Isolation and purification of spoilage microorganisms

Blueberries provided by Organic Food Co., Ltd (Dandong City, Liaoning Province, China) were squeezed (1:6 with water), centrifuged, filtered, and then divided into four groups. They were stored at room temperature for 0, 1, 2 and 3 days, respectively. The total number of isolates of different groups were calculated using data obtained on plate count agar (24 h at 37 °C).

Blueberry juice samples (1 mL) stored for different days were collected and added to 15–20 mL sterilized Luria–Bertani (LB), Bengal red agar, manitol salt agar (MSA), De Man, Rogosa, Sharpe (MRS) medium⁴⁸ and then those media were used for the isolation to select for total bacteria, total fungi, Staphylococcus and Micrococcaceae species, and lactic acid bacteria, respectively. Inoculated LB and MRS were cultivated at 37 °C for 24 h, MSA was cultivated at 30 °C for 48 h, Bengal red agar was cultivated at 28 °C for 5 days. In order to obtain pure isolates, single colonies were picked and further grown on the corresponding media at least once. These candidate isolates were preserved on an inclined plane at 4 °C.