Biotransformation of patulin by Gluconobacter oxydans

Abstract

A bacterium isolated from patulin-contaminated apples was capable of degrading patulin to a less-toxic compound, ascladiol. The bacterium was identified as Gluconobacter oxydans by 16S rRNA gene sequencing, whereas ascladiol was identified by liquid chromatography-tandem mass spectrometry and proton and carbon nuclear magnetic resonance. Degradation of up to 96% of patulin was observed in apple juices containing up to 800 microg/ml of patulin and incubated with G. oxydans.

FIG. 1.

RAPD-PCR patterns of patulin-degrading bacterial isolates from PDA plates. Lanes: M, DNA molecular weight marker (Sigma); 1 to 8, isolates M1, M2, M3, M4, M5, M6, M8, M13; 9, PCR negative control.

FIG. 2.

Dendrogram of Gluconobacter strains based on 16S rRNA gene sequence analysis, using the neighbor-joining algorithm and 0.75 as the maximum sequence difference with the “distance tree of results,” which is freely available at http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi.

FIG. 3.

HPLC-UV/DAD chromatograms of YPM liquid medium spiked at 200 μg/ml patulin (3) (upper trace) and YPM liquid medium spiked at 200 μg/ml patulin and inoculated with G. oxydans (lower trace), containing E ascladiol (1), Z ascladiol (2) and residual patulin.

FIG. 4.

Structures of patulin, E ascladiol, and Z ascladiol.

FIG. 5.

(A) ¹H NMR COSY spectrum showing the scalar coupling network for the E- and Z-ascladiol isomers. (B) ¹H NMR NOESY spectrum showing the dipolar coupling network for the E- and Z-ascladiol isomers. Cross-peaks due to the spatial proximity between CH₂(7) and CH₂(8) (a) and between CH(6) and CH₂(8) (b) are selectively observed for the E and Z isomers, respectively.

FIG. 6.

Time course of patulin reduction and ascladiol formation on commercial apple juice spiked with patulin at 100 μg/ml, inoculated with G. oxydans, and incubated for 72 h at 30°C at 175 rpm. Each result is the mean ± the standard error (SE) of three replicates.

FIG. 7.

Comparison between G. oxydans growth with and without patulin at 100 μg/ml for 72 h at 30°C on apple juice. The cell count was performed by plating diluted aliquots of the samples on YPM agar plates. Each result is the mean ± the SE of three replicates.