Identification And Analysis Of Alicyclobacillus From The Production Chain Of Kiwi Fruit And The Study Of Their Control By Radio Frequency

Alicyclobacillus are a group of spoilage bacteria of juice products which can cause spoilage of many kinds of juice and beverage products and produce offensive off-odor.Spoilage incidents caused by Alicyclobacillus have been reported constantly. To date there are more than 20 species of Alicyclobacillus have been reported, and the differences among them are huge. Only some species can cause spoilage. China is the largest production country of kiwi fruit in the world, and Shaanxi province is her main production area. The industry of kiwi fruit products(juice, wine, etc) is developing very fast recently in Shaanxi province,therefore the potential risk of Alicyclobacillus contamination in kiwi fruit products is increasing. There are still few reports about the isolation and identification of Alicyclobacillus from kiwi fruit products. Alicyclobacillus are spore-forming bacteria and their spores can make them survival the common pasteurization and therefore spoil the juice products subsequently.In this study the area around Mei county, Yangling, and Zhouzhi county were chosen as the sampling area, and all sampling sectors of orchards, production plant, and commercial products were included. After enrichment, Alicyclobacillus were isolated and their genomic DNA was extracted. Identification was performed through their 16 S r RNA gene amplification and sequencing. The biodiversity among isolates were studied through Phylogenetic tree construction and RAPD-PCR amplification, as well as API tests and genomic DNA hybridization. Their capacities of spoilage were studied through growth curves, the determination of D90℃values, and analysis of their taint productions. Finally, the inhibitory effects of nisin on their growth and heat resistance and the control method by radio frequency combined with nisin were studied.Main results of this thesis include:(1) A total of 401 samples were tested and 76 samples were positive(19.0%), from which 85 strains of thermo-acidophilic microbes were isolated and identified, including 1strain from commercial kiwi fruit products, 16 strains from the producer plant, and 68 strains from the orchards. Seventy six strains of the total 85 strains were identified as Alicyclobacillus, including 4 species: A. acidoterrestris(46 strains), A. contaminans(10strains), A. herbarius(19 strains), and A. cycloheptanicus(1 strain). To my knowledge,this is the first report about the isolation and identification of A. contaminans and A. herbarius from kiwi fruit orchards in China. In addition, 9 strains of Bacillus were also isolated and identified(3 species).(2) All 85 isolates and 4 reference strains fell into 7 clusters according to the neighbourjoining phylogenetic tree constructed based on the sequences data of their 16 S r DNA, which was generally agreed with the results of the random amplified polymorphic DNA tests, and reflected biodiversity to a certain extent among the isolates. Among the results of the 49 API tests, there were 3~19 different results between the test strains and the reference strains used in this study, and 7~22 different results between the test strains and the reference strains reported in other articles, which reflected the differences about the metabolism among the test strains. The results of the genomic DNA hybridization tests showed that the hybridization rates among the test strains and their reference strains were between 74.75% and 104.79%,reflecting the differences about the DNA homology among the test strains.(3) The growth curves of the test strains showed that all strains could grow in BAM, with the highest increment being 2.61 log10 units, while in kiwi fruit juice(p H 4.2), only 7 strains could grow, with the highest increment being 3.23 log10 units. In BAM, the D90℃values ranged from 16.95 min to 45.45 min, while in kiwi fruit juice, the D90℃values ranged from12.50 min to 38.46 min among test strains. Guaiacol was detected in the cultures of all strains,but there were no 2,6-DBP and 2,6-DCP detected among them. The productions of guaiacol were between 2.56 ppb and 42.74 ppb after the 21 days incubation.(4) Nisin showed a certain inhibitory effect on the growth as well as the heat resistance of the test strains. No strains could multiply under 20 IU/m L of nisin, no matter in BAM or kiwi fruit juice, with the curve of the strain which was unadapted to this experimental environment dropping to zero just at day 4. When the concentrations of nisin increased from zero to 100 IU/m L, the decrements of D90℃values among test strains ranged from 0.56 min(3.30%) to 6.21 min(25.46%) in BAM, and from 0.45 min(3.60%) to 17.11 min(53.04%) in kiwi fruit juice. The above results indicated that nisin could inhibit the growth of the test Alicyclobacillus strains, as well as reduce their heat resistance, with the effects being different among strains. There was a synergistic effect between nisin and radio frequency in killing Alicyclobacillus spores, and the treatment of 90 ℃ for 15 min could reduce the spore concentration up to 4.17 log10 units, with the reduction increasing to 5.19 log10 units at the nisin concentration of 100 IU/m L. The effect of the radio frequency heating combined with nisin was better than the traditional heating combined with nisin, and the treatment by the former at 90 ℃ for 15 min under 20 IU/m L of nisin could kill more spores up to 2.08 log10 units than the latter under the same experimental conditions, which indicated that radio frequency combined with nisin was a more effective method for killing Alicyclobacillus spores than the traditional way.