Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers
that are able to spoil acidic food and beverage products through the production of
guaiacol and other taint compounds, which causes a medicinal off-flavour and/or
odour in the products. This thesis reports on the comparison of methods used for the
isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol
production of different strains isolated from the South African fruit processing
environment. Two methods for guaiacol detection were also evaluated and
compared.
Three isolation methods frequently used by South African fruit processors
were compared with regards to their ability to isolate a strain of A. acidoterrestris
from diluted peach juice concentrate. Method 1, the International Federation of Fruit
Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus
acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified
potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and
incubation of the membrane on K agar. The IFU Method No. 12 was the most
effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%.
These results support the use of the IFU Method No. 12 as a standard international
method for the isolation and detection of species of Alicyclobacillus.
Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris
DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South
African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A.
acidocaldarius FB19, were analysed based on their growth characteristics and
guaiacol production under optimum conditions. Strains were inoculated into BAT
medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for
7 d. All the strains had similar growth patterns, with cell concentrations increasing
rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations
of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication
of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an
increase in spores as the cell density and competition for resources increased. All
the strains were able to produce guaiacol in detectable concentrations [as measured
by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the
potential to cause product spoilage. Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers
that are able to spoil acidic food and beverage products through the production of
guaiacol and other taint compounds, which causes a medicinal off-flavour and/or
odour in the products. This thesis reports on the comparison of methods used for the
isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol
production of different strains isolated from the South African fruit processing
environment. Two methods for guaiacol detection were also evaluated and
compared.
Three isolation methods frequently used by South African fruit processors
were compared with regards to their ability to isolate a strain of A. acidoterrestris
from diluted peach juice concentrate. Method 1, the International Federation of Fruit
Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus
acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified
potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and
incubation of the membrane on K agar. The IFU Method No. 12 was the most
effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%.
These results support the use of the IFU Method No. 12 as a standard international
method for the isolation and detection of species of Alicyclobacillus.
Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris
DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South
African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A.
acidocaldarius FB19, were analysed based on their growth characteristics and
guaiacol production under optimum conditions. Strains were inoculated into BAT
medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for
7 d. All the strains had similar growth patterns, with cell concentrations increasing
rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations
of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication
of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an
increase in spores as the cell density and competition for resources increased. All
the strains were able to produce guaiacol in detectable concentrations [as measured
by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the
potential to cause product spoilage.
iv
The influence of temperature on the growth and guaiacol production of the
Alicyclobacillus strains was also investigated and two guaiacol detection methods,
the PECA and headspace gas-chromatography mass-spectrometry (HS GC-MS),
were compared with regards to their ability to detect guaiacol. The strains were
incubated at 25°C and 45°C for 6 d and samples analysed every 24 h. Growth of the
A. acidoterrestris strains was slower at 25°C, and maximum cell concentrations were
lower than at 45°C. A decrease in cell concentrations was observed in the A.
acidocaldarius strains at 25°C, as this temperature is below their growth temperature
range. All the strains were able to produce guaiacol at 45°C, with guaiacol only
being detected once a cell concentration of 104-105 cfu.mL-1 had been reached. The
maximum guaiacol concentrations detected at 45°C in the samples containing A.
acidoterrestris were significantly higher than those detected in the A. acidocaldarius
samples. At 25°C there was a longer lag phase before guaiacol was detected in the
A. acidoterrestris samples, while no guaiacol was detected in the samples containing
A. acidocaldarius. Because guaiacol is produced at ambient temperatures, cooling of
products is recommended to control spoilage by A. acidoterrestris. The sensitivity of
the two guaiacol detection methods also differed significantly and, therefore, the
PECA is recommended for presence/absence detection of guaiacol, while HS GCMS
is recommended where accurate quantification of guaiacol is required.
Alicyclobacillus acidoterrestris FB2 was investigated for its ability to grow and
produce guaiacol in white grape juice supplemented with vanillin at different
concentrations. Alicyclobacillus acidoterrestris FB2 was inoculated into white grape
juice concentrate diluted 1:10 with distilled water containing 0-500 mg.L-1 vanillin and
incubated at 45°C for 6 d. Similar growth patterns were observed in all the samples,
except in the sample containing 500 mg.L-1 vanillin, which had a longer lag phase of
growth. Guaiacol concentrations, detected using the PECA, increased as the vanillin
concentration increased, with the exception of the sample containing 500 mg.L-1
vanillin, where less guaiacol was detected than in the sample containing 250 mg.L-1
vanillin, due to growth inhibition caused by the higher vanillin concentration. A
number of conditions need to be favourable for detectable guaiacol production to
occur and it could, therefore, be possible to minimise or prevent guaiacol production
by controlling or eliminating some of these factors. Good manufacturing practices
should be employed in order to minimise contamination and, therefore, spoilage, by
Alicyclobacillus species. / AFRIKAANSE OPSOMMING: Bakterieë wat aan die genus Alicyclobacillus behoort, is termo-asidofiliese
spoorvormers wat suur voedsel en drank produkte kan bederf deur die produksie van
guaiakol en ander bederf verbindings, wat ‘n medisinale geur en/of reuk in die
produkte veroorsaak. Hierdie tesis doen verslag oor die vergelyking van metodes
wat vir die isolasie van spesies van Alicyclobacillus gebruik word, sowel as die groei
kenmerke en guaiakol produksie van verskillende stamme wat uit die Suid-
Afrikaanse vrugte prosesseringsomgewing geïsoleer is. Twee metodes vir die
deteksie van guaiakol is ook geëvalueer en vergelyk.
Drie isolasie metodes wat algemeen deur Suid-Afrikaanse
vrugteprosesseerders gebruik word, is vergelyk ten opsigte van hul vermoë om H A.
acidoterrestris stam uit verdunde perskesap konsentraat te isoleer. Metode 1, die
Internasionale Federasie van Vrugtesap Produseerders (IFU) Metode No. 12, maak
gebruik van spreiplating op Bacillus acidoterrestris (BAT) agar plate; Metode 2
behels gietplating met aartappel dekstrose agar (PDA) and Metode 3 maak gebruik
van membraan filtrasie en inkubasie van die membraan op K agar. Die IFU Metode
No. 12 was die mees effektiewe metode vir die isolasie van A. acidoterrestris, met H
sel herwinning van 75.97%. Hierdie resultate ondersteun die gebruik van die IFU
Metode No. 12 as H standaard internasionale metode vir die isolasie en deteksie van
spesies van Alicyclobacillus.
Sewe Alicyclobacillus stamme, insluitende die tipe stamme A. acidoterrestris
DSM 3922T en A. acidocaldarius DSM 446T en vyf stamme geïsoleer uit ‘n Suid-
Afrikaanse vrugte prosesseringsaanleg, A. acidoterrestris FB2, FB14, FB32, FB38 en
A. acidocaldarius FB19, is geanaliseer met betrekking tot hul groei kenmerke en
guaiakol produksie onder optimum toestande. Stamme is in BAT medium by pH
4.00, aangevul met 100 mg.L-1 vanillin, geïnokuleer en geïnkubeer teen 45°C vir 7 d.
Al die stamme het soortgelyke groeipatrone getoon, met selgetalle wat vinnig
toegeneem het van 0-24 h, gevolg deur ‘n stabilisering rondom maksimum selgetalle
van 105-107 kve.mL-1. Selgetalle na hitte behandeling, gemeet as H aanduiding van
spoorvorming, het toegeneem tot maksimum waardes van 105-107 kve.mL-1, wat
aandui dat spore toegeneem het soos die seldigtheid en kompetisie vir
voedingsbronne toegeneem het. Al die stamme kon guaiakol in bespeurbare konsentrasies produseer [soos gemeet deur die peroksidase ensiem kolorimetriese
bepaling (PEKB)] en besit dus die potensiaal om produkte te bederf.
Die invloed van temperatuur op groei en guaiakol produksie van die
Alicyclobacillus stamme is ook ondersoek en twee guaiakol deteksie metodes, die
PEKB en topspasie gas-kromatografie massa-spektrometrie (TS GK-MS) is vergelyk
ten opsigte van hul vermoë om guaiakol op te spoor. Die stamme is geïnkubeer teen
25°C en 45°C vir 6 d en monsters is elke 24 h geanaliseer. Groei van die A.
acidoterrestris stamme was stadiger by 25°C en maksimum selgetalle was laer as by
45°C. H Vermindering in selgetalle is waargeneem in die A. acidocaldarius stamme
by 25°C, aangesien hierdie temperatuur buite hul groei temperatuur grense val. Al
die stamme kon guaiakol produseer by 45°C, met guaiakol deteksie wat eers H
aanvang geneem het nadat H sel konsentrasie van 104-105 kve.mL-1 bereik is. Die
maksimum guaiakol konsentrasies wat by 45°C in die monsters met A. acidoterrestris
opgespoor is, was beduidend hoër as die konsentrasies wat in die A. acidocaldarius
monsters opgespoor is. By 25°C was daar H langer sloerfase voor guaiakol
opgespoor is in die A. acidoterrestris monsters, terwyl geen guaiakol opgespoor is in
die monsters wat A. acidocaldarius bevat het nie. Aangesien guaiakol by
kamertemperatuur geproduseer word, word verkoeling van produkte aanbeveel ten
einde bederf deur A. acidoterrestris te beheer. Die sensitiwiteit van die twee guaiakol
deteksie metodes het ook beduidend verskil en dus word die gebruik van die PEKB
aanbeveel vir teenwoordigheid/afwesigheid deteksie van guaiakol, terwyl TS GK-MS
aanbeveel word waar akkurate kwantifisering van guaiakol vereis word.
Ondersoek is ingestel na die vermoë van A. acidoterrestris FB2 om te groei en
guaiakol te produseer in witdruiwesap aangevul met verskillende vanillin
konsentrasies. Alicyclobacillus acidoterrestris FB2 is geïnokuleer in witdruiwesap
konsentraat 1:10 verdun met gedistilleerde water wat 0-500 mg.L-1 vanillin bevat het
en is geïnkubeer teen 45°C vir 6 d. Soortgelyke groeipatrone is waargeneem in al
die monsters, behalwe die monster wat 500 mg.L-1 vanillin bevat het, wat H langer
sloerfase van groei gehad het. Guaiakol konsentrasies, soos gemeet deur die
PEKB, het toegeneem soos die vanillin konsentrasie toegeneem het, met die
uitsondering van die monster wat 500 mg.L-1 vanillin bevat het, waar minder guaiakol
opgespoor is as in die monster wat 250 mg.L-1 bevat het as gevolg van groei inhibisie
veroorsaak deur die hoër vanillin konsentrasie. H Aantal toestande moet gunstig
wees vir guaiakol produksie om plaas te vind en dit kan dus moontlik wees om guaiakol produksie te minimaliseer of te voorkom deur die beheer of uitskakeling van
sommige van hierdie faktore. Goeie vervaardigingspraktyke moet in plek gestel word
ten einde kontaminasie en bederf deur Alicyclobacillus spesies tot H minimum te
beperk.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2245 |
| Date | 12 1900 |
| Creators | Smit, Yvette |
| Contributors | Witthuhn, R. C., Venter, P., Le Roux, Y., University of Stellenbosch. Faculty of Agrisciences. Dept. of Food Science. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
Page generated in 0.0033 seconds