Isolation, Identification And Detection Alicyclobacillus Spp From Orange Juice And Citrus Groves

Alicyclobacillus spp can cause the pasteurized fruit juice to be corrupt, affect the quality of fruit juices, arousese the international concern, has become new content of technical barriers to international trade in fruit juice. In the international trade, strictly require the content of Alicyclobacillus spp is smaller than 1 per 10 mL concentrate fruit juice. But in our country, the research on isolation and detection for Alicyclobacillus spp are few, and only focus on the apple juice, research on the orange juice of Alicyclobacillus spp less. Therefore, it's necessary to research the isolation,and identification of Alicyclobacllus in orange juice and citrus groves, it's also great significance to orange juice production of our country and the import-export trade.In this paper, chose 13 acid-heat-resistant strains as the study material, which isolated from different orange juice and citrus groves, First, carried out the conventional detection,included colony morphology, medium odor, acid resistance, heat resistance, Gram stain; subsequent,16S rDNA sequence and cluster analysis for isolates; studied isolates of 16S rDNA PCR-RFLP characteristics and carried out cluster analysis; In addition, according to 16S rDNA designed a pair of specific and sensitive primers for Alicyclobacillus spp. Results were as follows:(1) Select BAT medium at 45℃2～5 d of culture conditions, isolated 8 strains and 2 stains Alicyclobacillus spp from orange juice, and orange groves,, respectively,. Therefore, BAT medium at 45℃2-5 d of culture conditions, can be used as selective medium to isolate Alicyclobacillus spp in orange juice.(2) Isolates NFC-1 as study material, studied it's growth curve, results showed that the bacteria entered the logarithmic growth phase after 18 h, cell concentration to achieve the highest at 25 h, and then entered stable phase. To determine the genome of bacteria DNA extraction conditions: 225 r/min 45℃air bath shaker culture 25 h.(3) Compare sequence and analyze phylogenetic 16S rDNA of 13 isolates and 3 standard Alicyclobacillus spp. The results showed that 8 acid-heat-resistant stains which isolate from orange juice all were Alicyclobacillus spp, including 1 Alicyclobacillus acidiphilus DSM14558,3 Alicyclobacillus acidoteresstris DSM3922,4 A.acidocaldcular; citrus groves have 2 acid-heat-resistant strains are Alicyclobacillus acidiphilus DSM14558.(4) Cluster analysis 13 strains isolated from acid-heat-resistant strains and 3 strains of Alicyclobacillus spp standard strains by 16S rDNA PCR-RFLP. The results showed that at 87% similarity level, all strains were divided into five spiece group and 10 branches, including P-1, P-5, K-1 are similar with standard strain A. acidiphilus DSM14558 in high levels, can be regarded as the same species, thermophilic Alicyclobacillus spp; H-1, ZH-1, ZH-2 and A.acidoterrestrius DSM3922 similar to the higher level, can be regarded as the same species, that isAlicyclobacillus acid soil bacteria; NFC-1, NFC-2, NFC-4, NFC-10 in other branches, by cloning and sequencing and sequence alignment of NFC-4, results show that the sequence homology of NFC-4 and A.acidocaldarus more than 99%, indicating that four strains were A.acidocaldarus. In addition,16S rDNA PCR-RFLP and 16S rDNA sequence analysis clustering clustering results are more consistent, this shows the results of this study is reliable(5) According to the 16S rDNA are highly variable between areas, inter-species conserved regions to design a pair of specific and sensitive primers for Alicyclobacillus spp. By optimizing the test, the optimal amplification conditions:25μL amplification system, ddH2O 17.75μL,10×buffer 2.5μL,25 mM Mg2+1.5μL,10 mM dNTP 1.5μL,35L 0.3μL,35R 0.3μL,5 U/ul Taq enzyme 0.13μL, DNA template 1μL Amplification conditions:94℃5min;94℃40 s,59.8℃40 s,72℃40 s,35 cycles; 72℃8 min. Under the best of the amplification process, PCR amplification of Alicyclobacillus spp minimum amount of genomic DNA was 10-4 pg, amplification of Alicyclobacillus spp minimum bacteria concentration of 3.0×103 CFU/mL(6) Successfully established PCR rapid detect Alicyclobacillus spp. The overall system:20 mL orange juice→80℃13 min→10 mL was inoculated in BAT medium→225 r/min 45℃bath under the conditions of gas by shaking culture for 25 h→CTAB method for extracted DNA→PCR amplification→agarose gel electrophoresis detect amplified products→judge the results. The whole process takes about 2～5 d, but traditional testing takes about 7-14 d.