Microbial species diversity, community dynamics, and metabolite kinetics of water kefir fermentation

Abstract

Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.

FIG 1

(A) The production of water kefir grain mass (g, •) and of water kefir grain dry mass (%, ○) as a function of time (h). (B) The pH evolution and the consumption of carbohydrates (g liter⁻¹) as a function of time (h). pH, ○; total carbohydrates, •; sucrose, ▲; fructose, ■; glucose, ⧫. (C) The production of metabolites (g liter⁻¹) as a function of time (h). Ethanol, ■; lactate, Δ; glycerol, ♢; acetate, ○; mannitol, □.

FIG 2

The microbial species diversity of the water kefir liquor and water kefir grains, represented by pooling all samplings. The closest relatives to the sequenced fragments are given. Left panel (bacterial species diversity): 1, Lactobacillus casei/paracasei (99% identity; accession no. KF500575/KF516078); 2, Lactobacillus hilgardii (99% identity; accession no. JX099894); 3, Lactobacillus nagelii (99% identity; accession no. AB370876); 4, Lactobacillus harbinensis (100% identity; accession no. KF418816); 5, Acetobacter lovaniensis/fabarum (99% identity; accession no. FJ157228/AB665084); 6, Lactobacillus mali (99% identity; accession no. AB326352); 7, unknown. Right panel (yeast species diversity): 1, Saccharomyces cerevisiae (LSU [99% identity; accession no. JQ914745] and ITS [99% identity; accession no. KC515374]); 2, Dekkera bruxellensis (LSU [100% identity; accession no.: JQ689028] and ITS [100% identity; accession no. FJ545249]).

FIG 3

The PCR-DGGE profiles of the water kefir liquor (L) and the water kefir grains (G) after 0, 24, 72, and 192 h (indicated as subscripts) of fermentation. The closest relatives to the fragments sequenced are given. (A) Use of the universal V3 primer pair. 1, 2, 3, 5, and 7: Lactobacillus hordei/mali (98% identity; accession no. NR044394/AB326352); 4, unspecified bacterium; 6, Lactobacillus harbinensis/brevis (100% identity; accession no. KF418816/AY974809); 8, 9, 13, 14, 19, and 20, Lactobacillus casei/rhamnosus/paracasei (100% identity; accession no. KF500575/KF554252/KF516078); 10 and 16, Lactobacillus hilgardii/diolivorans (100% identity; accession no. KF418826/KF149643); 11 and 17, Lactobacillus ghanensis/nagelii (97% identity; accession no. AB690235/AB370876); 12 and 18, Bifidobacterium psychraerophilum (98% identity; accession no. AB437351); 15, Acetobacteraceae. (B) Use of the LAC primer pair. 5, 7, 11, 12, 14, 18, and 19, Lb. casei/paracasei (99% identity; accession no. KF500575/KF516078); 4, 8, 9, 15, and 16, Lb. nagelii (99% identity; accession no. AB370876); 1, 2, and 3, Lb. hordei/mali (99% identity; accession no. NR044394/AB326352); 6 and 13, Lb. harbinensis/brevis (99% identity; accession no. KF418816/AY974809); 10 and 17, Lb. hilgardii/diolivorans (99% identity; accession no. KF418826/KF149643).

FIG 4

(A) The production of volatile short-chain aroma compounds (mg liter⁻¹) as a function of time (h). Isoamyl acetate, •; isoamyl alcohol, ○; ethyl acetate, ♢; 2-methyl-1-propanol, □. (B) The production of volatile long-chain aroma compounds (mg liter⁻¹) as a function of time (h). Ethyl octanoate, □; ethyl decanoate, Δ; ethyl hexanoate, ♢.

FIG 5

Presence of different carbon-containing constituents of the water kefir fermentation process, as a function of time (h), expressed as a percentage (%) of the total amount of carbon recovered. 1, substrates; 2, kefir grain mass; 3, ethanol; 4, carbon dioxide; 5, glycerol; 6, mannitol; 7, lactate; 8, acetate.