Diversity of Weissella confusa in Pozol and Its Carbohydrate Metabolism

Abstract

The genus Weissella is composed of a group of Gram-positive facultative anaerobe bacteria with fermentative metabolism. Strains of this genus have been isolated from various ecological niches, including a wide variety of fermented cereal foods. The present study aimed to determine the relative abundance and fermentation capabilities of Weissella species isolated from pozol, a traditional maya product made of lime-cooked (nixtamalized) fermented maize. We sequenced the V3-V4 regions of 16S rDNA; Weissella was detected early in the fermentation process and reached its highest relative abundance (3.89%) after 3 h of culture. In addition, we evaluated five Weissella strains previously isolated from pozol but reported as non-amylolytic, to define alternative carbon sources such as xylan, xylooligosaccharides, and sucrose. While no growth was observed on birch xylan, growth did occur on xylooligosaccharides and sucrose. Strains WcL17 and WCP-3A were selected for genomic sequencing, as the former shows efficient growth on xylooligosaccharides and the latter displays high glycosyltransferase (GTF) activity. Genomes of both strains were assembled and recorded, with a total of 2.3 Mb in 30 contigs for WcL17 and 2.2 Mb in 45 contigs for WCP-3a. Both strains were taxonomically assigned to Weissella confusa and genomic analyses were performed to evaluate the gene products encoding active carbohydrate enzymes (CAZy). Both strains have the gene content needed to metabolize sucrose, hemicellulose, cellulose, and starch residues, all available in pozol. Our results suggest that the range of secondary enzymatic activity in Weissella confusa strains confer them with wide capabilities to participate in fermentative processes of natural products with heterogeneous carbon sources.

Keywords: CAZy; Weissella; fermented foods; maize; pozol.

FIGURE 1

Change in the structure of pozol bacterial communities over fermentation time. Taxonomic assignation was made by metabarcoding (V3-V4, 16S r DNA). “Other” includes all genera with relative abundance lower than 0.05%.

FIGURE 2

Growth and profile of xylooligosaccharides (XOS) in cultures with five Weissella strains isolated from pozol. (A) Biomass accumulation (OD) during fermentation. (B) Biomass accumulation (OD) after 24 h of growth. Statistically significant differences (p < 0.5) for strain WcL17 growth were obtained with the ggpubr package in R v4.0.2. XOS consumption during strain WcL17 culture was evaluated by (C) HPAEC-PAD chromatograms and (D) TLC plates. XOS hydrolysis by xylosidase activity in samples from WcL17 culture at M1 = 3 h, M2 = 6 h, and M3 = 12 h were also evaluated at baseline (t = 0) and after 5 h (t = 5) of reaction by (E) HPAEC-PAD chromatograms and (F) TLC plates.

FIGURE 3

Culture of five Weissella strains isolated from pozol in MRS-sucrose medium. (A) Biomass evolution (OD) of the five strains. The three strains with the highest growth, namely (B) WcSnc40, (C) WcSnc45, and (D) WCP3a, were studied in terms of growth (OD) and evolution of pH. Metabolized sucrose, glucose, and fructose were also monitored by HPLC analysis in the three Weissella cultures: strains (E) WcSnc40, (F) WcSnc45, and (G) WCP3a. Finally, extracellular glycosyltransferase activity (U/mL) for the three strains in 6-h cultures was compared (H); statistically significant differences in activity (p < 0.5) were obtained with the ggpubr package in R v4.0.2.

FIGURE 4

Heatmap of Average Nucleotide Identity (ANI) values of representative Weissella genera reported in the literature (Table 1), including genomes for strains WcL17 and WCP3a. Strains with ANI values > 90% are shown within red squares.

FIGURE 5

Comparative analysis of CAZy families for representative Weissella strains (Table 1), including WcL17 and WCP3a genomes. Colors within the map indicate the number of families in each genome, from zero to eight copies (black to red, respectively). The right sidebar indicates the type of substrate for each family (yellow = starch, green = hemicellulose, and blue = sucrose).