D.Xu et ad Food Microbiolo2)103340 ofu.After fer tion an sed mierpbial count in the Sovbean observed. ficant decline in microbia count red afte Soymilk boiling ing the coagulation dien gel pressing cutting us f 3.2×3.2x1.6cm Tofu that can b tha ely on Inoculation As 3.2778 method for evalt (10'spores/mL,I mL/100g tofu) Pehtze g the 28℃.24h A24 e mi 二 y used t 90%humidity Salt-pehtze d 5days5day·s 1)investigate the dynamic c ofphysic saturated salt water Dressing mixture (red kojic rice,edible alcohol,sugar,chiang.spices) 2 Materials and methods 0 day 2.1.Sufu preparati putting in closed. ed at the War 32 2.month., M3 e n e presen on t ng mixture for a Redsufu s)edible 0 day (D).5 da (D5).1 month (MD),2 months (M2),3 months (M3). Sufu samples sufu forday (DO).5 ixed for physico t-pehtze(S).fe tation of s om five) M21 out n triplicate ndent batc n ice and stored at 2.3.DNA extraction were mixed with 25 ml.0.1 mol/I.Tris 2.2.Physico chemical analysis through three by GMO od DNA Ext I DNA centration and quality were checked ing a nanoDror etermin to SB/T10170-2007 standard.Sufua ple 2000(Thermo) er and agarose gel ele 2.4.16S rRNA gene amplicon sequencing and ITS amplicon sequencing with d Nac of fun V3-V4 obial 16s rRN nd ti h The mixture w inal exte on at 72'C for 5mi The microbial 16S rk NA gen e wer ehe 2tofu. After fermentation, an increased microbial count in the pehtze was observed. A significant decline in microbial count occurred after salting, and almost no fungal growth (< 1 log cfu/g) could be detected (Ma et al., 2013a). Feng et al. evaluated the bacterial flora during the ripening of Kedong sufu (a typical bacteria-fermented sufu) using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and culturing methods. They found that Enterococcus avium, Enterococcus faecalis, and Staphylococcus carnosus were the dominant microflora throughout the fermentation of sufu (Feng et al., 2013). Since the results from other studies are only for microorganisms that can be easily cultivated, methods that rely on culture have been insufficient to fully understand the microbial population. PCR-DGGE is a common method for evaluating the composition of microorganisms, but it is time consuming and has limited ability to detect rare or un￾cultivable microorganisms (Hong et al., 2016). High-throughput sequencing has been widely used to characterize the composition of the microbial community of fermented food, such as wine, vinegar, soy sauce, etc. (Sulaiman et al., 2014; Tang et al., 2017; Wang et al., 2016). Based on this technology, the aims of this study were as follows: 1) investigate the dynamic changes of physicochemical properties and fungal structure during sufu fermentation and their correlation with process procedures, 2) identify the relative abundance and diversity of bacteria taxa in sufu samples, which is crucial for the flavor and security of fermented food. 2. Materials and methods 2.1. Sufu preparation and sample collection Red sufu samples were prepared and collected at the Wangzhihe Food Co. Ltd. A diagram of the production and sampling points with sample names are presented in Fig. 1. Simply, pehtze is prepared by inoculating Actinomucor elegans on the surface of tofu cubes, then salting the pehtze for about 5 days, and dispense the salt-pehtze into wide-mouthed glass bottles and ripens in the dressing mixture for a period of 3 months. The dressing mixture of red sufu mainly consists of red kojic rice (cooked rice inoculated with Monascus purpureus), edible alcohol, sugar, chiang (flour paste fermented by Aspergilus oryzae) and spices. Sufu samples were collected periodically at 9 different stages of fermentation: tofu (T), pehtze which inoculated with A. elegans for 24 h (A24), 48 h (A48), salt-pehtze (S), fermentation of sufu for 0 day (D0), 5 days (D5), 1 month (M1), 2 months (M2), 3 months (M3). Samples were collected from five independent batches and used as replicates. A total of 45 samples were transported into the lab on ice and stored at −80 °C until further use. 2.2. Physicochemical analysis Five grams of the sufu samples were homogenized with 50 mL of distilled water followed by pH measurements using a pH meter (Mettler Toledo). The amino nitrogen content and NaCl concentration were determined according to SB/T10170-2007 standard. Sufu samples (20 g) were boiled with distilled water (80 mL) with gentle stirring. Boiled sufu slurry was diluted to 200 mL with distilled water. The sufu solution was filtered with dry filter paper and then the filtrate was used to measure amino nitrogen content and NaCl concentration. 10 mL of the filtrate was mixed with 50 mL water and titrated to pH 8.2 with 0.05 mol/L NaOH and then 10 mL of 36% (w/v) formalin solution was added. The mixture was titrated to pH 9.2 with 0.05 mol/L NaOH. The volume of consumed NaOH for raising pH (from 8.2 to 9.2) was re￾corded to determine amino nitrogen content. To determine the NaCl concentration, 2 mL of the filtrate was mixed with 50 mL water and titrated with 0.100 mol/L AgNO3 using 5% (w/v) K2CrO4 solution (1 mL) as an indicator. The titration was terminated when the solution appeared orange. The content of reducing sugar was determined according to previous study (Van Waes et al., 1998). Samples from the same stage of the five independent batches were mixed for physico￾chemical analysis. All tests were carried out in triplicate. 2.3. DNA extraction Five grams of sufu samples were mixed with 25 mL 0.1 mol/L Tris￾HCl (pH 8.0), shaken well, and filtered through three layers of sterile gauze. The filtrate was centrifuged at 10,000×g for 20 min at 4 °C. The pellets were used for DNA extraction by GMO food DNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer's protocol. The total DNA concentration and quality were checked using a NanoDrop 2000 (Thermo) spectrophotometer and agarose gel electrophoresis. 2.4. 16S rRNA gene amplicon sequencing and ITS amplicon sequencing Variable regions V3–V4 on microbial 16S rRNA gene of bacteria and the ITS2 region of fungi were amplified using PCR (95 °C for 3 min, followed by 30 cycles at 98 °C for 20 s, 58 °C for 15s, 72 °C for 20 s and a final extension at 72 °C for 5 min). The microbial 16S rRNA gene were amplified by forward primer F341 5′- ACTCCTACGGGRSGCAGCAG -3′ and reverse primer R806 5′- GGACTACVVGGGTATCTAATC -3′ (Klindworth et al., 2013). ITS2 were amplified with forward primer F2045 5′-GCATCGATGAAGAACGCAGC-3′ and reverse primer R2390 5′-TCCTCCGCTTATTGATATGC-3′ (Hirokazu et al., 2012). PCR reac￾tions were performed in 30 μL mixture containing 15 μL of 2 × KAPA Fig. 1. The production diagram of sufu used in this study with the sampling points indicated. Tofu (T), pehtze which inoculated with A. elegans for 24 h (A24), 48 h (A48), salt-pehtze (S), fermentation of sufu for 0 day (D0), 5 days (D5), 1 month (M1), 2 months (M2), 3 months (M3). D. Xu, et al. Food Microbiology 86 (2020) 103340 2