Food Microbiology 86 (2020)103340 Library Amplification ReadyMix,1uL of each primer (10 umol/L). 3.2.ITS2 rRNA gene sequencing of the fungal community in sufu the i ed space y obta )A th of 335 bp we nd139 fo 2 5.Bioinformatics analysis 9.61%-78.7% the ain phyl tags after se ut- that follo t20020 es (Fi 29.49% N.Bioinf ne an alyse were perfo st ab aligned us g PyNAST(C ring 2010)to obtain the ur at the time Asp 32.129 t06.22 D5 I lyat the end of r s(46 the er 0.85at 3)(Fig mily.genus and species e ive abund ved th ches .The first branch es of thr ave ti DO D5 M1 M2 and M3.The changes the different on by b ovo tax was co g Fast ure (Fig.4) and be ated 3.3.16S rRNA gene ni的ynf Knight.2005 eighted pri ength of 42 C. wer u into 466 OTUs at 97 chemical pre 92 ith the d by G s were 3.Results ntation(A)Pro e the most the ferm 3.1.Physic ical properties of the sufu auch le affected by the hte()than in the peh 2 proced res and on timn initial pH 24 24%)than in the pehtze(10.81 stages.At the end of the fe From the Do until the end,the H valt showed a gradual ely.At the genus le alt-pehtze stage (13.6%)and dec g3fom190%o51.46%an the ond ed to drop until M1(6 4306)and then increased to 8 30%6d the o D5,but dramatically inr ed to 24.66%at Ml.Contra ng st (0.65 ino nitrog he 0.64%atM1. was 7%from A24 to D5 ontin to M3. they ing sugar was ng g la were more abundant in the during the pre-fermentation stage(.and 5) Library Amplification ReadyMix, 1 μL of each primer (10 μmol/L), 10 ng of template DNA and ddH2O. PCR products were detected using 2% agarose gel electrophoresis, and then purified by AxyPrep DNA gel Extraction Kit (AXYGEN). Amplicon libraries were quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Amplicons were then sequenced using Illumina HiSeq PE250 at Realbio Genomics Institute (Shanghai, China). 2.5. Bioinformatics analysis The paired-end reads were assembled into longer tags after se￾quencing and then quality-filtered. Tags were restricted between 220 bp and 500 bp and the average Phred score of bases was not < 20 (Q20) and not > 3 ambiguous N. Bioinformatic analyses were performed using QIIME (v1.7.0) on the extracted high-quality sequences (Caporaso et al., 2010). First, the sequences were aligned using PyNAST (Caporaso et al., 2009) and clustered under 100% sequence identity using UCLUST (Edgar, 2010) to obtain the unique sequence set and clustered into operational taxonomic units (OTUs). Then these representative se￾quences were further classified into operational taxonomic units (OTUs) with a 97% similarity using UCLUST. Each representative sequence was assigned to a taxa by Ribosomal Database Project, (RDP, Release 11.5) (Cole et al., 2006) and Greengenes (Release 13.8) (DeSantis et al., 2006) to obtain the taxonomy information of phylum, class, order, fa￾mily, genus and species. 2.6. Statistics analysis Differences in the relative abundances of taxonomic levels between samples were evaluated using the Mann-Whitney test. Values of P < 0.05 were considered significantly different between different groups. Alpha diversity (Shannon index) analyses were analyzed using QIIME. A de novo taxonomic tree was constructed using FastTree (Price et al., 2009) for alpha and beta diversity calculations. To evaluate alpha diversity, the Shannon Wiener and the Chao1 were calculated. UniFrac metrics were calculated to evaluate beta diversity (Lozupone and Knight, 2005). Both weighted and unweighted principal coordinate analysis (PCoA) were performed. Redundancy analysis (RDA) was performed using CANOCO (canonical community ordination) 4.5 soft￾ware. The graphic presentations were generated by the R package version 3.1.2 and the origin software package version 8.5. Significant differences of physicochemical properties were calculated with the Duncan's multiple range test using SAS (version 9.1.2) and the graphic presentations were generated by GraphPad prism 7. Differences were considered statistically significant when P < 0.05. 3. Results 3.1. Physicochemical properties of the sufu The physicochemical properties were mainly affected by the pro￾duction procedures and fermentation time. The initial pH was 6.07 which significantly increased to 6.39 at salt-pehtze stage (P < 0.05). From the D0 until the end, the pH value showed a gradual decrease (from 6.44% to 6.33%) (Fig. 2A). The NaCl concentration peaked at the salt-pehtze stage (13.6%) and decreased significantly after adding dressing mixture (8.02%) (P < 0.05). Concentrations of NaCl con￾tinued to drop until M1 (6.43%) and then increased to 8.30% during the following stages (Fig. 2B). The amino nitrogen content peaked at the end of the pehtze stage (0.65%) and decreased significantly to 0.26% at D0 (P < 0.05). Thereafter, the amino nitrogen continued to rise from 0.26% to 0.59% during the ripening fermentation stage (Fig. 2C). Re￾ducing sugar was not detected before pouring dressing mixture. It peaked at M1 (10.20%) and was significantly higher than that of other stages during the post-fermentation (P < 0.05) (Fig. 2D). 3.2. ITS2 rRNA gene sequencing of the fungal community in sufu The Illumina Hiseq platform was employed to sequence the internal transcribed spacer 2 (ITS2) of 45 samples, thereby obtaining 1.55 million clean tags (34,606 ± 5201 on average) after concatenation and quality control. The clean reads with an average length of 335 bp were clustered into 334 OTUs at 97% similarity level. These OTUs were clustered into 3 phyla and 139 genera. Ascomycota (accounting for 5.13%–46.15%), Basidiomycota (accounting for 16.09%–55.79%) and Zygomycota (accounting for 9.61%–78.77%) were the main phyla during the pehtze and salt-pehtze stage. Then Ascomycota became the dominant phyla that accounts for 90.27%–99.42% in the following stages (Fig. 3A). In the genus level, Trichosporon (29.4%) at A24 and Actinomucor (78.8%) at A48 were the most abundant respectively. The Cryptococcus (40.0%) and Actinomucor (29.8%) were the most abundant genera at salt-pehtze stage. But after pouring dressing mixture, Mon￾ascus and Aspergillus became the main genera during the ripening fer￾mentation stage. Monascus increased from D0 (36.87%) to M1 (70.42%) and at the same time Aspergillus declined from 32.12% to 6.22%. Meyerozyma, Millerozyma and Pichia were also the main genera at D0 and D5. Interestingly, at the end of ripening fermentation stage (M3), Monascus (46.91%) and Aspergillus (40.00%) were at a similar ratio as they were at D0 - the beginning of ripening fermentation stage (the Aspergillus/Monascus ratio was 0.87 at D0 and 0.85 at M3) (Fig. 3B). The similarity in analysis among different sufu samples based on the relative abundance of fungal OTUs showed that besides T (not included in the fermentation period), the samples could be organized into two main branches. The first branch was composed of samples of three time points, A24, A48 and S. The second branch was composed of samples of five time points: D0, D5, M1, M2 and M3. These changes were in accord with the production procedures. Since the sufu was mainly fermented by fungus, we can divide the whole fermentation process into pre-fer￾mentation and post-fermentation by before or after pouring the dressing mixture (Fig. 4). 3.3. 16S rRNA gene sequencing of the bacterial community in sufu The Illumina HiSeq platform was employed to sequence the V3–V4 regions of 16S rRNA gene of 45 samples, thereby obtaining more than 1.57 million clean tags (35,049 ± 1721 on average) after concatena￾tion and quality control. The clean reads with an average length of 427 bp were clustered into 22,466 OTUs at 97% similarity level (Information of the sample statistic has been listed in the Supplementary Table S1). These OTUs were clustered into 20 phyla, 47 classes, 71 orders, 153 families, and 355 genera. In terms of the relative abundance, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla throughout all the stages of the fermentation (Fig. 5A). Proteobacteria were the most abundant phylum at every stage; especially at the end of the fermentation where its re￾lative abundance was 90.65%. Firmicutes were much less abundant in the salt-pehtze (7.88%) than in the pehtze (32.71%) stages. Contrast￾ingly, Bacteroidetes were much more abundant in the salt-pehtze (24.24%) than in the pehtze (10.81%) stages. At the end of the fer￾mentation, the relative abundance of Firmicutes and Bacteroidetes de￾clined to 4.21% and 4.26%, respectively. At the genus level, Acineto￾bacter were the most abundant classified genus from the pehtze stage to M2 (ranging from 11.90% to 51.46%) and the second most abundant at M3 (11.46%). The relative abundance of Enterobacter was < 4% from A24 to D5, but dramatically increased to 24.66% at M1. Contrastingly, the relative abundance of Empedobacter was > 7% from A24 to D5, but dramatically declined to 0.64% at M1. Meanwhile Lactococcus declined rapidly from M1 to M3. Streptococcus and Weissella were also the main genera during the pehtze stage, but they became less abundant in the following stages. Pseudomonas and Klebsiella were more abundant in the ripening fermentation stage than the pehtze stage. Bacillus was not detected during the pre-fermentation stage (A24, A48, and S). D. Xu, et al. Food Microbiology 86 (2020) 103340 3