Cultured Chinese Giant Salamander Skin and Skin Secretions as a Source of Bioactive Peptides for Food and Medicine

Despite being widely recognized as edible amphibians, data on the global trade value of CGSs are limited. A comprehensive investigation combining market surveys and interviews was conducted to explore and address the domestic trade of salamanders. Reports indicate that the overexploitation of CGSs for food consumption has led to habitat destruction, particularly in China, where it has been listed as a Critically Endangered species by the IUCN Red List of Threatened Species (Rosa et al., 2023). Consequently, conservation efforts have been launched through collaborations between local governments and farmers to meet market demands while mitigating overexploitation. An example of such collaboration occurred in the Qinling region of China, where the government provided subsidies to initiate salamander farms, which also attracted investments from local stakeholders. CGS meat is typically exported as frozen deskinned meat, with other body parts, such as the head, trunk, and skin, discarded without further use. Hence, implementing diverse coordinated strategies is crucial for sustainably managing and preserving CGS populations while ensuring animal welfare standards and minimizing overexploitation levels.

Salamanders, particularly CGSs, are more highly esteemed in China than in other regions. CGS meat offers a delicate texture akin to that of fish, rendering it a favored choice for consumers. Its nutritional value and palatability are comparable to those of other terrestrial animals. Table 1 illustrates the nutritional breakdown of CGSs, as documented in previous studies. The variability in their nutritional profiles is influenced by factors such as biodiversity, age, sex, diet, farming methods, breeding practices, and anatomical region (Hu et al., 2019). CGS meat typically comprises moisture (ranging from 79.0% to 82.3%), protein (14.0% to 16.4%), fat (2.5% to 3.5%), and ash (0.7% to 1.1%), although its carbohydrate content remains unreported (Geng et al., 2019). Zhu et al. (2021) noted that while cultural and environmental factors may lead to variations in yield, the fundamental composition, nutritional benefits, and medicinal properties of these plants remain consistent. The nutritional profile of CGS meat aligns closely with that of chicken and fish per serving.

Researchers have studied the secretion of mucus, molting, and protein composition of giant salamander skin and identified 155 proteins in mucus, 97 in molting, and 249 in the skin. Notably, mucus contains 105 unique proteins, second only to the skin, whereas molting has the fewest unique proteins (Pan et al., 2020). Gene Ontology analysis revealed that some proteins play roles in various physiological processes, such as the extracellular matrix, cytoskeleton, cell junctions, cell adhesion, wound healing, immune response, and inflammation (Wang et al., 2023b). Tables 1 and 2 provide further details, and Table 3 compares proteins found in meat, eggs, and milk with those from the giant salamander, highlighting differences between them and common protein sources. However, the proteins isolated and purified from salamander skin mucus remain limited and mainly consist of glycoproteins and lectins. Glycoproteins are compounds formed by the covalent binding of short, branched oligosaccharides and peptide chains (Fairbanks, 2019). They serve as crucial components of cell membranes, plasma, hormones, and the intercellular matrix, contributing significantly to various life processes, inflammatory reactions, abnormal proliferation of cancer cells, and the immune system (Nalehua and Zaia, 2022). The secretion of mucus from giant salamander skin involves multiple processing steps, including hot water extraction, filtration, concentration, the Sevage method to remove free protein, alcohol precipitation, dissolution, dialysis, vacuum freeze-drying, separation, and purification, to obtain a pure glycoprotein matrix. One identified glycoprotein with a molecular weight of 58 kDa is γ-carboxyglutamic acid protein III (MGP III; Jin et al., 2019).

Lectin, a nonimmune-derived glycoprotein or protein, facilitates cell aggregation and glycoconjugate precipitation and is associated with cell growth, differentiation, development, and immunity (Hendrickson and Zherdev, 2018). Wang et al. (2019) successfully extracted a 17 kDa lectin from the mucus produced by giant salamander skin, which is rich in amino acids.

Glycopeptides refer to compounds formed by linking short sugar chains and short peptides. When glycoproteins are hydrolyzed by proteases, they breakdown into various peptide segments with sugar chains, known as glycopeptides, which are often utilized for analyzing their sugar chain structures (Doelman and van Kasteren, 2022). Wang et al. (2023a) employed marine acidic proteases to break down the mucus secreted by giant salamander skin, yielding oligosaccharide peptides with molecular weights less than 3.5 kDa. Additionally, Li et al. (2024) utilized the skin mucus secretion of giant salamanders as the raw material and subjected it to composite enzyme hydrolysis to obtain oligopeptides with molecular weights less than 4 kDa.

Antimicrobial peptides are small peptide compounds that are widely found in natural organisms and are important components of the body’s innate immune system (Zhang and Gallo, 2016). Zhu et al. (2018) used a 5% acetic acid extraction method followed by Sephadex G50/25 gel chromatography to isolate an antimicrobial peptide with a molecular weight of 4.3 kDa from the skin mucus of giant salamanders. Similarly, Yuan Rui used 5% acetic acid extraction coupled with Sephadex G200 gel chromatography to obtain antibacterial peptides with a molecular weight of approximately 10 kDa.

Polysaccharides are derived from the skin mucus of giant salamanders through various extraction methods, including alkaline, acid, water, alkaline, and acidic protease methods. The total sugar content in the extracts ranged from 1.54% to 4.23%, indicating variations based on the extraction process employed. The monosaccharide composition also varies among the mucopolysaccharides obtained through different extraction techniques. Hyaluronic acid, a polymer comprising N-acetylglucoside and D-glucuronic acid disaccharides, is predominantly found in the connective tissues of both humans and animals (Sagbas Suner et al., 2019). Although the structure of hyaluronic acid remains consistent across different sources, differences exist in its relative molecular weight. HA plays crucial roles in cell proliferation and differentiation, alleviating knee joint pain, regulating vascular wall permeability, and protecting the corneal endothelium. Yu Haihui successfully obtained hyaluronic acid through enzymatic hydrolysis and separation using diethylaminoethyl cellulose DE52, yielding 1.7041 mg/g.

The antioxidative properties of natural substances primarily involve inhibiting lipid oxidative degradation, scavenging free radicals, hindering pro-oxidants (such as chelating transition metals), and exhibiting reducing power (Vuong, 2021). In vitro antioxidant assessments conducted on salamander skin-secreted mucus and its derivatives demonstrated significant scavenging potential against superoxide anion radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and hydroxyl radicals. Moreover, this scavenging capacity was observed to increase with concentration (Fu et al., 2024a).

Animal mucus typically exhibits antimicrobial properties. Using the agar disk diffusion method, Wu et al. (2024) evaluated the antimicrobial activity of salamander agglutinin. These findings indicated that salamander agglutinin inhibited the growth of Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Schizosaccharomyces pombe (Wu et al., 2024). In a separate study, Yang et al. (2017) investigated the inhibitory effects of antimicrobial peptide solutions on E. coli, Pseudomonas aeruginosa, S. aureus, Streptococcus hemolyticus, Staphylococcus epidermidis, and Candida albicans via the ring-of-inhibition method. The minimum effective inhibitory concentrations were 12 μg/mL, 16 μg/mL, 16 μg/mL, 22 μg/mL, 16 μg/mL, and 18 μg/mL. Furthermore, Akter et al. (2020) assessed the impact of antimicrobial peptides on Aeromonas hydrophila, Aeromonas sobria, Citrobacter freundii, Edwardsiella tarda, and Hafnia alvei, revealing varying degrees of inhibitory effects on these bacteria.

According to historical records such as the “Collected Notes on the Classic of Materia Medica” compiled by Tao Hongjing from the North and South Dynasties, salamanders have been known for secreting skin mucus for approximately 1,600 years and are traditionally used to treat burns. Additionally, folk remedies often involve salamander skin powder mixed with tung oil for burn treatments (Zheng et al., 2021). Recent studies have confirmed the wound-healing properties of salamander skin mucus. In the experiments, lyophilized mucus powder from giant salamander skin was hydrated to form a gel. This gel exhibited excellent adhesion to adipose tissue when applied between layers of pig skin. Moreover, it demonstrated flexibility suitable for wound contact, as evidenced by a three-point bending test. In a rat skin incision model, the salamander gel effectively promoted skin wound healing and reduced scarring, as observed through histological analysis (Zhai et al., 2020). Compared with conventional surgical bioadhesives such as cellulose glue and cyanoacrylate glue, salamander skin mucus facilitates faster wound healing, boasts greater biocompatibility, and is entirely biodegradable without causing harm to the organism (Deng et al., 2019).

Chen et al. (2021) illustrated that salamander oligopeptides exhibit antiplatelet aggregation properties through a platelet aggregation test utilizing the whole blood resistance method. The hypothesis posits that salamander oligopeptides interact with adenosine diphosphate, consequently impeding its binding to cell surface receptors. This research holds clinical significance for treating conditions such as hypertension, thromboembolic disease, and atherosclerosis associated with increased platelet aggregation (Chen et al., 2021).

Hepatic glycogen and creatine lactate are key indicators for assessing fatigue levels in the body (Zhou and Jiang, 2019). Increased liver glycogen stores are correlated with increased resistance to fatigue, whereas increased lactic acid accumulation indicates increased fatigue. The results revealed a 65% reduction in liver glycogen consumption in the mice in the salamander oligosaccharide peptide group compared with that in the blank control group following weight-bearing swimming fatigue. Additionally, the muscle lactic acid content of the mice was 24.4% lower than that of the blank control group (Zhang et al., 2016a). These data highlight the significant improvement in the anti-fatigue capacity of mice following the administration of salamander oligosaccharide peptide.

Geng et al. (2020) investigated the impact of salamander oligopeptides on liver injury induced by CCL₄. The findings revealed that the middle- and high-dose salamander oligosaccharide peptides notably suppressed the activities of serum ghrelin induced by CCl₄, reduced malondialdehyde levels, and increased hepatic superoxide dismutase activity in mouse liver tissues. Histological examination revealed that hepatocytes in the CCl₄ model group exhibited significant swelling and deformation, disorganized hepatocyte cords, and inflammatory cell infiltration. Conversely, compared with those in the model group, the salamander oligosaccharide peptide dosage groups presented an orderly arrangement of hepatocyte cords, significantly mitigating liver tissue damage (Geng et al., 2020).

The mice were orally administered various doses of salamander oligosaccharide peptide (0.01 g/kg, 0.05 g/kg, or 0.1 g/kg) for 8 days. Serum immunoglobulin IgG levels, macrophage activity in engulfing chicken erythrocytes, and T lymphocyte function were assessed. The findings revealed notable increases in the IgG levels and macrophage counts in the mice that received salamander oligosaccharide peptide compared with those in the control group. These results suggest that giant salamander oligopeptides exert a regulatory influence on mouse immune function (Jiang et al., 2021).

Amphibian skin is an agricultural byproduct that is rich in proteins and lipids and serves as a valuable source for collagen, gelatin, and their hydrolysates. Gelatin and its hydrolysates have been widely used in food products as functional additives or supplements (Yokoyama et al., 2018). BPs derived from these sources have been developed and integrated into food preservatives, edible films, and coatings. Additionally, the incorporation of collagen hydrolysate (CH) into biodegradable films adds further value to the CH in the food supply chain, aligning with sustainability goals (Terauchi et al., 2023). Protein hydrolysates containing BPs contribute to biopolymer films, improving the mechanical properties and extending the shelf-life of packaged foods (Wong and Chai, 2023). BPs present in protein hydrolysates serve as functional ingredients in the food and medicine industries (Wang et al., 2022). For example, gelatin extracted from Asian bullfrog skin has a gel strength comparable to that of bovine gelatin, with studies confirming its safety for human consumption (Karnjanapratum et al., 2017). Gelatin derived from the skin of CGSs enhances foam expansion and stability, making it suitable for novel applications in food emulsion systems (Terauchi et al., 2023). CHs demonstrate bioactivities such as antioxidant and antimicrobial effects, making them suitable as daily food supplements for collagen production or regeneration in the human body. Understanding the bioaccessibility of BPs during digestion is crucial, as peptide modifications occur, generating smaller peptides with various bioactivities (Wang et al., 2023a). Proline-containing peptides, such as CH, are presumed to have high resistance to hydrolysis by digestive enzymes.

In wine production, marine microbial acidic proteases are utilized to generate low-molecular-weight glycopeptides from SSAD. These glycopeptides were incorporated into Cabernet Sauvignon grapes, which were cofermented and aged with brewing yeast to yield giant salamander wine enriched with low-molecular-weight glycopeptides (Zheng et al., 2021). The inclusion of CGS oligosaccharide peptides not only enhances sugar utilization efficiency during yeast fermentation but also enriches wine with 30 aroma components. Furthermore, the CGS oligosaccharide peptide wine exhibited potent scavenging abilities against hydroxyl and superoxide anion radicals.

In the biomedical sector, amphibian skin and secretions hold immense potential for various applications. For example, collagen derived from frog skin and CGSs represents an alternative source for tissue engineering agents (Ong et al., 2021). Collagen exhibits accelerated wound healing capabilities and enhances immunomodulation, demonstrating its function as a biomaterial scaffold. Studies involving alternative wound dressings employing collagen, particularly from marine sources, have demonstrated significant advancements in rapid wound healing in rat models (Wang et al., 2023b). Owing to their diverse bioactivities, the widespread use of collagens extends beyond the food industry to encompass the pharmaceutical sector. The use of collagen from industrial byproducts as an alternative source holds promise for preparing wound dressings (El Masry et al., 2019). Additionally, the BPs found in various frog skins exhibit antimicrobial, anticancer, antidiabetic, and immunomodulatory properties (Cancelarich et al., 2020). This underscores their potential as multifunctional supplements for specific consumer groups, such as elderly individuals or those with chronic diseases. Moreover, future cellular studies utilizing BPs in topical formulations for antiaging pharmaceuticals or as neuroprotectors hold promise for developing therapeutic strategies targeting oxidative stress in neurological disorders (Xia et al., 2021). Evaluations of the anticancer activity of BPs have further expanded their potential as future agents for cancer treatment (Wang et al., 2017). Collaborative formulations of BPs from amphibians with other bioactive compounds could pave the way for the development of novel drugs or serve as effective drug delivery agents.

Scientists have developed a production method for skincare products containing SSAD glycoprotein, determined the fundamental formula for these products, and performed stability assessments on the finalized SSAD glycoprotein skincare items (Chen et al., 2018). These products adhere to quality standards and exhibit notable moisturizing properties. SSAD has antioxidant and antiphotoaging properties, and skincare formulations incorporating SSAD as a primary ingredient have demonstrated notable effectiveness in combating skin aging.

In capacitive material development, SSAD serves as a biological carbon substrate, which is uniformly mixed with cellulose nanocrystals and cellulose nanofibrils. Through unidirectional lyophilization and high-temperature carbonization, researchers have developed honeycomb-structured nanofiber carbon aerogels (Zhang et al., 2016a). Compared with alternative carbon aerogels, those incorporating SSAD-derived materials exhibit remarkable resilience in repeated compression and release evaluations. The carbon aerogel composite exhibited symmetrical properties. In addition, improvements in the electrochemical capacitance performance and cycling stability of the adhesive-free supercapacitor were observed. After 500 cycles of compression and release, the supercapacitor retained a high capacitance, indicating its robustness and electrochemical consistency.

The CGS (A. davidianus s.l.) represents the largest extant amphibian population, reaching lengths of nearly 2 meters. Tracing back to the Middle Jurassic era, they are considered “living fossils.” Despite being critically endangered in the wild, CGSs are abundantly bred on commercial farms. Striking a balance between conservation and utilization is pivotal for their future. The development of bioactive products from these salamanders can bolster the industrial cultivation of the species, thereby contributing to conservation efforts.

Currently, the use of mucus secreted from the skin of giant salamanders remains limited, largely due to insufficient exploration of its physiological mechanisms and functional components. Moreover, the high viscosity of the mucus and the use of traditional extraction methods often lead to low yields of active ingredients. Hence, there is an urgent need to adopt modern extraction and separation techniques to increase the extraction efficiency of individual components and comprehensively analyze the constituents of skin secretions.

Furthermore, by employing genomics, proteomics, and other omics approaches, this study aimed to elucidate the mechanisms underlying the secretion of skin mucus from giant salamanders via cell models, animal models, and clinical studies. These investigations offer theoretical support for the extensive exploration of salamander skin secretions. As a renewable resource with a rich history of medicinal application, SSAD holds promise for the widespread development and promotion of functional skincare products, medical materials, healthy foods, and health supplements in the future.