Two species of land snail, L. fulica and H. distincta, were collected from the wild in Thailand. The snails (n=20 each species/anatomical location) were reared at 24 °C for 2 days. then, the mucus was extracted by gently poking on foot and mantle lobes. The harvested mucus was filtered through 0.45-µm membrane (Millipore) and stored at 4 °C before use. This process did not harm the snails and the experiments was carried out in accordance with the approved guidelines of the Animal Care and Use Committee of Faculty of Science, Chulalongkorn University (Protocol Review No. 1223003).
Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, and Acinetobacter spp. L9, PK3, and Y3 were cultured in Luria-Bertani broth at 37 °C, while Staphylococcus aureus ATCC 25923 was cultured in tryptic soy broth with 2% (w/v) NaCl at 30 °C with shaking overnight. The bacteria were re-inoculated in the fresh media and cultured until they reached OD600 of 0.2 and were diluted about hundred-fold in poor broth [1% (w/v) tryptone, 0.5 % (w/v) NaCl, pH 7.5] to an OD600 of 0.001. the selected bacteria are prevalent species of gram negative and positive bacteria and most common nosocomial pathogens.
SDS-PAGE and LC-MS/MS
The protein concentration of slime was determined using the Bradford assay , and then 25 µg of the sample was mixed with 5X reducing SDS loading dye and heated at 100 °C for 10 min. The samples were then subjected to 12.5% SDS-PAGE. One gel was stained with Coomassie blue and the other gel was developed by silver staining .
The selected protein bands were cut from the stained SDS-PAGE and analyzed using LC-MS/MS at the Research Instrument Center, Khon Kaen University, Thailand. Briefly, the protein bands were destained, reduced, and digested with Sequencing Grade Modified Trypsin. The tryptic peptides were extracted and analyzed using LC-MS/MS. The obtained data were searched in Protein database using the MASCOT program.
Antimicrobial activity assay
The antimicrobial activity of snail mucus was analyzed using the minimal inhibitory concentration assay . The snail mucus (12.5, 25, 50, 100, and 200 µg of total protein in 100 µl phosphate-buffered saline (PBS)) was added and then inoculated with 20 µl of the fresh bacterial culture (1 x 104 cells, OD600 = 0.001). The reactions were incubated overnight and then the bacterial growth was measured at OD600. The negative controls were performed using PBS.
Anti-tyrosinase activity assay
Each 25-µg total protein of mucus adjusted with PBS to 70 µl was incubated with 10 µl of mushroom tyrosinase (Sigma-Aldrich) in potassium phosphate buffer pH 6.5 (5 U/reaction), and then 20 µl of 1 mg/ml L-DOPA (Sigma-Aldrich) in buffer was added. The reactions were monitored the absorbance at A475. Distilled water was used as a negative control and kojic acid as positive control. The inhibition was calculated from the formula: Inhibition (%) = [1 − (A475 in sample/A475 in control)] × 100%.
Antioxidant activity assay
A 100-µl of the snail mucus in various concentration was added into the each well and incubated with 100 µl of 0.2 mM DPPH in absolute ethanol in dark place for 30 min. Then, the absorbance was measured at 517 nm (A517)  . Distilled water was used as a negative control and ascorbic acid as a positive control. The DPPH scavenging effect was calculated by the following formula: %Effective = (ABlank – ASample)/Asample x 100. %Effective were plotted against concentration by GraphPad Prism 8. The EC50 values were calculated as the concentration to cause half-maximal inhibition of DPPH radical scavenging.
Results were represented as mean with standard deviation in triplicate. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s test. Significance was accepted at the p < 0.05 level.