Comparative study of physicochemical composition and microbial community of Khoormog, Chigee, and Airag, traditionally fermented dairy products from Xilin Gol in China

Abstract Due to their outstanding nutritional and functional properties, the traditionally fermented dairy products (TFDP) from camel, mare, and cow gained universal praise during their long history of production. In this study, the physicochemical composition and microbial communities of Khoormog, Chigee, and Airag, the TFDP from Xilin Gol in China, were investigated and compared. The physicochemical analysis revealed a higher content of total solid content, protein, and fat in Khoormog (12.5 ± 1.6%; 4.6 ± 0.7%; 4.4 ± 1.3%) compared to Chigee (7.8 ± 1.3%; 2.1 ± 0.2%; 0.8 ± 0.2%) and Airag (8.9 ± 0.7%; 3.7 ± 0.4%; 1.4 ± 0.5%). All three types of TFDP shared 41.2% of bacterial and 25.4% of fungal OTUs, and 95.34% of bacterial and 95.52% of fungal sequence reads. The bacterial and fungal community consisted of four phyla and five genera, and three phyla and seven genera, respectively. Lastly, Lactobacillus predominated in Khoormog, Chigee, and Airag at the genus level, while the dominant fungal genera varied among the samples. In conclusion, the microbial community structures of TFDP from camel, mare, and cow were not significantly different in a definite area (Xilingol region), and Khoormog, Chigee, and Airag bred the common “core microbiota”.

up and down with a wooden stick to increase oxygen and ensure homogeneity. An ancient aseptic technique of natural fermentation was used to extend storage time of the milk and to produce TFDP with enriched nutritional properties and probiotics. The TFDP have a longstanding traditional consumption in many countries across the world due to their exceptional organoleptic properties and numerous health benefits. Several beneficial properties of the probiotics have been reported, such as anticancer, antihypertensive, antidiabetic, and antioxidant activities for fermented camel's milk (Ayyash et al., 2018), and angiotensin I-converting enzyme (ACE) inhibitory activity for the traditionally fermented mare's milk (Chen et al., 2010). Moreover, the cholesterol-lowering activity of Lactobacillus helveticus from fermented cow's milk and L. fermentum from fermented mare's milk was previously documented (Damodharan et al., 2016;Pan et al., 2011). More interestingly, certain aspects of research indicate that the physicochemical attributes and microbiota determine the versatile functions of these TFDP.
The physicochemical composition and microbial community were investigated in TFDP from mare and cow Sun et al., 2014). However, there has been none of the researches on both nutritional properties and microbiota of Khoormog from camel's milk. Additionally, none of the studies reported the comparative examination of the physicochemical composition and microbial community of Khoormog, Chigee, and Airag. In this study, Khoormog, Chigee, and Airag from Xilingol region were gathered to investigate their physicochemical composition and microbial community.

| Physicochemical analysis
The microbial metabolic pathways analysis from the fermented dairy products was predicted based on the 16S rRNA and ITS data using phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2). The relationship between microbial genera and physicochemical indexes was assessed by Pearson correlation heatmap and considered statistically significant at p < .05.

| Physicochemical analysis of Khoormog, Chigee, and Airag
The physicochemical analysis was applied to evaluate the nutritional properties of TFDP from three animals in Xilin Gol, China.
During the process of spontaneous dairy fermentation, the lactic acid bacteria (LAB) use lactose as a source of carbon and produce lactic acid, which increases the acidity of fermented products, while yeasts enhance the alcohol content. The acidity of Khoormog, Chigee, and Airag were 244.1 ± 62.5°T, 168 ± 50.6°T, and 238.5 ± 89.1°T, and the content of alcohol were 0.2 ± 0.2% for Khoormog, 0.5 ± 0.7% for Chigee, and 0.1 ± 0.1% for Airag (Table 1).

A relative abundance of macromolecular compounds in
Khoormog may contribute to higher antioxidant activity, angiotensin-converting enzyme inhibition, and anticancer activity in comparison to compounds from fermented bovine milk (Ayyash et al., 2018). Thus, the derivatives and metabolites of protein and fat in TFDP are considered indicators of executive functions, such as antihypertension, antioxidant activity, and anticancer activity. The samples of spontaneously fermented products, Khoormog, Chigee, and Airag were obtained from Mongolian herdsmen in yurts across the entire grassland from five administrative divisions of Xilin Gol,

Sample
Total solid content (g/100 g) Protein (g/100 g) Fat (g/100 g) Lactose (g/100 g) Acidity a (°T) China. According to the obtained values of SD (Table 1), we observed large differences in acidity, lactose and alcohol content, between Khoormog, Chigee, and Airag. Presumably, the diversity of starter culture, operational habit of individual, and humiture and hygiene of yurt may determine the consumption of lactose by lactic acid bacteria and yeasts, and thus, the quality features of Khoormog, Chigee, and Airag, such as acidity and alcohol content. Therefore, these huge differences reflected the natural quality of homemade fermented milk in a wide range of variations.

| Bacterial and fungal sequence reads analysis
The 16S rRNA gene was amplified from three Khoormog, three Chigee, and three Airag samples. A total of 566,227 of high-quality bacterial sequence reads were generated from the nine traditionally fermented samples, with an average of 62,914 (SD = 5,816) ( Table 2).
Afterward, the ITS gene was amplified from these samples, and a total of 994,354 of high-quality fungal sequence reads were generated, with an average of 110,484 (SD = 16,831) ( Table 2). The highquality sequence reads were clustered into 68 OTUs for bacteria and 59 OTUs for fungi, with an average of 29.8 ± 6.8 and 17.6 ± 6.4 per sample, respectively (Table 2).
Regarding the differences between the samples of three different animals, the number of bacterial OTUs was 29.3 ± 8.1 for Khoormog, 26.3 ± 3.8 for Chigee, and 33.7 ± 8.1 for Airag. A total of 48, 41, and 55 OTUs identified in these dairy products is shown in Figure 2a. In addition, the number of fungal OTUs was 14.3 ± 3.8 for camel samples, 21.7 ± 9.1 for mare samples, and 16.7 ± 4.9 for cow samples, and a total of 31, 42, and 33 OTUs identified in these samples are presented in Figure 2c. We found that 70.6% of bacterial and 54.2% of fungal OTUs were shared by at least two types of these dairy products, whereas 41.2% of bacterial and 25.4% of fungal OTUs were shared by all three types (Figure 2b,d). Interestingly, the two of these dairy products shared 99.91% of bacterial and 97.74% of fungal sequence reads, while all three types shared 95.34% and 95.52% of bacterial and fungal sequence reads, respectively (Figure 2b,d).
The three types of analyzed dairy products from camel, mare, and cow shared microbial OTUs to a certain extent, while the OTUs sequence reads predominantly originated from bacterial and fungal reads. Regarding the similarity of geographical origin (Xilingol region in Inner Mongolia) and artisanal technology of these traditional dairy products, we hypothesized that these naturally fermented dairy products within a certain geographic area could share the common functional "core microbiota" in the process of spontaneous fermentation. This also implies that the distinct components in different animal milk may contribute to the enrichment of specific microorganisms (OTUs).
The analysis of alpha diversity for the bacterial and fungal reads was performed on Khoormog, Chigee, and Airag. As shown in Figure S1, the rarefaction curves maintained an exponential in-  The microbial richness between bacterial and fungal communities of TFDP was compared using the number of OTUs and Chao index. The bacterial number of OTUs (29.8 ± 6.8) and Chao index (32.1 ± 8.2) were significantly higher than in fungal samples (17.6 ± 6.4; 20.7 ± 9.0) (p < .01; p < .05). On the other hand, the microbial diversity was estimated using Shannon and

| Bacterial community structure analysis
The relative abundance analysis of bacterial taxa, based on the 16S rRNA sequences, showed that in these nine samples four phyla could Airag from Xilingol region, respectively. In the previous researches of traditionally fermented products (vrum, koumiss, and tarag), the identical four phyla had been identified as major phyla, along with the predominance of Firmicutes Sun et al., 2014;Yamei et al., 2019).
As shown in Figure 3a, five bacterial genera were identified in these nine samples, with an average relative abundance >1%.
The relative abundances of genera were 11.78% for Lactococcus,  (Yamei et al., 2019), koumiss , tarag (Sun et al., 2014), hurood cheese, and jueke (Gao et al., 2017). In addition, a very small amount of contamination with Citrobacter, Raoultella, and Escherichia-Shigella was detected in several samples. This instance of bacterial contamination may result due to nonpasteurization of raw milk, insanitation of tools, or artisanal fermentation.
The bacterial community structures of TFDP from camel, mare, and cow were analyzed using the unweighted and weighted UniFrac PCoA. The results showed that nine samples from three animals were largely separated in the both unweighted (corresponding to 39.11% and 25.41% of the total variance by the two principal components, respectively) and weighted (corresponding to 66.24% and 24.64% of the total variance by the two principal components, respectively) analysis (Figure 4a,b). In addition, the Anosim analysis, based on the unweighted and weighted UniFrac, was also utilized to evaluate the differences in the bacterial community of fermented dairy products from three animals. As shown in Figure 5a,b, the results demonstrated that there was no difference in the bacterial community between the samples from three animals (R = −0.029; p = .561). Thus, the existence of "core microbiota" in these three types (camel, mare, and cow) of TFDP was supported with the results obtained from UniFrac PCoA and Anosim analyses.

| Fungal community structure analysis
The analysis of fungal taxa relative abundances, based on the ITS sequences, showed that in these nine samples three phyla could  (6.17%), Dekkera (5.72%), and Pichia (1.28%). These genera are common components of the fungal TFDP community Sun et al., 2014;Yamei et al., 2019;Gao et al., 2017). The results obtained from the relative abundances of genera in fungal communities showed that the dominant fungal genera varied among different samples.

F I G U R E 3
The relative abundance of bacterial (a) and fungal (b) sequence in the nine traditionally fermented dairy samples of Khoormog, Chigee, and Airag. The color version is available online The analysis of the fungal communities of TFDP (from camel, mare, and cow) was performed using the unweighted and weighted UniFrac PCoA. The obtained values of nine samples from three animals diverged largely in both unweighted (accounting for 36.27% and 25% of the total variance by the two principal components, respectively) and weighted (accounting for 61.16% and 17.45% of the total variance by the two principal components, respectively) analysis (Figure 4c,d). In addition, the unweighted and weighted UniFrac Anosim analysis of the sequence reads was used to evaluate the differences between the fungal communities of TFPD from camel, mare, and cow. The results showed that there was no difference between the three types of naturally fermented dairy products (R = 0.268; p = .062) (Figure 5c,d). Hence, these results support the previously stated assumption of the "core microbiota" in three types of TFDP from camel, mare, and cow.

| Metabolic pathways and correlation analyses
The microbial metabolic pathways of the traditional fermented dairy products were predicted to compare the different microbial pathways among the nine samples from camel, mare, and cow. The metabolic pathways were adenosine deoxyribonucleotides de novo biosynthesis II, guanosine deoxyribonucleotides de novo biosynthesis II, gondoate biosynthesis (anaerobic), pyruvate fermentation to acetate and lactate II, cis-vaccenate biosynthesis, and so on for 16S rRNA data prediction (Table S1) and aerobic respiration I (cytochrome c), aerobic respiration II (cytochrome c) (yeast), glyoxylate cycle, TCA cycle II (plants and fungi), adenosine ribonucleotides de novo biosynthesis, and so on for ITS data prediction (Table S2), and these above predicted metabolic pathways were enriched in each type of samples. To explore the potentially functional microbial profile in traditional fermented dairy products, we calculated all F I G U R E 4 Unweighted (a and c) and weighted (b and d) UniFrac principal coordinate analyses of the bacterial (a and b) and fungal (c and D) diversity in Khoormog, Chigee, and Airag from Xilin Gol in China. The blue (trilateral), red (roundness), and green (tetragonal) symbols represent Khoormog, Chigee, and Airag, respectively. The color version is available online pairwise Spearman's correlations between different bacterial and fungal genera and physicochemical indexes, and constructed a correlation heatmap. We found some bacterial and fungal genera to do with total solid content, protein, fat, lactose, acidity, and alcohol ( Figure 6). Nevertheless, only 3 samples in each group are not enough to elucidate the correlations between microbial genera and physicochemical indexes. In future, we will try our best to collect enough samples to investigate the differences of the physicochemical composition and microbial communities between Khoormog, Chigee, and Airag from Xilin Gol in the same family (yurt) at the same time in order to study the influence of the types of milk on microbial community, and we use homemade starters to inoculate pasteurized raw milk from camel, mare, and cow to study the influence of the types of milk on microbial community. OTUs. The results demonstrated that these naturally fermented dairy products within a certain geographic area could share the common functional "core microbiota" in the fermentation process, and the distinct components in different animal milk may contribute to the enrichment of specific microorganisms.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.