Few studies are concerned with the effect of the conjugat protein on the bioactivities of the abalone gonad polysaccharide (AGP). In this study, a series of treatments, including raw material (female and male) defatting, extraction temperature (25–121 °C), proteolysis, ultrafiltration, and ethanol precipitation, was conducted to investigate the role of the conjugate protein on AGP anticoagulant activity. All AGP extracts significantly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT). The strongest was observed in the female AGPs prepared at 50 and 121 °C. The most active is located at 30–300 kDa by ultrafiltration. After being exposed to neutral protease, quick shortening of APTT and TT was found in all AGPs. Further ethanol precipitating of found the longest APTT in the sediment, which contains most polysaccharides and proteins. Defatting lowered the activity of female AGP but increased that of males. Proteolysis also significantly weakened the clotting factor inhibition effect of the 50 °C female AGP, but heating seemed not affect the effect. Five fractions were obtained after the 50 °C female AGP was subjected to ion exchange column. Fraction V, with the highest protein and medium polysaccharide content, showed the strongest anticoagulant effect and was also much higher than AGSP, which was obtained by multi-step proteolysis. The findings supported positive effect of the conjugate protein in AGP anticoagulant activity.

Autolysis in sea cucumber has long been a threat to raw material storage and product processing. The involvement of endogenous cysteine protease in sea cucumber autolysis has been proved extendedly. However, as an essential part of the mechanism of autolysis, the role of its endogenous inhibitor has seldom been reported. To investigate the role of cysteine protease inhibitors in the early stage of hypoxic exposure-induced autolysis, a novel cystatin gene (SjCyt) belonging to the subfamily of cystatin C was cloned from Apostichopus japonicus by homology cloning and rapid amplification of cDNA ends. The affinity of SjCyt to cysteine protease (cathepsin L and cathepsin B) was investigated by molecular dynamics simulations. Pertinent metrics, including the root mean square deviation, radius of gyration, Gibbs free energy, binding free energy, and bond-forming frequency, showed that the conformation of SjCyt–SjCL was more stable and confirmed a stronger interaction of SjCyt with cathepsin L than with cathepsin B. Thus, cathepsin L (SjCL) was selected to further study its co-expression with SjCyt over a period of 9 h at an early stage of hypoxic exposure. Quantitative RT-qPCR revealed a ubiquitous transcriptional profile of SjCyt and SjCL in all the tested tissues, with the highest abundance in the dorsal epidermis, tube feet, and coelomocytes. Temporal transcription of them showed an overall up-regulated co-expression in the dorsal epidermis and tube feet. However, up-regulated SjCyt and down-regulated SjCL were observed at the protein level. Further immunofluorescence double labeling also found increased staining of SjCyt and SjCyt–SjCL complexes and decreased SjCL. Additionally, recombinant SjCyt was prepared and demonstrated an evident autolysis-inhibiting effect. The results of this study indicated that the anti-autolytic regulation of SjCyt functions at the very early stage of hypoxic exposure, exerting effects at both the transcriptional and translational levels. The above finding offers new insights into the mechanisms of sea cucumber autolysis.

Collagen-derived products are widely applied in functional foods; however, limited information is available regarding how different preparation methods, particularly thermal degradation and enzymatic hydrolysis, affect their anti-aging efficacy and biological functions. In this study, sturgeon skin was used as raw material to prepare gelatin–peptide complexes via thermal degradation (GPC-TD) and enzymatic hydrolysis (GPC-EH), and their comparative anti-aging and biological effects were evaluated in D-galactose-induced aging mice. Female ICR mice were divided into eight groups: a blank control group (normal saline), an aging model group (D-galactose, 500 mg/kg), three GPC-TD and three GPC-EH groups (D-galactose supplemented with 100, 200, 400 mg/kg GPC-TD or GPC-EH). After eight weeks of administration, various physiological parameters were evaluated. Throughout the experiment, no statistically significant difference in body weight (BW) was observed among the groups; however, the blank and model groups consistently maintained the highest BW. The medium- and high-dose GPC-TD groups showed relatively faster weight gain, whereas the 100 mg/kg GPC-TD group and all three GPC-EH groups exhibited the slowest BW gain. Notably, the gastric indices of these latter groups were significantly lower than those of other groups (p < 0.05), which might be a key factor affecting BW gain. Behavioral tests revealed that the model group exhibited significantly reduced swimming speed and weakened nesting ability (p < 0.05), both of which were alleviated to varying degrees by treatment with GPC-TD and GPC-EH. Furthermore, both complexes markedly decreased malondialdehyde content in liver tissue (p < 0.05). Compared with the model group, high-dose GPC-TD and GPC-EH effectively increased acetylcholine content and inhibited acetylcholinesterase activity (p < 0.05). Masson staining revealed abnormal collagen fibers accumulation in certain tissues of model mice, a condition that was clearly ameliorated by GPC-TD and, to a greater extent, by GPC-EH. In addition, medium and high doses of both complexes significantly protected against D-galactose-induced loss of Nissl bodies in brain neurons; in the high-dose GPC-EH group, the density and number of Nissl bodies approached those observed in the blank group. These findings suggest that both GPC-TD and GPC-EH possess potential anti-aging effects, with GPC-EH exhibiting superior efficacy. This study provides theoretical support for consumers, the catering industry, and manufacturers in selecting appropriate processing techniques for the preparation of sturgeon skin GPC.