Detection and isolation of thermophilic acidophilic bacteria from fuit juices
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Fruit juices were until recently considered to only be susceptible to spoilage by yeasts, mycelial fungi and lactic acid bacteria. Spoilage by these organisms was prevented by the acidic pH of fruit juices and the heat-treatment applied during the hot-fill-hold process. Despite these control measures, an increasing number of spoilage cases of fruit juices, fruit juice products and acidic vegetables due to contamination by thermophilic acidophilic bacteria (TAB) have been reported. The genus Alicyclobacillus, containing TAB were first classified as Bacillus, but were reclassified in 1992. Species of Alicyclobacillus are Gram-positive, rod-shaped, endospore-forming bacteria. The unique characteristic of these organisms is the presence of ω-alicyclic fatty acids, such as ω-cyclohexane and ω-cycloheptane, as the major components of the cellular membrane. This organism has been shown to survive pasteurisation conditions of 95°C for 2 min and grows within a pH range of 2.5 to 6.0 and temperatures between 25° and 60°C. The genus currently consists of 11 species, with A. acidoterrestris, A. acidocaldarius and A. pomorum being the only species associated with the spoilage of fruit juices and fruit juice products. The aim of this study was to evaluate culture-dependent and culture-independent approaches for the detection and isolation of Alicyclobacillus spp. from pasteurised South African fruit juices and concentrates. The culture-dependent approach was evaluated by comparing five different growth media, for growth and recovery of A. acidoterrestris, A. acidocaldarius and A. pomorum at different incubation temperatures, from sterile saline solution (SSS) (0.85% (m/v) NaCl), diluted and undiluted fruit juice concentrates. The five media evaluated included potato dextrose agar (PDA), orange serum agar (OSA), K-agar, yeast extract (YSG)-agar and Bacillus acidocaldarius medium (BAM). The culture-independent approach was used to identify the micro-organisms present in fruit juices and concentrates from different South African manufacturers before and after pasteurisation, using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. Spread plates of PDA at pH 3.7 and incubation temperature of 50°C for 3 days was found to be the best isolation media for species of Alicyclobacillus from fruit juice and fruit juice concentrate. With the inclusion of a heat shock treatment at 80°C for 10 min the growth media of preference for spores of Alicyclobacillus from fruit juice concentrates was OSA at pH 5.5 and an incubation temperature of 50°C for 3 days. The culture-dependent approach could detect cells or endospores at a minimum concentration of 104 cfu.ml-1 in SSS and diluted fruit juices. PCR-based DGGE analysis was more sensitive and detected cells of Alicyclobacillus spp. from fruit juices and concentrates at a minimum concentration of 103 cfu.ml-1. Alicyclobacillus acidoterrestris was found to be present in South African apple juice, pear juice, white grape juice and aloe vera juice. White grape juice was also found to contain A. pomorum. Other organisms present in the orange, apple, mango and pear juices were two uncultured bacteria that were identified as members of the genus Bacillus, and one uncultured bacterium closely related to Alcaligenus faecalis. This study confirmed the presence of TAB in pasteurised South African fruit juices and concentrates and emphasises the need for the rapid and accurate detection of TAB in food products.
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