Abstract
Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S. cerevisiae and Lactobacillus bacteria in TKGs.
Highlights
Tibetan kefir grains (TKGs), the so-called Tibetan mushroom, are unique dairy starters in Tibet, China, and belong to the same kefir family originating from the Caucasian mountains [1]
Fine structure of TKGs TKGs are composed of a polysaccharide/protein matrix, containing a complex microbial community of lactic acid bacteria, yeasts, and sometimes acetic acid bacteria, and represent one of the unusual specific symbiotic systems in nature [1]
Given the fact that TKGs are produced in milk, it is conceivable to speculate that the hollow globular structures contribute to the uptake of nutrients by their designers and residents of bacteria and yeasts as well as to the structural stability of the grains
Summary
Tibetan kefir grains (TKGs), the so-called Tibetan mushroom, are unique dairy starters in Tibet, China, and belong to the same kefir family originating from the Caucasian mountains [1]. Tibetan kefir (TK) is a fermented milk made using TKGs. Tibetan kefir (TK) is a fermented milk made using TKGs It is a kind of popular yogurt in Tibet, which is characterized by a considerably acidic and somewhat alcoholic taste, a slightly yeasty flavor, and very creamy consistency [1,3]. It is a traditional beverage in Tibet, currently TK is becoming a kind of home-made yogurt in numerous families across the mainland of China. Besides its higher nutritional value, kefir appears to possess dramatic health benefits including enhancing the immune system, improving digestive health, as well as having antimicrobial, antitumoral, and antioxidant effects [14,15,16,17,18]
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