Problems and prospects of current studies on the microecology of tongue coating

Total bacterial count on the tongue coating and the content of lysozyme were decreased after cure of acute pancreatitis (AP) [18]. The microbiota imbalance on the lingual surface was related to the changes in the tongue coating. AP patients had a thick tongue coating and increased in Gram-negative anaerobic bacilli [18]. Zhu et al. [19] prepared tongue coating smears for microscopic examination after Gram-staining to set the quantitative criteria for analyzing the microbiota on the tongue coating of patients with damp-heat syndrome. They discovered that the total bacterial count was higher in damp-heat syndrome’s yellow-dense tongue coating than in the normal white-greasy coating. Another study [20] diagnosed patients suffering from diarrhea-predominant irritable bowel syndrome, through microscopic examination of tongue coating smears to be a pi and wei’s shi-re (damp-heat) syndrome. Denaturing gel gradient electrophoresis (DGGE) was increasingly used in tongue coating studies. Fei et al. [21] discovered high similarity in DGGE analysis among different samples from the patients with lung cancer, indicating that the type of tongue coating was similar in the composition of microbial flora. 16S rRNA-DGGE was used to investigate the microbial changes in chronic gastritis patients’ greasy tongue coating, and found a new species of bacteria closely associated with the generation of greasy tongue coating [22]. The new species has a nearest neighbor Moraxella catarrhalis with a similarity of 96.2%. The microbial changes in the oral cavity could be one of the causes of the generation of greasy tongue coating. In addition, research on the relationship between TCM and tongue coating using the tongue coating rating scale found that a Jianpishenshi decoction changed the intestinal microecology and tongue coating of the patients with pi-xue (spleen-deficiency) syndrome [23].

First, few modern experimental techniques ‘have been employed by the studies of the microecology of tongue coating such as bacteria cultures and microscopic examination. Conventional bacteria cultures and microscopic examination of smears are inadequate to analyze large sizes of samples [24]. Second, the sample collection was not systematic [25]. The lack of objective judgment of “zheng” makes eliminating individual differences and differences between TCM doctors difficult. The diseases of different internal organs would be reflected in different regions (Figure 1), such as the tongue tip for xin (heart) and fei (lung) that belong to upper-jiao, middle tongue for pi and wei that belong to middle-jiao, tongue root for shen (kidney) that belongs to lower-jiao, and lateral tongue for gan (liver) and dan (gallbladder). It is necessary to establish a standard process for collecting tongue coating samples to ensure the comparability of samples collected from different patients.

First, inclusion and exclusion criteria for sampling collection should be established. Second, non-culture-based bacteriological technology, like 16sRNA-DGGE [26], 2S-DGGE [27], RFLP-PCR [28], and RAPD-PCR [29], should be used for bacterial component analyses, and floral structural relation analyses, and flora network analysis (Figure 2).

Bai et al. [30] analyzed tongue-coating microbiomes and their relationships with tongue diagnosis, proposing a method of zheng discrimination before Kanawong et al.[31], by a machine learning algorithm with a color space set of features. Lo et al. [32] proposed standardization of zheng by determining the regions in which the samples were distributed on the tongue dorsa and fixing the sample collection region. Bai et al. [30] applied the next-generation sequencing methods to analyze tongue coating samples via bacterial 16sRNA V6 regions and analyzed the network of operational taxonomic units and microecology of zheng.