Evaluation of the optical density reading for the determination of shelf life of whole chicken carcasses by the enumeration of the microbial contamination levels
Loading...
Date
2003-11
Authors
Banda, Thabo
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
English: Increased media and public interest over meat contamination have highlighted the need for continual improvements in this regard in the poultry industry. In spite of all the effort and money placed into research on microbial contamination of poultry carcasses, it is still not possible to produce carcasses, which are free of spoilage and pathogenic microorganisms. Carcasses are mostly contaminated during processing, contributing to the presence of high number of mesophilic microorganisms. The spoilage microorganisms cause consumers to reject the product due to appearance, off odour or undesirable flavour whereas the pathogenic microorganisms may lead to health hazards.
The Standard plate counts methods have been the method of choice for the enumeration of contaminating microbial populations in the poultry industry. However, these methods are laborious and time consuming. In this study, optical density (OD) reading as a microbial enumeration method on poultry carcasses was established and evaluated for its application as a rapid, alternative method.
In chapter 2, the dominant microorganisms were isolated and identified from eviscerated poultry carcasses. The carcasses were sampled by whole carcass rinse method and bacteria were isolated on Plate Count Agar at 27oC for 48 h. A total of five different species were identified, based on their prevalence in the sample and were identified by conventional methods. The identification of the isolates was further confirmed by API techniques. The dominant species were Escherichia coli, Shewanella putrefaciens, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus. Mean counts (log10 cfu/g) of these organisms were 3.08, 1.48, 0.85, 1.74, and 1.21 respectively.
In chapter 3, OD readings as an enumeration method was established. This required the establishment of a number of parameters, which include:
1) Determining the wavelength to be used throughout this study. This achieved by scanning the medium through the spectra of 200-700 nm and a wavelength of 420 nm was selected as the optimal wavelength.
2) Determination of incubation time, which was achieved by the construction of separate growth curves for each isolates and determine the time when all isolates were in the exponential phase of growth. A time of 4 h was selected.
3) Evaluation of the repeatability of the OD readings of each isolate. Both pure culture and mixed culture were evaluated. The coefficients of variation for repeatability evaluations had coefficients of variation less than 15% for pure culture. Similar coefficients of variation values was obtained with mixed cultures and most values were even below 10%.
4) The correlation of OD reading evaluated after 4 h incubation at 37oC to standard plate counts (incubated in PCA at 37oC for 24 h). The scatter plots graph obtained in this study had had a positive strong correlation, which was above 0.9 for all isolates, except for Serratia marcescens, which had the correlation coefficient of 0.886. The high repeatability and correlation showed high potential of the OD in enumeration, hence the further objective of the involved evaluating the method on chicken carcasses.
In chapter 4, carcasses were sterilized, followed by artificially inoculation with known microorganisms at different load. Sterilization was evaluated by examining different concentrations of Virukill solution at different concentration.
Virukill is a non-toxic, highly effective disinfectant. High concentration (1, 2 & 3 v/v%) of this disinfectant was effective in eradicating all microbial population under the applied conditions. It was established that at these high levels, substantial residual effects were found which prevented the re-inoculation of chicken carcasses. With lower concentrations, it was established that a 0.3 v/v% of Virukill eradicated all microorganisms and did not have residual effect, thus allowing re-inoculation of the carcasses with known concentrations of bacteria. A correlation coefficient between standard plate counts and the OD reading method of 0.903 and 0.968 were found for mixed cultures determined by whole carcass rinse and neck skin sampling methods, respectively. A. hydrophila, had correlation coefficient of 0.849 and 0.985 for whole carcass rinse and neck skin respectively. Similarly Serratia marcescens had 0.993 and 0.940 for whole rinse and neck skin respectively.
The results found in this study clearly show that bacterial enumeration through the use of OD readings is capable of reducing time and labour required to obtain the initial microbial load after processing of the carcasses. The OD method evaluated on artificially contaminated carcasses is promising. The method shows great potential for enumeration of bacteria during routine evaluation at the poultry processing plant.
Afrikaans: Toenemende belangstelling deur die media en publiek na vleis-kontaminasie het die nodigheid vir toenemende verbetering in hierdie verband in die pluimvee industrie aan die lig gebring. Ten spyte van al die moeite en geld aan navorsing na mikrobiese kontaminasie van pluimvee karkasse is dit steeds nie moontlik om karkasse, vry van bederf en patogeniese mikroörganismes, te produseer nie. Karkasse word meestal gekontamineer gedurende die ontwikkeling, wat bydra tot die teenwoordigheid van hoë getalle mesofiliese mikroörganisme. Die bederf mikroörganismes veroorsaak dat verbruikers die produk verwerp weens voorkoms, af-reuke of onaanvaarbare geur, waar patogeniese mikroörganismes kan lei tot gesondheidsgevare. Die standaard plaat-telling metodes was die metode van keuse vir die bepaling van kontamininerende mikrobe populasies in die pluimvee industrie. Maar hierdie metode is arbeid intensief en tydrowend. In hierdie studie is optiese digheidslesings (OD) as ‘n mikrobiese bepaling metode op pluimvee karkasse ontwikkel en geëvalueer as ‘n vinnige alternatiewe metode. In hoofstuk 2, is die dominante mikroörganismes geïsoleer en identifiseer uit geslagte pluimvee karkasse. Monsters is geneem vanaf karkasse deur die heel karkas-spoel metode en die bakterieë is geïsoleer op “plate count agar” (PCA) by 37oC vir 24 uur, ‘n totaal van vyf verskillende spesies is geselekteer, gebaseer op voorkoms in die monster en is geïdentifiseer deur konvensionele metodes. Die identifikasie van die isolate is verder bevestig deur API tegnieke. Die dominante spesies was Escherichia coli, Shewanella putrefaciens, Aeromonas hydrophila, Serratia marcescens en Staphylococcus aureus. Die gemiddelde tellings (log10 cfu/g) van hierdie mikroörganismes was 3.08, 1.48, 0.85, 1.74, en 1.21 respektiewelik. In hoofstuk 3, is die OD lesings as bepalings metode ontwikkel. Dit was dus nodig om ‘n aantal parameters vas te stel: 1) Bepaling van die golflengte wat deur die studie gebruik is. Dit is gedoen deur media te skandeer deur die spektrum 200-700 nm en ‘n golflengte van 420 nm is geselekteer as die optimum golflengte. 2) Bepaling van inkubasie tydperk wat gedoen is deur afsonderlike groeikurwes vir elke isolaat te doen en vas te stel wanneer alle isolate in die eksponensiële groei fase is. ‘n Tyd van 4 ure is geselekteer. 3) Bepaling van die herhaalbaarheid van die OD lesings van elke isolaat. Beide pre-isolasie suiwer kulture en gemengde kulture is geëvalueer. Die koëffisiënt van variasie van minder as 15% is getoon vir suiwer kulture. Soortgelyke koëffisiënt van variasie waardes is verkry vir gemengde kulture en die meeste waardes was minder as 10%. 4) Die korrelasie van OD-lesings (geëvalueer na 4 uur inkubasie by 37oC) met standaard plaattellings (geïnkubeer in PCA by 37oCvir 24 ure). Die “scatter plots” grafiek verkry het positiewe korrelasie getoon, wat hoër as 0.9 was vir alle isolate behalwe Serratia marcescens wat ‘n korrelasie koëfisiënt van 0.886 gehad het. Die hoë herhaalbaarheid en korrelasie toon baie potensiaal vir die OD-lesings in die bepaling, waarna die metode geëvalueer is op hoender-karkasse. In hoofstuk 4 is karkasse gesteriliseer, gevolg deur kunsmatige inokulasie met bekende mikroöganismes teen verskillende hoeveelhede. Sterilisasie is geëvalueer deur verskillende konsentrasies Virukill oplossings te ondersoek. Virukill is ‘n nie toksiese, hoogs effektiewe ontsmettingsmiddel. Hoë konsentrasies (1, 2 en 3%) van die ontsmettingsmiddel was effektief om alle mikrobiese populasie te ontwortel onder die gegewe kondisies. Daar is vasgestel dat by hierdie hoë konsentrasie daar substansieele residuele effekte was wat her-inokulasie van die hoenderkarkasse verhoed het. By laer konsentrasie is daar vasgestel dat in 0.3% van Virukill alle mikroörganimes verwyder is en dat daar geen residuele effekte was nie, dus was her-inokulasie van die karkasse met bekende konsentrasies bakterieë moontlik. ‘n Korrelasie koëffisiënt tussen die standaard plaat-tellings en die OD lesing metode was 0.903 en 0.968 vir gemengde kulture bepaal deur heel karkas spoel en nekvel monstering metode respektiewelik. A. hydrophila het ‘n korrelasie koëffisiënt van 0.849 en 0.985 vir heel karkas spoel en nekvel respektiewelik getoon. So ook het Serratia marcescens ‘n 0.993 en 0.940 vir heel karkas spoel en nekvel respektiewelik gehad. Die resultate gevind in hierdie studie wys duidelik dat bakteriese bepaling deur die gebruik van OD-lesings dit moontlik maak om die tyd en werk lading nodig om die aanvanklike mikrobiese lading na prosessering van die karkasse te verminder. Die evaluering metode van OD op self gekontamineerde karkasse is belowend. Die metode wys groot potensiaal vir die bepaling van bakterie gedurende roetine evaluasie by die pluimvee plantasie.
Afrikaans: Toenemende belangstelling deur die media en publiek na vleis-kontaminasie het die nodigheid vir toenemende verbetering in hierdie verband in die pluimvee industrie aan die lig gebring. Ten spyte van al die moeite en geld aan navorsing na mikrobiese kontaminasie van pluimvee karkasse is dit steeds nie moontlik om karkasse, vry van bederf en patogeniese mikroörganismes, te produseer nie. Karkasse word meestal gekontamineer gedurende die ontwikkeling, wat bydra tot die teenwoordigheid van hoë getalle mesofiliese mikroörganisme. Die bederf mikroörganismes veroorsaak dat verbruikers die produk verwerp weens voorkoms, af-reuke of onaanvaarbare geur, waar patogeniese mikroörganismes kan lei tot gesondheidsgevare. Die standaard plaat-telling metodes was die metode van keuse vir die bepaling van kontamininerende mikrobe populasies in die pluimvee industrie. Maar hierdie metode is arbeid intensief en tydrowend. In hierdie studie is optiese digheidslesings (OD) as ‘n mikrobiese bepaling metode op pluimvee karkasse ontwikkel en geëvalueer as ‘n vinnige alternatiewe metode. In hoofstuk 2, is die dominante mikroörganismes geïsoleer en identifiseer uit geslagte pluimvee karkasse. Monsters is geneem vanaf karkasse deur die heel karkas-spoel metode en die bakterieë is geïsoleer op “plate count agar” (PCA) by 37oC vir 24 uur, ‘n totaal van vyf verskillende spesies is geselekteer, gebaseer op voorkoms in die monster en is geïdentifiseer deur konvensionele metodes. Die identifikasie van die isolate is verder bevestig deur API tegnieke. Die dominante spesies was Escherichia coli, Shewanella putrefaciens, Aeromonas hydrophila, Serratia marcescens en Staphylococcus aureus. Die gemiddelde tellings (log10 cfu/g) van hierdie mikroörganismes was 3.08, 1.48, 0.85, 1.74, en 1.21 respektiewelik. In hoofstuk 3, is die OD lesings as bepalings metode ontwikkel. Dit was dus nodig om ‘n aantal parameters vas te stel: 1) Bepaling van die golflengte wat deur die studie gebruik is. Dit is gedoen deur media te skandeer deur die spektrum 200-700 nm en ‘n golflengte van 420 nm is geselekteer as die optimum golflengte. 2) Bepaling van inkubasie tydperk wat gedoen is deur afsonderlike groeikurwes vir elke isolaat te doen en vas te stel wanneer alle isolate in die eksponensiële groei fase is. ‘n Tyd van 4 ure is geselekteer. 3) Bepaling van die herhaalbaarheid van die OD lesings van elke isolaat. Beide pre-isolasie suiwer kulture en gemengde kulture is geëvalueer. Die koëffisiënt van variasie van minder as 15% is getoon vir suiwer kulture. Soortgelyke koëffisiënt van variasie waardes is verkry vir gemengde kulture en die meeste waardes was minder as 10%. 4) Die korrelasie van OD-lesings (geëvalueer na 4 uur inkubasie by 37oC) met standaard plaattellings (geïnkubeer in PCA by 37oCvir 24 ure). Die “scatter plots” grafiek verkry het positiewe korrelasie getoon, wat hoër as 0.9 was vir alle isolate behalwe Serratia marcescens wat ‘n korrelasie koëfisiënt van 0.886 gehad het. Die hoë herhaalbaarheid en korrelasie toon baie potensiaal vir die OD-lesings in die bepaling, waarna die metode geëvalueer is op hoender-karkasse. In hoofstuk 4 is karkasse gesteriliseer, gevolg deur kunsmatige inokulasie met bekende mikroöganismes teen verskillende hoeveelhede. Sterilisasie is geëvalueer deur verskillende konsentrasies Virukill oplossings te ondersoek. Virukill is ‘n nie toksiese, hoogs effektiewe ontsmettingsmiddel. Hoë konsentrasies (1, 2 en 3%) van die ontsmettingsmiddel was effektief om alle mikrobiese populasie te ontwortel onder die gegewe kondisies. Daar is vasgestel dat by hierdie hoë konsentrasie daar substansieele residuele effekte was wat her-inokulasie van die hoenderkarkasse verhoed het. By laer konsentrasie is daar vasgestel dat in 0.3% van Virukill alle mikroörganimes verwyder is en dat daar geen residuele effekte was nie, dus was her-inokulasie van die karkasse met bekende konsentrasies bakterieë moontlik. ‘n Korrelasie koëffisiënt tussen die standaard plaat-tellings en die OD lesing metode was 0.903 en 0.968 vir gemengde kulture bepaal deur heel karkas spoel en nekvel monstering metode respektiewelik. A. hydrophila het ‘n korrelasie koëffisiënt van 0.849 en 0.985 vir heel karkas spoel en nekvel respektiewelik getoon. So ook het Serratia marcescens ‘n 0.993 en 0.940 vir heel karkas spoel en nekvel respektiewelik gehad. Die resultate gevind in hierdie studie wys duidelik dat bakteriese bepaling deur die gebruik van OD-lesings dit moontlik maak om die tyd en werk lading nodig om die aanvanklike mikrobiese lading na prosessering van die karkasse te verminder. Die evaluering metode van OD op self gekontamineerde karkasse is belowend. Die metode wys groot potensiaal vir die bepaling van bakterie gedurende roetine evaluasie by die pluimvee plantasie.
Description
Keywords
Dissertation (M.Sc.Agric. (Mibrobial, Biochemical and Food Biotechnology))--University of the Free State, 2003, Poultry carcasses, Mesophiles, Optical density, Plate count, Sampling, Isolation, Identification, Repeatability, Correlation, Virukill, Poultry -- Microbiology, Poultry -- Preservation, Microbial contamination