Abstract
Snail secretion is a complex mixture of several components, including proteins, glycoproteins, mucopolysaccharides and smaller molecules. Its growing use in nutraceutical, cosmetic and biomedical applications, as well as a component of edible and green packaging to replace chemical plasticizer, implies more affordable and sustainable extraction methods. We chose four extracts obtained from Cornu aspersum snails, different by origin, extraction medium (namely, citric acid, lactic acid or none) and additives and we performed a series of characterizations including the SDS-page, the measure of pH and density, the evaluation of dry matter and of protein content, supported by structural determinations by means of UV-visible and infrared spectroscopy, X-Rays diffraction and thermogravimetric measurements. Biological assays comprising evaluation of cytotoxicity and antibacterial activity were also carried out. All the tests were performed both on the as received snail filtrates and on the samples after proper dialysis to remove preservatives added by manufacturers. The obtained results put into evidence that the properties and composition of the final extract are strongly influenced by the collection method, that can be relevant for the proper use of snail filtrate in specific applications.
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Introduction
Snails are members of the Mollusca phylum and are part of the Gastropoda class, which includes over 80% of all mollusks, comprising snails and slugs. There is a huge variety comprising over 30,000 living species, both aquatic and terrestrial: indeed, snails can be found everywhere around the world, and live in very diverse types of habitats1. Their abundance and widespread distribution have made them an important resource for humans throughout history. Snail farming, also called ‘heliciculture’, is a currently growing agricultural reality in a number of countries, recognized by both public and institutional bodies. Cornu aspersum (Helix aspersa)2, also known as ‘small grey snail’ (or French ‘petit gris escargot’)3, is one of the most farmed species.
The slime is the mucus that covers the entire external surface of the snail and it is secreted by salivary epidermal glands (pedal glands) to accomplish different functions4: it is primarily used for locomotion due to its lubricant and adhesive properties, but it also prevents the desiccation of tissues, protects the animal against the environment, and provides an efficient nutrient uptake.
Human beings have always considered snails as a source of numerous therapeutic or biological properties: since ancient Greece snails of different species were prepared in various ways, such as chopped, powdered, grilled or infused5 and have been considered of medicinal value in traditional Chinese medicine. Nowadays, animal-derived ingredients are less frequently used for various reasons, including the infectious risk of some derivatives, and ethical issues and concerns regarding the protection of biodiversity and endangered species. However, snail slime or other animal derivatives (as bee-derivatives) are obtained without causing suffering or harm to the animals, and their use is widely accepted and encouraged, as their unique composition confer peculiar properties to materials both in the cosmetic and pharmaceutical field.
In particular, snail slime has become a popular natural product in the cosmetic industries to treat, for example, acne and scars, because of its reported benefits for human skin. These products are indeed claimed to have healing, rejuvenating, moisturizing and protective properties6,7 thanks to their slime content or, more specifically, to the natural occurrence of hyaluronic acid, proteoglycans, glycoprotein, enzymes and antimicrobial peptides. Indeed, the slime from Cornu aspersum snails confirmed the occurrence of an unusual combination of natural ingredients with beneficial and therapeutic qualities for human skin8 and thus it is also used in the para-pharmaceutical for the management of skin wounds and to treat burn injuries, as well as to treat chronic bronchitis3. In literature, it is possible to find papers related to the use of snail slime (filtrate/extract) as an ingredient in the preparation of biomaterials3,9, and some of the authors also patented the formulation of biopolymers-based films enriched with this natural extract10. In particular, films obtained by mixing carboxymethyl cellulose and snail slime have been deeply investigated and proved to be effective as sustainable and active food packaging11.
Recently, a paper has studied and shown that oral administration of snail mucin before sun exposure is effective in slowing the formation of UV-induced wrinkles and moisture loss, as well as improve skin elasticity of hairless SKH mice12.
Although snail meat and slime have been consumed for thousands of years in many countries, information on the nutritional value and chemical composition of edible parts of snails has so far been limited13. At first, glycolic acid and allantoin, the most abundant molecules in snail slime, were believed to be the essential component for the biological activities of snail slime14. However, it has been recently demonstrated that slime has a greater effect than that of the individual molecules7.
Snail slime is therefore a peculiar compound not reproducible in the laboratory: this may rely on the synergy in activity of the different molecules present in the snail secretion, but also in the specific relationship of these components in the natural secretion of the snail15. Moreover, the composition of snail slime could strongly depend on several variables such as snail feeding, seasonal harvesting and collection method. Indeed, snails can be stimulated to produce an excess of slime without causing any damage to their health by means of natural methods or by using an aqueous stimulating solution inside a specific machine, conditioned with ozone to preserve the final product. Natural methods involve manual or mechanical massage of the animal, which is gently stimulated to produce the slime. This method is considered the most gentle and respectful towards the animal. On the other hand, methods utilizing stimulating solutions involve the application of substances such as diluted natural organic acids or sodium chloride on the snails. Indeed, all these methods are claimed to be cruelty free for the animal. The resulting solution is then settled and filtered according to different procedures to obtain the commercially available snail secretion filtrates (SSFs). The filtered samples can be stored within sterile vials or by adding preservatives.
In literature some papers reported the characterization of films or scaffolds obtained by mixing SSF and biopolymers: the slime addition even conferred antibacterial properties to these materials, allowing their application both in the biomedical sector and as packaging materials16,17,18,19,20,21.
However, although the field of application of SFF is increasingly expanding, a study correlating composition and properties of the material as a function of the collection method, comprising both the extraction and the filtration procedures, is still missing. With the aim of obtaining a deeper understanding of the compositions of SSFs and the relation to their collection method, we selected snail slimes from different Italian retailers to understand their differences in terms of density, dry residue, structural characterization, protein concentration and biological properties as antioxidant, cytotoxicity and antibacterial activities.
Results and discussion
The SSFs used in this work were purchased from different Italian retailers and collected with different methods, eventually involving the use of ozone in specific chambers to purify the product. All the information available about the SSFs before starting the characterization, were derived from what the farmers reported, both through their websites and on the product labels, and are reported in the Materials section.
Commercial slimes may also contain several additives and are obtained by diluting the extracted slime with water. SSF A label does not declare any addition of preservatives, while SSFs B and C are added with sodium benzoate and potassium citrate, acting as preservatives.
The SSFs batches associated to different retailers, extraction and filtration methods were investigated to better understand their properties and to correlate them with their chemical composition. Moreover, to evaluate the properties of SSFs free from additives, some analyses were conducted on samples previously submitted to dialysis. The evaluation of metals present in SSFs was performed only on dialyzed samples, to avoid considering elements from external additives: the results are reported in SI, Tables S1-S4.
Density and dry residue evaluation
The SSF is a mixture of active compounds such as proteins, glycoproteins, glycosaminoglycans (GAGs), fatty acids, polyphenols, vitamins, glycolic acid, allantoin and minerals15,22,23; thus, different collection methods can influence the active compounds content. All the SSFs appear clear and yellowish, the measured pHs are strongly dependent on the extraction method applied: the most acidic pHs were found for samples B and C1, extracted by means of citric acid.
SSF A is the only extract to have a pleasant mild-rose aroma, characteristic of Feniol that is a well-balanced compound able to inhibit bacterial and fungal growths. Indeed, it has a wide spectrum antimicrobial activity and represents an alternative to traditional cosmetic preservatives, allowing to create self-preserving formulations with reduced irritating and sensitizing potential. Feniol is often added to natural extracts but, as it does not contain preservatives listed in EU Annex VI or allergens, allows the claims preservative-free and fragrance-free. From these characteristics and further evidences reported below, we hypothesized Feniol addition to SSF A.
Table 1 reports the dry residue and the density of SSFs both before and after dialysis (cut-off of 12–14 kDa). All the density values are close to 1 since the slimes are widely diluted with aqueous solutions. The dry residues of as received snail slimes are in the range 1–3%, similar to the values reported in the literature15, and the dry matter content of samples B and C1 is significantly higher (****p < 0.001) than that of A and C2 (Figure S1). After dialysis the amount of dry matter drops abruptly, being up to thirty times lower, and no statistical differences are evidenced among the samples. During the dialysis step, all the compounds with a molecular weight lower than 14 kDa (e.g., Vitamins E, C, B12, B13, glycolic acid, allantoin, small proteins and minerals, but also additives used during and after snail slime collection) pass through the membrane and are lost.
According to the obtained results, most chemicals in the snail slime are under the molecular weight cut-off. However, during storage the precipitation of a brown residue occurred in both as received SSFs B and C1. A digital picture of the residue is reported in Figure S2, together with the infrared spectrum collected on the material after freeze-drying, clearly showing the presence of proteins. Probably, the strong acidity of these SSFs (compare pH values in Table 1) induces proteins aggregation during storage.
Protein content assay
The first attempt to assess the protein content in SSF samples was carried out by using the Bradford assay, a method widely reported in literature for the protein quantification in natural substances including snail slime15,22. The Bradford assay is based on the use of the Coomassie Blue dye22. The resulting complex shows a maximum absorption at a wavelength of 595 nm. Nevertheless, this method is highly sensitive to some interferent and to the presence of glycosylated proteins that reduce the formation of the blue complex giving a lower and incorrect protein quantification24. The Bradford test was repeated several times and the values obtained were different each time: the non-reproducibility of the protocol confirmed the presence of glycosaminoglycans and glycoproteins in the snail slimes analyzed.
The bicinchoninic acid assay (BCA) is a highly sensitive and tolerant to interfering species method that allows the quantification of both proteins and glycoproteins24. The assay is based on two chemical reactions: the reduction of cupric ions (Cu2+) to cuprous ions (Cu1+) by the peptide bonds (Biuret reaction) and by some specific residues (cysteine, cystine, tryptophan and tyrosine) in an alkaline environment followed by the chelation of one ion Cu1+ by two BCA molecules, forming an intense purple complex with a maximum at a wavelength of 562 nm.
Since BSA (bovine serum albumin) is known to give misleading results in many tests25 fish gelatin has been used as a standard protein for the calibration curve and to correlate the protein content as much as possible to the presence of the peptide bond and not to other residues, as the composition of the slime is unknown. The assay was performed on as received SSF samples and on the samples after dialysis (both liquid and freeze-dried). The amount of proteins in the different SSF samples is reported in Table 2, and the results of statistical analysis in Figure S1.
According to the results reported in Table 2, the protein content of different SSFs seems to be very low compared to other studies reported in literature15,26,27. Pitt et al.26 and Gugliandolo et al.15 evaluated the protein content of undiluted snail mucus, manually extracted by cotton swab, which represented around 50% of the dry residue. The values found by these authors were 15,4 and 4,8 mg/mL, respectively: the significant different protein content they obtained can be explained considering that materials of natural origin are characterized by a great variability, which depends on environmental factors and processing. Furthermore, it is known that the properties and composition of snail slime are strongly affected by the breeding of animals and their feeding. Indeed, it is reported that snails raised on a leaning surface produce a greater number of GAG than those grown on a flat area28,29. However, our results are in tune with those obtained by Trapella et al., who extracted the slime with an acid-free method and analyzed the resulting solution (not the pure slime)7. In fact, the protein content they claimed varies between 3.3 and 12.5 g/100 g, while in the samples we analyzed the values are in the range 4–14 g/100 g. In addition, it must be considered that the reproducibility of this protein model could vary with each seasonal collection of samples22. For this reason, we characterized all the SSF collected in the same season. From our studies, there is striking evidence of the influence of the extractive method on the properties of the slime: by comparing the protein content, it is evident that slime A, extracted manually without the use of a stimulating solution, contains more proteins (****p < 0.0001, Figure S1) compared to the other SSF (B, C1 and C2). It is interesting to point out that, comparing the SSF (C1 and C2) produced from snails raised in the same location and fed in the same way by the same retailer, the use of lactic acid significantly increases the amount of proteins extracted (C1 vs. C2, ****p < 0.0001). Filtration of the extract represents a further crucial step during the slime processing: a comparison among our data and those reported in literature is more difficult, due to the variety of filters and filtration sequencies used.
After the dialysis process, all the compounds under the membrane threshold are lost since the dry residues of the dialyzed samples are much lower than those of the samples as received, it is reasonable to assume that most of the chemicals present in the slime have a molecular weight lower than 12–14 kDa. According to the literature15, Vitamin C, E, B12 and B3, allantoin, glycolic acid and minerals account at least for 20–30% of the dry residue7, and their washing out during the dialysis, together with other preservatives added by the manufacturers, corresponds to a weight loss of about 95% (see Table 1). As a consequence, the protein content of the freeze-dried residue is higher in all the SSFs after dialysis, being A and C2 the samples with the highest amount of proteins, (97% and 93%, respectively), while this value is significantly lower in B and C1 filtates (****p < 0.0001, see Table 2 and Figure S1). From these results, an interesting correlation between the amount of protein and the collection method can be drawn: acidic extraction decreases the protein content when citric acid is used (compare sample A to samples B ****p < 0.0001 and C1 ****p < 0.0001, Table 2), while lactic acid seems to have the same effect of the stimulation without additives (no significant difference in protein content of freeze-dried SSF between samples A and C2, see Figure S1).
FT-IR and X-Rays diffraction
The infrared spectra acquired on freeze-dried samples of snail slimes before and after dialysis are reported in Fig. 1.
FT-IR spectra of snail slimes samples: (A) as received (red line) and after dialysis (pink); (B) as received (blue line) and after dialysis (light blue); (C1) as received (brown line) and after dialysis (orange); (C2) as received (green line) and after dialysis (light green).
All the samples show a broad band centered at 3400 cm−1 attributable to -OH stretching, with a contribution of acid CO-OH stretching and NH moieties. Samples collected after dialysis also show a shoulder at lower wavelengths (centered at 3300 cm−1) typical of amide A30,31. The double band centered at 2950 cm−1 is attributed to the -CH2 stretching.
FTIR spectra of as received SSFs reveal absorption bands in the range 1650 cm−1-1500 cm−1, indicating the presence of amide I and Amide II bonds that, in samples B, C1 and C2 partially overlap with some bands centered around 1700 cm−1 ascribable to carboxylic moiety20. The presence of this band is due to the acidic stimulating solution used for the slime extraction. Furthermore, Amide I and Amide II bands become very strong in IR spectra obtained on all the dialyzed samples, clearly showing the increase of protein content in the residue20.
The IR spectra of samples B and C1 as received show two more bands at 1400 and 1080 cm−1 attributed to the asymmetric stretching of C = O and to the symmetric stretching of C-OH of the citric acid, respectively32,33, while the absorption bands of C2 in the region between 1200 and 950 cm−1 are mainly due to C-C and C-O stretching of lactic acid34,35. On the other hand, in the IR spectrum of A, the absorption bands between 1453 –500 cm−1 due to the slime components are covered with those belonging to Feniol (a mix of caprylyl glycol and phenyl ethyl alcohol) added by manufacturers, thus representing further evidence of the presence of additives at very high concentration. As Feniol is not classified as a preservative according to the current cosmetic legislation, its amount in the self-preservative formulation may not be reported in the final product.
X-Ray diffraction patterns obtained from freeze-dried samples before and after dialysis are reported in Figure S3. The patterns are characterized by diffuse halos typical of amorphous materials. However, in the pattern of SSF A as received the presence of some crystalline material is detected, and could be mainly attributed to the addition of antimicrobials molecules following the extraction and are eliminated during the dialysis: in fact, the pattern collected from dialyzed slime is only characterized by a broad band attributed to macromolecular amorphous structures.
UV-Vis spectroscopy
Spectroscopy is a very powerful technique for the determination and quantification of chemicals in solutions like proteins. The absorption pattern of snail slime samples in the UV-Vis region are shown in Fig. 2.
UV-vis spectra of SSFs as received (dark line) and after dialysis (pale line).
Both the UV-Vis spectra of the pure and dialyzed SSFs show absorption only at wavelengths below 300 nm. This could be related to the presence of proteins: amino acids with conjugated groups and aromatic groups absorb mainly in the UV region between 250 and 300 nm wavelength. In particular, absorption between 240 and 300 nm may be due to the π->π* of aromatic amino acids, while at shorter wavelengths there is the π-> π* of the peptide bond (between 190 and 240 nm) and the σ-> σ* of all bonds (< 190 nm). However, different amino acids absorb at different wavelengths and with varying intensities: for example, tryptophan has an absorption about 7 times higher than that of phenylalanine. In addition, sulfur-containing amino acids contribute to the UV spectrum: for example, cystine, which occurs in high proportion in many proteins, absorbs between 250 and 310 nm while methionine and cysteine generally at wavelengths below 250 nm. Hence, not knowing the relative quantities of the different amino acids contained in the slime samples, we can only make qualitative comparisons. Even the preservatives added by manufacturers or small molecules present in SSF, such as glycolic acid, absorb in the UV region.
Overall, the absorption of dialyzed samples is lower than that of the non-dialyzed ones, due to the loss of small molecules passing through the membrane.
SDS-PAGE
Differently collected SSF samples were analysed in SDS-PAGE for their protein profiles (Fig. 3).
SDS-PAGE analysis of freeze-dried SSF samples (lanes 1–4) and dialyzed plus freeze-dried SSF samples (lanes 5 to 8). M: Precision Plus Protein™ Standards (Bio-Rad). Lanes 1 and 5: SSFs from (A); lanes 2 and 6: SSFs from (B); lanes 3 and 7: SSFs from (C1); lanes 4 and 8: SSFs from (C2).
Considering that the SSFs analysed in the present work were obtained from the same snail species (Cornu aspersum), the presence of different protein profiles among the samples suggests that different extraction methods alter the protein composition of the SSFs. These changes may be due both to a different response of the snail to the stimulus used to induce mucus production, but also to the different stability of the proteins to the mixtures used for mucus preservation.
After dialysis, all samples show a more marked banding pattern than non-dialysed samples and in the case of sample A bands become visible (Fig. 3, lane 1 and lane 5, respectively). This could be due to the removal of preservatives and aromatic molecules, as it is known that phenyl ethyl alcohol (PEA, one of the components of Feniol), induces protein fibrillation, thus affecting the sample entry into the gel matrix36. Among the different SSFs, the lactic acid-collected protein mucus (Fig. 3, lane 8) appeared less damaged when analysed in SDS-PAGE, as evidenced by the pattern of sharper bands suggesting higher integrity of the extracted proteins.
These results put into evidence that during collection of SFFs a part of proteins is lost, and that the extraction with lactic acid better keeps the material properties.
Thermogravimetric analysis
Thermogravimetric analysis (TGA) was then performed on all freeze-dried SSF samples before and after dialysis to assess differences in thermal stability (see Fig. 4).
TGA analysis of SSFs as received and after dialysis.
For the samples A, B and C1 as received, the thermal behavior is quite similar. The weight loss below 120 °C is due to the adsorbed and structural water while the one between 120 and 250 °C could be attributed to small molecules, preservatives and stimulating solutions. The thermal behavior over 250 °C can be attributed to the macromolecules and proteins present in all the samples.
The thermal behavior of samples A, B and C1 after dialysis present a sudden weight loss close to 60 °C attributed to water molecules: this loss occurs at temperature much lower with respect to non-dialyzed samples because the water molecules are probably adsorbed and are not present as structural molecules. The further degradation can be attributed to the macromolecules and proteins present in the snail slime having a molecular weight higher than 14 kDa: their decomposition starts at temperature values higher than those previously recorded for the as received samples. This finding is in tune with the enrichment of the dialyzed sample in macromolecules with higher molecular weight.
Antioxidant activity
The DPPH assay is the primary method reported in literature to evaluate the antioxidant activity of natural materials and was used to have a deeper understanding of the antioxidant behavior of the SSFs. Nevertheless, this test has some limitations: DPPH• is very different from the radicals responsible for the autoxidation, therefore it is difficult to prove if the sample can interrupt the chain reaction of autoxidation or simply reduce the probe. Furthermore, this method shows more the “radical trapping power” or the “reducing power” rather than true antioxidant activity37.
Obtained measures are reported in Fig. 5: the as received samples have a higher scavenging activity compared to the corresponding dialyzed ones, probably because of the chemicals added by the suppliers to the extracted substance. Lactic acid and citric acid are reported in literature as dose dependent free radical scavenger and antioxidant, respectively38,39. As well sodium benzoate, added in samples B and C as a preservative, acts as hydroxyl radical scavenger and benzoates inhibit the mechanisms that generate free radicals40: for this reason, the antioxidant activity of samples B and C is higher with respect to other slimes.
Inhibition ratio (%) at 30 min evaluated on SSF as received and after dialysis.
The inhibition ratio of as received and dialyzed sample A is very similar and lower with respect to the negative control: this slime is extracted manually without the use of any acidic solution and no preservative with scavenging activity is added. Slime B is the only sample showing a poor antioxidant activity after dialysis.
Cytotoxicity assay
Vero cells were used as model system to evaluate the effects of the different SSFs on mammalian cell metabolism. Cell viability results were measured following 24 h of incubation with the samples as received and after dialysis. Data are reported in Fig. 6.
Cell viability and proliferation assay performed on Vero cells after 24 h of incubation with the different SSF samples at 100 µg/mL (A) and 10 µg/mL (B). Data are expressed as percentage values (means ± SD) relative to the positive growth controls. Differences between as received and dialyzed samples were determined by unpaired student’s t test (p value < 0.05).
The as received samples displayed a high cytotoxicity when tested at 100 µg/mL; indeed, they all reduced Vero metabolism at different extent compared to the positive control. At 10 µg/mL, all SSF samples did not interfere with cell proliferation with the only exception of sample A that maintained its cytotoxic effect. This finding may be due to the presence of certain compounds added by the retailer (such as preservatives or additives) previously observed by X-rays diffraction (see Figure S2). A further confirmation of this hypothesis is given by the values obtained for dialysis samples: a high cellular viability was found for samples regardless of the dilution factor. Considering that snail slime is normally used in cosmetic and para-pharmaceutical products at a very high dilution, our results demonstrate its biocompatibility, regardless of the collection method.
Overall, these results are promising especially considering that the dilution factor in which snail slime is normally used in cosmetic and para-pharmaceutical products is very high.
Antibacterial activity
The antibacterial activity of all the SSFs was assessed in vitro against S. aureus and E. coli, as reference model systems for Gram-positive and Gram-negative bacteria, respectively.
SSFs were firstly tested by the disk diffusion method: in this test, only sample A (p < 0.001) as received proved to interfere with bacterial proliferation leading to a clear inhibition zone of 12 ± 1 mm for S. aureus and 7 ± 1 mm for E. coli. However, the corresponding dialysed sample was not able to inhibit bacterial growths. This result indicates that dialysis completely and effectively removes preservatives contained in the sample that have only a generic toxic effect on cells rather than an antibacterial selective activity.
As it is impossible to quantify and/or predict the active compounds able to diffuse into the agar medium, all SSFs regardless of the results obtained in the standardized agar well diffusion test, were assayed by a broth microdilution method. None of the tested samples proved to inhibit E. coli, even at the highest concentration of 100 µg/mL. As for S. aureus, the as received sample A remarkably inhibited bacterial growth with a MIC of 12.5 µg/mL (Fig. 7). However, sample A following dialysis proved to be ineffective against the tested strain confirming the removal of preservatives as well as unknown antimicrobial slime components with a molecular weight lower than 14 kDa.
Bacterial growths of S. aureus when treated with SSF samples as received (A) and following dialysis (B), at different concentrations. Data are expressed as percentage values (means ± SD) relative to the positive growth controls (bacteria grown in regular medium).
Among the biologically active natural substances identified in the mucus of the snails, the antimicrobial peptides (AMPs) are of great interest for their broad-spectrum activity against a wide range of pathogens including bacteria, fungi, and viruses, and their reduced development of antibiotic resistance41. Many identified AMPs in the mucus of H. aspersa have molecular masses lower than 30 kDa, and it has been hypothesized the presence of a synergistic effect between peptides with < 3 kDa and polypeptides with higher molecular weights42,43. It is likely that the removal of the peptide fraction lower than 14 kDa herein obtained by dialyses makes polypeptides with high molecular weight ineffective against the tested strains.
Conclusion
In the field of snail secretion utilization, a fundamental discrepancy arises between the industrial cosmetic interpretation of “snail slime” and its scientific research counterpart. While in cosmetic product labeling, “snail slime” typically refers to snail secretions filtrates, scientific literature denotes the term for pure mucus secreted by snails reared in laboratory conditions. This article outlines the properties of snail extract, due to the different extraction method used. Such comprehension is imperative for informed decision-making and the selection of the most suitable extract for a specific application.
Four commercial slime filtrates were examined, extracted by acidic stimulation of snails (sample B, C1 and C2), or without the use of stimulating solutions (sample A). All the examined extracts contain diluted snail slime, as verified by evaluating the dry residue after freeze drying, ranging from 0.8 to 2.48%, where the highest value corresponds to SSFs extracted by using citric acid (C1 sample).
A higher protein content was found in the SSF extracted without the use of stimulating solutions (SSF A) and in the SSF sample extracted with lactic acid (SSF C2); after dialysis the total dry residue of both these samples consists almost exclusively of proteins, thus highlighting the influence of extraction conditions on the material properties. SSFs extracted by citric acid (B and C1) are characterized by lower pHs that could be responsible for the aggregation and precipitation of a colloidal protein residue observed over time.
As stated by manufacturers, SSF commercial solutions contain preservatives and additives, responsible for certain properties such as antibacterial and antioxidant effects. However, not all added additives must be declared in the product label, as in the case of Feniol in SSF A.
In conclusion, snail slime emerges as a versatile and intriguing ingredient for the formulation of products in various applications. However, it is crucial to recognize the significant variability associated with its properties, which requires careful characterization before use. Despite this variability, the unique properties inherent in snail slime present promising opportunities for a sustainable innovation in areas ranging from cosmetics to nanotechnology. Through understanding this variability, researchers and industries can effectively exploit the potential of snail slime, contributing to the development of solutions tailored to different needs.
Experimental part
Raw materials preparation
The SSFs used in this comparison were purchased by three independent helicicole farms operating in different Italian regions and extracted by using different methods:
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SSF ‘A’ was obtained from snails grown in southern Italy from animals fed only and exclusively with chosen vegetables to ensure a unique end-product. The slime was collected from live snails by manual stimulation, thus allowing retailers to have a pure product without the addition of water or irritating agents. Retailer claimed that the process guarantees a slime with a pH from 5 to 7 rich in mucopolysaccharides and proteins. The extract was filtered through 0.45 μm filters before packaging.
SSFs B and C were produced in northern and central Italy, respectively, and both were obtained using a stimulating solution within the extraction chamber. This method consists of two steps: the snails are subjected to a gentle shower of osmotized water mixed with ozone, then they are stimulated by spraying an acidic solution. In particular, citric acid was used to obtain samples B and C1, while lactic acid is used to obtain sample C2.
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SSF B, C1 and C2 were filtered with 40 μm depth filters and potassium sorbate and sodium benzoate were added as preservatives in a concentration of 0.1% w/w, as reported by retailers.
Once received from the suppliers, SSFs were stored in the refrigerator at 4 ºC until use.
Each SSF was characterized as received and after dialysis. Samples both before and after the dialysis step were also freeze-dried (Labconco, T = -45 °C, P = 0.850 mbar, t = 24 h) and the results were compared.
Dialysis
Preservatives and stimulating solutions, often present in snail slime samples, were removed by dialysis performed with a 12–14 kDa cut-off membrane (Merk, flat width 25 mm). The membrane was previously conditioned by immersion for 24 h in abundant MilliQ water under stirring, frequently replacing water. Dialysis was conducted for 24 h at room temperature against 1 L of MilliQ water (samples volume: 20 mL) under stirring. Water was replaced three times. Dialyzed samples were stored at -19 °C until use.
ICP analysis
ICP analysis of SSFs after dialysis was conducted by means of Agilent Technologies 4210 Molecular Plasma Atomic Emission Spectroscopy (MP-AES), on properly diluted samples, by using a multielement standard (Ultrascientific) for the calibration curves.
Density and dry residue evaluation
The density of the SSFs was calculated using the following equation:
Where Ws is the weight of a known volume of slime and Vs the corresponding volume.
The dry residues were calculated by weighting the SSF before and after lyophilization using the following formula:
Where Wf is the final weight of the lyophilized sample and Wi the one of the liquid SSF. The values are an average of at least 5 samples.
Protein content evaluation
Bicinchoninic acid assay (BCA) was used to evaluate protein concentration in both liquid and freeze-dried samples, before and after dialysis. Dialyzed SSFs were diluted 1:10 with distilled water, while lyophilized samples were weighted (0.5-2 mg) and dissolved in a known volume of distilled water. 1 mL of aqueous SSF and 2 mL of BCA reagent were mixed. The solutions were kept at 37 °C for 30 min. Then, 2 mL of phosphate buffer (PB 0.1 M, pH 7.4) were added and the absorption values were immediately recorded at a wavelength of 562 nm. PB was used as spectrophotometric blank. Fish gelatin dissolved in PB (0.5 mg/mL) was used as standard for the calibration curve. Each value reported in Table 2 is a mean of five determinations.
Infrared spectroscopy
The Fourier transform infrared spectra (FT-IR) were recorded using a Thermo Scientific Nicolet iS10 FTIR spectrometer on KBr tablets. 1 mg of lyophilized sample was carefully mixed in a mortar with 300 mg of KBr (infrared grade) and pelletized under a pressure of 5 Bar for 3 min. All the spectra were recorded in transmission mode between 4000 and 400 cm−1 with a resolution of 2 cm−1, as an average of 70 scans.
X-Rays diffraction
X-ray diffraction patterns in reflection mode were recorded on lyophilized samples by using a Philips X’Celerator diffractometer equipped with a graphite monochromator. The 2θ range was from 3° to 50° /2 theta with a step size of 0.100° and a time per step of 120 s. CuKα radiation (40 mA, 40 kV, 1.54 Å) was used.
UV-Vis spectroscopy
The UV-Vis spectra were recorded in absorbance mode using a Cary 60 UV-Vis (Agilent) spectrophotometer between 200 and 800 nm wavelengths. To avoid spectra saturation, pure snail slimes were properly diluted with distilled water: 60 µL of snail slime were poured in 5 mL of distilled water, while 600 µL were used for the dialyzed samples. Distilled water was used as blank.
SDS-PAGE
SSF samples for SDS-PAGE analyses were prepared by weighing freeze-dried powders of SSFs and dialysed SSFs. Weighed powders were suspended in the appropriate volume of denaturing and reducing protein-loading buffer (2% SDS, 2% β-mercaptoethanol, 10% glycerol, 100 mM Tris-HCl, pH 6.8, 0.01% bromophenyl blue) to obtain a final concentration of 10 mg/mL based on the percentage of protein content in each sample (see Table 2). Once suspended, 35–140 µg of protein samples were transferred into clean tubes and heat denatured at 100 °C for 7 min. The samples were then loaded into a 12.5% polyacrylamide gel. The gel was run at 120 V for approximately 100 min using a Mini-PROTEAN system (Bio-Rad). At the end of the run, the gel was stained with Coomassie Blue R-250 solution (50% methanol, 10% acetic acid, 0.25% Coomassie Blue R-250) and de-stained with 25% ethanol and 8% acetic acid de-staining solution. The molecular weight standard was Precision Plus Protein™ Standards (Bio-Rad).
Thermogravimetric analysis (TGA)
Thermogravimetric analysis (TGA) was conducted by means of a SDT Q600 thermogravimetric analyzer (TA Instruments TM) on 5–10 mg of lyophilized slimes. The analysis was performed in air flow (100 mL/min) at a rate of 3.5 °C/min up to 120 °C and then 10 °C/min to 800 °C.
Antioxidant activity
The antioxidant activity of snail slimes as received and dialyzed was evaluated with DPPH method, following the literature. A photo-sensible solution 0.2 mM of DPPH in pure ethanol was prepared and kept at room temperature for two hours prior to use, covered with an aluminum foil to avoid light exposure. Tris-HCl buffer (0.1 M; pH 7.4) was prepared and the pH was adjusted to 7.4 with concentrated HCl. 200 µL of snail slime, 800 µL of tris-HCl buffer, 100 µL of ethanol and 900 µL of DPPH solution were added in a tube covered with an aluminium foil and accurately shaken. The absorbance values at a wavelength of 517 nm were acquired 30 min after the addition of DPPH. A mixed solution of 1.2 mL of ethanol and 800 µL of Tris–HCl buffer was used as a blank21, [47]. To calculate the inhibition ratio, a reference solution was prepared by mixing 300 µL of ethanol, 800 µL of Tris-HCl buffer and 900 µL of DPPH following the above procedure and the values were obtained by using the following formula:
Biological assays: cytotoxicity and antibacterial activity
For biological assays, the freeze dried SSF samples, before and after dialysis, were dissolved in ultrapure distilled water at 10 mg/mL and then stored at 4 °C. If necessary, the pH of the samples was adjusted to 6.5–7.5 before experiments.
Cell viability of Vero cells (African green monkey kidney epithelial cells, ATCC CCL-81) was assessed by a colorimetric WST-8 method (CCK-8, Cell Counting Kit-8, Dojindo Molecular Technologies, Rockville, MD, USA) and using a well-established protocol [48]. Briefly, cells were seeded into a 96-well tissue culture plate at 104 cells/well and incubated for 24 h at 37 °C and 5% CO2, in a regular cell culture medium (RPMI-1640 supplied with 10% Fetal Bovine Serum, 100 U/mL penicillin, and 100 µg/mL streptomycin). Following washes with PB, the cell monolayer was incubated with 100 µL of culture medium containing 100 µg/mL of the different samples, and subsequent 2-fold serially dilutions. Untreated Vero cells were included in the assays. After 24 h of growth, the medium was removed and 100 µL of fresh medium containing 10 µL of CCK-8 solution were added. The absorbance value at 450 nm was measured using a microplate photometer after 2 h of incubation at 37 °C. Data were expressed as mean percentage values relative to the untreated control, obtained in at least two independent experiments performed in triplicate.
The in vitro antibacterial activity was evaluated by testing Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), and by using two different methodologies such as an agar-disk diffusion test and a broth microdilution method, according to previously well-established procedures [49], [50] and in compliance with the international guidance documents [51]. The reference bacterial strains were routinely grown on 5% blood agar plate (Biolife Italiana S.r.l., Milan, Italy) at 37 °C. As for the agar-disk diffusion method, Mueller-Hinton (MH) agar plates (Biolife Italiana S.r.l., Milan, Italy) were inoculated with a standardized suspension of the test microorganisms (0.5 McFarland), then, sterile paper disks (6 mm in diameter) were placed on the agar surface and filled with 10 µL of the SSF samples (corresponding to 10 mg of sample). After 24 h of incubation at 37 °C the agar plates were observed, and the diameter of the inhibition zone was measured to the nearest whole millimeter with a ruler. All experiments were performed on duplicate in different days.
In addition, the standardized broth dilution procedure was carried out to determine the Minimum Inhibitory Concentration (MIC) of the active SSF samples. Briefly, the standardized suspension corresponding to 0.5 McFarland was incubated in a MH broth containing 100 µg/mL of the different samples, subsequently 2-fold serially diluted. The following controls were also included in each assay: bacterial suspensions incubated in MH broth (positive control), dilutions of the tested samples (background controls) and MH broth without bacterial inoculum (negative control) to check the turbidity of reagents and the sterility of the procedure. After incubation for 24 h at 37 °C, the absorbance value at a wavelength of 630 nm was spectrophotometrically measured, and MIC was defined as the SSF concentration that inhibits the growth of bacteria.
Statistical analysis
Statistical analysis was performed with Graph Pad Prism 8. One-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison Test, was employed to assess statistical significance of the data. Statistically significant differences were determined at p < 0.05.
Data availability
The data supporting our findings are available in the manuscript, in the supplementary materials or from the corresponding authors upon request.
References
Ibrahim, A. M., Hamed, A. A. & Ghareeb, M. A. Marine, freshwater, and terrestrial snails as models in the biomedical applications. Egypt. J. Aquat. Biol. Fish. 25(3), 23–38. https://doi.org/10.1016/B978-0-12-385028-7.00011-1 (2021).
Conte, R. Heliciculture: purpose and economic perspectives in the European community. Official J. Inst. Sci. Technol., pp. 25–37, (2015).
Liudmyla, K., Olena, C. & Nadiia, S. Chemical properties of Helix aspersa mucus as a component of cosmetics and pharmaceutical products. Mater. Today Proc. Mar.https://doi.org/10.1016/J.MATPR.2022.02.217 (2022).
Greistorfer, S. et al. Snail mucus – glandular origin and composition in Helix pomatia. Zoology. 122, 126–138. https://doi.org/10.1016/j.zool.2017.05.001 (2017).
Bonnemain, B. Helix and Drugs: Snails for Western Health Care From Antiquity to the Present, Evidence-based Complementary and Alternative Medicine, vol. 2, no. 1, p. 28, Mar. doi: (2005). https://doi.org/10.1093/ECAM/NEH057
Chinaka, N. C. et al. Snail slime: evaluation of anti-inflammatory, phytochemical and antioxidant properties. J. Complement. Altern. Med. Res. 13(1), 8–13. https://doi.org/10.9734/jocamr/2021/v13i130214 (2021).
Trapella, C. et al. HelixComplex snail mucus exhibits pro-survival, proliferative and pro-migration effects on mammalian fibroblasts. Sci. Rep. 8(1), 1–10. https://doi.org/10.1038/s41598-018-35816-3 (2018).
El Mubarak, M. A. S., Lamari, F. N. & Kontoyannis, C. Simultaneous determination of allantoin and glycolic acid in snail mucus and cosmetic creams with high performance liquid chromatography and ultraviolet detection, J Chromatogr A, vol. 1322, no. August pp. 49–53, 2013, doi: (2017). https://doi.org/10.1016/j.chroma.2013.10.086
Perpelek, M. et al. Bioactive snail mucus-slime extract loaded chitosan scaffolds for hard tissue regeneration: the effect of mucoadhesive and antibacterial extracts on physical characteristics and bioactivity of chitosan matrix. Biomed. Mater. 16, 65008. https://doi.org/10.1088/1748-605X/ac2352 (2021).
Albertini, B., Passerini, N., Panzavolta, S. & Dolci, L. WO2020201439A1 - polymer films comprising material secreted by gastropods, WO2020/201439 A1, 2020.
Di Filippo, M. F. et al. July., Cellulose derivatives-snail slime films: New disposable eco-friendly materials for food packaging, Food Hydrocoll, vol. 111, no. p. 106247, 2021, doi: (2020). https://doi.org/10.1016/j.foodhyd.2020.106247
Kim, Y., Sim, W. J., Lee, J. & Lim, T. G. Snail mucin is a functional food ingredient for skin. J. Funct. Foods. 92, 105053. https://doi.org/10.1016/j.jff.2022.105053 (2022).
Matusiewicz, M. et al. In Vitro influence of extracts from snail Helix aspersa Müller on the Colon Cancer Cell Line Caco-2. Int. J. Mol. Sci. 19, 1064. https://doi.org/10.3390/ijms19041064 (2018).
Tsoutsos, D., Kakagia, D. & Tamparopoulos, K. The efficacy of Helix aspersa Müller extract in the healing of partial thickness burns: A novel treatment for open burn management protocols, Journal of Dermatological Treatment, vol. 20, no. 4, pp. 219–222, Aug. doi: (2009). https://doi.org/10.1080/09546630802582037
Gugliandolo, E. et al. Protective effect of snail secretion filtrate against ethanol-induced gastric ulcer in mice. Sci. Rep. 11 (1). https://doi.org/10.1038/S41598-021-83170-8 (Dec. 2021).
Perpelek, M. et al. Bioactive snail mucus-slime extract loaded chitosan scaffolds for hard tissue regeneration: the effect of mucoadhesive and antibacterial extracts on physical characteristics and bioactivity of chitosan matrix. Biomedical Mater. (Bristol). 16 (6). https://doi.org/10.1088/1748-605X/ac2352 (2021).
López Angulo, D. E. et al. do Fabrication, characterization and in vitro cell study of gelatin-chitosan scaffolds: New perspectives of use of aloe vera and snail mucus for soft tissue engineering, Mater Chem Phys, vol. 234, no. May, pp. 268–280, doi: (2019). https://doi.org/10.1016/j.matchemphys.2019.06.003
Mello Zamudio, R. et al. Obtaining a freeze-dried biomaterial for skin regeneration: reinforcement of the microstructure through the use of crosslinkers and in vivo application. Mater. Chem. Phys.290, https://doi.org/10.1016/j.matchemphys.2022.126544 (July, 2022).
Mane, P. C. et al. Terrestrial snail-mucus mediated green synthesis of silver nanoparticles and in vitro investigations on their antimicrobial and anticancer activities. Sci. Rep. 11(1), 1–16. https://doi.org/10.1038/s41598-021-92478-4 (2021).
Gubitosa, J. et al. Dec., Biomolecules from snail mucus (Helix aspersa) conjugated gold nanoparticles, exhibiting potential wound healing and anti-inflammatory activity, Soft Matter, vol. 16, no. 48, pp. 10876–10888, doi: (2020). https://doi.org/10.1039/D0SM01638A
Rizzi, V. et al. Snail slime-based gold nanoparticles: An interesting potential ingredient in cosmetics as an antioxidant, sunscreen, and tyrosinase inhibitor, J Photochem Photobiol B, 224, no. 112309, doi: https://doi.org/10.1016/j.jphotobiol.2021.112309. (2021).
Noothuan, N., Apitanyasai, K., Panha, S. & Tassanakajon, A. Snail mucus from the mantle and foot of two land snails, Lissachatina fulica and Hemiplecta distincta, exhibits different protein profile and biological activity. BMC Res. Notes. 14 (1), 1–7. https://doi.org/10.1186/S13104-021-05557-0/FIGURES/3 (Dec. 2021).
Onzo, A. et al. Untargeted analysis of pure snail slime and snail slime-induced au nanoparticles metabolome with MALDI FT-ICR MS. J. Mass Spectrom. 56 (5), e4722. https://doi.org/10.1002/JMS.4722 (May 2021).
Noble, J. E. & Bailey, M. J. A. Quantitation of protein, in Methods in Enzymology, vol. 463, (eds Noble, C. J. E. & Bailey, M. J. A.) Elsevier Inc., 73–95. doi: https://doi.org/10.1016/S0076-6879(09)63008-1. (2009).
Huang, T., Long, M. & Huo, B. Competitive binding to Cuprous ions of protein and BCA in the Bicinchoninic acid protein assay. Open. Biomed. Eng. J. 4(1), 271–278. https://doi.org/10.2174/1874120701004010271 (2010).
Pitt, S. J., Graham, M. A., Dedi, C. G., Taylor-Harris, P. M. & Gunn, A. Antimicrobial properties of mucus from the brown garden snail Helix aspersa. Br. J. Biomed. Sci. 72(4), 174–181. https://doi.org/10.1080/09674845.2015.11665749 (2015).
Laneri, S., Di Lorenzo, R., Sacchi, A. & Dini, I. Dosage of bioactive molecules in the nutricosmeceutical Helix aspersa muller mucus and formulation of new cosmetic cream with moisturizing effect. Nat. Prod. Commun. 14(8), 1–7. https://doi.org/10.1177/1934578X19868606 (2019).
Newar, J. & Ghatak, A. Studies on the Adhesive Property of snail adhesive mucus. Langmuir. 31, 12155–12160. https://doi.org/10.1021/acs.langmuir.5b03498 (2015).
Zhong, T., Min, L., Wang, Z., Zhang, F. & Zuo, B. Controlled self-assembly of glycoprotein complex in snail mucus from lubricating liquid to elastic fiber. RSC Adv. 8(25), 13806–13812. https://doi.org/10.1039/c8ra01439f (2018).
Barth, A. Infrared spectroscopy of proteins, Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol. 1767, no. 9, pp. 1073–1101, Sep. doi: (2007). https://doi.org/10.1016/J.BBABIO.2007.06.004
Kong, J. & Yu, S. Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures. Acta Biochim. Biophys. Sin (Shanghai). 39 (8), 549–559. https://doi.org/10.1111/j.1745-7270.2007.00320.x (2007).
Kumar, M. et al. Combating food pathogens using sodium benzoate functionalized silver nanoparticles: synthesis, characterization and antimicrobial evaluation. J. Mater. Sci. 52, 8568–8575. https://doi.org/10.1007/s10853-017-1072-z (2017).
Rǎcuciu, M., Creangǎ, D. E. & Airinei, A. Citric-acid-coated magnetite nanoparticles for biological applications. Eur. Phys. J. E. 21 (2), 117–121. https://doi.org/10.1140/epje/i2006-10051-y (2006).
Chen, Y. K., Lin, Y. F., Peng, Z. W. & Lin, J. L. Transmission FT-IR study on the adsorption and reactions of lactic acid and poly(lactic acid) on TiO2. J. Phys. Chem. C. 114 (41), 17720–17727. https://doi.org/10.1021/jp105581t (2010).
Pǎucean, A. et al. Monitoring lactic acid concentrations by infrared spectroscopy: a new developed method for lactobacillus fermenting media with potential food applications. Acta Aliment. 46(4), 420–427. https://doi.org/10.1556/066.2017.0003 (2017).
Seraj, Z., Groves, M. R. & Seyedarabi, A. Cinnamaldehyde and Phenyl Ethyl Alcohol promote the entrapment of intermediate species of HEWL, as revealed by structural, kinetics and thermal stability studies. Sci. Rep. 9(1), 1–11. https://doi.org/10.1038/s41598-019-55082-1 (2019).
Amorati, R. & Valgimigli, L. Advantages and limitations of common testing methods for antioxidants. Free Radic Res. 49(5), 633–649. https://doi.org/10.3109/10715762.2014.996146 (2015).
Ciriminna, R., Meneguzzo, F., Delisi, R. & Pagliaro, M. Citric acid: emerging applications of key biotechnology industrial product. Chem. Cent. J. 11(1), 1–9. https://doi.org/10.1186/s13065-017-0251-y (2017).
Lampe, K. J., Namba, R. M., Silverman, T. R., Bjugstad, K. B. & Mahoney, M. J. Impact of lactic acid on cell proliferation and free radical-induced cell death in monolayer cultures of neural precursor cells. Biotechnol. Bioeng. 103(6), 1214–1223. https://doi.org/10.1002/bit.22352 (2009).
Andersen, F. A. Final report on the safety assessment of Benzyl Alcohol, Benzoic Acid, and Sodium Benzoate, Int J Toxicol, vol. 20, no. SUPPL. 3, pp. 23–50, doi: (2001). https://doi.org/10.1080/10915810152630729
Xuan, J. et al. Antimicrobial peptides for combating drug-resistant bacterial infections. Drug Resist. Updates. 68, 100954. https://doi.org/10.1016/j.drup.2023.100954 (2023). January.
Dolashki, A. et al. Antimicrobial activities of different fractions from mucus of the Garden Snail Cornu Aspersum. Biomedicines. 8, 1–17. https://doi.org/10.3390/biomedicines8090315 (2020).
Topalova, Y. et al. Effect and mechanisms of antibacterial peptide fraction from mucus of C. Aspersum against Escherichia coli NBIMCC 8785. Biomedicines. 10 (3). https://doi.org/10.3390/biomedicines10030672 (2022).
Funding
Project funded under the National Recovery and Resilience Plan (NRRP), Mission 04 Component 2 Investment 1.5 – NextGenerationEU, Call for tender n. 3277 dated 30/12/2021. Award Number: 0001052 dated 23/06/2022.
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M.F.D.F., L.S.D., S.P., A.B. and F.B. conceived and designed the research. M.F.D.F., A.B., F.S .and F.B. conducted the experiments. All authors analysed the data. M.F.D.F., L.S.D., S.P., A.B., F.S. and F.B. drafted the manuscript. All authors reviewed and contributed to the final manuscript which was approved by all.
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Di Filippo, M.F., Dolci, L.S., Bonvicini, F. et al. Influence of the extraction method on functional properties of commercial snail secretion filtrates. Sci Rep 14, 22053 (2024). https://doi.org/10.1038/s41598-024-72733-0
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DOI: https://doi.org/10.1038/s41598-024-72733-0
