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Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

ABSTRACT: Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

SUBMITTER: Rizo J

PROVIDER: S-EPMC7414965 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cereal-based traditional fermented beverages (TFBs) are prevalent among India's ethnic community, and lugri is one such TFB popular among the tribal people of the Lahaul valley in North-Western Himalaya. Previous studies have reported that lugri harbors probiotics and contains amino acids and vitamins but comprehensive substrate-specific exploration of lugri for probiotic attributes is unexplored. The present study selected three substrate-based lugri (wheat, rice, and barley) to study their biochemical properties and explore potential probiotics. This study screened the best probiotic strains for antioxidant studies and the fermentative process. A biochemical analysis determined that rice-based lugri had a higher alcohol content, electric conductivity, crude protein, and lower pH than barley and wheat-based lugri. A total of 134 distinct morphotypes were screened, and 43 strains were selected based on their qualitatively superior acid and bile tolerance. Rice-based undistilled lugri harbored the most probiotics, with 22 out of 43 strains isolated. All 43 bacterial isolates exhibited properties like cell surface hydrophobicity, cell-auto aggregation, β-galactosidase, and exopolysaccharide production, supporting them as possible probiotics. Based on antibiotic susceptibility, hemolytic activity, and biofilm formation, all the bacterial strains were found to be non-pathogenic. Taxonomically, they ranged among eight distinct genera and 10 different species. Statistically, 12 isolates were found to be the most promising probiotic, and eight strains were isolated from rice-based undistilled lugri. Furthermore, the antioxidant activity of the promising isolates was tested, based on free-radical scavenging ability toward 2,2-diphenyl-1-picrylhydrazyl (4.39-16.41%) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (15.29-57.74%). The strain Lacticaseibacillus paracasei LUL:01 showed the best antioxidant activity and probiotic attributes, and hence was used for the production of fermented milk. The strain LUL:01 fermented the sterile milk within 18 h, and the viable count remained above the legal requirement of 6 log10 CFU/ml during 28 days storage at 4°C. The strain represents a suitable candidate for applying probiotic functional food formulation with several health benefits.

Project description: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.

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Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

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