| Alicyclobacillus gen is the common spoilage bacteria in apple juice and other fruit juice, and it is the urgent safety hazard in fruit juice processing right now. The spoilage of Alicyclobacillus gen, residues of pesticides and heavy metal, contamination of patulin and post-haze were the four difficulty problems of the concentrated apple juice industry in China. As the typical stain of Alicyclobacillus gen, Alicyclobacillus acidoterrestris spp is the most important species that greatly polluted the apple juice ptoduct. At the same time, Alicyclobacillus acidoterrestris spp was the urgent safety problem of apple juice product. As a result, it is necessary to determine and detect the Alicyclobacillus acidoterrestris spp as quickly as possible in the processing, so we can detect and monitor its spoilage on-line and ensure the quality of the apple juice product.In order to accomplish the rapid determination and detection of Alicyclobacillus acidoterrestris in apple juice processing , we took the characteristic material in the cell as the target, after physiology and biochemistry analysis, medium optimization, growth and metabolic kinetic study and components analysis of whole cell, we found thatω-cyclic fatty acids were the mono-component of the Alicyclobacillus acidoterrestris. Then took theω-cyclic fatty acids as the target, using GC-MS to analyze the fatty acids, the result showed that GC-MS analysis perfectly determined the two strains. On the basis of GC-MS analysis, we used FT-IT spectroscopy to rapidly determine and detect the Alicyclobacillus acidoterrestris. All the result showed as below:1. The standard strain was able to grow from 28℃to 60℃, and pH form 2.7 to 6.0; the isolated strain was able to grow from 24℃to 60℃, and pH from 2.7～6.7.2. Got the optimized medium on the basis of NO. 402 medium of DSMZ, the including chemicals as following: CaCl2.2H2O 0.25 g, MgSO4.7H2O 0.50 g, (NH4)2SO4 0.20 g, MnSO4.2H2O 1.00 g, yeast extraction 2.00 g, peptone 5.00 g, glucose 5.00 g, KH2PO4 3.00 g,distilled water 1 L. And the optimized medium was used to study the kinetic model in the following experiment.3. The optimum conditions for the growth of Alicyclobacillus acidoterrestris: 2% for the inoculation volume, 150 r/min for the shaking speed and 45℃for the incubation temperature. Got the biomass model, glucose consumption model and the guaiacol production model, and the models reflected the growth and metabolic circumstances of the two strains very well.4. The standard stain and the isolated strain were typically correlated, and the canonical correlation coefficient was 0.995 7, which explained the result of 16S rDNA sequence analysis perfectly.5. The guaiacol production was growth-associated for the two strains, the correlation coefficient of standard strain was 0.937 8 between biomass and metabolite, and 0.958 5 for the isolated strain.6. The optimum conditions to get the protein in the cell were 500W, 3 s: 2 s intermission for 30 min disruption.7. The result of protein SDS-PAGE electrophoresis showed that the protein of Alicyclobacillus acidoterrestris were mostly low molecular weight protein. The protein molecular weight distribution of standard stain converged at two regions: one was between 30 to 35 kDa, the other between 44 to 58 kDa. The isolated strain converged at 31 to 34 kDa。8. Got the fatty acid of Alicyclobacillus acidoterrestris using saponification and acidulation. Analyzed the fatty acids by GC-MS after methyl esterification by BF3-CH3OH, and determined thatω-cyclic fatty acids were the characteristic components for the Alicyclobacillus acidoterrestris, and it can be used for the determination of Alicyclobacillus acidoterrestris.9. Carried out the rapid analysis ofω-cyclic fatty acids of Alicyclobacillus acidoterrestris by FT-IR spectroscopy. On the basis of the characteristic spectroscopy, we can perform the rapid determination and detection of Alicyclobacillus acidoterrestris.10. Got the rapid detection model of Alicyclobacillus acidoterrestris by FT-IR: 0.45μm film filtration, washing, drying, liquid paraffin grind, and FT-IR transmission analysis. The technology greatly improved analyzing speed, and ensure the accuracy and reliability of the analysis.
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