Media preparation
Several media were used throughout the isolation and screening process.
- Basal medium: 1% glucose, 1% yeast extract, 3% (v/v) absolute ethanol, adjusted to pH 4.5.
- Isolation medium: basal medium supplemented with 2% agar and 2% calcium carbonate, with 3% (v/v) absolute ethanol.
- Slant preservation medium: basal medium supplemented with 2% agar and 1% calcium carbonate, with 3% (v/v) absolute ethanol.
- Acid-production medium: basal medium sterilized and cooled first, then supplemented with 7% (v/v) absolute ethanol, and dispensed into 250 mL Erlenmeyer flasks at 50 mL per flask.
All media were sterilized at 121°C for 20 minutes. Calcium carbonate was sterilized separately by dry heat at 165°C for 30 minutes. After sterilization, calcium carbonate and absolute ethanol were added when the medium had cooled to about 70°C.
Workflow for isolation and screening
The overall procedure followed this sequence:
Preparation of microbial sources → enrichment culture → dilution and isolation → strain purification → slant preservation → qualitative acid-production test → primary screening → secondary screening → stability testing
Experimental procedure
1. Preparation of microbial sources
Small amounts of vinegar starter and decayed apple tissue were soaked in sterile saline for 1 hour, then used for enrichment culture.
2. Enrichment culture
Samples were taken from naturally fermented apple vinegar liquid, vinegar starter liquid, and liquid from spoiled apples. For each enrichment, 10 g of sample was added to a 500 mL Erlenmeyer flask containing 100 mL of basal medium. Cultures were incubated at 30°C with shaking at 120 r/min for 48 hours.
3. Dilution and isolation
One milliliter of culture was transferred into a test tube containing 9 mL sterile water, then serially diluted with sterile water to obtain suspensions at 10^-4, 10^-5, 10^-6, and 10^-7. From each dilution, 0.2 mL was spread onto isolation medium plates and incubated at 30°C for 72 hours.
After incubation, single colonies were selected based on consistent colony morphology, a large transparent zone, dense colony growth, and clear growth dominance on the plate.
4. Purification of isolates
The selected colonies were repeatedly streaked and cultured on plate medium until morphologically uniform single colonies were confirmed. Purified colonies were then inoculated onto slant preservation medium, incubated at 30°C for 72 hours, and stored at 4°C.
5. Gram staining
The preserved strains on slants were reactivated twice before Gram staining was carried out.
6. Qualitative test for acid production
Each preserved slant culture was inoculated into basal medium containing 3% (v/v) ethanol and incubated as a shallow static culture at 30°C for 72 hours. Then 5 mL of cell-free culture supernatant was collected and neutralized to pH 7.0 with 2.5 mol/L NaOH. After that, 5 to 6 drops of 5% FeCl3 solution were added. Formation of a reddish-brown precipitate was taken as evidence that the strain produced acetic acid.
7. Primary screening of strains
Single-colony isolates that met the results of Gram staining and the qualitative acid-production test were further separated on plate medium. Screening at this stage was based on the HC value, defined as the ratio of the discoloration-zone diameter to the colony diameter.
8. Secondary screening
Strains selected in the primary screen were inoculated into acid-production medium. Each strain was tested in triplicate and incubated statically at 30°C for 120 hours. Acidity was then measured and compared among strains.
9. Determination of acetic acid yield and alcohol conversion
For acetic acid determination, 2 mL of fermentation broth was diluted with 50 mL distilled water. Then 3 to 5 drops of 0.5% phenolphthalein in alcohol were added, and the sample was titrated with standardized 0.1 mol/L NaOH until a faint pink endpoint appeared. The acid content of the sample was calculated from the volume of NaOH consumed.
Results of isolation and screening
Gram staining
After dilution plating and repeated streak purification of samples from vinegar starter used for edible vinegar, four single-colony isolates with consistent morphology were obtained. These strains were preserved on slants, reactivated, and then subjected to Gram staining.
Qualitative acid-production test
The qualitative acid-production test was performed according to the stated procedure, followed by the addition of 5% FeCl3 solution to evaluate acetic acid production.