Salt Aggregation Test

1. Bacterial cell suspension preparation. The cultures were centrifuged at 2000g for 10 min, and the cells were washed with saline and resuspended in phosphate buffer (0.02 M, pH 6.8) to a final concentration of 1 x 109 CFU/mL (see Note 2).

2. Salt aggregation assay. The aggregating properties of lactobacilli in (NH4)2SO4 were tested by mixing on a glass slide 25 ^L of the bacterial suspension and the same volume of each one of the salt solutions. They were mixed with a circular movement for 2 min, and the formation of aggregates was observed.

A negative control was prepared to compare the characteristics of the bacterial suspension with and without (NH4)2SO4. The control was prepared by mixing 25 ^L of the bacterial suspension with 25 ^L of phosphate buffer 0.02 M, pH 6.8 (see Note 3). A positive control was prepared by mixing the same volume of bacterial suspension and 25 ^L of the 4 M (NH4)2SO4 solution. The results of the SAT were expressed as positive or negative, indicating the salt concentration. Bacteria that were able to form aggregates even after mixing with diluted (NH4)2SO4 solutions were termed highly aggregating. Lactobacilli that were able to aggregate in the presence or absence of (NH4)2SO4 were termed as self-aggregating.

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