packaging. Packaging Micro-organism (bacteria and fungi species) SPBC Bacillus cereus, Streptococcus lactis, Staphylococcus aureus, Proteus vulgaricus, Micrococcus acidiophilus, 5 bacteria + 2 fungi species Aspergillustamari, Aspergillusniger OMPBC Staphylococcus aureus, Bacillus ce reus, Micrococcus luteus, Pseudomonas aureginosa, Streptococcus lactis, 5 bacteria + 2 fungi species Rhizopusnigricans, Aspergillusniger STPBC Staphylococcus aureus, Bacillus cereus, Micrococcus acidiophilus, Proteus vulgaricus, Streptococcus lactis, 5 bacteria + 2 fungi species Aspergillustamari, Aspergillusniger SPBO Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Proteus vulgaricus, Pseudomonas aureginosa, 5 bacteria + 2 fungi species Rhizopusnigricans, Aspergillusniger OMPBO Streptococcus lactis, Micrococcus acidiophilus, Bacillus cereus, Streptococcus lactis, Proteus vulgaricus, Pseudomonas aureginosa, 6 bacteria + 2 fungi species Rhizopusnigricans, Aspergillusniger STPBO Micrococcus acidiophilus, Streptococcus lactis, Proteus vulgaricus, Bacillus cereus, Staphylococcus aureus, 5 bacteria + 2 fungi species Aspergillusniger, Fusariumoxysporum SPBM Staphylococcus aureus, Micrococcus luteus, Bacillus macerans, Streptococcus lactis, Proteusmorganii, 5 bacteria + 2 fungi species Rhizopusnigricans, Aspergillustamari OMPBM Staphylococcus aureus, Bacillus cereus, Proteus vulgaricus, Bacillus subtilis, Streptococcus lactis, Micrococcus acidiophilus, 6 bacteria + 2 fungi species Aspergillustamari, Fusariumoxysporum STPBM Micrococcus acidiophilus, Micrococcus leteus, Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis, 5 bacteria + 2 fungi species Rhizopusnigricans, Aspergillusniger 4. Discussion The highest bacteria load (TVC) of 12.4 × 104 was re- corded in the fresh C. gariepinus followed by 10.6 × 104 in O. niloticus and lastly 9.8 × 104 in the fresh M. rume. However, initial hot smoked reduced th e bacteria load of O. niloticus to 8.4 × 104 and M. rume to 7.0 × 104 while the initial hot smoked C. gariepinus TVC of 12.6 × 104 was not affected by hot-smoking since a slight increase of 0.2 × 104 was recorded after hot smoking. Packaging had a significant (P < 0.05) effect at 12 weeks storage/packaging of smoked fish for C. garie- pinus which reduced from 12.6 × 104 TVC to OMPBC (8.6 × 104)—SPBC (8.3 × 104)—STPBC (8.2 × 104) and M. rume with TVC reducing from 7.0 × 104 (in the initial hot smoked fish) to 6.6 × 104 (OMPBM)—6.4 × 104 (SPBM)—6.4 × 104 (STPBM). However all the OMPB packages (Open Mouth Polythene Bag)—OMPBC (8.6 × 104), OMPBO (7.6 × 104) and OMPBM (6.6 × 104) had the highest bacteria load in each of the 3 fish species. This is also revealed in Figure 1. Total Coliform Count (TCC) generally increased from the fresh fish sample 0.4 × 104 to 9.8 × 104 (C. garie- pinus) 0.8 × 104 to 4.2 × 104 (O. niloticu s) and 0.3 × 104 to 3.0 × 104 in the initial smoked (M. rume). Total Coli- form Count (TCC) dropped significantly (P < 0.05) from 9.8 × 104 in the initial hot smoked C. gariepinus to a range of 4.0 × 104 - 4.3 × 104 in all the 3 C. gariepinus packaging, while TCC virtually remained the same for the M. rume packaging and dropped from 4.2 × 104 to a range of 3.3 × 104 - 3.8 × 104 for O. niloticus at the end of 12 weeks. This is shown in Figure 2. No Fungi count was recorded in the fresh fish sample 0 2 4 6 8 10 12 TOTALCOLI FO R MCO UNT(TCC) C.gariepinus O.niloticus M.rume Figure 2. Total Coliform Count (TCC) for the three fish species for the fresh fish, initial hot smoked and final/ smoked packaged at the end of 12 weeks. for the 3 fish species. However a value of 0.2 × 104 fungi count was recorded for the 3 fish species after initial hot smoking. This value increased; highest for M. rume (0.6 × 104 to 0.7 × 104) next is 0.4 × 104 to 0.6 × 104 in C. gariepinus and lastly 0.3 × 104 - 0.5 × 10 4 in O. nilo ticus. Since micro-organisms are ubiquitous the type of pack- aging (as shown in this study and Figure 3) will not limit their existence. The bacterial load (TVC) count for all the three spe- cies of fish in the nine packages used for this study are below the maximum bacteria count of 5 × 105 cfu for good fish product according to the International Com- mission on Microbiology Safety for Food [14]. For C. gariepinus significant (P < 0.05) decreases were observed in the TCC 9.8×104 in the initial smoked fish which reduced to a range of 3.1 × 104 - 3.2 × 104 at the end of 12 weeks. This was in conformity with Wil- Open Access FNS
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials 1207 0 0.2 0.4 0.6 0.8 FreshFish Initial SPB OMPB STPB TOTALFUNGICOUNT (T FC) C.gariepinus O.niloticus M.rume Figure 3. Total Fungi Count (TFC) for the three fish species for the fresh fish, initial hot smoked and final/smoked packaged at the end of 12 weeks. liam, C.F and Dennis, C.W [15] who reported that the faecal coliforms count of fresh C. gariepinus fillets were similarly low after 8 days of cold storage. Table 3 shows that there was absence of fungi in the fresh sample of the three fish species, while in Table 4 only one species of fungi (Rhizopusnigrica) was present in the initially smoked C. gariepinus. At the end of the 12 weeks of storage/packaging three (3) more fungi spe- cies (Aspergillusniger, Aspergillustamari and Fusaru- moxysporum) were represented at th e rate of 2 fung i spe- cies per packaging. That is fungi species were repre- sented in all the 9 packages. The results obtained were similar to those observed by Adebayo-Tayo et al. and Fafioye, O.O et al . [9,16]. During storage of smoked fish product there was significant (P < 0.05) increase in the fungi count with length of storage as seen in this study. This is in line with Oyebamiji, O. F et al. and Wogu, M.D et al [11,12] who worked on stored smoked fish products marketed in the open market. The presence of fungi may be due to the difference in the chemical com- position of th e fish species and to which different moulds react differently [16,17]. Only the OMPBM and OMPBO had 6 bacteria species represented while the remaining 7 packages had 5 bacte- ria species. The prominent bacteria species represented in all the nine (9) packages are Micrococcus sp (acidiophi- lus and luteus), Bacillus sp (aureus, cereus and luteus), staphylococcus aureus (is present in 8 packages) except in OMPBO. Streptococcus lactis also in 8 packages ex- cepting SPBO. Others are Proteus vulgaricus, P. mor- ganii and Pseudomonas aureginosa. 5. Conclusion Highest Bacteria Count (TVC) was recorded in C. garie- pinus packages among the nine packages at the end of 12 weeks. The 3 packaged fishes for C. gariepinus had the highest bacteria load with OMPBC (Open Mouth Poly- thene Bag Being the Highest). Highest tendency for heavy bacteria load (TVC) is in the Open Mouth Poly- thene Bag which has been confirmed in the OMPB for all the 3 fish species. Highest ranged Total Fungi Count (TFC) was recorded in M. rume followed by O. niloticus. Total Coliform Count (TCC) was highest in C. garie- pinus followed by O. niloticus. Packaging did not limit the existence of micro-organisms. There were 5 bacteria species and 2 fungi species represented in each of the packages (with the exception of OMPBM and OMPBO with 6 bacteria species). The prominent fungi species represented in the 9 packages at the end of 12 weeks are Aspergillus species (niger and tamari), Rhizopusnigri- cans and Fusariumoxysporum. Prominent bacteria spe- cies represented in all 9 packages are Micrococcus spe- cies (acidiophilus and luteu s), Bacillus species (aureus, cereus and luteus). Staphylococcus aureus is present in 8 packages (excepting OMPBO) and also Streptococcus lactis (excepting SPBO). Proteus species (vulgaricus and morganii) (in 7 packages excepting OMPBC and STPBM) and lastly Pseudomonas aureginosa are present in only 3 packages (SPBO, OMPBO and OMPBC). REFERENCES [1] A. A. Eyo, “Fish Processing Technology in the Tropic National Institute for Freshwater Fisheries Research (NIFFR),” New Bussa, 2001, pp. 10-170. [2] H. I. Ibrahim, A. A. Kigbu and R. Muhammed, “Wo- men’s Experiences in Small Scale Fish Processing in Lake Fafenwa, Fishing Community, Nasa rawa State,” Ni- geria livestock Research for Rural Development, Vol. 23, 2011, p. 3. [3] G. Hobbs, “Fish Handling and Processing,” Ministry Of Agric, Fisheries and Food, Torry Counter, Edinburg, London, 1965, pp. 20-23. [4] H. H. Huss, “Fresh Fish Quantity and Quality Changes,” F.A.O Fisheries Series, Danish Rome, 1988, pp. 15-29. [5] J. M. Shewan, “Bacteriological Standards for fish and Fishery Products,” Chemical Industrial, Vol. 2, 1970, pp. 299-302. [6] J. J. Conell, “Control of Fish Quality,” 4th Edition, Churchhill Livingstone, Edinburg, 1995. [7] B. W. Fleming, S. L. Cochi, K. L. MacDonald, J. Bron- dum, P. S. Hayes, B. D. Phkaytes, M. B. Holmes, A. Audurier, C. V. Broome and A. L Reingold, “Pasteurized Milk as a Vehicle of Infestation in an Outbreak of Luste- riosis,” New England Journal of Medicine, Vol. 312, 1985, pp. 404-407. http://dx.doi.org/10.1056/NEJM198502143120704 [8] P. K. Ben Embarek, “Presence, Detection and Growth of Lusteramonocytogenes in Seafoods: A Review,” Interna- tional Journal of Food Microbiology, Vol. 23, No. 1, 1994, pp. 17-34. http://dx.doi.org/10.1016/0168-1605(94)90219-4 [9] B. O. Adebayo-Tayo, A. A. Onilude and U. G. Patrick, “Micro Floral of Smoked-Dried Fishes Sold in Uyo, East- Open Access FNS
Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials Open Access FNS 1208 ern Nigeria,” World Journal of Agriculture Science, Vol. 43, 2008, pp. 346-350. [10] O. J. Abolagba and E. C. Uwagbu, “A Comparative Ana- lysis of the Microbial Load of Smoked-Dried Fishes (Ethmalosafunbruata and Pseudotolithuselongatus) Sold in Oba and Koko Markets in Edo and Delta States, Nige- ria at Different Season,” Australian Journal of Basic and Applied Science, Vol. 5, 2011, pp. 500-544. [11] O. F. Oyebamiji, T. R. Fagbohun and O. O. Olubanjo, “Fungal Infestation and Nutrient Quality of Traditionally Smoke Dried Freshwater Fish,” Turkish Journal of Fish- eries and Aquatic Science, Vol. 8, 2008, pp. 7-13. [12] M. D. Wogu and A. D. Iyayi, “Micro-Flora of Some Smoked Fish Varieties in Benin City, Nigeria,” Ethiopian Journal of Environmental Studies and Management, Vol. 4, No. 1, 2011, pp. 36-38. [13] AOAC, “Official Methods of Analysis of AOAC Interna- tional,” 17th Edition, Association of Analytical Commu- nities, Gaithersburg, 2002. [14] ICMSF, “International Commission on Microbiological Specifications for Foods. Micro-Organisms in Foods 7. Microbiological Testing in Food Safety Management,” Kluwer Academic Plenum Publishers, New York, 2002, p. 199. [15] C. F. William and C. W. Dennis, “Food Microbiology,” 4th Edition, Food Science Series, Mac Grow-Hill Book Company, Singapore, 1998, pp. 243-252. [16] O. O. Fafioye, M. O. Efuntoye and A. Osho, “Study on the Fungal Infestation of Five Traditionally Smoked Fish,” 2002. [17] W. Reed, J. Burchard, J. Hopson, A. J Jeness and I. Yaro, “Fish and Fisheries of Northern Nigeria,” Gaskiya Cor- poration, Zaria, 1967, 226 p. Abbreviations TVC: Total Viable Count TCC: Total Coliform Count TFC: Total Fungi Count SPBC: Sealed Paper Bag-Clariasgariepinus OMPBC: Open Mouth Polythene Bag-Clariasgariepinus STPBC: Sealed Transparent Polythene Bag-Clariasgari- epinus SPBO: Sealed Paper Bag-Tilapia (Oreochromisniloticus) OMPBO: Open Mouth Polythene Bag-Tilapia (Oreo- chromisniloticus) STPBO: Sealed Transparent Polythene Bag-Tilapia (Ore- ochromisniloticus) SPBM: Sealed Paper Bag-Mormyrusrume OMPBM: Open Mouth Polythene Bag-Mormyrusrume STPBM: Sealed Transparent Polythene Bag-Mormyrus- rume
|