Biocontrol of Aspergillus flavus in Ensiled Sorghum by Water Kefir Microorganisms

Abstract

The capacity of microorganisms from water kefir (WK) to control Aspergillus flavus growth during the aerobic phase of ensiled sorghum grains was determined. Sorghum inoculated with A. flavus was treated with filter-sterilized and non-sterilized water kefir, ensiled, and incubated 7 days at 25 °C. A. flavus growth was quantified by qPCR after incubation. Mold growth was inhibited in the presence of water kefir while no inhibition was observed when filter-sterilized water kefir was applied, demonstrating the relevant role of the microorganisms in the kefir water in the biocontrol process. Fungal and bacterial diversity in treated sorghum mini-silos was analyzed by high-throughput sequencing. Firmicutes was the predominant bacterial phyla and Lactobacillus represented the most abundant genus, while Ascomycota was the predominant fungal phyla with Saccharomyces and Pichia as the major genera. Bacterial and yeast counts before and after incubation indicated that the microbial community obtained from WK was able to grow in the sorghum mini-silos in the presence of A. flavus. Results of the present work indicate that the use of a mixed inoculum of microorganisms present in WK may represent an alternative management practice to avoid the growth of A. flavus in ensiled sorghum grains and the concomitant contamination with aflatoxins.

Keywords: Aspergillus flavus; high moisture sorghum silage; high-throughput sequencing; water kefir.

Figure 1

Standard curve for the real time PCR assay of A. flavus DNA. Bars represent confidence intervals (α = 0.05).

Figure 2

Nanograms (ng) of A. flavus DNA per sorghum weight obtained from the control (sterile water), T1 (water kefir), and T2 (filter-sterilized water kefir) treatments. Values labeled with different letters are significantly different, as calculated by a LSD Fisher Test α = 0.05.

Figure 3

Experimental high moisture sorghum grain mini-silos opened after 7 days of incubation at 25°C. (a) Control (sterile water); (b) T1 (water kefir consortium); (c) T2 (filter-sterilized water kefir). Arrows indicate A. flavus growth.

Figure 4

(a) Bacterial plate count per sorghum weight in aerobic and anaerobic condition. (b) Yeast plate count per sorghum weight in aerobic and anaerobic conditions. Values labeled with different letters are significantly different, as calculated by a LSD Fisher Test α = 0.05.

Figure 5

Phylogenetic trees based on D1/D2 sequences from (a) Pichia membranifaciens. (b) Saccharomyces cerevisiae isolates. Trees were constructed using the neighbor-joining method. Bootstrap values (1000 tree interactions) are indicated at the nodes. ^T- means type strain.

Figure 6

Phylogenetic tree based on the partial sequence of the 16S rRNA gene of Lactobacillus spp. isolates. The tree was constructed using the neighbor-joining method. Bootstrap values (1000 tree interactions) are indicated at the nodes. ^T- means type strain.

Figure 7

(a) Rarefaction curves for the partial sequences for the gene that encodes 16S rDNA of bacteria before incubation (A) and after incubation (B). (b) Rarefaction curves for the partial sequences for the ITS 2 region of fungal rRNA before (A) and after (B) incubation.

Figure 8

(a) Relative abundance of bacteria to Phylum level. (b) Relative abundance of bacteria to Genera level.

Figure 9

Phylogenetic tree based on OTUs sequences associated with Lactobacillus genus. The tree was constructed using the neighbor-joining method. Bootstrap values (1000 tree interactions) are indicated at the nodes. ^T- means type strain

Figure 10

Percentage of sequences assigned to different OTUs in the total of Lactobacillus sequences in T1 mini-silos before and after incubation.