JIA-2018-09

2129 WANG Ying et al. Journal of Integrative Agriculture 2018, 17(9): 2126–2136 standard deviation (SD). Statistical analysis of data was performed by using SPSS 13.0 software (SPP Inc., Chicago, USA). 3. Results 3.1. Isolation of pellicle-forming microbes in black- berry wine On YPD agar plates, one kind of colonial morphology was found and named as Hmp-1. Its colonial and cellular morphology were shown in Fig. 1-A and A1. The colonial and cellular morphology of Hmp-1 were significantly different from those of S . cerevisiae FM-S-115 strain (Fig. 1-B and B1). On MRS agar plates, one kind of bacteria was found and named as Hmp-2. The colonial and cellular morphology of Hmp-2 were shown in Fig. 1-C and C1. Hmp-1 predominated in the pellicle formation of blackberry wine, and its population density was (8.21±0.23)×10 9 CFU g –1 . The population density of Hmp-2 was only (5.21±0.49)×10 4 CFU g –1 . 3.2. Confirmation of the isolate Hmp-1 associated with pellicle formation The pellicle-forming ability of two isolates (Hmp-1 and Hmp-2) and S . cerevisiae strain FM-S-115 in blackberry wine was examined, respectively. After 15 d of incubation, Hmp-1 could form pellicle on the surface of blackberry wine (Fig. 2), whereas Hmp-2 and FM-S-115 could not form pellicle on the surface of blackberry wine. 3.3. Molecular identification of Hmp-1 The expected sequence of 26S rDNA D1/D2 domain (576 bp) was successfully amplified from Hmp-1. The sequence was deposited at GenBank database under an accession number KJ914549. Based on this sequence, a phylogenetic tree was constructed using neighbor-joining method, in which Hmp-1 was clustered with P . membranifaciens (Fig. 3). BLASTn analysis showed that the D1/D2 domain sequence of Hmp-1 shared 99% identity with those of P . membranifaciens (JX188208.1, DQ198957.1, EF564396.1 1 cm A B C A1 B1 C1 1 cm 1 cm 10 μm 10 μm 10 μm Fig. 1 The colonial morphology and cellular morphology of the isolate Pichia membranifaciens Hmp-1 (A, A1), Saccharomyces cerevisiae FM-S-115 strain (B, B1), and the isolate Hmp-2 (C, C1). Fig. 2 The pellicle-forming ability of Pichia membranifaciens Hmp-1 (A), Saccharomyces cerevisiae FM-S-115 (B), and Hmp-2 (C) on the surface of blackberry wine. Hmp-1, S. cerevisiae FM-S-115, and Hmp-2 were cultivated for 15 d in 50 mL of blackberry wine at 28°C without shaking. A B C