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

Smit, Yvette (2009-12)

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009.

Thesis

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.

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