JIA-2018-09

2127 WANG Ying et al. Journal of Integrative Agriculture 2018, 17(9): 2126–2136 2014). Many of those yeasts could form a pellicle on the surface of bulk wines during storage, which caused cloudiness, haziness, and sediments (Sponholz 1992; Nakagawa et al . 2011). Wines with pellicle would lose commercial value. Pichia membranifaciens is a kind of pellicle-forming yeast, and could form ascospores, which might cause a serious problem when temperature treatment is carried out in the industrial production of fruit juice and wine (Veiga and Madeira-Lopes 2000). In addition, P . membranifaciens could produce volatile phenols, such as 4-ethylphenol and 4-ethylguaiacol. When these compounds are above certain levels, wine would produce abject odours (Saez et al . 2011). To the best of our knowledge, though some researches had found that P . membranifaciens was a kind of frequent spoilage yeast in the wine industry (Loureiro and Malfeito- Ferreira 2003; Saez et al . 2011), the growth dynamics of P . membranifaciens during fermentation period and its control measures had not been elucidated, especially in the blackberry wine. Blackberry ( Rubus fruticosus sp.) is known to contain high levels of phenolic compounds including anthocyanins, flavonols, chlorogenic acid and procyanidins, which have strong antioxidant activity (Elisia et al . 2007; de Souza et al . 2014). Blackberry wine was produced by blackberry juice and yeast. This popular fruit wine is of particular interest because of its high phenolic contents and antioxidant properties (Johnson and Gonzalez de Mejia 2012; Ortiz et al . 2013). In recent years, we studied the brewing technique of blackberry wine, and obtained a Saccharomyces cerevisiae FM-S-115 strain (patent no. ZL 201310098108.4, China). The strain was isolated from the spontaneous fermentation of blackberry wine, and had been used as an alcohol fermentation strain in the small-scale commercial production of blackberry wine. In the previous work of our laboratory, the pellicle-forming phenomenon was found on the surface of blackberry wine at the late stage of fermentation and during storage. The pellicle formation led to quality deterioration of the wine. In order to control the pellicle-forming spoilage during fermentation and storage of wine, the objectives of this study were: (a) to identify the pellicle-forming yeasts in the blackberry wine; (b) to study the effects of fermentation environments on their growth; and (c) to evaluate the negative effect of the spoilage yeast on the wine quality. 2. Materials and methods 2.1. Isolation of spoilage microbes in pellicles of blackberry wine The blackberry wine was fermented by S. cerevisiae FM-S- 115 strain, and stored in stainless steel vats in a traditional cellar (annual average temperature of 18.0°C) located in Jiangsu Academy of Agricultural Sciences, Nanjing, China. The pellicle samples were collected in sterile conditions, and transported to the laboratory at 4°C. The collected sample (5 g) was homogenized in a sterile porcelain mortar with 5 mL of sterile water. Diluted homogenates were plated on YPD agar plates (yeast extract 1% w/v, peptone 2% w/v, glucose 2% w/v, and agar 1.8% w/v; pH 5.8) and MRS agar plates (Oxoid Microbiology Co., Ltd., Beijing, China) plus 100 mg L –1 natamycin, respectively. The number of colony forming units (CFU) on each plate was counted after incubating for 48–72 h at 28°C. Isolates with distinct colony morphology were recovered fromYPD and MRS agar plates, respectively. The cellular morphology of isolates was viewed under the light microscope (YS100, Nikon Inc., Japan). In addition, the isolates were stored at –70°C in 20% sterile glycerol for further studies. 2.2. Confirmation of isolates associated with pellicle formation This experiment was carried out to investigate the pellicle- forming ability of the isolates. The blackberry wine was prepared (pH 3.35, 8.05% v/v alcohol, 0.85 g L –1 reducing sugar) and sequentially filtered through 0.8-μm and 0.45-μm sterile membranes (Merck Millipore Ltd., Billerica, MA, USA). Two isolates (Hmp-1 and Hmp-2) and S . cerevisiae strain FM-S-115 were transferred to 10 mL YPD broth (Hmp-1 and FM-S-115) or MRS broth (Hmp-2), respectively, and incubated for 12 h at 28°C. Cells were harvested by centrifugation at 3 000×g for 10 min. The cells were inoculated in 100 mL flasks containing 50 mL of blackberry wine, and the cell concentration of each strain was adjusted to 10 6 CFU mL –1 . Then the asks were incubated for 15 d at 28°C without shaking. All experiments were carried out in triplicate. 2.3. Molecular identification of the pellicle-formation isolate Hmp-1 Genomic DNA of the pellicle-forming yeast Hmp-1 was extracted with a DNA extraction Kit (Tiangen, China), and the D1/D2 domain of the large-subunit (26S) ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) as previously described (Van der Aa Kühle and Jespersen 2003). The following primers were used: NL-1 (5´-GCA TAT CAA TAA GCG GAG GAAAAG-3´) and NL-4 (5´-GGT CCG TGT TTC AAG ACG G-3´). Then the PCR products were directly sequenced (Invitrogen Inc., Shanghai, China). Multiple sequence alignment was performed based on the partial nucleotide sequence of 26S rDNA D1/D2 domain using ClustalX 1.83 software (Thompson et al .