JIA-2018-09

2128 WANG Ying et al. Journal of Integrative Agriculture 2018, 17(9): 2126–2136 1997). A phylogenetic tree was constructed using the neighbor-joining method (MEGA 5.05 software) (Tamura et al . 2011). Kluyveromyces lactis (HE660074.1) was used as an outgroup. 2.4. Population dynamics of FM-S-115 and Hmp-1 during blackberry juice fermentation S . cerevisiae strain FM-S-115 and the isolate Hmp-1 were cultured in YPD broth for 12 h at 28°C for the following experiment. Blackberry juice (pH 3.25, 8.51°Brix) was prepared from fresh blackberry, 10.01% (w/v) sugar and 75 mg L –1 sulfur dioxide (SO 2 ) were added, and sequentially filtered through 0.8- and 0.45-μm sterile membranes (Merck Millipore Ltd., Billerica, MA, USA). The population dynamics of FM-S-115 and Hmp-1 during blackberry wine fermentation was assessed using the dilution plate method as follows: 100 mL flasks containing 50 mL of blackberry juice was inoculated with FM-S-115 (the final concentration (2.01±0.25)×10 6 CFU mL –1 ), Hmp-1 ((2.01±0.31)×10 6 CFU mL –1 ), or both FM-S-115 and Hmp-1 ((1.02±0.25)×10 6 and (1.02±0.17)×10 6 CFUmL –1 ), respectively, and the flasks were incubated under static conditions at 28°C. The population dynamics of FM-S-115 and Hmp-1 were investigated after 0, 2, 4, 6, 8, 10, 12, and 14 d, respectively. Because the colony morphologies of FM-S-115 and Hmp-1 were remarkably different, two kinds of yeasts on each YPD agar plate could be counted according to their colony morphologies, respectively. All experiments were carried out in triplicate. 2.5. Effects of fermentation conditions on growth dynamics of Hmp-1 and FM-S-115 In order to obtain a better adaptation to the test conditions, the Hmp-1 and FM-S-115 were cultured in 20 mL of YPD broth medium adjusted to pH 4.0 with citric acid (1 mol L –1 ) and supplemented with 50% of blackberry juice (pH 3.25, 8.51°Brix, sequentially filtered through 0.8- and 0.45-μm sterile membranes). This medium was incubated for 3 d at 28°C without shaking. Cells were harvested by centrifugation at 3 000×g for 10 min and then inoculated in 100-mL flasks containing 50 mL of blackberry juice supplemented with 25 mL of sterile YPD broth. The cell concentration of each strain was adjusted to 10 6 CFU mL –1 . All flasks were incubated at 28°C without shaking. Then the fermentation conditions were modified with the variables described below. To investigate the impact of ethanol on growth dynamics of Hmp-1 and FM-S-115, varying amounts of ethanol were added to flasks containing FM-S-115 or Hmp-1 culture to make up respective final concentrations of 8, 10, 12, and 14% (v/v). To investigate the impact of SO 2 on growth dynamics of Hmp-1 and FM-S-115, varying amounts of potassium metabisulphite were added to flasks containing FM-S-115 or Hmp-1 culture to make up respective final concentrations of 25, 50, 75, 100 and 125 mg L –1 SO 2 . The concentration of SO 2 was calculated based on the technical specifications of potassium metabisulphite. To investigate the impact of sugar on growth dynamics of Hmp-1 and FM-S-115, varying amounts of sugar were added to flasks containing FM-S-115 or Hmp-1 culture to make up respective final concentrations of 10, 15, 20, 25% and 30% (w/v). To investigate the impact of temperature on growth dynamics of Hmp-1 and FM-S-115, the flasks containing FM-S-115 or Hmp-1 culture were respectively cultured at 15, 20, 25, 30 or 35°C. The population densities of FM-S-115 and Hmp-1 were respectively evaluated using the dilution plate method after 1, 5 and 10 d of incubation. All experiments were carried out in triplicate. 2.6. GC-MS analysis This experiment included two treatments: (1) blackberry wine fermented by single S . cerevisiae FM-S-115; (2) blackberry wine fermented by both FM-S-115 and the isolate Hmp-1. Flavor compounds of each kind of blackberry wine were respectively extracted by headspace solid-phase microextraction (HS-SPME) technique (Jiang et al . 2013). In brief, 5 mL of wine sample was placed in a 15-mL SPME glass vial together with 3 g of NaCI. The vial was tightly capped, and incubated for 15 min at 50°C to equilibrate. Then the sample was extracted for 40 min at 45°C. The gas chromatographic-mass spectrometry (GC-MS) analysis of the sample was carried out by using an Agilent 6890 GC equipped with an Agilent 5975 mass selective detector (MSD). Separations were performed using a DB-Wax column (30 m length, 0.25 mm i.d. , 0.25 μm lm thickness) (J&WScienti c, Folsom, CA, USA). Spectra were obtained on electron impact at 70 eV, scanning from 15 to 250 m/z at 2 scans s –1 . The temperature program ranged from 40 to 250°C as following: 40°C for 10 min, from 40 to 200°C at 5°C min –1 , 200°C for 1 min, from 200 to 250°C at 5°C min –1 , and finally at 250°C for 10 min. The carrier gas was helium at a ow rate of 1 mL min –1 . A total of 1 µL of each extract fromwine was injected into splitless mode. The volatile compounds were identified using the software library of mass spectra database Willey 6.1 (NY, USA). 2.7. Statistical analysis Results were means of three independent experiments±