Snail Mucus from the Mantle and Foot of Two Land

Noothuan et al. BMC Res Notes (2021) 14:138 https://doi.org/10.1186/s13104-021-05557-0 BMC Research Notes RESEARCH NOTE Open Access Snail mucus from the mantle and foot of two land snails, Lissachatina fulica and Hemiplecta distincta, exhibits diferent protein profle and biological activity Nattaphop Noothuan1, Kantamas Apitanyasai1, Somsak Panha2 and Anchalee Tassanakajon1* Abstract Objective: Snails secrete diferent types of mucus that serve several functions, and are increasingly being exploited for medical and cosmetic applications. In this study, we explored the protein pattern and compared the biological properties of the mucus secreted from the mantle collar and foot of two snail species, Lissachatina fulica and Hemi- plecta distincta. Result: Protein profle showed a diferent pattern between the two species and between the two secretory parts. The mantle-specifc protein bands were further characterized and among them was an antibacterial protein, achacin. Accordingly, the mucus from the mantle exhibited the higher antibacterial activity than that from the foot in both snail species. The mucus from H. distincta, frst reported here, also showed antibacterial properties, but with a lower activity compared to that for L. fulica. Snail mucus also exhibited anti-tyrosinase activity and antioxidant activity but with no signifcant diference between the foot and mantle mucus. These results indicate some diferent protein compositions and biological activities of snail slime from the mantle and foot, which might be associated with their specifc functions in the animal and are useful for medical applications. Keywords: Land snail, Snail mucus, Antimicrobial activity, Anti-tyrosinase activity, Antioxidant activity Introduction compounds have been found in snail mucus, such as Land snails have been used as food and for various allantoin, hyaluronic acid, peptides and proteins [3, 4, medical treatments for centuries [1–3]. Te snail slime 14]. Moreover, it has been reported that diferent kinds of (mucus) has many functions in the animal, such as adhe- mucus are released from the diferent types of secretory sive, emollient, moisturizing, lubricant, and defense glands in a snail, depending on the way it is stimulated [4–7]. Recently, snail slimes have been applied in human [15]. However, little work on the biochemical properties medical and cosmetics [8–11]. Studies have shown that give rise to these functional diferences has been that snail mucus exhibits various biological activities, elucidated. such as antimicrobial, antioxidant, anti-tyrosinase, and In this study, the mucus was collected from the man- antitumoral activities [10, 12, 13]. In addition, many tle and foot of two snail species, H. distincta and L fulica, in which H. distincta is distributed in deciduous forest in Tailand [16], but L. fulica is a very famous invasive *Correspondence: [email protected] 1 Department of Biochemistry, Faculty of Science, Chulalongkorn alien species that occurring everywhere at anthropogenic University, Bangkok 10330, Thailand areas in many parts of the world [17]. Te protein pat- Full list of author information is available at the end of the article tern and biological properties (antioxidant, antibacterial, © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Noothuan et al. BMC Res Notes (2021) 14:138 Page 2 of 7 and anti-tyrosinase activities) of the mucus were then Te snail mucus (12.5, 25, 50, 100, and 200 µg of total compared. protein in 100 µl phosphate-bufered saline (PBS)) was Te results revealed somewhat diferent protein com- added and then inoculated with 20 µl of the fresh bacte- 4 positions and biological properties that are probably rial culture (1 × 10 cells, OD 600 = 0.001). Te reactions associated with their functions and are useful for cos- were incubated overnight and then the bacterial growth metic and medical applications. was measured at OD600. Te negative controls were performed using PBS. Materials and methods Sample preparation Anti‑tyrosinase activity assay Two species of land snail, L. fulica and H. distincta, were Each 25-µg total protein of mucus adjusted with PBS collected from the wild in Tailand. Te snails (n = 20 each species/anatomical location) were reared at 24 °C to 70 µl was incubated with 10 µl of mushroom tyrosi- for 2 days then, the mucus was extracted by gently pok- nase (Sigma-Aldrich) in potassium phosphate bufer pH ing on foot and mantle lobes. Te harvested mucus was 6.5 (5 U/reaction), and then 20 µl of 1 mg/ml L-DOPA fltered through 0.45-µm membrane (Millipore) and (Sigma-Aldrich) in bufer was added. Te reactions stored at 4 °C before use. Tis process did not harm the were monitored the absorbance at A475. Distilled water snails and the experiments was carried out in accordance was used as a negative control and kojic acid as positive with the approved guidelines of the Animal Care and Use control. Te inhibition was calculated from the formula: Committee of Faculty of Science, Chulalongkorn Univer- Inhibition (%) = [1 − (A475 in sample/A475 in control)] × sity (Protocol Review No. 1223003). 100%. Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, and Acinetobacter spp. L9, PK3, and Y3 were cul- Antioxidant activity assay tured in Luria-Bertani broth at 37 °C, while Staphylococ- A 100-µl of the snail mucus in various concentration cus aureus ATCC 25923 was cultured in tryptic soy broth was added into the each well and incubated with 100 µl with 2% (w/v) NaCl at 30 °C with shaking overnight. Te of 0.2 mM DPPH in absolute ethanol in dark place for bacteria were re-inoculated in the fresh media and cul- 30 min. Ten, the absorbance was measured at 517 nm tured until they reached OD of 0.2 and were diluted 600 (A ) [21]. Distilled water was used as a negative control about hundred-fold in poor broth [1% (w/v) tryptone, 517 and ascorbic acid as a positive control. Te DPPH scav- 0.5% (w/v) NaCl, pH 7.5] to an OD of 0.001. Te 600 enging efect was calculated by the following formula: selected bacteria are prevalent species of gram negative %Efective (A – A )/A 100. %Efective and positive bacteria and most common nosocomial = Blank Sample sample × were plotted against concentration by GraphPad Prism pathogens. 8. Te EC50 values were calculated as the concentra- SDS‑PAGE and LC‑MS/MS tion to cause half-maximal inhibition of DPPH radical scavenging. Te protein concentration of mucus was determined using the Bradford assay [18], and then 25 µg of the sample was mixed with 5X reducing SDS loading dye and Statistical analysis heated at 100 °C for 10 min. Te samples were then sub- Results were represented as mean with standard devia- jected to 12.5% SDS-PAGE. One gel was stained with tion in triplicate. Statistical analysis was performed using Coomassie blue and the other gel was developed by silver a one-way analysis of variance (ANOVA) followed by staining [19]. Tukey’s test. Signifcance was accepted at the p < 0.05 Te selected protein bands were cut from the stained level. SDS-PAGE and analyzed using LC-MS/MS at the Research Instrument Center, Khon Kaen University, Tailand. Briefy, the protein bands were destained, Results reduced, and digested with Sequencing Grade Modifed Snail mucus extraction and protein profles Trypsin. Te tryptic peptides were extracted and ana- Te protein pattern of the two snails analyzed by 12.5% lyzed using LC-MS/MS. Te obtained data were searched SDS-PAGE exhibited quite a diferent banding pattern in Protein database using the MASCOT program. despite some commons bands that were observed (Addi- tional fle 1: Fig. S1). Te L. fulica mucus showed major Antimicrobial activity assay bands at about 13, 37, 70, and > 200 kDa, whereas H. dis- Te antimicrobial activity of snail mucus was analyzed tincta showed major bands at approximately 11, 12, 14, using the minimal inhibitory concentration assay [20]. 25, and 120 kDa (Additional fle 2: Fig. S2). Noothuan et al. BMC Res Notes (2021) 14:138 Page 3 of 7 Protein pattern of snail mucus from the mantle and foot (Additional fle 3: Table S1) suggested that each band of L. fulica and H. distincta might be a mixture of proteins. Interestingly, the 30 and Te snail mucus from the mantle collar and foot of L. 60 kDa proteins were matched with achacin, the anti- fulica and H. distincta was separately collected and microbial peptide in L. fulica, while the 56 and 60 kDa analyzed the protein profles. Te results showed com- protein band from H. distincta and L. fulica, respec- mon protein bands with some distinct bands (Fig. 1a). tively, matched with diferent proteins including a sar- In addition, a gel with silver staining for L.

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