MICROBIAL OCCURENCE ON DIFFERENTLY PRESERVED …aquaticcommons.org/24094/2/28_opt.pdfupandallowed togel.TheMcConkey treated plates wereinverted andplates were incubated at37QC[or24

1.01NTRODl:CTIO~ i;

Tilapia fish are widely distributed and now cultured inmost part of the world (Adeparusi et of. 2Q07).Nigeria is the second largest producer of farm-raised tilapia in Africa, after Egypt (Adesulu, 1997;Fagbenro,2002; EI-Sayed, 2006; Fagbenro et al., 2010). As human food, fish protein contains mOSIof theessentialamino acids in particularly lysine, methionine and tryptophan. Due LO fish low cholesterol levelcoupledwith quality nutrient profile it is most desirable (VlFC, 2003). However, fish, being a perishableproduct and a good substrate suitable for microbial growth, arc widely exposed to microbialcontamination through contact with soil, dust and water and by hancUing at harvest or during postharvestprocessing(De Rover 1999, Venugopa12002, Jimoh et al, 2009). They, therefore, harbour a diverse rangeofmicroorganisms including plant and human pathogens. Plausible explanation to the origin of thesemicro-organisms may be through contaminated surfaces of the processing equipment (Reij et al. 2004).Transfer of microorganisms by personnel during handling and preparation can also be one of the ways bywhichfish and its products gct contaminated (Chen et al. 200 1,Montville et al. 2001, Bloomfield, 2003).Bacteria are transferred from contaminated hands of food workers to food and subsequently to othersurfaces(Montville et al. 2002). When fish are live, muscle tissue is considered to be sterile, but after deaththebarriers to microbiological invasion begin to break dO\VIland bacteria are able to grow more freely,althoughwill be rarely found within deep muscle tissue. Bacteria wi11tend to be confined to the surface ofthedead fish and this is where growth will occur and spoilage begins (Betts and CCFRA, 1996). Theprimary objective of food processing industries is to provide safe, wholesome and acceptable food to theconsumerand control of microorganisms is essential to meet this objective (Baggen-Ravn et aZ. 2003:).

. . '.. ,:

ABSTRACTThisstudy reports the microbial flora of differently processed tilapiafish obtainedfrom Olomoore market,Abeokuta,Nigeria. Samples were purchased, differently processed (using smoking, salting.freezing) andanalyzedfor [he presence 0/ bacteria. Microbial loads and characterisation were examined on the-gillsandthe skill using standard microbiological procedures. The progression of growth. was also monitoredwithin ]O-day storage period jar the differently processed fish. Proteus vulgaris, Leuconostoc:meseritiroides, Klebsiella planticola, Pseudomonas spp, Staphylococcus aurcus, Bacillus subtiUs,· .Enterobacter spp, Proteus vulgaris, Lactobacillus spp. were the bacteria isolated from the differentlyprocessedfish. There was no significant difference (PC 0.05) in the bacteria load ofthe gill during thefirstday ofprocessing. However, significant variation (PO O.05) existed in the bacteria load of the skin of thefish during the first day of processing. Although no Significant difference (POO.05) was. recorded in thebacteriaload of {he gill and the skin of the fish during the first day 0.(processing in nutrient agar medium.Inthe tenth dayfrozenjish gill had the highest bacteria load while smoked fi~h gill had the lowest bacterialoadthere was Significant difference (P= O.05) in the bacteria load 01'1 the gill of differently processedfishgill. Similarly. there was significant variation (PO O.05) in {he bacteria load ofthe skin 0/ the fish during~~~~~~ ~.Keywords: Bacteria, microbial occurrence, tilapio., smoking, salting, boiling ..

Department of Fisheries Technology, Federal College of Animal Health and ProductionvTechnology, Moor Plantation, P\.ffi 5029, Ibadan

IDcpartment ofBiologicaJ Sciences, Crescent University, PMB 2104, Sapon Abeokuta, Ogun State, NigerialDepart!:nent of.Marine Sciences, University of Lagos, Akoka, Nigeria

* Corresponding Authors E-Mail: jawabus§l:.rma:l.com

MICROBIAL OCCURENCE ON DIFFERENTLYPRESERVED TILAPIA FISH ~ ABEOKUTA NORTH

LOCAL GOVERNMENT, NIGERIA

2.5 Statistical analysisResults are expressed as mean ± SD. All data were subjected to one way ANOYA using SPSS 13.0 forwindow software. Where significant differences occurred, the group means were further compared withDuncan's multiple range test using SPSS 13.0 (SPSS, Il., USA).

2.4 Microbial Count: Colonies which developed after incubation were subjected to counting and wereexpressed in Colony forming Unii/g (CRT).

3.0RE8ULTSTable 3.1 shows the identification of bacteria isolated from the skin and gills of differently processedOreochromisniloticus obtained from Olomoorc market, Abeokuta. The bacteria isolated include Proteusvulgaris, Leuconostoc meseritiroides, Klebsiella planticola. Pseudomonas spp, Staphylococcus aureus,Enierubacler spp,Proteus vulgaris,Lactobacillus spp.~~~~~~~~~~~~~~------~~~~~~

2.3 Isolation and characterization of'rnicrofloraEach organ was placed insterile bottle containing 5ml sterile distilled water and vigorously shaken to allowthe content dissociate in water and serially diluted to 10-;. Then from each suspension, 0.1m! was pourplated using fresbly prepared :McConkcy. The plates after being coveted were gently swirled to evenly mixup and allowed to gel. The McConkey treated plates were inverted and plates were incubated at 37 QC [or 24hours. Representative colonies emerging from the plates were grouped according to their culturalcharacteristics, purified by repeated sub-culturing and maintained on appropriates agar slams as stockcultures. The bacterial isolates were tested for Gram reaction (Claus 1992), morphology, motility, catalaseand oxidase reactions, citrate utilization, coagulase production, starch hydrolysis and sugar fermentation(Harrigan and McCance, 1976,Seeley and Van Demark 1972).

2.2 Processing of the Fish SamplesThe processing methods carried out on tile samples were freezing, salting and smoking. The frozensamples were kept in freezer, smoked samples were obtained using smoke from smothering wood, the thirdsct was heavily salted. Samples were taken from gill and flesh for culture on the i" day, 3nd day, 5\b day, 7&0day, 9'h day and 11(,days. Each of the samples was dissected aseptically to remove the gill and the flesh andmashed gently with pestle and mortal respectively. The glass wares used were sterilized inan oven at 160°c for 90 minutes. Absolute alcohol was used to sterilize the surface of the working table.

Salrin27S±SAverage Weight , 265:::;::15"

2.1 Collection of the Fish SamplesThe fish samples (Oreochromis niloticus) were obtained from fishermen at Olomoorc market, Abeokuta,Nigeria. They \~ere caught from Ogun river which covers the upper Ogun to lower Ogun, flowing fromOyan dam area 'to Adigbe-Saraki area. They were transported to the market in baskets, and plasticscontainers. The fish were transported to the laboratory where they were properly washed and weighed.They were divided into three parts and the following trearments were given to each of them as follows asshown in table 1.

Table 1: Average Weight of Processed fishI Freezing

Fish are processed inmany different ways in different parts of the world. Heavy salting freezing, drying,hot smoking, canning and pasteurisation are all recognised methods of fish preservation. All affect themicro-organisms on the fish indifferent ways and will result in a different type ofmicrofl ora and differentrisks from spoilage organisms and pathogens. Similarly, the fish microflora load and diversity changeduring storage (Lund et al, 2000). TIlls study therefore examines the bacteria load and diversity ondifferently processed tilapia stored for a period often days.2.0 MATRRIALSAND METHODS

-_:.1' -,t.

Coagulase-

1+I - - - - - + I . I -I -

SII"AR UTILIZATION--

Glucose - + + + + I + + + + - +-

Fructose + + - - I + + - + + "T +

Maltose .+ + - - + + .' + + + + + +"

Sucrose-... + -l + + + + + + II + +

•I.actose - + + + + + + + + + II,Probable Proteus Leuconostoc Klebsiella Pseudomonas Staphylococcus Enterobacter Bacillus Proteus Lactobacillus Pseudomonas Proteus Tactobacillusbacterium \'U7garis meseritiroides planticola aureus spp .. subtilis " vulgari« SPP vulgaris.. JfJP SPP spp

+_- • .... ' ". ..' , __I

+.i.

+ ++ + ++

+~'olility +

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Catalase + +

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od Rod

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, While Wl.itc

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Entire Irregular

Flat Fntire

rial Raised

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Samples DayO Da}O gill IDay2 IDay2 gill IDay-l Skin IDay" gill IDay6 Day6 DayS SkSkin Skin Skill gill

COLO;'ol YIhIORl'ROLOGY-

Colour \nile Dirty white \\ hi'e Creamy Creamy Creamy White WLite Whit..:

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Rough 'MOOlh Glittering Smooth ( llirtering Rough Rougb Rough RoughI

Share Ent.re Ro.md Circular ROll'ld Circular Irregular ITree.1I1M Entire Irregul

Flc\ution Flat Hat EO'ifo: i Eulin: Entire R~is('d Entire Flat Entire-

Fdgc Plat l_,obute Raised Raised R01ise;r-- Entire Raised nat Rds<.:u

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Table 3.1: 11> }!;~TlFICATIOl'\OFTIIE DACfERIA rSOT ..\"Jjo;SON THE DIFFERENTLYPROCESSED TlLAPIA OBTAINED FROM Ol.OMOORE ]\{AR.K.k:T.ABEOKUTA.

4.0 DISCUSST01"Microbial tests of fish and fish products arc used by the industry for contractual and internal purposes andby the authorities to check that the microbiological status is satisfactory (Jay 1992). In this present study,all the processed samples'gills and skin haboured Proteus vulgaris, Leuconostoc meseritroides, Klebsiellaplanticola r Pseudomonas spp, Staphylococcus aureus, Enterobacter spp., Bacillus subtilis. Lactobacillusspp. Most olthc organisms found on these processed fish are those commonly .ound in SOil and water. Themicroflora isolated in tills present study were similar to the microorganisms reported by Olawale et al(2005) and Adesokan et al (2005). The presence of Bacillussp.va this samples is also comparable with thefindings of Fagade et al (2005) .Aspergillus niger obtained in the fish samples were in accordance withMartin (1994) when he stated that these organisms were the commonest microorganisms associated withprocessed fish and these microorganisms were also reported by Abolagba and Igbincvoo, (2010) insmoked fish (Clarias sp) sold in Benin metropolis. The results could not establish whether contaminationtook place before the raw material was being processed. Plausible explanation that could be given is thatcontamination did take place in the processing area, as this is supported by other studies. According toVenugopal (2002) contamination of fish particularly by pathogens may occur prior to harvest, duringcapture, processing, distribution and/or storage. Huss et al. (2000) have pointed out that some pathogenic

Column means with the different superscript are significnntty different (P>O.OS)from one Another

OayO nay 2 Day 4 I Day 6 Day 8 Day 10

Gill Skin Uill Skin Gill Skin Gill Skin Gill Skin Gill Skin

Smoked 1.1 1.6 :I: 2.4:1: 3.1 + 1.2 :I: 1.2.i. 2.1 = J.l :I: 3.9 :I: 1.6 ± 4.61. 4.4 ±x 10~ + o.or' o.or 0.01' O.Olc o.or 0.01 0.01 o.oi" o.cr o.oi' O.Olc

0.01

Salted x 1.1.i. 2.5= 2.6:1: 1.4+ 7.8 :I: 1.21. 2.1 :I: 2.9:1: 2.7+ 1.3 = 5.6.i. 5.1 :I:JO~ 0.01 oms 0.01" o.or 0.018 0.01° 0.01 0.01 o.or o.or' O.Olb 0.0(11

Frozen 1.1:1: 12:1: G.8 :t 7.5 - '1.95 1.6 :: 2.2:1: 3.1 + 2.5 ±J4i 165=~x 10-6 0.01 O.Olc 10.01ll O.OIA :I: 0.01 b 0.01 0.01 c.or o.oi> 0.01· 0.018

0.01i>

Tah1e 3.2 shows the bacteria load of the differently processed tilapia obtained from Olomoore market,Abeokuta. There was no significant difference (pOO.05) in the bacteria load of the gill during the first dayof processing. However, significant variation (p _ 0.05) existed in the bacteria load of the skin of the fishduring the first day of processing, In the second day frozen fish skin had the highest bacteria load whilesalted fish skin had the lowest bacteria load there is significant diflerence in the bacteria load on the skin ofdifferently processed fish skin. Similarly, there was significant variation (poO.OS) in the bacteria load ofthe gill of the fish during the second day of processing. Inthe fourth day the salted fish gill had the highestbacteria load while smoked fish gill bad the lowest bacteria load there was significant difference (P;]0.05)in the bacteria load on the gill of differently processed fish gill during the fourth day processing. However,significant variation (poO.05) existed in the bacteria load of the skin of the fish during the fourth dayprocessing. In the sixth day there was 110 significant difference (pOO.OS) in the bacteria load of the gill andthe skin during the sixth day processing. In the eighth day smoked fish gill had the highest bacteria load.there was significant difference in the bacteria load of the gill of the fish during the eighth day processing.However, there is significant variation (pOO.OS) in the bacteria load of the skin of the fish during the eightday processing. In the tenth day frozen Iish gill nad the highest bacteria load while smoked fish gill had thelowest bacteria load there is significant difference in the hacteria load on the gill of differently processedfish gill. However there is significant variation (pO 0.05) in the bacteria load or the skin of the fish duringthe tenth day processing.

Table 3.2: THT. BACTERIA LOAD (CFU/g) OF THF. DIFFERENTLY PROCESSED TIIJA 'PIAOBTAJI'ED FRO)l OLOMOORE MARKf,T,ADEOKUT A, CSIl'iG MCCONKEY AGAR ASl\{EDTh""f:

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Oreochromis niloticus and Clarias gariepinus collected from OwenaAdvances in Food, 29 (2):162-166Bagge-Ravn, D., Ng, Y., Hjelm, M., Christiansen, .:--.I.J., Johansen, C .and Gram, L.(2003). Themicrobial ecology of processing equipment in different fish industries-analysis of the micro floraduring processing and following cleaning and disinfection. international Journal of EoodMicrobiology. 87: 239-250.Bloomfield, S.P. (2003). Home IIygiene: a risk approach. International Journal of Hygiene andEnvironmental Health. 206: 1-8.Berts, G. D. code of practice for the manufacture of vacuum and modified atmosphere packaged

chilled foods with particular regard to the risks of botulism. 11:108-109.Chen, YR., Jackson, K.M., Chea, F.P. and Schafltner, D.\V. (2001). Quantiflcation and variability

REFERE~CES ".Abolagba, OJ. and E.E. Igbinevbo (2010). Microbial Load of Smoked Fish (clarias spy Marketed inBenin Metropolis, Nigeria. Research Journal of Fisheries and Hydrobiology,5(2): INSlnct Publicatiori.pp: 99-104. 'Adesokan lA, Ogunbanwo ST, Odetoyinbo BB (2005). Microbiological quality of selected :brands of beer in Nigeria. In: the Book of Abstract of the 29th Annual Conference & General ... ~Meeting (Abeokuta 2005) on Microbes As Agents of Sustainable Development, organized by :' ;'~;Nigerian Society for Microbiology (NSM), University of Agriculture, Abeokuta, from 6-l0lhNovember, p. 21 .Adesulu, E.A. (1997). Current status of tilapia in Nigerian aquaculture. Proceedings of thefourth International Symposium on Tilapia Aquaculturc, pp.577-583 (K. Fitzsimmons, cd.). Orlando,USA. ':Adeparusi, E. 0.,Afolabi, G. O. and Jeff-Agboola, Y, A. (2007). Bacteria and Fungi Flora of Wild

bacteria are naturally present in the aquatic (Clostridium botulinum type E, pathogenic Vibrio sp.,Aeromonasi and the general environment (C botulinum, type A and B,Listeria monocytogenesy and maytherefore be found on live or raw fish. Studies done by Vogel et al. (2001) on L. monocytogenes, indicatedthat contamination occurred along the processing line. Other studies dealing with different processingoperations have similarly concluded that the plant and processing environment is the SOUTceof productcontamination rather than the raw material. However, this does not exclude the possibility that the raw fishor material is an important initial source for contaminating processing equipment and environment (Vogelet al. 2001).Abo, water, like food, is a vehicle for the transmission of many agents of diseases (Kirby etal.2003, Jimoh et al, 2009). The presence oiStaphylococcus aureus in fish samples according to Okonko etal., (2008) might have been through handling. Many studies such as the one done by Montville et al.(2002) have similarly concluded that, during handling and preparation, bacteria may be transferred fromcontaminated hands of food workers to food and subsequently to other surfaces (including food contactsurfaces). Snyder (1998) also found that low infectious doses from organisms such as Shigella and thepathogen Ecoh were linked to hands as a source of contamination. Other studies suchasdone by Reij et al.(2004) identified equipment as a major source ofmicrobial contamination. The total number of organismsvaries enormously; Liston (1982) states a normal range of 102_10' cfu (colony forming unitsj/cm' on theskin surface. The gills and the intestines both contain between 103 and 10"cfu/g (Shewan, 1962). This isconsonance with the values reported in this report. It is therefore suggested that consumers should beeducated on the adverse effect of using untreated or polluted water lor processing as these could serve assources of microbial contamination. However, the processors/handlers/sellers should observe stricthygienic measures so that they will not serve as source of chance inoculation of microorganisms andcontamination of these processed frozen seafood products. So also caution should be taken in consumingprocessed fish shaded openly because such fish could contain microbial cells and reheating may' tie . :necessary to destroy or inactivate such cells. ; -',

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Venugopal, V. (2002). Biosensors in fish production and quality control. Biosensors andBioelectronics. 17: 147-157.WorldFish Centre (\VFC) (2003): An issue for everyone. A concept paper for 'Fish for All'produced by World fish Centre, Penang, Malaysia, 2-4.

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Reij, M.W., Den Aantrckker, E.D. and TLSI (2004). Europe Risk Analysis in Microbiology Taskforce. Recontamination as a source of pathogens inprocessed foods. International Journal ofFood]v£icrobi%gy.91:1-11.Seeley 11.W.,(Jr.) & P.I VanDemark. (1972). Microbes in action- a laboratory manual ofMicrobiology. Freeman, San Francisco.361 p.Shcwan, ].),1., (1962). The bacteriology of fresh and spoiling fish and some related chemicalchanges. In: J. Hawthorn & J. Muil Leitch (eds.), Recent advances infood science, I, 167-193,.Snyder, O'P (1998). Hand washing for retail food operations-a review, Diary Food EnvironmentSanitation. 18: 149-162. The International Commission on Microbiological Specifications for foods.(1998). Micro-organisms inFoods, Book 6. Microbial Ecology ol'Food Commodities. BlackieAcademic and Professional. London.Vogeln.r., Huss H.H.: Ojcniyi B., (2001). Elucidation oiListeria monocytogenes contamination routesin cold-smoked salmon processing plants detected by DNA-based typing methods. Applied andEnvironmental Microbiology 67 (6): 2586-2595. '.