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

[摘要] ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that causespoilage 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 juicesleading 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, fruitjuice containing drinks and tomato products, such as tomato juice and cannedtomatoes. 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 to6.0. Knowledge of this organism is limited, both locally and internationally and the routeof contamination to the final product is not well established.In this study the fruit concentrate processing environment was investigated as apotential source and route of contamination for the final product. Species ofAlicyclobacillus were isolated from orchard soil, various stages during processing andfrom fruit juice and concentrates. The isolates were identified based on morpholological,biochemical and physiological properties. Identification to species level was done by16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Resultsindicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were foundin orchard soil and throughout the processing environment. This is the first report on theisolation of these species from orchard soil, vinegar flies and the fruit processingenvironment. The 16 isolates identified as A. acidoterrestris grouped into four clustersbased on RAPD-PCR banding patterns, suggesting that they belong to at least fourgenotypic groups. Isolates from the fruit concentrate, wash water and soil locatedoutside of the fruit processing plant grouped into one cluster. Concluded from theseresults, A. acidoterrestris found in the wash water and soil outside of the factory couldact as a potential reservoir of organisms for the contamination of the final fruitconcentrate. Thus good manufacturing practices play an essential role in controllingincidence of spoilage caused by these bacteria.Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillusacidoterrestris spores were inoculated into tap water, used wash water from a fruitprocessing 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 log10reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated withA. acidoterrestris. The applied novel UV technology could serve as an alternative tothermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in thetreatment of contaminated processing wash water.Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestrisisolated 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 thermalinactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that asthe microbial population is exposed to a specific high temperature, the sporesinactivated at a constant rate. D-values determined in the buffer solution werecalculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be2.25 min and 2.58 min for the two strains tested. From this study it is clear that theD-value is dependant on the strain tested, but also on the soluble solids of the solutionthe cells are suspended in. The results indicated that the spores of A. acidoterrestrisisolated from South African fruit juice concentrate may survive after the pasteurisationtreatment commonly applied during manufacturing.

[发布机构] Stellenbosch University