Identification of precursors present in fruit juice that lead to the production of guaiacol by Alicyclobacillus Acidoterrestris

Thesis (MSc Food Sc)--University of Stellenbosch, 2011.

ENGLISH ABSTRACT: Alicyclobacilli are endospore-forming, thermophilic, acidophilic bacteria (TAB) that survive the pasteurisation process and spoil acidic fruit juices through the production of the taint compound guaiacol. Guaiacol causes an undesirable odour with an unpleasant smoky, medicinal or phenolic-like taste. This thesis reports on the precursors, vanillin and vanillic acid metabolised to guaiacol by Alicyclobacillus spp. in fruit juice, the pathway of guaiacol production and the spoilage potential of contaminated fruit juices supplemented with these precursors. A high performance liquid chromatography method with UV-diode array detection (HPLC-DAD) was developed for the simultaneous detection and quantification of guaiacol and its precursors. Alicyclobacillus acidoterrestris FB2 was incubated at 45 °C for 7 d in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid. The cell concentrations were determined every 24 h and the concentration of the precursors and the production of guaiacol was determined using HPLC-DAD. The guaiacol production was also determined using the peroxidase enzyme colourimetric assay (PECA). Alicyclobacillus acidoterrestris produced guaiacol from vanillin and vanillic acid, confirming both vanillin and vanillic acid as precursors for guaiacol production by A. acidoterrestris FB2. Furthermore, a metabolic pathway directly from vanillin to guaiacol was identified in this study. However, guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid and it is, therefore, not considered a direct precursor for guaiacol production by A. acidoterrestris. The spoilage potential of apple juice supplemented with either 10 mg L-1 or 100 mg L-1 vanillin or vanillic acid by A. acidoterrestris FB2 (106 cfu mL-1) was also evaluated. The production of guaiacol increased with the increase in vanillin or vanillic acid concentrations (in BAT broth and apple juice) indicating that the concentration of vanillin and vanillic acid present in fruit juice will influence the spoilage potential of the juice. Guaiacol concentrations in apple juice well above the best estimated threshold value of guaiacol for taste (0.24 – 2.00 μg L-1) and odour (0.50 - 2.32 μg L-1) was produced by A. acidoterrestris FB2 in the apple juice supplemented with 10 mg L-1 vanillin or vanillic acid. This indicates that fruit juice with a vanillin or vanillic acid concentration as low as 10 mg L-1 has the potential to spoil if the juice is contaminated with A. acidoterrestris. The concentrations of vanillin and vanillic acid in different fruit juices can be used to indicate if a specific fruit juice is susceptible to guaiacol spoilage by Alicyclobacillus spp. In the development of juice products and different blends of fruit juices, special care must be taken not to concentrate the amount of vanillin and vanillic acid present in the fruit juices.

AFRIKAANSE OPSOMMING: Alicyclobacilli is endospoor-vormende, termofiliese, asidofiliese bacterie (TAB) wat die pasteurisasie proses oorleef en suur vrugtesappe bederf met die produksie van ‘n taint komponent guaiakol. Guaiakol veroorsaak ‘n ongewensde reuk en onaangename rookagtige, medisinale of fenoliese smaak. Hierdie tesis doen verslag oor die voorloper komponente, vanillien en vanilliensuur in vrugtesappe wat gemetaboliseer word na guaiakol deur Alicyclobacillus spesies, die padweg van guaiakol produksie en die bederfbaarheid van gekontamineerde vrugtesap aangevul met hierdie voorloper komponente. ‘n Hoëprestasie vloeistof chromatografie metode met UV-deteksie (skanderend) (HPVC) is ontwikkel vir die gesamentlike deteksie en kwantifisering van guaiakol en die voorloper komponente. Alicyclobacillus acidoterrestris FB2 is geïnkubeer by 45 °C vir 7 d in Bacillus acidoterrestris (BAT) medium aangevul met feruliensuur, vanillien of vanilliensuur. Die sel konsentrasies is elke 24 h bepaal en die aangevulde komponente en die geproduseerde guaiakol is bepaal deur van HPVC gebruik te maak. Die guaiakol konsentrasies is ook bepaal deur van die peroksidase ensiem kolorimetriese bepaling (PEKB) gebruik te maak. Alicyclobacillus acidoterrestris het guaiakol geproduseer vanaf vanillien en vanilliensuur, dus is beide vanillien en vanilliensuur bevestig as voorlopers van guaiakol produksie deur A. acidoterrestris FB2. A padweg direk van vanillien na guaiakol is in hierdie studie geïdentifiseer. Guaiakol is nie geproduseer deur A. acidoterrestris FB2 in the monsters wat met feruliensuur aangevul is nie en feruliensuur is dus nie ‘n direkte voorloper van guaiakol produksie deur A. acidoterrestris. Die bederf potensiaal van appelsap aangevul met 10 mg L-1 of 100 mg L-1 vanillien of vanilliensuur deur A. acidoterrestris (106 kve mL-1) is ook geëvalueer. Die produksie van guaiakol het toegeneem met die toename in vanillien of vanilliensuur konsentrasies (in beide BAT en appelsap) wat aandui dat die konsentrasie vanillien en vanilliensuur teenwoordig in vrugtesap die bederfbaarheid van die sap sal beïvloed. Guaiakol konsentrasies in appelsap hoog bo die drumpel waardes van guaiacol vir smaak (0.24 – 2.00 μg L-1) en reuk (0.50 - 2.32 μg L-1) is geproduseer deur A. acidoterrestris FB2 in die appelsap monsters aangevul met 10 mg L-1 vanillien of vanilliensuur. Hierdie verskynsel dui aan dat vrugtesap met ‘n vanillien of vanilliensuur konsentrasies van so laag as 10 mg L-1 die potensiaal het om te bederf indien die sap gekontamineer is met A. acidoterrestris. Die konsentrasies van vanillien en vanilliensuur in verskillende vrugtesappe kan gebruik word om aan te dui of ‘n spesifieke vrugtesap ‘n hoë risiko het vir guaiakol bederf deur Alicyclobacillus spesies. Tydens die ontwikkeling van vrugtesap produkte en verskillende mengsels van vrugtesappe moet seker gemaak word dat die hoeveelhede vanillien en vanilliensuur in die sappe nie gekonsentreer word nie.

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