Purpose: To evaluate the impact of harvest and hulling practices on the microbiota of inshell walnuts using a non-culture-based classification methodology.
Methods: Water from the “float tank” (used to separate inshell walnuts from rocks) and inshell walnuts from several locations before and after the float tank were collected from a commercial walnut huller over multiple days during the walnut harvest. Duplicate or triplicate samples were agitated in 0.1% peptone and centrifuged to obtain a bacterial pellet. Total genomic DNA was extracted and the V4 domain of bacterial 16S rRNA genes was amplified by PCR and subsequently sequenced on an Illumina MiSeq instrument. The 16S rRNA gene sequences were assigned to operational taxonomic units (OTUs) at the 97% similarity level, and a representative sequence from each OTU was assigned a taxonomic identity. Alpha-diversity and beta-diversity analyses were performed in QIIME.
Results: Walnuts received at the huller had proportionately lower amounts of Enterobacteriaceae (31%) than the float tank water (66%) or the walnuts collected after passing through the float tank water (54%). Bacterial communities on individual pre-float tank walnut samples were highly variable but beta-diversity analysis showed significantly different microbiota associated with incoming walnuts than with walnuts exposed to the float tank. Several OTUs were recovered at proportionally higher levels in float tank water and on walnuts collected after passing through this water than on walnuts collected at receipt.
Significance: Beta-diversity analyses of inshell walnut microbiota provides evidence that passage through a float tank can alter the microbiota of walnuts.