Growth and guaiacol production of species of Alicyclobacillus isolated from the South African fruit processing environment

[摘要] ENGLISH ABSTRACT: Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formersthat are able to spoil acidic food and beverage products through the production ofguaiacol and other taint compounds, which causes a medicinal off-flavour and/orodour in the products. This thesis reports on the comparison of methods used for theisolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacolproduction of different strains isolated from the South African fruit processingenvironment. Two methods for guaiacol detection were also evaluated andcompared.Three isolation methods frequently used by South African fruit processorswere compared with regards to their ability to isolate a strain of A. acidoterrestrisfrom diluted peach juice concentrate. Method 1, the International Federation of FruitJuice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillusacidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidifiedpotato dextrose agar (PDA); and Method 3 makes use of membrane filtration andincubation of the membrane on K agar. The IFU Method No. 12 was the mosteffective 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 internationalmethod for the isolation and detection of species of Alicyclobacillus.Seven strains of Alicyclobacillus, including the type strains A. acidoterrestrisDSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a SouthAfrican fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A.acidocaldarius FB19, were analysed based on their growth characteristics andguaiacol production under optimum conditions. Strains were inoculated into BATmedium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for7 d. All the strains had similar growth patterns, with cell concentrations increasingrapidly from 0-24 h, followed by a stabilisation around maximum cell concentrationsof 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indicationof spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating anincrease in spores as the cell density and competition for resources increased. Allthe strains were able to produce guaiacol in detectable concentrations [as measuredby the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess thepotential to cause product spoilage. Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formersthat are able to spoil acidic food and beverage products through the production ofguaiacol and other taint compounds, which causes a medicinal off-flavour and/orodour in the products. This thesis reports on the comparison of methods used for theisolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacolproduction of different strains isolated from the South African fruit processingenvironment. Two methods for guaiacol detection were also evaluated andcompared.Three isolation methods frequently used by South African fruit processorswere compared with regards to their ability to isolate a strain of A. acidoterrestrisfrom diluted peach juice concentrate. Method 1, the International Federation of FruitJuice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillusacidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidifiedpotato dextrose agar (PDA); and Method 3 makes use of membrane filtration andincubation of the membrane on K agar. The IFU Method No. 12 was the mosteffective 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 internationalmethod for the isolation and detection of species of Alicyclobacillus.Seven strains of Alicyclobacillus, including the type strains A. acidoterrestrisDSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a SouthAfrican fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A.acidocaldarius FB19, were analysed based on their growth characteristics andguaiacol production under optimum conditions. Strains were inoculated into BATmedium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for7 d. All the strains had similar growth patterns, with cell concentrations increasingrapidly from 0-24 h, followed by a stabilisation around maximum cell concentrationsof 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indicationof spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating anincrease in spores as the cell density and competition for resources increased. Allthe strains were able to produce guaiacol in detectable concentrations [as measuredby the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess thepotential to cause product spoilage.ivThe influence of temperature on the growth and guaiacol production of theAlicyclobacillus 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 wereincubated at 25°C and 45°C for 6 d and samples analysed every 24 h. Growth of theA. acidoterrestris strains was slower at 25°C, and maximum cell concentrations werelower 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 temperaturerange. All the strains were able to produce guaiacol at 45°C, with guaiacol onlybeing detected once a cell concentration of 104-105 cfu.mL-1 had been reached. Themaximum guaiacol concentrations detected at 45°C in the samples containing A.acidoterrestris were significantly higher than those detected in the A. acidocaldariussamples. At 25°C there was a longer lag phase before guaiacol was detected in theA. acidoterrestris samples, while no guaiacol was detected in the samples containingA. acidocaldarius. Because guaiacol is produced at ambient temperatures, cooling ofproducts is recommended to control spoilage by A. acidoterrestris. The sensitivity ofthe two guaiacol detection methods also differed significantly and, therefore, thePECA is recommended for presence/absence detection of guaiacol, while HS GCMSis recommended where accurate quantification of guaiacol is required.Alicyclobacillus acidoterrestris FB2 was investigated for its ability to grow andproduce guaiacol in white grape juice supplemented with vanillin at differentconcentrations. Alicyclobacillus acidoterrestris FB2 was inoculated into white grapejuice concentrate diluted 1:10 with distilled water containing 0-500 mg.L-1 vanillin andincubated 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 ofgrowth. Guaiacol concentrations, detected using the PECA, increased as the vanillinconcentration increased, with the exception of the sample containing 500 mg.L-1vanillin, where less guaiacol was detected than in the sample containing 250 mg.L-1vanillin, due to growth inhibition caused by the higher vanillin concentration. Anumber of conditions need to be favourable for detectable guaiacol production tooccur and it could, therefore, be possible to minimise or prevent guaiacol productionby controlling or eliminating some of these factors. Good manufacturing practicesshould be employed in order to minimise contamination and, therefore, spoilage, byAlicyclobacillus species.

[发布机构] Stellenbosch University