Studies On The Guaiacol Production Pathway, Detection And Control Methods For Alicyclobacillus Acidoterrestris

Alicyclobacillus acidoterrestris has recently received much attention due to its implication in the spoilage of pasteurized fruit juices. The spoilage is characterized by the formation of a distinct medicinal or antiseptic off-odor attributed to guaiacol, which at a low level can be detected by sensory means in fruit juices. The purpose of this study was to try to find the precursors of guaiacol, elucidate details about the conversion of vanillic acid and vanillin to guaiacol by A. acidoterrestris, and characterize the vanillic acid decarboxylase(VAD) responsible for guaiacol production. Then, the methods for the detection and control of A. acidoterrestris in apple juices were established. The main results of this study were as the follows:(1) Four potential substrates including ferulic acid, catechol, phenylalanine and tyrosine were analyzed, but they could not be metabolized to guaiacol by all the thirty A. acidoterrestris strains tested. Resting cell studies and enzyme assays demonstrated that vanillin was reduced to vanillyl alcohol by NADPH-dependent vanillin reductase and oxidized to vanillic acid by NAD(P)+-dependent vanillin dehydrogenases in A. acidoterrestris DSM 3923. Vanillic acid underwent a nonoxidative decarboxylation to guaiacol. The reversible vanillic acid decarboxylase involved was oxygen insensitive and pyridine nucleotide-independent. The addition of ATP(5 mmol/L) did not affect the rate of carboxylation.(2) Specific activity of VAD in vanillic acid-induced A. acidoterrestris DSM 3923 cells was highest in the early stage of log phase, and almost could not be detectable in stationary and death phases. Of the four techniques used to extract VAD, sonication was found to be the most effective and recovered 3.23 U/mg of VAD. Through optimization of the crucial parameters for sonication, the recovery of VAD had more than doubled(7.68 U/mg). The crude enzyme extract was purified by ammonium sulfate precipitation and an 8.75-fold purification was obtained. The partially purified VAD exhibited optimum activity at pH 6.0-6.5, 45 °C and was stable at pH 5.0-7.5, 20-45 °C. VAD activity was stimulated by Co2+ and Mn2+, but was inhibited by Ni2+, Cu2+, Ba2+ and Fe3+. Cinnamic acid, ferulic acid, resveratrol, quercetin and rutin at the concentration of 1 mmol/L could completely inhibit the activity of VAD.(3) The VAD encoding gene(vdc) was amplified, sequenced; then the obtained gene sequence was analyzed. The results showed that the size of the vdc gene in A. acidoterrestris DSM 3923 was 3309 bp, consisting of four open reading frames, vdcA(894 bp), vdcB(602 bp), vdcC(1424 bp) and vdcD(239 bp). Protein sequence comparisons suggested that the product of vdcA(297 aa) was LysR family transcriptional regulator; the putative products of vdcB(198 bp), vdcC(474 aa) and vdcD(76 aa) were similar to subunits B, C and D of phenolic acid decarboxylase. Based on the conserved sequence of vdcC gene, a pair of degenerate primer was designed. The partial vdcC gene was appeared in all the guaiacol-producing Alicyclobacillus spp., but absent in guaiacol non-producing strains. The similarity of the partial vdcC gene between A. acidoterrestris, A. herbarius and A. acidiphilus was 87.3%, while the value were 99.5% and 99.1% in A. acidoterrestris and A. herbarius, respectively, which indicated that the vdcC genes in the same species of Alicyclobacillus was highly conservative.(4) The vdcC genes from different species were analyzed using Clustal X software, and the specific primers for A. acidoterrestris were designed by Primer Premier 6.0. Then, a SYBR Green I real-time PCR detection method for A. acidoterrestris in apple juices was established. The specificity of the assay was confirmed using 15 A. acidoterrestris strains and 28 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers was found in any microorganisms but A. acidoterrestris. The detection limit of the established real-time PCR was 2.6×101 colony forming unit(CFU)/mL. When used in the apple juice samples, the correlation coefficient of standard curves was 0.987, and the calculated PCR efficiency was 105.86%. A positive correlation(R2 = 0.833) was found between A. acidoterrestris content and guaiacol concentration in 36 apple juice samples. This work offers a quick alternative to conventional methods of guaiacol quantification and and A. acidoterrestris detection in fruit juices.(5) Fuji apples were inoculated with a five-strain suspension of A. acidoterrestris spores and treated by ClO2, ultrasound and shaker, individually or combined. The results showed that ClO2 in combination with shaker was the most effective in reducing A. acidoterrestris spores on apples. After treatment with 200 mg/L ClO2 plus shaker(200 rpm) for 20 min, the viable spores remaining on the surface of the apple was reduced to undetectable levels(<1.7 log10 CFU/apple). The inactivation efficiency of ClO2 was significantly decreased with the increase of pH in the range of 2.5-8.5, whereas the alkaline condition was favorable to the spore detachment from apple surfaces. As for the artificially A. acidoterrestris-contaminated samples with spore concentrations of 3, 4 and 5 log10 CFU/apple, spores were reduced below the level of detection after combined treatment with ClO2(50 mg/L, pH 2.5) and shaker(200 rpm) for 5, 10 and 20 min, respectively.(6) The individual effects of potassium sorbate, sodium benzoate, potassium metabisulfite, dehydroacetic acid, ethyl 4-hydroxybenzoate, cinnamic acid and ε-polylysine on A. acidoterrestris growth and guaiacol production were firstly evaluated in a laboratory medium. Of the seven preservatives investigated, only dehydroacetic acid, cinnamic acid and ε-polylysine were effective both in controlling growth and guaiacol formation by A. acidoterrestris. Then, these three antimicrobials were applied to apple juice. Through the addition of 270 mg/L dehydroacetic acid, 108 mg/L cinnamic acid or 100 mg/L ε-polylysine, the A. acidoterrestris counts were reduced by 3.43, 3.17 and 4.78 log10 CFU/mL, respectively, and no guaiacol was detected after 14 days storage. Sensory evaluation revealed that the addition of these three preservatives did not affect the organoleptic properties of the apple juice.