Chemical and microbiological characterization of two traditional Mongolian high‐fat dairy products produced in Xilin Gol, China

Abstract Mongolian butter and Tude are traditional high‐fat dairy products produced in Xilin Gol, China, which have unique chemical and microbiological characteristics. Mongolian Tude is made from Mongolian butter, dreg, and flour. In this study, the traditional manufacturing process of Mongolian butter and Tude was investigated for the first time. Mongolian butter was characterized by high‐fat content (99.38 ± 0.63%) and high acidity (77.09 ± 52.91°T), whereas Mongolian Tude was considered a high‐fat (21.45 ± 1.23%) and high‐protein (8.28 ± 0.65%) dairy product obtained by butter, dreg, and flour. Mongolian butter and Tude were proven to be safe for human consumption in terms of benzopyrene content. In addition, Listeria monocytogenes, Staphylococcus aureus, Salmonella, coliforms, and aflatoxin M1 were not detected in the samples. Bacteria and molds were not isolated from Mongolian butter; in contrast, the total count of bacteria and molds in Mongolian Tude was within the range of 4.5 × 102 to 9.5 × 104 and 0 to 2.2 × 105, respectively. Moreover, Lactococcus (41.55%), Lactobacillus (11.05%), Zygosaccharomyces (40.20%), and Pichia (12.90%) were the predominant bacterial and fungal genera, and Lactobacillus helveticus (15.6%), Lactococcus raffinolactis (9.6%), Streptococcus salivarius (8.5%), Pantoea vagans (6.1%), Bacillus subtilis (4.2%), Kocuria rhizophila (3.5%), Acinetobacter johnsonii (3.5%), Zygosaccharomyces rouxii (46.2%), Pichia fermentans (14.7%), and Dipodascus geotrichum (11.7%) were the predominant species in the microbiota of Mongolian Tude. Thus, it can be stated that the microbiota of food products produced by different small families varied significantly. Collectively, the findings presented herein are the first report of chemical and microbiological characterization of products of geographical origin and highlight the need for standardization of manufacturing procedures of Mongolian butter and Tude in the future.


| INTRODUC TI ON
The traditional processing of Mongolian butter was different from that of commercial butter . To produce the former, filtered fresh milk was spontaneously fermented for 1-2 days  Figure 1h) and then mixed with butter and dreg (at a ratio of 1:1:1, w/w) to obtain a slightly elastic dough, which was subsequently placed in molds ( Figure 1i). After processing, Mongolian butter and Tude can undergo long-term storage by refrigeration, during which special organoleptic qualities were developed.
Considering the steps used in the processing of Mongolian butter and Tude, it can be concluded that spontaneous/natural fermentation and cooling storage are the main factors that are responsible for the development of special properties of these products, such as fermented smell, sourness, the taste of milk with wheat aroma.
However, since this artisanal process is based on natural fermentation, it may comport risks for food safety due to the contamination with pathogenic bacteria and molds. In addition, mycotoxin production and/or benzopyrene formation can occur, respectively, as a result of mold growth and excessive temperature.
Thus, the present study aimed to comprehensively evaluate the chemical composition and microbiological safety of traditional Mongolian butter and Tude based on chemical, microbiological, and microbial community analyses. Benzopyrene and aflatoxin M1 were determined by the HPLC method. Acidity was determined by the titration method using sodium hydroxide (0.1 mol/L) with a phenolphthalein indicator (Sinopharm; China National Food Safety Standard, 2016f). The data obtained from the experiment were analyzed by ANOVA in SPSS20.0.

| Quantification of coliform, TBC, Mold, Listeria monocytogenes, Staphylococcus aureus, and Salmonella
The enumeration of coliforms was determined using Violet Red The data obtained from the experiment were analyzed by ANOVA in SPSS20.0.
The final reads were obtained by trimming the chimeric tags and were clustered into the operational taxonomic units (OTUs) by using the UPARSE pipeline (Edgar, 2013). The OTUs were classified into organisms with the Naive Bayesian Model by using RDP classifier (Wang et al., 2007) based on the SILVA database for 16 S rRNA gene sequencing (Pruesse et al., 2007) and UNITE database for ITS gene sequencing (Koljalg et al., 2005).

| Chemical composition of Mongolian butter and Tude
As shown in Table 1, the contents of fat and solids-not-fat in Mongolian butter were 99.38 ± 0.63% and 0.46 ± 0.64%, respectively; the moisture content was 0.16 ± 0.09%. Overall, no significant difference was found in the evaluated parameters (fat, solids-notfat, and moisture) between different producing regions (p > .05).
Nevertheless, the acidity of Mongolian butter produced in different producing regions varied significantly (p < .05). Although Mongolian butter is derived from naturally fermented cream, its composition is roughly identical to commercial butter. However, the acidity of Mongolian butter is 77.09 ± 52.91°T, which is significantly higher than that of commercial butter. High acidity confers unique qualities to butter, such as distinct flavor, and also acts as a natural preservative.
Mongolian Tude is produced from Mongolian butter, dreg, and flour, thus containing milk-like and wheat-like odors. As shown in

| Evaluation of chemical and microbiological safety of Mongolian butter and Tude
The chemical and microbiological safety of Mongolian butter and Tude has been proven by their long history of production and consumption. However, natural fermentation, high temperature, familysized production, and the absence of preservatives increase the risk associated with the consumption of Mongolian butter and Tude, mainly involving pathogenic bacteria and molds, and benzopyrene formation. As shown in Table 3, benzopyrene content was 0 in Mongolian butter. In contrast, benzopyrene was found in two out of 10 samples of Mongolian Tude (1.7 and 1.2 μg/kg), at levels below the criteria established in European food safety standards (2 μg/kg).
The above results indicated that the high temperature employed during the production of Mongolian butter did not lead to benzopyr-   Although molds were detected in Tude, aflatoxin M1 could not be found in none of the samples. Collectively, Mongolian butter and Tude were shown to be safe for consumption based on the findings of the current study.

| Composition of the bacterial community of Mongolian Tude
After the removal of low-quality reads, 472,147 reads  (1.21%), and Acinetobacter guillouiae (1.14%). Most genera and species found in Tude samples have been previously identified in naturally fermented dairy products produced in Xilin Gol, China Yamei et al., 2019). Nevertheless, certain bacterial genera (e.g., Pantoea, Ralstonia, Kocuria, and Bacillus) were described for the first time in Tude, but were not found in traditional koumiss , traditionally fermented vrum (Yamei et al., 2019), Khoormog, Chigee, and Airag produced in Xilin Gol .
In addition, a higher number of bacterial genera and species in Tude was found compared to other naturally fermented dairy products.
Considering that Tude contains flour, it can be easily contaminated with a wider variety of microorganisms.

| Composition of fungal community of Mongolian Tude
After the removal of low-quality reads, a total of 585,641 reads (Average ± SD: 58,564 ± 11,680) assigned to fungi were generated from Tude samples collected from ten family-sized production sites.
OTUs, as well as Shannon, Simpson, Chao1, and Good's coverage indices, were used to evaluate the richness and diversity of fungal communities in samples. Shannon curves (Figure 3a) but not rarefaction curves (Figure 3b) enabled data saturation in ITS gene sequencing (Table 4) Yamei et al., 2019), the diversity of fungal genera and species in Mongolian Tude was notably higher, and the relative abundance of certain fungi was increased. Considering the familysized production conditions and nutrient-rich matrix, a wide variety of molds can grow in Tude. Fungal contamination in traditional dairy products obtained by natural fermentation is common, which may also confer distinct flavors to these dairy products.
In addition, multivariate analysis was used to compare the composition and abundance of fungal communities in Mongolian Tude samples from two producing regions. Figure 3d

| CON CLUS IONS
Mongolian butter and Tude are traditional high-fat dairy products, and their traditional processing steps, chemical composition, and microbiological characteristics have not been reported so far.
In this study, nutritional composition (e.g., fat, protein, moisture, and acidity), safety (e.g., benzopyrene, L. monocytogenes, S. aureus, Salmonella, coliform, and aflatoxin M1), and microbiological (e.g., TBC, mold, bacterial and fungal community) parameters were determined. Overall, Mongolian butter could be considered a traditional acidic butter with fermented odor, whereas Tude could be characterized as a flour-containing high-fat and high-protein suspension.

F I G U R E 3
Shannon diversity curves (a) and Rarefaction curves (b) for ITS sequencing analysis. Relative abundance of fungal sequences at genus level in the 10 individual Tude samples obtained from Mongolian family-sized production plants resided in Huang Banner and Lan Banner (c). Unweighted (d) and weighted (e) UniFrac principal coordinate analyses of the fungal diversities in the 10 individual Tude samples. OTU, operational taxonomic units.