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| Establishment of Ethidium Monoazide and Quantitative PCR Method to Detect Viable Lactic Acid Bacteria (Lactobacillus plantarum) in Kimchi |
| , , , , , , ,韦露450103198306212562李丽婷120103198310162928 |
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Abstract Kimchi is a kind of traditional fermented vegetable, and it is also one well-know beneficial food. The number of viable lactic acid bacteria (LAB) is an important indicator to assess the nutritional value of kimchi. Ethidium monoazide in combination of quantitative PCR (EMA-qPCR) has been considered as a rapid and effective method to enumerate viable cell. In this study, EMA-qPCR method was established to detect viable LAB(Lactobacillus plantarum) rapidly and precisely in kimchi. For non-viable LAB, the maximum ΔCt (with EMA - without EMA) was achieved at an EMA concentration of 10 μg/mL, and there were no significant differences (P>0.05) in the ΔCt values for LAB treated with different EMA concentrations of 10, 25, 50 and 100 μg/mL. Moreover, the ΔCt increased significantly (P<0.05) as the photoactivation time of EMA increased from 0 to 20 min at 10 μg/mL EMA. But this effect was not observed among viable LAB, so EMA treatment could not affect the enumeration of viable cells. Therefore, optimum EMA treatment (10 μg/mL and 20 min light activation) lead to effective discrimination between viable and dead LAB. Under these conditions, results from EMA-qPCR (viable LAB of 109, 108, 107, 106, 105 and 104 CFU/mL) correlated well with that of plate counting (R2=0.999), and PCR efficiency reached to 104%. Due to sublethally injury of LAB with fermentation proceeding, EMA could penetrate into sublethal injured cell, and EMA-qPCR method consequently underestimated cell counts. Incubating cells in MRS medium for 30 min before detection could offset this error. That because incubation for suitable time could recover the sublethally injured cells but could not increase cell number. That indicated that EMA-qPCR could not only discriminate viable and non-viable cells, but also detect the sublethally injured cells. The viable LAB counts detected by EMA-qPCR increased with fermentation time increasing in the early stage of kimchi fermentation. As fermentation proceeded, lactic acid produced by LAB accumulated, leading to high environmental pressure to the survival of microorganisms. This gave rise to the sublethal injure of cells. Eventually LAB died and the cell number decreased. Viable LAB could be successfully enumerated at different fermentation times in kimchi through the establishment of EMA-qPCR method. Through exploration of suitable conditions, this method differentiating viable and non-viable cells can also be applied in other foods and environments in the future investigation.
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Received: 14 July 2012
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Corresponding Authors:
韦露450103198306212562李丽婷120103198310162928
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