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PCR-RFLP typification of microbes used in the production of a fermented fish productSpengler, C. J. 12 1900 (has links)
Thesis (MScFoodSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The preservation of various fresh fish products is achieved by either smoking,
salting, canning, freezing or fermenting a highly perishable raw product. Since
many of these facilities are not readily available, the use of fermentation as a
means of preserving the product has been extensively practiced. However, the
fermentation of fish is a time consuming practise and only by accelerating the
process would it be possible to ensure the production of a more cost effective and
readily available safe end-product.
The quality of the fermented fish product is partially determined by the
fermentation conditions and the metabolic activity of the microbes present. The
rapid identification of the microbes present during the fermentation would enable
the selection of possible starters to ensure an accelerated production of high
quality fermented fish products. This study was thus undertaken to develop
identification fingerprints for bacteria isolated from fermented fish products. A
1300 bp fragment of the 16S rRNA genes of each of the bacteria previously
isolated was successfully amplified using the PCR technique. The isolates
included strains of the genera Bacillus, Staphylococcus, Sphingomonas, Kocuria,
Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas and Agrobacterium. The
data obtained can, therefore, be used in the identification of these microbes
isolated from other similar fermented fish products. The fingerprints could also be
used to assist in determining the dominant microbial populations responsible for
the characteristic qualitative changes occurring in the fish product during
fermentation.
The microbial composition of a fermenting fish product partially determines
the quality of the end-product, therefore, the use of selected bacterial starters
could result in the accelerated production of a microbial safe fermented fish
product. A further objective of this study was to accelerate the production of a
fermented fish product by inoculating macerated trout with either selected lactic
acid bacteria (LAB) or with selected bacteria with high proteolytic activity over a 30
day fermentation period. The LAB included a combination of Lactobacillus
plantarum, Lactococcus diacetylactis and Pediococcus cerevisiae strains, whereas
the bacteria with high proteolytic activity included strains of Kocuria varians,
Bacillus subtilis, two strains of B. amyloliquefaciens and a combination of these bacterial species. The quality of the fermented product was determined using
changes in product pH, titratable acidity (%TA) and free amino nitrogen (FAN)
formation as efficiency parameters.
The data obtained during the fermentation of the macerated trout showed
that the selected starters did not have a significant effect on the pH decrease in
the product over a 30 day fermentation period. The LAB strains did not have a
significant effect on the %TA of the fermenting fish product, yet the presence of
these bacteria appeared to limit the FAN production in the product. The bacteria
with high proteolytic activity resulted in slightly enhanced %TA values and a higher
FAN content in the fermented product. It was also determined that the LAB and
Kocuria varians, in contrast to the Bacillus spp. inoculums, did not survive the
fermentation conditions well, possibly due to the low pH environment. The
presence of the starter bacteria in the fermenting fish mixture at the end of the
fermentation was also successfully determined with the use of the PCR-RFLP
technique.
The fermented fish product, obtained at the end of the fermentation period,
had a good aroma and compared favourably to similar commercially available
fermented fish products. The use of different microbial starters could in future
enable the production of a diverse range of high quality products, which could be
produced and marketed locally. / AFRIKAANSE OPSOMMING: Die preservering van ‘n verskeidenheid vars vis produkte word bereik deur die
hoogs bederfbare produk te rook, te sout, te blik, te vries of te fermenteer.
Aangesien baie van hierdie fasiliteite nie geredelik beskikbaar is nie, is die gebruik
van fermentasie as ‘n preserverings metode al ekstensief beoefen. Die
fermentasie van vis is egter 'n tydsame proses en slegs deur die versnelling van
die proses sal dit moontlik wees om die produksie van ‘n meer koste effektiewe en
geredelike beskikbare veilige eindproduk te verseker.
Die kwaliteit van die gefermenteerde vis produk word gedeeltelik bepaal
deur die fermentasie kondisies en die metaboliese aktiwiteit van die mikrobes
teenwoordig. Die vinnige identifikasie van die mikrobes teenwoordig gedurende
die fermentasie sal die seleksie van moontlike suursels om die versnelde
produksie van hoë kwaliteit gefermenteerde vis produkte moontlik maak. Hierdie
studie is dus onderneem om identifikasie vingerafdrukke vir bakteriee wat
gei'soleer is van gefermenteerde vis produkte moontlik te maak. ‘n 1300 bp
fragment van die 16S rRNA gene van elkeen van die bakteriee wat voorheen
gei'soleer is, is suksesvol geamplifiseer deur die PCR tegniek. Die isolate sluit in
stamme van die genera Bacillus, Staphylococcus, Sphingomonas, Kocuria,
Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas en Agrobacterium. Die
data kan dus gebruik word in die identifikasie van hierdie mikrobes as dit gei'soleer
word van ander gefermenteerde vis produkte. Die vingerafdrukke kan ook gebruik
word om die dominante mikrobiese populasies wat verantwoordelik is vir die
kenmerklike kwalitatiewe veranderinge wat plaasvind in die vis produk gedurende
die fermentasie, te identifiseer.
Die mikrobiese samestelling van ‘n fermenterende vis produk bepaal
gedeeltelik die kwaliteit van die eindproduk, daarom kan die gebruik van
geselekteerde bakteriese suursels die versnelde produksie van ‘n mikrobies
veilige gefermenteerde vis produk teweeg bring. ‘n Verdere doel van hierdie
studie was om die produksie van ‘n gefermenteerde vis produk te versnel deur
fyngemaakte forel met of geselekteerde melksuurbakteriee of met geselekteerde
bakteriee met hoë proteolitiese aktiwiteit te inokuleer oor ‘n 30 dag fermentasie
periode. Die melksuurbakteriee het ingesluit ‘n kombinasie van Lactobacillus
plantarum, Lactococcus diacetylactis en Pediococcus cerevisiae, terwyl die bakterieë met hoë proteolitiese aktiwiteit stamme van Kocuria varians, Bacillus
subtilis, twee stamme van Bacillus amyloliquefaciens en ‘n kombinasie van hierdie
bakteriese stamme ingesluit het. Die kwaliteit van die gefermenteerde produk is
bepaal deur die veranderinge in die pH, titreerbare suur (%TS) en vrye amino
stikstof (VAS) vorming van die produk as effektiwiteits parameters te gebruik.
Die data wat verkry is gedurende die fermentasie van die fyngemaakte forel
het gedui daarop dat die geselekteerde suursels nie ‘n merkwaardige effek op die
afname in pH in die produk oor ‘n 30 dag fermentasie periode het nie. Die
melksuurbakteriee het nie ‘n merkwaardige effek op die %TS van die
gefermenteerde vis produk gehad nie, terwyl dit geblyk het dat die
teenwoordigheid van hierdie bakterieë die produksie van VAS in die produk
belemmer het. Die bakteriee met hoe proteolitiese aktiwiteit het ‘n effense
verhoogde %TS en ‘n hoër VAS inhoud in die gefermenteerde produk veroorsaak.
Dit is ook bepaal dat die melksuurbakteriee en Kocuria varians, in teenstelling met
die Bacillus spp. inokulums, nie die fermentasie kondisies goed oorleef het nie,
moontlik as gevolg van die lae pH omgewing. Die teenwoordigheid van die
suursel bakteriee in die fermenterende vis mengsel aan die einde van die
fermentasie is ook suksesvol bepaal met die PKR-RFLP tegniek.
Die gefermenteerde vis produk, verkry aan die einde van die fermentasie
periode, het ‘n goeie aroma gehad en het goed vergelyk met soortgelyke
kommersieel beskikbare gefermenteerde vis produkte. Die gebruik van
verskillende mikrobiese suursels kan in die toekoms die produksie van ‘n diverse
reeks hoë kwaliteit produkte wat plaaslik geproduseer en bemark kan word
moontlik maak.
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