The Study On Isolation And Identification Of Alicyclobacillus In The CAJ Industry Chain And The Inhibition Of Nisin On Alicyclobacillus

Twenty-two thermo-acidophilic bacteria were isolated from the orchards,apple storagehouseand Concentrated AJC production environment in Shaanxi province,China.The samples were soil,leaves,air,apple,production water,condensed water,fresh juice,clear juice,enzymatic juice,pre-concentrated juice,concentrated juice and air of various sections.By observation of the cell and colony morphology of the reference strain A.acidoterrestris DSM3922T and isolated strains,and also by determination of their growth conditions,physiological and biochemical characteristics tests,and genetic identification in the 16S rDNA region,all the strains were identified into species level.The thermal resistance of spores(D-value),biochemical characteristics(glycolysis reaction),and enzymatic spectra of 9 of 22 strains were subjected to in-depth research and analysis.The inhibition of Nisin on Alicyclobacillus was assayed from three factors(concentration,temperature,and pH),as well as minimum inhibitory concentration(MIC)and the effect of Nisin on the thermal resistance of the bacteria in apple juice.The results showed nisin had a certain inhibition on Alicyclobacillus in apple juice,and the destruction of cell morphology was observed by scanning electron microscopy,which provides a method for controling the hazards posed by Alicyclobacillus in concentrated apple juice.The main results of this study are as follows:1.Twenty-two strains of thermo-acidophilic bacteria were isolated including five from orchard(apple),eight from library(apple)and nine from AJC production environment(five from water,one from air of fresh juice,on from enzymatic juice and one from concentrated juice).The colony morphology with all the twenty-two isolates were generally round,milky white or cheese color,not quite glossy,moist,bulging in the center,approximately 2-5mm.The vegetative cells of isolates were rod shaped with terminal that are approximately 0.4-0.8μm wide and 2.0-5.0μm long.Swelling of the cells was sometimes observed.Gram stain was positive.2.By determination of their growth condition,physiological and biochemical characteristics,the most strains had a certain similarity with reference strain.PCR products(approximately 1500bp)of the 16S rRNA were successfully amplified and subsequent phylogenetic analysis revealed that the majority of strains(15/22)belonged to the genus Alicyclobacillus.There were only seven strains that belonged to the genus Bacillus.The results of 16S rRNA analysis indicated that the closest relative of 15 strains is Alicyclobacillus acidoterrestris,with a 16S rRNA sequence similarity of 99%.Other isolates belonged to B.ginsenghuimi(99%).3.For more in-depth understanding of characteristics of Alicyclobacillus,we study the spore thermal resistance(D-value),glycolysis capacity and enzymatic patterns of the selected nine strains,which provides basis for further control.The D-value(90℃)for the nine strains were 39.28,38.04,39.61,37.39,33.17,40.96,33.42,38.38 and 39.48min,respectively.4.Nisin has inhibitory effect on both vegetative cells and spores of Alicyclobacillus.at 100IU/mL.No significant influence on microbial inhibition by Nisin was observed when thermal treatment was applied,and better performance was achieved in acidic conditions.The D-value of strains dropped largely after thermal treatment at 90℃ relative to control(without nisin).By scanning electron microscopy,morphology of treated vetegative cells shows cell damage to certain extent.Nisin can be added into acidic juice.