Lactobacillus hordei dextrans induce Saccharomyces cerevisiae aggregation and network formation on hydrophilic surfaces

doi:10.1016/j.ijbiomac.2018.04.068

International Journal of Biological Macromolecules

Volume 115, August 2018, Pages 236-242

https://doi.org/10.1016/j.ijbiomac.2018.04.068 Get rights and content

Abstract

Water kefir granules are supposed to mainly consist of dextrans produced by Lactobacillus (L.) hilgardii. Still, other microorganisms such as L. hordei, L. nagelii, Leuconostoc (Lc.) citreum and Saccharomyces (S.) cerevisiae are commonly isolated from water kefir granules, while their contribution to the granule formation remains unknown. We studied putative functions of these microbes in granule formation, upon development of a simplified model system containing hydrophilic object slides, which mimics the hydrophilic surface of a growing kefir granule. We found that all tested lactic acid bacteria produced glucans, while solely those isolated from the four different L. hordei strains induced yeast aggregation on the hydrophilic slides. Therefore, structural differences between these glucans were investigated with respect to their size distributions and their linkage types. Beyond the finding that all glucans were identified as dextrans, those of the four L. hordei strains were highly similar among each other regarding portions of linkage types and size distributions. Thus, our study suggests the specific size and structural organization of the dextran produced by L. hordei as the main cause for inducing S. cerevisiae aggregation and network formation on hydrophilic surfaces and thus as crucial initiation of the stepwise water kefir granule growth.

Introduction

Water kefir is a traditional fermented beverage made from sucrose, water, water kefir grains, and dried or fresh fruits. The microbiota isolated from water kefir grains has been described to be a stable symbiotic multispecies community, which generally consists of lactic acid bacteria (LAB), acetic acid bacteria (AAB), bifidobacteria and yeasts [[1], [2], [3], [4]]. Common LAB isolated from kefir grains are (among others) Leuconostoc (Lc.) mesenteroides, Lactobacillus (L.) hilgardii, L. hordei, L. casei. Furthermore, yeasts of the genera Saccharomyces, Hanseniaspora, Zygotorulaspora and Candida are commonly present in water kefir [2,[4], [5], [6], [7]].

The key organisms involved in water kefir preparation are embedded in the kefir granules. The backbone of the kefir granule is made from a dextran matrix mainly produced by L. hilgardii, which is the basic layer for biofilm formation by the embedded microorganisms [5,[7], [8], [9]]. Dextran is produced by dextransucrases from sucrose and contains consecutive α-1,6-linkages in its major chains, which usually make up >50% of the total linkages [10,11]. These glucans also contain side chains, which are mainly attached through α-1,3-branched linkages and occasionally through α-1,4- or α-1,2-branched linkages. Gulitz et al. [2] and Stadie [12] showed that the majority of LAB strains (81%) from water kefir belonging to the species of L. hordei, L. nagelii, L. hilgardii, Lc. mesenteroides/citreum were able to produce exopolysaccharides (EPS) from sucrose.

Since microbial interaction processes are important for the establishment of the typical kefir microbiota, metabolic interactions among kefir organisms have been explored in different studies. Stadie et al. [13] studied metabolic activity and symbiotic interactions of LAB (L. hordei and L. nagelii) and yeasts (S. cerevisiae and Zygotorulaspora florentina) isolated from water kefir. Although these works described some of the microbial metabolic interactions in kefir fermentation, little is still known about how key microbes build up and adhere to the growing hydrophilic kefir granule. Consequently, any attempts to reconstitute the naturally formed kefir granules in single or co- cultivation experiments failed in the past. Therefore, we set up a simple model system made up of hydrophilic slides and investigated the possible adhesion of water kefir born S. cerevisiae upon co-cultivation with different glucan-producing, water kefir LAB and finally correlated the observed aggregation results with the produced glucan types.

Section snippets

Strain and pre-culture medium

L. hordei TMW 1.1817, TMW 1.1821, TMW 1.1822, TMW 1.1907, L. nagelii TMW 1.1827, Lc. citreum TMW 2.1194, and S. cerevisiae TMW 3.221 isolated from water kefir were chosen for this experiment [2]. All LAB were pre-cultured anaerobically for 24 h at 30 °C in modified MRS medium described by Stolz et al. [14]. S. cerevisiae was pre-cultured anaerobically for 24 h at 30 °C in YPG medium made of peptone from casein (10 g/L), yeast extract (5 g/L), glucose (20 g/L).

EPS production activity test and isolation

Each LAB strain was spread onto

Aggregation and network formation of S. cerevisiae in co-cultivation experiments and after supplementation of different isolated dextrans

LAB, yeast and polysaccharides are the main components of the gelatinous kefir grains, which provide the spot for cell-cell physical contact. Previous works demonstrated that L. hordei, L. nagelii, Lc. citreum are the predominant LAB in water kefir granules at our institute, while S. cerevisiae was one of the most dominant yeasts species [2,12]. Moreover, preliminary experiments revealed that L. hilgardii TMW 1.828, which is supposed to be the main producer of the water-kefir dextran, did not

Discussion

The most dominant species isolated and identified from the water kefir source studied in this work were L. hordei, L. nagelii, Lc. citreum, and S. cerevisiae [2]. In order to get insight into their possible interactions in the complex granule system, they were investigated in a newly set-up hydrophilic slide model system. S. cerevisiae were co-cultivated with L. hordei, L. nagelii, Lc. citreum in petri dishes containing hydrophilic slides, respectively. The slides should mimic the hydrophilic

Acknowledgement

Part of this work was supported by the China Scholarship Council in grant no. 201306820010 and by the German Ministry of Economics and Technology (via AiF) and the Wifoe (Wissenschaftsförderung der Deutschen Brauwirtschaft e.V., Berlin) projects AiF 16454 N and AiF 19180 N. The authors are grateful to Felix Urbat and Andreas Becker for experimental support.

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Lactobacillus hordei dextrans induce Saccharomyces cerevisiae aggregation and network formation on hydrophilic surfaces

Abstract

Introduction

Section snippets

Strain and pre-culture medium

EPS production activity test and isolation

Aggregation and network formation of S. cerevisiae in co-cultivation experiments and after supplementation of different isolated dextrans

Discussion

Acknowledgement

Food Chem.

Int. J. Food Microbiol.

J. Dairy Sci.

Carbohydr. Polym.

Food Microbiol.

Carbohydr. Res.

Carbohydr. Polym.

Food Microbiol.

Int. J. Food Microbiol.

J. Microbiol. Methods

Anal. Biochem.

Phytochemistry

Carbohydr. Res.

Carbohydr. Polym.

Food Microbiol.

Food Microbiol.

Carbohydr. Polym.