The Type and Concentration of Inoculum and Substrate as Well as the Presence of Oxygen Impact the Water Kefir Fermentation Process

Abstract

Eleven series of water kefir fermentation processes differing in the presence of oxygen and the type and concentration of inoculum and substrate, were followed as a function of time to quantify the impact of these parameters on the kinetics of this process via a modeling approach. Increasing concentrations of the water kefir grain inoculum increased the water kefir fermentation rate, so that the metabolic activity during water kefir fermentation was mainly associated with the grains. Water kefir liquor could also be used as an alternative means of inoculation, but the resulting fermentation process progressed slower than the one inoculated with water kefir grains, and the production of water kefir grain mass was absent. Substitution of sucrose with glucose and/or fructose reduced the water kefir grain growth, whereby glucose was fermented faster than fructose. Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei), Lentilactobacillus hilgardii (formerly known as Lactobacillus hilgardii), Liquorilactobacillus nagelii (formerly known as Lactobacillus nagelii), Saccharomyces cerevisiae, and Dekkera bruxellensis were the main microorganisms present. Acetic acid bacteria were present in low abundances under anaerobic conditions and only proliferated under aerobic conditions. Visualization of the water kefir grains through scanning electron microscopy revealed that the majority of the microorganisms was attached onto their surface. Lactic acid bacteria and yeasts were predominantly associated with the grains, whereas acetic acid bacteria were predominantly associated with the liquor.

Keywords: inoculum; kinetics; modeling; oxygen; substrate; water kefir.

FIGURE 1

Scanning electron microscopy images of water kefir grains. Visualization of two different locations on the water kefir grain surfaces with a magnification level of 5,000 (A), 10,000 (B), and 3,000 (C,D), and visualization of the inside of a water kefir grain with a magnification level of 2,000 (E) and 5,000 (F). The sizes are indicated with horizontal white bars.

FIGURE 2

Culture-dependent species diversity for the water kefir grain and liquor inocula (INO), and for the water kefir liquors of fermentation series 2S-2G-An, 2S-2L-An, and 2S-2G-Ae after 4 days of fermentation. The number of isolates are indicated between brackets. Isolates from YPD agar medium: (1) Saccharomyces cerevisiae [LSU (99% identity; GenBank accession no. CP011558) and ITS (99% identity; accession no. KC515374)]; and (2) Dekkera bruxellensis [LSU (99% identity; accession no. GU291284) and ITS (99% identity; accession no. FJ545249)]. Isolates from MRS agar media: (1) Lacticaseibacillus paracasei (100% identity with Lactobacillus paracasei; accession no. AP012541); (2) Lentilactobacillus hilgardii (100% identity with Lactobacillus hilgardii; accession no. LC064898); and (3) Liquorilactobacillus nagelii (99% identity with Lactobacillus nagelii; accession no. NR112754). Isolates from mDMS agar media: (1) Gluconobacter roseus/oxydans (100% identity for both species; accession no. NR041049/NR026118); (2) Acetobacter fabarum (100% identity; accession no. NR113556); and (3) Acetobacter indonesiensis (99% identity; accession no. NR113847). LSU, large subunit rRNA gene; ITS, internal transcribed spacer. Abbreviations are as in Table 1.

FIGURE 3

Culture-independent species diversity for the grain (G) and liquor (L) inocula (INO), and for the water kefir liquors (L) of fermentation series 2S-2G-An, 2S-2L-An, 2S-2G-Ae, 2GF-2G-An, 2G-2G-An, and 2F-2G-An after 4 days of fermentation. The numbers indicate the bands that were sequenced and the closest known type strains of the sequenced fragments are given. With the V3 primer pair: (1) Lacticaseibacillus casei/paracasei/rhamnosus (99% identity with Lactobacillus casei/paracasei/zeae/rhamnosus for all species; GenBank accession nos. LC064894/AB289229/AB289313/JQ580982); (2) Lentilactobacillus hilgardii/diolivorans (100% identity with Lactobacillus hilgardii/diolivorans; accession nos. LC064898/NR037004); (3) Liquorilactobacillus nagelii/ghanensis (99% identity with Lactobacillus nagelii/ghanensis; accession nos. NR119275/NR043896); (4) Oenococcus sicerae (99% identity; accession no. CP029684); (5) Bifidobacterium aquikefiri (100% identity; accession no. LN849254); (6) Acetobacteraceae sp. (100% identity). With the LAC primer pair: (1) Lacc. casei/paracasei (99% identity with Lb. casei/paracasei/zeae; accession nos. LC064894/AB289229/AB289313); (2) Lenl. hilgardii (100% identity with Lb. hilgardii; accession no. LC064898); and (3) Liql. nagelii (99% identity with Lb. nagelii; accession no. NR119275). With the Yeast primer pair: (1) Saccharomyces cerevisiae (100% identity; accession no. NG042623); and (2) Dekkera bruxellensis (100% identity; accession no. AY969049). Abbreviations are as in Table 1.

FIGURE 4

The pH (■) and concentrations of water kefir grain wet mass (∙), ethanol (□), glycerol (◆), lactic acid (Δ), acetic acid (∘), and mannitol (□) as a function of time for the anaerobic water kefir fermentation series 2S-2G-An with sucrose as substrate and started with a grain inoculum (left), and for the anaerobic water kefir fermentation series 2S-2L-An with sucrose as substrate and started with a liquor inoculum (right). The model lines (solid lines) describe the modeled concentrations of water kefir grain wet mass, ethanol, glycerol, lactic acid, acetic acid, and mannitol during the first 72 h of fermentation. Abbreviations are as in Table 1.