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Bioactive Earthworm Peptides Produced by Novel Protease-Producing Bacillus velezensis PM 35 and Its Bioactivities on
Pimphan Wasunan1, Chutamas Maneewong1, Wichittra Daengprok2
1Program in Biotechnology, Faculty of Science, Maejo University, Chiang Mai, Thailand.
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.
Insights
Bioactive peptides from Amynthas arenulus earthworms (PAAEs) show promise for liver cancer prevention, antioxidant effects, and immune cell activation. These peptides, derived using Bacillus velezensis PM 35, demonstrate significant therapeutic potential.
Area of Science:
- Biochemistry
- Pharmacology
- Immunology
Background:
- Earthworms (Amynthas arenulus) possess high protein content (55.39%) and traditional medicinal uses.
- Bacillus velezensis PM 35, isolated from earthworm carcasses, is a potent protease-producing bacterium.
- Bioactive peptides derived from natural sources are increasingly explored for therapeutic applications.
Purpose of the Study:
- To create bioactive peptides from Amynthas arenulus earthworm (PAAEs).
- To evaluate PAAEs for liver cancer bioprophylactic activity, antioxidant properties, oxidative stress protection, and immune cell activation.
- To optimize protein hydrolysis using response surface methodology.
Main Methods:
- Proteins from Amynthas arenulus were hydrolyzed using Bacillus velezensis PM 35 protease.
- Response surface methodology optimized enzyme/substrate ratio (3% v/v) and hydrolysis time (3 h).
- PAAEs were tested for anticancer activity (HepG2 cells), antioxidant capacity (DPPH, ABTS, FRAP), oxidative stress protection (L929 cells), and immune cell proliferation (B and T lymphocytes).
Main Results:
- Optimal hydrolysis yielded 24.62% protein hydrolysate with 85.45% degree of hydrolysis.
- PAAEs (<3 and 3-5 kDa) induced apoptosis in liver cancer cells (HepG2).
- PAAEs (<3 kDa) demonstrated significant antioxidant activity (IC50 values: DPPH 0.94, ABTS 0.44, FRAP 6.34 mg/ml), protected fibroblast cells from oxidative stress, and stimulated B and T lymphocyte proliferation.
Conclusions:
- PAAEs exhibit potent anticancer, antioxidant, and immunomodulatory activities.
- PAAEs are non-cytotoxic and protect against oxidative stress.
- PAAEs hold significant potential for applications in the food and pharmaceutical industries.
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