Comparative analysis of the microbial community and nutritional quality of sufu

Abstract Sufu is a type of fermented food with abundant nutrients and delicious taste. It is made from the fermentation of tofu by various microorganisms. In this study, three types of sufu were prepared through natural fermentation: (NF), single‐strain fermentation (SF), and mixed‐strain fermentation (MF). Microbial species, amino acids, and fatty acids were identified to investigate dynamic changes in nutritional quality and microbial flora in sufu. The results showed that the number of microbial species in NF sufu was the highest (n = 284), whereas that in SF sufu was the lowest (n = 194). Overall, 153 microbial species were found in all three types of sufu. Relative abundance analysis also revealed that Tetragonococcus, Bacillus, Acinetobacter, and Staphylococcus were the main bacteria in sufu. However, there was a large number of harmful bacteria such as Enterococcaceae in NF sufu. The levels of various nutrients were low in SF sufu, whereas the contents of protein and soy isoflavones were higher in NF and MF sufu. Seventeen kinds of amino acids were detected, comprising seven essential amino acids and ten other amino acids. The contents of essential amino acids and essential fatty acids were higher in MF sufu than the other two types, resulting in its high nutritional value. The sufu produced through the three fermentation methods differed significantly (p < .05) in terms of microbial flora and nutritional quality.

when the environmental temperature exceeds 28℃. Recently, the Rhizopus oryzae fungus has been used in the production of sufu; because of its high-temperature tolerance, it can grow favorably at temperatures close to 40℃ (Ma et al., 2015).
Sufu is rich in numerous nutrients in addition to those found in tofu. After fermentation, the protein and starch in tofu are decomposed into amino acids, peptides, and other nutrients by fungal enzymes, which can give a unique flavor to sufu. At the same time, the bitter taste, gas-producing effect, and anti-nutritional properties of soybeans were weakened, and the production of soy isoflavones was enhanced. The concentrations of antioxidants and active substances as well as biovalence are also greatly increased (Xu et al., 2015). In the fermentation process, bacterial strains produce peptidase or protease, which can effectively decompose the macromolecules in tofu into small-molecule substances. These small-molecule substances are easily absorbed by the human body (Chen et al., 2016;González et al., 2018).
The microbial flora in fermented foods determines their safety, odor, and nutritional quality. Many studies have investigated the nutrients in sufu. However, due to the complexity fermentation process of sufu, how its nutritional quality is affected by the microflora remains unclear (Lv et al., 2015). Traditionally, sufu is produced through natural fermentation (NF). Currently, Mucor are usually used in single-strain fermentation (SF). However, NF sufu contains a multitude of microorganisms, even including many pathogenic bacteria. In the SF production process, the overgrowth of a single strain inhibits the growth and reproduction of other beneficial bacteria, which affects the nutritional quality of sufu. In order to enrich the nutrients and improve the odor of sufu, in this study, NF sufu, SF sufu, and mixed-strain fermentation (MF) sufu were produced using different microorganisms, and the bacterial population and nutrient changes were analyzed.

| Preparation of fungi
Mucor racemosus (CICC40481) and R. oryzae preserved in potato dextrose medium were washed with 20 ml of sterile water and filtered thorough sterile gauze. The filtrate was collected, and cells were counted using a hemocytometer. The spore suspension concentration was adjusted to 10 6 (cfu/mL) as the seed.

| Sample preparation
Soybeans were screened, soaked, milled, filtered, boiled, spotted, and chopped into 2.8 × 2.8 × 1.4-cm 3 white bars. The water content of the white bars was controlled at approximately 70% (Qiu et al., 2018), and each white bar was gently placed in a salver. The white bars were fermented under natural conditions without any seed in NF, while Mucor spore suspension was dropped on the white bars in SF and a mixed suspension of Mucor and R. oryzae spore with a ratio (1.5:1) was dropped on the white bars in MF (Feng, Gao, Ren, Chen, & Li, 2013). These samples were maintained in an environment with 28℃ temperature and 90% relative humidity for two days.
After the surface of the white bars was covered with mycelium, they were marinated with salt and then packaged into 300-mL glass bottles, which were filled with a solution of 12% ethanol and each auxiliary material. Finally, the bottles were aged at room temperature for 90 days to obtain sufu (Han et al., 2004) (Figure B1 and Figure B2).

| DNA extraction, amplicon, and sequencing
In this study, 5.0 g of sufu was mixed with 25 ml of phosphate buffer (pH 7.2). The mixture was centrifuged at 5,000 rpm for 10 min, and then, 5 ml of the supernatant was subjected to genomic DNA extraction using a DNA kit (Sangon Biotech, Shanghai) (Almeida et al., 2018;Knob et al., 2018). The extracted genomic DNA was separated through 1% agarose gel electrophoresis. The V3-V4 domain of the 16S rRNA gene was amplified using the primers 338F and 806R (Table A1, ABI, Foster City, CA, USA) using TransGen AP221-02 (TransStart Fastpfu DNA Polymerase) and a PCR machine (ABI GeneAmp Model 9700). Three replicates of each sample were prepared. The PCR products of each sample were mixed and detected using 2% agarose gel electrophoresis (Jarocki et al., 2016).
The PCR products were recovered using the AxyPrep DNA gel recovery reagent and were eluted with Tris-HCl before undergoing 2% agarose electrophoresis. With reference to the preliminary electrophoretic quantification results, the PCR products were quantified using a QuantiFluorTM-ST blue fluorescence quantitation system (Promega) (Naegele et al., 2018). Amplicons were submitted to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for Illumina paired-end library preparation, cluster generation, and 300-500 bp paired-end sequencing on the MiSeq instrument.

| Data management and species annotation and assessment
MiSeq sequencing results were obtained from double-ended sequence data. According to the overlap relationship between PE reads, the paired reads were merged into a sequence, and the quality of the reads and the effects of the merged sequence were quality-controlfiltered on the basis of the barcodes at each end of the sequence.
Primer sequences were used to distinguish the samples to obtain valid sequences and the correct orientation of the sequence for generating optimized data. Clustering of the sequences was conducted at 97% similarity levels, and one group contained OTU representative sequences (Gryganskyi et al., 2018). Taxonomic analysis of the samples was conducted at 97% similarity levels using the RDP classifier Bayesian algorithm and OTU representative sequences. The samples were compared with the sequences in the SILVA database, and the statistics of each sample at each taxonomic level were used to determine the community composition. One-way analysis of variance was used to detect significant differences. Fisher's exact test was used to determine the differences in the abundance of two fermented sufu species across two or three sufu samples (Kulkarni et al., 2018).

| Nutritional quality analysis
The sufu sample was weighed (5.0 g, accurate to 0.001 g) in a filter paper cylinder. The filter paper cylinder was placed in a Soxhlet extraction tube for Soxhlet extraction. After the extraction, the fat content was calculated using the dry weight. Phenolphthalein was then added to a receiving flask, and then titration was performed with a 10% sodium hydroxide solution. The volume of sodium hydroxide solution consumed was recorded to calculate the fatty acid content (Rusu et al., 2018).
The sufu sample (2.0 g, accurate to 0.001 g) was accurately weighed in Kjeldahl bottles, and 0.5 g of a copper sulfate and potassium sulfate mixture was added, followed by 12 ml of concentrated sulfuric acid. The sample was placed on a digestion rack at a temperature of 400℃. After 3-4 hr of digestion, the crude protein content in the sample was determined on an automated Kjeldahl nitrogen analyzer.
A high-performance U3000 liquid chromatograph (Thermo Fisher) was used to determine the amino acid content. Total amino acids (TAAs) were determined under the following chromatographic conditions: mobile phase A was 0.1 mol/L sodium acetate solution and mobile phase B was acetonitrile-water (8:2), a 10 cm column with octadecylsilane-bonded silica gel as a filler (4.6 × 250 mm, 5 μm) was used, the flow rate was 1.0 ml per minute, the column temperature was 40℃, the injection volume was 10 μL, and the wavelength was 254 nm (Liu, Han, Deng, Sun, & Chen, 2018). The lyophilized samples were then homogenized and dissolved in sulfosalicylic acid, and free amino acid (FAA) detection was performed using sodium citrate buffer systems and ninhydrin detection columns (pH 2.2, 3.3, 4.3, and 5.4) .

| Analysis of microflora in sufu
In Figure 1, the Venn diagram presents the numbers of common and unique bacterial species (at the species and genus levels) in sufu.
Different colors represent different sufu samples produced using the various fermentation methods (NF, MF, and SF). Nonoverlapping parts represent the unique species in each sufu type, and the numbers denote the corresponding number of species. As shown in   (Jung et al., 2016). Therefore, more research attention should be paid to their roles in sufu production in the future.

| Analysis of nutritional quality in sufu
As shown in Figure 4a, the fat content in NF sufu (21.38 g/100 g) was much higher than the fat contents in MF (12.02 g/100 g) and SF sufu (10.08 g/100 g). The fatty acid content in SF sufu was the highest (16.73 g/100 g). The overall protein content in the three types of sufu was the same, and the average content was approximately 10%. Fat and protein are not only important nutritional components in sufu but also important indicators of the maturity of sufu during fermentation. The soy isoflavone content was the same in NF and MF sufu; in comparison, it was slightly lower in SF sufu (Figure 4b).
Texture analysis results and scanning electron microscopy images of the structure of sufu during fermentation are shown in Figure A2 and Figure A3. The results showed that approximately 30 hr after inoculation, the hardness of sufu reached the maximum level and then decreased ( Figure A2). The hardness of NF sufu was slightly higher than that of SF and MF sufu. The adhesiveness of sufu increased gradually during fermentation. MF sufu exhibited the least change in elasticity. There was no significant difference among the texture analysis. But differences in structure were observed in three types of sufu. As shown in Figure A3 the structure in SF sufu was looser than that in NF and MF sufu.  Table 2. In SF sufu, the FAA content was higher than that in NF and MF sufu, and the EAA content was also significantly higher (p <.05) in SF sufu than in NF and MF sufu.

| D ISCUSS I ON
In this study, the sufu produced through three fermentation methods exhibited significant differences (p <.05) in terms of bacterial species and nutritional quality. NF sufu contained the largest number F I G U R E 2 Analysis of the bacterial community composition in sufu. NF: Natural fermentation, MF: Mixed fermentation, SF: Single strain fermentation. The ordinate is relative abundance of the species in sufu, the abscissa is the sufu types; the columns of different colors represent different species, and the length of the column represents the relative abundance of the species of bacterial species (n = 284), whereas SF sufu had the least num- The activities of protease, amylase, and lipase are high in Bacillus F I G U R E 3 Analysis of species differences in sufu. NF: Natural fermentation, MF: Mixed fermentation, SF: Single strain fermentation. (a) Species difference analysis among NF, SF and MF sufu; (b) Species difference analysis between NF and SF sufu; (c) Species difference analysis between SF and MF sufu; (d) Species difference analysis between NF and MF sufu. One-way analysis of variance was performed among three types of sufu. The vertical axis represents the species name under the genus level. The column length corresponds to the relative abundance of the species in each sample. Different colors indicate different samples. Pvalues: * 0.01 < p≤.05, ** 0.001 < p≤.01, and *** p ≤.001 (Cha et al., 2018). In the current study, the Bacteroides phylum and the Firmicutes phylum were found in MF sufu. The enzyme activity of the species of the Firmicutes phylum is higher than that of the species of the Bacteroidetes phylum, which enables the more effective absorption of nutrients from food, reducing the risk of obesity.
Wang et al. detected that the purine content of fermented sufu was high, which was related to Acinetobacter . Gout is a purine metabolism disorder, so patients with gout should not eat sufu. Enterococcaceae is a normal inhabitant of the intestine that is innately resistant to many antibacterial drugs. Enterococcus is the most important nosocomial Gram-positive pathogen, except for Staphylococcus. NF sufu is produced using the diverse microorganisms from environment. There may be some harmful microorganisms, which could lead to food safety risks. In comparison, in MF and SF sufu, few harmful bacteria are present during fermentation (Schön et al., 2016). Studies have shown that the content of nutrients differs in three types of sufu. In the current study, the fat content in NF sufu was higher than that in MF sufu and SF sufu. No difference in protein content was observed among the three types of sufu, and the ratio of fatty acids to amino acids in SF sufu was much higher than that in MF sufu. This difference was mainly due to the uniform distribution of microbial species in MF sufu; for example, Bacillus can produce a variety of digestive enzymes to improve the digestion and absorption of nutrients. The activities of protease, amylase, and lipase in Bacillus are high. During fermentation, microorganisms secrete proteases, lipases, and other enzymes. These enzymes promote the decomposition of macromolecular substances into small molecules, so the nutrients in sufu were enriched. The nutrients in NF sufu are similar to those in MF sufu. NF sufu is rich in methionine (Met), which can be converted into cysteine under the action of many types of microbial flora (Palaric et al., 2018;Speranza et al., 2017). Cysteine can damage endothelial cells in the arterial wall, leading to the deposition of cholesterol and triglycerides in the arterial wall and the development of atherosclerosis; thus, it is not safe. The nutritional quality of SF sufu was lower due to inoculation with a single strain, which inhibited the growth and reproduction of other microorganisms, and the enzyme system was relatively simple.
To investigate the effects of bacteria on nutritional quality, we

| CON CLUS ION
In this study, dynamic changes in nutrients and bacterial communities were analyzed, and the differences in nutritional quality and microbial diversity in sufu were explored. The results provide a comprehensive understanding of the biochemical process of sufu. In MF sufu, the contents of EAA and essential fatty acids are high. The distribution of bacteria and nutrients in sufu is uniform, which is beneficial to the enrichment of sufu. Using different fungi in the production of sufu is a favorable approach to improve the flavor of sufu. In addition, comprehensive studies on the correlation among microbial survival, metabolism, and flavor substances in sufu fermentation should be conducted.

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

S TATE M E NT O F D ECL A R ATI O N FO R H U M A N S U BJ EC TS
This study has no involvement of human or animal subjects.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

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.