Taxonomy of species of Alicyclobacillus from South African orchards and fruit concentrate manufacturing environments and the prevention of fruit juice contamination

Groenewald, Willem Hermanus (2009-12)

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

Thesis

ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that cause spoilage of heat-treated fruit juices stored at room temperature. During the past decade, Alicyclobacillus spp. have become a major cause of spoilage in pasteurised fruit juices leading to significant economic losses world-wide. Spoilage has been reported in apple, pear, orange, peach, mango and white grape juice, as well as in fruit juice blends, fruit juice containing drinks and tomato products, such as tomato juice and canned tomatoes. Spoilage is characterised by a medicinal smell and guaiacol production. These endospore-formers have been shown to survive pasteurisation conditions of 95 °C for 2 min, grow at temperatures between 25° and 60 °C and a pH range of 2.5 to 6.0. Knowledge of this organism is limited, both locally and internationally and the route of contamination to the final product is not well established. In this study the fruit concentrate processing environment was investigated as a potential source and route of contamination for the final product. Species of Alicyclobacillus were isolated from orchard soil, various stages during processing and from fruit juice and concentrates. The isolates were identified based on morpholological, biochemical and physiological properties. Identification to species level was done by 16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Results indicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were found in orchard soil and throughout the processing environment. This is the first report on the isolation of these species from orchard soil, vinegar flies and the fruit processing environment. The 16 isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Isolates from the fruit concentrate, wash water and soil located outside of the fruit processing plant grouped into one cluster. Concluded from these results, A. acidoterrestris found in the wash water and soil outside of the factory could act as a potential reservoir of organisms for the contamination of the final fruit concentrate. Thus good manufacturing practices play an essential role in controlling incidence of spoilage caused by these bacteria. Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of 254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillus acidoterrestris spores were inoculated into tap water, used wash water from a fruit processing plant and grape juice concentrate. Ultraviolet dosage levels (J L−1) of 0, 61, 122, 183, 244, 305 and 367 were applied using a novel UV-C turbulent flow system. The UV treatment method was shown to reliably achieve in excess of a 4 log10 reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated with A. acidoterrestris. The applied novel UV technology could serve as an alternative to thermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in the treatment of contaminated processing wash water. Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestris isolated from contaminated fruit juice concentrates were investigated in a 0.1% (m/v) peptone buffer solution (pH 7.04) and grape juice (pH 4.02, 15.5 °Brix). The thermal inactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that as the microbial population is exposed to a specific high temperature, the spores inactivated at a constant rate. D-values determined in the buffer solution were calculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be 2.25 min and 2.58 min for the two strains tested. From this study it is clear that the D-value is dependant on the strain tested, but also on the soluble solids of the solution the cells are suspended in. The results indicated that the spores of A. acidoterrestris isolated from South African fruit juice concentrate may survive after the pasteurisation treatment commonly applied during manufacturing.

AFRIKAANSE OPSOMMING: Spesies van Alicyclobacillus is suur-tolerante en hittebestande bakterieë wat bederf veroorsaak in hitte-behandelde vrugtesappe wat teen kamertemperatuur gestoor word. Gedurende die afgelope dekade het Alicyclobacillus spp. ‘n belangrike oorsaak van bederf in gepasteuriseerde vrugtesappe geword en beduidende ekonomiese verliese wêreldwyd veroorsaak. Bederf is aangeteken in appel-, peer-, lemoen-, perske-, mango- en witdruiwesap, sowel as in vrugtesapversnitte, vrugtesapbevattende drankies en in tamatieprodukte soos tamatiesap en ingemaakte tamaties. Bederf word gekenmerk deur ’n medisinale reuk en guaiacol produksie. Daar is gevind dat hierdie endospoorvormers pasteurisasie teen 95 °C vir 2 min kan oorleef en kan groei by temperature tussen 25° en 60 °C en ‘n pH van 2.5 to 6.0. Plaaslik sowel as internasionaal is kennis van hierdie organisme beperk en die roete van kontaminasie van produkte is nog nie goed vasgestel nie. In hierdie studie is die vrugtekonsentraat-verwerkingsmilieu ondersoek as ‘n moontlike bron en roete van kontaminasie van die finale produk. Spesies van Alicyclobacillus is vanuit vrugteboordgrond, verskeie verwerkingstadia en van vrugtesap en vrugtesapkonsentraat geïsoleer. Die isolate is op grond van morfologiese, biochemiese en fisiologiese eienskappe geïdentifiseer. Identifikasie tot spesiesvlak is deur 16S rDNS sekwensering gedoen en stam differensiasie deur RAPD-PKR. Resultate het aangetoon dat A. acidoterrestris en A. acidocaldarius in vrugteboordgrond sowel as in alle stadia van die verwerkingsmilieu voorkom. Dit is die eerste verslag van die isolering van hierdie spesies uit die Suid-Afrikaanse vrugteverwerkingsmilieu, vrugteboordgrond en asynvlieë. Die 16 isolate, geïdentifiseer as A. acidoterrestris en in vier groepe geplaas op grond van hul RAPD-PKR bandpatrone, dui aan dat hulle aan minstens vier genotipiese groepe behoort. Isolate afkomstig van die vrugtekonsentraat, waswater en die grond buitekant die vrugteverwerkingsaanleg het een groep gevorm. Uit hierdie resultate kan afgelei word dat A. acidoterrestris, wat in die waswater en grond buite die aanleg voorkom, as ‘n moontlike bron van organismes vir die kontaminering van die finale vrugtekonsentraat kan dien. Goeie vervaardigingspraktyke speel dus ‘n noodsaaklike rol in die beheer van bederf veroorsaak deur hierdie bakterieë. Vrugtesappe kan behandel word met ultravioletlig (UV-C) met ‘n golflengte van 254 nm wat ‘n dodende effek op mikro-organismes het. Kraanwater, gebruikte waswater van ‘n vrugtesapvervaardigingsaanleg en druiwesapkonsentraat is met A. acidoterrestris spore geïnokuleer. Ultraviolet toedieningsvlakke (J L−1) van 0, 61, 122, 183, 244, 305 en 367 is aangewend met behulp van ‘n nuwe UV-C drukvloei stelsel. Daar is aangetoon dat die UV-behandelingsmetode ‘n betroubare vermindering (99.99%) van meer as 4 log10 per 0.5 kJ L-1 van ‘n UV-C dosis gee in al die vloeistowwe wat geïnokuleer is met A. acidoterrestris. Die toegepaste nuwe UV-tegnologie kan gebruik word as ‘n alternatief tot die hittebehandeling van vrugtesap vir die deaktivering van Alicyclobacillus spore of in die behandeling van gekontamineerde waswater. Ten slotte is hitte-deaktivering teen 95 °C van twee stamme van A. acidoterrestris, geïsoleer uit gekontamineerde vrugtesapkonsentraat, in ‘n 0.1% (m/v) peptoonbufferoplossing (pH 7.04) en druiwesap (pH 4.02, 15.5 °Brix), ondersoek. Die hitte-deaktivering van A. acidoterrestris spore het eerste-orde kinetika gevolg, wat aandui dat die mikrobe-populasie teen ‘n konstante tempo afsterf, wanneer blootgestel aan ‘n spesifieke hoë temperatuur. Die D-waardes in die bufferoplossing is bereken as 1.92 min en 2.29 min, terwyl daar gevind is dat die D-waardes in druiwesap 2.25 min en 2.58 min is vir die twee betrokke stamme. Vanuit hierdie studie is dit duidelik dat die D-waardes afhang van die betrokke stam, maar ook van die oplosbare vaste stowwe van die oplossing waarin die selle opgelos is. Die resultate dui daarop dat die spore van A. acidoterrestris, wat geïsoleer is uit Suid-Afrikaanse vrugtesapkonsentraat, die pasteurisasiebehandeling wat algemeen tydens vervaardiging toegepas word, kan oorleef. Aangesien die toepassing van strenger hittebehandeling om spore van A. acidoterrestris te deaktiveer onaanvaarbare organoleptiese veranderinge in die produk tot gevolg het, word dit aanbeveel dat die risiko van bederf verminder behoort te word deur die gebruik van goeie vervaardigingspraktyke gedurende vrugteverwerking.

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