Presence of an ultra-small microbiome in fermented cabbages

Abstract

Background: Ultramicrobacteria (UMB), also known as ultra-small bacteria, are tiny bacteria with a size less than 0.1 µm³. They have a high surface-to-volume ratio and are found in various ecosystems, including the human body. UMB can be classified into two types: one formed through cell contraction and the other that maintains a small size. The ultra-small microbiome (USM), which may contain UMB, includes all bacteria less than 0.2 µm in size and is difficult to detect with current methods. However, it poses a potential threat to food hygiene, as it can pass through sterilization filters and exist in a viable but non-culturable (VBNC) state. The data on the USM of foods is limited. Some bacteria, including pathogenic species, are capable of forming UMB under harsh conditions, making it difficult to detect them through conventional culture techniques.

Methods: The study described above focused on exploring the diversity of USM in fermented cabbage samples from three different countries (South Korea, China, and Germany). The samples of fermented cabbage (kimchi, suancai, and sauerkraut) were purchased and stored in chilled conditions at approximately 4 °C until filtration. The filtration process involved two steps of tangential flow filtration (TFF) using TFF cartridges with different pore sizes (0.2 µm and 100 kDa) to separate normal size bacteria (NM) and USM. The USM and NM isolated via TFF were stored in a refrigerator at 4 °C until DNA extraction. The extracted DNA was then amplified using PCR and the full-length 16S rRNA gene was sequenced using single-molecule-real-time (SMRT) sequencing. The transmission electron microscope (TEM) was used to confirm the presence of microorganisms in the USM of fermented cabbage samples.

Results: To the best of our knowledge, this is the first study to identify the differences between USM and NM in fermented cabbages. Although the size of the USM (average 2,171,621 bp) was smaller than that of the NM (average 15,727,282 bp), diversity in USM (average H' = 1.32) was not lower than that in NM (average H' = 1.22). In addition, some members in USM probably underwent cell shrinkage due to unfavorable environments, while others maintained their size. Major pathogens were not detected in the USM in fermented cabbages. Nevertheless, several potentially suspicious strains (genera Cellulomonas and Ralstonia) were detected. Our method can be used to screen food materials for the presence of USM undetectable via conventional methods. USM and NM were efficiently separated using tangential flow filtration and analyzed via single-molecule real-time sequencing. The USM of fermented vegetables exhibited differences in size, diversity, and composition compared with the conventional microbiome. This study could provide new insights into the ultra-small ecosystem in fermented foods, including fermented cabbages.

Keywords: Fermented cabbage; Kimchi; Sauerkraut; Single molecule real time sequencing; Suancai; TFF; Tangential flow filtration; USM; Ulramicrobacteria; Ultra-small microbiome.

Figure 1. Rarefaction curve (A) and Shannon index (B) of microbial and ultramicrobial communities detected in fermented cabbages.

Relationship between number of operational taxonomic units (OTUs) and sequences was applied to a rarefaction curve, and each sample was plateaued. Shannon index was expressed for each sample (left), cabbage type (middle), and community (right).

Figure 2. Relative abundance profiling (A), principal coordinate analysis (PCoA) plot (B), and hierarchical cluster analysis (HCA) dendrogram (C) reflected the species-level abundance.

OTUs with an abundance below 10 as determined via relative abundance profiling were expressed as others. The statistical significance of the clustering pattern in the PCoA plot was evaluated through permutational ANOVA (PERMANOVA). The distance measure and clustering algorithm of HCA were applied to the Bray–Cutis index and Ward, respectively.

Figure 3. A heat tree used to compare and sum the microbial and ultramicrobial communities for each sample at the genus level.

(A–C) The total microbiome of kimchi, sauerkraut, and suancai, respectively; (D–F) comparison between the NM and USM of kimchi, sauerkraut, and suancai, respectively.

Figure 4. Random forest classification analysis.

Analysis confirmed the top 15 OTUs with greatest variability between communities. The mean reduced accuracy represents the accuracy that communities lose by excluding each variable. The lower the accuracy, more important the variable species for a successful classification.

Figure 5. Transmission electron micrographs for ultramicrobial communities after ultra-section of fermented cabbages.

Transmission electron micrographs of the ultramicrobial community of a size < 0.2 µm in (A–D) kimchi (Kimchi_USM), (E–H) sauerkraut (Sauerkraut_USM), and (I–L) suancai (Suancai_USM). DT, dichotomy; IM, inner membrane; PS, periplasmic space: OM, outer membrane.