Table 1.
PCR primers used in this study.
Table 2.
FISH probes used in this study.
Figure 1.
Stereomicroscope images of the Tibetan kefir grains.
(A) Tibetan kefir grains and a single grain unit marked with a circle in (A) and observed from the side (B), the top (C), and the bottom (D). Scale bar = 1.2 mm.
Figure 2.
The microflora in TKGs observed on thin sections stained with SYBR Green I.
(a–e) Series thin sections, from outside towards inside, of a single hollow globular unit. Scale bar = 500.0 µm. (A–C) The polyhedron-like net structure, from outside towards inside, of a grain unit. (D–F) The distribution of yeasts on the outside wall of a grain unit. The arrows indicate the representative yeast cells. The rod-shaped cells (A–F) are bacteria as marked with the arrowheads. Scale bar = 10.0 µm.
Figure 3.
FISH analysis of yeast species in TKGs.
Epifluorescence micrographs of bacteria and yeasts stained with DAPI (A, D); Saccharomyces hybridized with probe Sacch (B, E); Kluyveromyces hybridized with probes Kluyv 1 and 2 (C); and Yarrowia hybridized with probe Ylip (F). Others represent merged photographs. The bacilliform signals are the DAPI stained bacteria, and the bright dots are the yeasts. Scale bar = 10.0 µm.
Figure 4.
Scanning electron micrographs of TKGs.
(A) The outer surface of TKGs; (B) The high magnification of (A); (C) The cross-section of TKGs; (D) The high magnification of (C); (E) The inner surface of NCTKGs; (F) The high magnification of (E); (G) The inner surface of ACTKGs; (H) The high magnification of (G). The arrows, shown in (A), (B), (F), (G), and (H), indicate the representative yeasts in KGs. The outside and inside surfaces of KGs are indicated by the arrows in (C). Scale bar = 10.0 µm.
Table 3.
Phylotypes of yeast community in TKGs.
Figure 5.
The shift of yeast species in TKGs during a long-term subculture as revealed by real-time qPCR assay.
N-1: NCTKG-1, N-2: NCTKG-2, A-1: ACTKG-1, A-2: ACTKG-2. NCTKG-2 and ACTKG-2 are the samples cultivated for 10 months more than NCTKG-1 and ACTKG-1. Standard deviations are shown by error bars. Differing letters within each subfigure (i.e., a, b, c) indicate statistically significant differences (p<0.01). The statistical analysis was performed based on the experimental data of 3 repeats, each in 3 parallels, and a total of 9 grains were analyzed for each sample.