Bioactive Earthworm Peptides Produced by Novel Protease-Producing Bacillus velezensis PM 35 and Its Bioactivities on Liver Cancer Cell Death via Apoptosis, Antioxidant Activity, Protection Against Oxidative Stress, and Immune Cell Activation

Abstract

Earthworms have long been used as traditional medicine. The purposes of this research were to create bioactive peptides from the unique Amynthas arenulus earthworm (PAAEs) and test their potentials on liver cancer bioprophylactic activity, antioxidant, oxidative stress protection, and immune cell activation. This earthworm had a high protein content ratio, at 55.39%. Besides, PM 35 is one out of 58 bacteria isolated from the earthworm carcasses that exhibited the highest protease and yield protein production which was chosen as the protease-producing bacteria to hydrolyze the protein. The genera were identified by 16S rRNA and 16S–23S rRNA comparison and confirmed as Bacillus velezensis PM 35. The response surface methodology was applied to optimize these hydrolysis parameters, i.e., the enzyme/substrate (E/S) concentration ratio [1%–3% (v/v)] and time (1–3 h) of the hydrolyzing earthworm’s proteins. The optimal hydrolyzing conditions were 3% (v/v) of E/S concentration ratio and 3 h of hydrolysis time, which found protein-hydrolysate yield (24.62%) and degree of hydrolysis (85.45%) as the highest. After being challenged in the gastrointestinal tract-resistant model, these PAAEs (MW <3 and 3–5 kDa) induced liver cancer cell (HepG2) death via apoptotic action modes (cell morphological change and DNA fragmentation). The PAAEs (MW <3 kDa) exhibited significant antioxidant activity via DPPH, ABTS, and FRAP with IC50 values of 0.94, 0.44, and 6.34 mg/ml, respectively. The PAAEs (MW < 3 kDa) were non-cytotoxic and protected the mouse fibroblast cells (L929) against oxidative stress. These PAAEs (MW < 3 kDa, 0.2 mg/ml) stimulated the B lymphocytes (122.3%), and T lymphocytes (126.7%) proliferation. This research suggests that PAAEs can be used in a variety of applications, especially in the food and pharmaceutical industries.

Potential bioactivities via anticancer, antioxidant, and immunomodulatory properties of cultured mycelial enriched β-D-glucan polysaccharides from a novel fungus Ophiocordyceps sinensis OS8

“…DNA fragmentation in HT-29 cells treated with OS8P at 200 µg/ml was assessed using a modified method based on Wasunan et al. ( 24 ). Cells were seeded in 6-well plates, treated with either 1 nM Paclitaxel or 200 µg/ml of OS8P, and incubated for 48 h. Genomic DNA was extracted, and electrophoresis was performed on a 1.5% agarose gel for 45 minutes at 80 V.…”

Effect of earthworm and papain enzyme concentration on antioxidant activity of earthworm (Eudrilus eugeniae) protein hydrolysate

“…According to Halim et al (2018), amino acids in protein hydrolysates are critical water-binding capacity, emulsifying properties, solubility, and fat binding capacity. In addition, the amino acid composition in earthworm hydrolysate is important in the antioxidant activity (Wasunan et al, 2022). Table 5 shows that protein hydrolysate using enzymes can produce higher amino acid content than without adding enzymes.…”

Research advance of Bacillus velezensis: bioinformatics, characteristics, and applications

“…T h e p r o t e a s e -p r o d u c i n g s t r a i n s B . v e l e z e n s i s B 1 6 a n d B. velezensis PM 35 were employed in fermentation for polypeptide production [17][18] . Therefore, B. velezensis has the potential to produce a range of high-value products from marine proteins, including fermented food, proteases, and bioactive peptides.…”