Microbial community dynamics during production of the Mexican fermented maize dough pozol

Abstract

The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A Streptococcus species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of Lactobacillus fermentum, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of L. plantarum, L. casei, and L. delbrueckii) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity-according to the Shannon-Weaver index-was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of Bifidobacterium, Enterococcus, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.

FIG. 1

Evolution of microorganisms in a pozol ball as estimated by plate counting using culture media. Results are the means of three repetitions (triplicate plating). Error bars are not shown to facilitate reading of the results. Symbols: ○, periphery; ●, center. EPS, exopolysaccharide.

FIG. 2

DGGE analysis of PCR-amplified 16S rDNA fragments from pozol bacterial communities. DNA was derived from two concentric fractions from the same balls of pozol. The positions and numbers of bands discussed in the text are indicated.

FIG. 3

Relationships of partial 16S rDNA sequences derived from pozol DGGE bands or organisms isolated from pozol to those of reference organisms obtained from GenBank. Sequences from this study (bands B1 to B17) and from a previous study (bands A1 to A6, strains LEU and MRS) (8) are included. A neighbor-joining analysis with bootstrap (1,000 samples) was performed by using the ClustalX and NJPlot programs. Zea mays chloroplast and related sequences were chosen as an outgroup. Bootstrap values are given at nodes when they exceed 50%. The scale bar represents an estimated 5% difference in nucleotide sequence.

FIG. 4

DGGE analysis of PCR-amplified 16S rDNA fragments from pozol eukaryotic communities. DNA was derived from two concentric fractions from the same balls of pozol. The position of maize nuclear DNA is indicated.

FIG. 5

Dendrogram derived from DGGE analysis of the bacterial community (Fig. 4) on the basis of the Dice's coefficient of similarity with the clustering algorithm of Ward.

FIG. 6

Shannon-Weaver index of diversity (H′) and Simpson index of dominance (D) calculated from two independent DGGE analyses of the bacterial community and two independent DGGE analyses of the eukaryotic community (examples of denaturing gels are given in Fig. 2 and 4). Symbols: ○, periphery; ●, center.

FIG. 7

Quantification of rRNA with phylogenetic oligonucleotide probes. Results are given as percentages of the total rRNA quantified with universal probe Univ1390. Results are the means of three to five repetitions. Error bars are not shown to facilitate reading of the results. Symbols: ○, periphery; ●, center. Lb+Ln+Pc+W+Ec, Lactobacillus plus Leuconostoc plus Pediococcus plus Weissella plus Enterococcus.

FIG. 8

Sugar and fermentation product concentrations. The results are given in micromoles per gram (wet weight) except for soluble starch (milligrams per gram [wet weight]). Symbols: ○, periphery; ●, center.

FIG. 9

Comparison of quantification of four organisms during fermentation of pozol as determined by using either the relative intensities of DGGE bands (○) or the relative amounts of 16S rRNAs determined with phylogenetic oligonucleotide probes (●).