Novel intervention strategies to control Alicyclobacillus acidoterrestris spores in fruit juice and fruit-based beverages

Alicyclobacillus acidoterrestris is a thermo-acidophilic and spore-forming bacterium which was recently nominated as a reference microorganism for quality control in the fruit juice industry. This extremophile is described as having an unusual heat tolerance and being able to grow and produce in fruit-based products. Alicyclobacillus spores can withstand commercial juice pasteurisation processes, and subsequently germinate and grow in juice products during storage and distribution at suitable temperature. Although not hazardous, the presence of Alicyclobacillus in fruit juice and fruit juice concentrates can lead to significant economic losses and adversely impact manufacturers' brand names due to undesired flavours and/or odours of spoiled products, typically identified as guaiacol and halophenols. The prevalence of Alicyclobacillus spp. is reported worldwide with a diverse range of strains and species derived from raw material and final products. However, the organism is insufficiently studied. There are research gaps in genomic information on the genus and important spoilage species including A. acidoterrestris. There are current needs for alternative processing treatments to replace methods generally ineffective at eliminating spores. Finally, the increasing demand for food products with premium quality drives research to develop innovative strategies to produce new products. This study was set up to assess the genotypic and phenotypic diversities of a wide range of Alicyclobacillus spp. isolates. The study included analysis of important characteristics and searched for the correlation of such features with genomic information from whole genome sequencing analyses. Lastly, an innovative processing method which combines emerging highpressure technology and mild heat application was investigated for its effectiveness and versatility in inactivating spores of A. acidoterrestris in acidic fruit-based beverages. Spores of Alicyclobacillus spp. isolates (n = 215) which were derived from a wide range of fruit- and vegetable-based products from Australia and neighbouring countries were used to investigate diversity of the thermo-acidophilic spoiler present. Molecular methods including DNA rapid amplification polymerase chain reaction (RAPD-PCR) and next generation sequencing were performed to assess genetic material and diversity of the spoilage organisms. The spoilage ability of representative isolates was determined by an assay of guaiacol, a dominant taint compound, using gas chromatography and mass spectrometry. D-values were examined to evaluate heat resistance of genetically representative isolates. Finally, the synergy of high-pressure and thermal processing (HPTP) was evaluated in potassium buffer and in commercial apple juice using laboratory scale high-pressure and thermal units. Physical and physiological conditions of HPTP treated spores were also assessed by flow cytometry; images of damaged spores were captured by electron microscopy in order to elucidate further insights behind the effectiveness of this innovative strategy against A. acidoterrestris spores. The study's results revealed genetic diversity among Alicyclobacillus spp. in fruit juice, fruit juice concentrates and fruit- and vegetable-based products. Two distinct genotypic groups of Alicyclobacillus spp. were identified by DNA fingerprints and taint metabolism ability. The use of F-64 primer provided more detailed DNA-signatures than by BA-10 primer which is useful for species differentiation. Beside the identification of guaiacol and non-guaiacol groups, variation in guaiacol production was also observed among guaiacol-producing strains regardless of product type or origin and diverse genetic evidence. Whole genome sequencing analyses confirmed the presence of vanillate decarboxylase clustered genes (vdcBCD) in sequences of guaiacol-producing strains. The sequence analyses also revealed a putative vanillin dehydrogenase encoding gene (vdh) which bio-converts vanillin to vanillic acid, suggesting a two-step pathway of guaiacol metabolism by Alicyclobacillus. ANI and dDDH values from whole genome sequences further divided the guaiacol producing group into two genotypic groups; and revealed the presence of another Alicyclobacillus spoilage species which is closely related to A. acidoterrestris. While the two genotypic groups demonstrated similar capability in taint metabolism, they differed in heat resistance in which genotypic group 1 was more resistant to heat than genotypic group 2. Non-synonymous changes in squalene-hopene cyclase (shc) amino acid sequences were corresponded to the distinct heat resistance of the two groups. In particular, the replacement of glutamine residues with basic amino acids arginine and lysine possibly reduced the reinforcement of heat stability of the shc-coded enzyme structure. High pressure and thermal processing (HPTP) effectively inactivated spores from two distinct genotypic groups of spoilage Alicyclobacillus. Despite different heat resistance, the same susceptibility in HPTP was observed for the two genotypic groups. HPTP at 600 MPa and 70, 80 and 90°C resulted in high levels of spore inactivation, between 2.8 to 6.2 log-reductions depending on pressure dwell time. The most effective treatments were achieved by two HPTP combinations of 600 MPa at 80°C for 3 min and 90°C for 1 min which reduced inoculated spores to undetectable levels (< 1 CFU/mL). In addition, HPTP at 70°C also effectively inactivated spores with 2.8 and 5.2 log-reduction in 1 and 3-minutes pressure dwell time, respectively. Although viable spore count data indicated equal inactivation by HPTP for genotypic group 1 and 2 strains, flow cytometric analyses elucidated further information on different membrane integrity of treated spores. Strain AJA 66 strain of genotypic group 1 may possess more stable cell membranes that that of ATCC 49025, the type strain of A. acidoterrestris and member of genotypic group 2 in this study.