Determination of fungal diversity of acidic gruel by using culture‐dependent and independent methods

Abstract Traditional fermented cereals are a rich source of naturally derived, diverse microorganisms. Illumina MiSeq high‐throughput sequencing was used to investigate thoroughly fungal microflora in Western Inner Mongolian acidic gruel. A total of 589,495 sequences were obtained from 16 acidic gruel samples. Ascomycota was found to be the predominant phylum with a relatively abundance of 97.54%, followed by Basidiomycota (2.26%) and Chytridiomycota (0.1%). The dominant genera obtained from the acidic gruel were Candida, Galactomyces, Hanseniaspora, Guehomyces, Zygosaccharomyces, Trichosporon, Rhodosporidium, Penicillium, and Blastobotrys. Candida and Galactomyces were predominant genera, and their relative abundances were 57.59% and 34.95%, respectively. A total of 50 yeast strains were isolated and identified. Statistical analysis indicated that P kudriavzevii and Geo. silvicola affiliated with Ascomycota were the dominant yeasts in acidic gruel, accounting for 28% and 22%, respectively. This study provides an unequivocal theoretical basis for the study of fungal diversity and the identification and preservation of yeasts in traditional fermented cereals. It also provides validated strain resources for further exploration of the effect of yeasts on acidic gruel quality.

gruel, foods made with it can calm both the stomach and spleen and reduce fever and elevated body temperature, but is yet to achieve industrial production. Research on acidic gruel has proved fruitful. Li, Chang, and Wang (2016) and Chang (2016) used pure culture approach to isolate and identify the yeast and lactic acid bacteria in acidic gruel from Northwest Shanxi, while Qin (Qin et al., 2019) and Zhang (Zhang, Li, Song, & Luo, 2020) explored traditional acidic gruel preparation on the basis of strains, fermentation temperature, time, and amount of physalis. However, most studies have focused on the separation and identification of lactic acid bacteria and yeast by traditional culture methods or explored acidic gruel production under different conditions. There are few reports detailing the comprehensive analysis of fungal diversity within traditional fermented acidic gruel.
Fungi are essential microorganisms in the production of Chinese fermented, with different fungi are present in different raw materials and production methods, contributing for example to the unique flavors and texture of traditional acidic gruel (Stephanie, Burton, & Reid, 2015). Many studies have highlighted the importance role which yeasts play in the fermentation products. They also have an important impact on food preservation include inhibiting of the growth of spoilage microorganisms (Angmo, Kumari, Savitri, & Bhalla, 2015;Zhang, 2010). Further study of fungal microorganisms in acidic gruel using advanced technology may offer an enhanced understanding of its intrinsic microbial community and improve both the production process and the ultimate quality of acidic gruel.
Many advanced molecular ecology methods have been developed and used widely to analyze rapidly and efficiently microbial communities in fermented food (Liang, Yin, Zhang, Chang, & Zhang, 2018;Sun et al., 2018). Illumina MiSeq is a powerful and attractive molecular biological technique able to evaluate microbial diversity (Nutan, Changotra, Grover, & Vashistt, 2018;You et al., 2016). It is simple to use, low in cost, and highly suitable for use in small labs (Melanie et al., 2015). High-throughput sequencing technology has been used widely to study the microbial diversity of fermented foods. However, there are few relevant studies of the fungal diversity of acidic gruel.
In this study, Illumina MiSeq sequencing was used to evaluate the fungal microbial communities in acidic gruel, and traditional microbial culture was used to isolate and identify yeasts. The objective of this research was to analyze the diversity of fungal microflora in acidic gruel. It also provides a suitable basis for further study of the microbial community within it and thereby improves the quality of acidic gruel.

| Samples collection and DNA extraction
Research team collected 16 samples of traditional fermented acidic gruel from different households in Ordos, Bayan Nur, and Baotou in western Inner Mongolia. These samples were numbered A1-A16. The raw material in the samples was primarily millet. After washing, six times the volume of water was added, and the resulting traditional fermentation gruel soup was left as primer to ferment for 24 hr at room temperature. The fermented mature acidic gruel was milky white, the physalis was thick, and the pH value was approximately 4.0.
Total genomic DNA was extracted from acidic gruel samples used the QIAGEN DNeasymericon Food Kit (QIAGEN, Germany) following to the manufacturer's instructions. The integrity of genomic DNA was analyzed using 0.8% (w/v) agarose gel electrophoresis in 1 × TAE buffer. DNA concentration was measured using a Micro-ultraviolet spectrophotometer (Nano Drop 2000c, Thermo, America).
SILVA (Quast et al., 2012) was used to assign these sequences to their respective phylum, class, family, order, and genus (Cole et al., 2013).
The α-diversity of 26S rDNA sequences from the samples including Chao 1, the observed species, and the Shannon and Simpson indices calculated using the QIIME platform (Caporaso et al., 2010) Cluster analysis was undertaken using weighted UniFrac distances and variance analysis (Hamady, Lozupone, & Knight, 2009).

Nucleotide sequences of evaluated yeasts have been deposited at
GenBank with accession numbers MH880134-MH880183.

| Sequence data analysis and diversity by highthroughput sequencing
After filtering and preprocessing, a total of 589,495 sequences were obtained from 16 traditional fermented acidic gruel samples, 10,895 different OTUs were obtained under 97% similarity. The abundance and diversity of acidic gruel are reflected by Chao 1 (representing species richness), Simpson index (representing species diversity), and Shannon index ( Table 1). The Chao 1 index of sample A16 (5,850) was higher than that of other samples, and the observed species of sample A3 (1,192) was the highest. Sample A7 (0.81) had the highest Simpson index, while sample A15 (3.89) had the highest Shannon index. These results indicate that the fungal abundance of samples A16 and A3 was the highest among the samples, investigated. The fungal diversity of samples A7 and A15 was the highest. The abundance and diversity of sample A13 were the lowest, seen. This is consistent with it having low fungal diversity.
All of sequences were classified into 6 phyla, 15 classes, 30 orders, 52 families, and 85 genera. Ascomycota, Basidiomycota, and Chytridiomycota were identified as dominant fungal phyla ( Figure 1). Among these three phyla, Ascomycota (97.54%) was the most abundant group, followed by Basidiomycota (2.26%) and Chytridiomycota (0.1%). These results indicate Ascomycota to be the predominant fungal phylum in acidic gruel. A total of 9 main genera were detected, with only 0.37% being unclassified. Those with relative abundance less than 0.1% were merged with other genera (Figure 2). The average relative abundance of each genera in acidic gruel was as follows: Candida (57.59%), Galactomyces  F I G U R E 1 Comparative analysis of the relative abundance of fungal phyla in acidic gruel. A fungal phylum with average relative content >1% was defined as a dominant phylum. Fungal phyla with an average relative content of <1% were defined as others F I G U R E 2 Comparative analysis of the relative abundance of dominant fungal genera in acidic gruel. A fungal genus with an average relative content >1% was defined as dominant genus. Fungal genera with an average relative content of <1% were defined as others

| Sample difference analysis
The cluster analysis based on the weighted UniFrac distance metric indicated differences for all samples (Figure 3). Taking sequence abundance into account to further quantify the variation in different lineages between samples, it was found that the 16 samples of acidic gruel could be classified into three clusters. Cluster I contained 7 samples (A2, A3, A8, A11, A14, and A16). Sample A4, A5, A6, A7, A9, A10, and A13 formed cluster II, while cluster III contained only sample A1 and A15. The results indicate that the fungal community of each cluster was different. As the number of samples in cluster III was small, only samples from cluster I and cluster II were analyzed for variance. The main fungal genera causing differences between the two clusters were Galactomyces and Candida (p < .01) ( Table 2).

| Isolation and identification of yeast
Based on the above analysis, it was found that the cumulative relative content of the fungal genera belonging to yeasts in the detected fungal genera exceeds 98%, and 6 out of the 9 dominant fungal genera were yeasts, indicating that yeasts in the traditional fermented acidic gruel were rich and varied.

F I G U R E 5
The relative abundance of all yeasts isolated from acidic gruel. 50 yeast strains were isolated from the samples of acidic gruel analyzed in the study This may be due to the growth conditions or inherent limitations of pure culture approach. P kudriavzevii (previously called C krusei) plays a crucial role in the production of traditional fermented food, and it may have potential probiotic effects (Greppi et al., 2013(Greppi et al., , 2017Pedersen, Owusu-Kwarteng, Thorsen, & Jespersen, 2012). Other work suggests P kudriavzevii and Geo. silvicola can also enhance food flavor and improve the freshness of food. Eric (Grondin et al., 2017) evaluated the ability of 30 strains of yeast belonging to Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, and Saprochaete to produce volatile flavor substances with HSSPME-GC/MS analysis and found that Geotrichum has good ester production capacity. Citric acid metabolism is an advantage of P kudriavzevii, which can reduce the acidity of cocoa pulp, promote the endogenous proteolysis of raw materials and other enzymatic activities in the process of cocoa fermentation, thereby affecting the quality of cocoa beans and chocolate and possibly affecting the microbial ecology of the entire fermentation process (Soccol et al., 2017).
Traditional methods used to produce fermented food are greatly affected by weather, temperature, and microorganisms in the environment, resulting in the inconsistent taste and flavor of food.
To understand its diversity and function of microorganisms in the production of acidic gruel, several researchers have studied yeast.
Bai (Bai et al., 2010) T asahii, S cerevisiae, Geotrichum sp., and I orientalis were detected in acidic gruel from western Inner Mongolia (Zhang, 2010). In a recent study, a total of 41 yeast strains were isolated from six fermentation stages of northwestern Shanxi acid gruel (Li et al., 2016).
Physiological and biochemical experiments combined with ITS4 and ITS5 analysis showed that all strains could be classified into 5 types according to their morphological characteristics. These had similar it to P kudriavzevii (99%, homology), I orientalis (100%), and S cerevisiae (99%). Such studies indicate that elucidation of fungal diversity within acidic gruel and the accurate of yeast has been a longstanding research interest and that clear differences arise from analysis of different regional samples and the use of different research methods. This is a principal reason for using MiSeq high-throughput sequencing technology, so that the fungal diversity in acidic gruel can be analyzed rigorously and completely. As well as acidic gruel, the researchers also investigated fungi and yeasts found in similar traditional fermented cereal foods from different regions and countries. Idil is a traditional fermented food from India and Sri Lanka,

S cereuisiae, Debaryomyces hansenii, Hansenula anomala, and T beigelii
were common yeasts involved in the fermentation of Idil (Durgadevi & Shetty, 2014;Soni & Sandhu, 1989). Ogi, made from maize, sorghum, or millet and produced by fermenting maize grains, is popular in several West African countries (Adegoke & Babalola, 1988). S cerevisiae, C krusei, Geo. candidum, Geo. fermentans, C tropicalis, and R graminis were isolated during ogi fermentation (Omemu, Oyewole, & Bankole, 2007). S cerevisiae, C pelliculosa, and Candida tropicalis have also been isolated from togwa, a Tanzanian fermented food (Mugula, Nnko, Narvhus, & Sørhaug, 2003). These innate mycological communities are similar to those found in this study. However, for the yeast species isolated, there were appreciable differences between I orientalis and S cerevisiae. These may be caused by differences in local geography, and climate, as well as grain types and producers.

| CON CLUS IONS
To summarize, this work was a first approach to analyze the fungal community structure of traditional fermented Inner Mongolian acidic gruel with high-throughput sequencing and pure culture technology. Multivariate statistical analysis indicated that the fungi in acidic gruel were abundant. Although there were certain differences between the samples, Pichia and Candia were the core fungal genera. Some in the isolated yeasts were found to have potential utilization value. Future research will further characterize these and other yeast strains, while also exploring the mechanism underlying how these and other fungi influence the physical and chemical properties of acidic gruel.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.