Discover innovative antitumor peptides from pea protein hydrolysis, offering new cancer therapy insights through enzymatic extraction and purification techniques.

Key words: Pea peptides, Antitumor activity, Enzymatic preparation, Isolation and purification, Structure identification

Abstract

This study focuses on the extraction and identification of antitumor peptides from pea protein through enzymatic hydrolysis, followed by an evaluation of their antitumor activities against various human cancer cell lines. The process involves pea protein’s enzymatic hydrolysis, isolation, and purification of peptides, structural identification, and assessment of antitumor activity, offering insights into potential therapeutic applications.

Materials and Methods

Experimental Materials

Pea protein powder was obtained commercially, and both alkaline protease and trypsin were purchased from Solabao Biotech, Beijing. The cell lines used in the experiments included human liver cancer (HepG2), breast cancer (MCF-7), stomach cancer (MGC803), lung cancer (A549), and normal liver (L-02) cells.

Main Experimental Reagents

The study utilized various analytical grade reagents including glucose, lactose, yeast extract, sodium phosphate dibasic, beef extract, Tween 80, magnesium chloride, potassium dihydrogen phosphate, sodium chloride, proteinase K, agar powder, ethanol, sulfuric acid, magnesium sulfate, magnesium turnings, glycerol, sodium phosphate dibasic, calcium chloride, potassium chloride, formaldehyde, fetal bovine serum (FBS), DMEM high-glucose culture medium, EMEM culture medium, 1640 culture medium, trypsin digestion solution, PBS, MTT, dimethyl sulfoxide (DMSO), and 5-fluorouracil (5-FU).

Main Instruments and Equipment

The experiment utilized a wide range of instruments including an autoclave, clean bench, pH meter, temperature-controlled oscillation incubator, water bath, electronic balance, anaerobic incubator, drying oven, ultra-low temperature freezer, ice maker, refrigerator, high-speed refrigerated centrifuge, CO2 incubator, biosafety cabinet, full-wavelength enzyme marker, liquid chromatograph, and automatic fraction collector.

Experimental Procedure

Pea Protein Hydrolysis Process

The pea protein was enzymatically hydrolyzed to obtain peptide fractions. The hydrolysis process included pre-treating pea protein powder in deionized water, adjusting the pH, and adding alkaline protease for reaction. Post-reaction, the mixture was heated to deactivate the enzyme, centrifuged to separate the supernatant, and then lyophilized to obtain pea peptide powder.

Determination of Hydrolysis Degree and Peptide Yield

The degree of hydrolysis was calculated based on the amount of free amino nitrogen using the formol titration method, while the peptide yield was determined using the TCA-soluble nitrogen content method.

Optimization of Hydrolysis Conditions

Single-factor experiments were conducted to optimize the hydrolysis conditions, including temperature, pH, substrate concentration, and enzyme-to-substrate ratio. An orthogonal experiment was designed based on these single-factor experiments to determine the optimal hydrolysis conditions.

Antitumor Activity of Pea Peptides

The antitumor activity of the peptides was evaluated using cell revival, culturing, passage, and freezing techniques on various human cancer and normal cell lines. The MTT assay method was used to measure the inhibition rate against cancer cells.

Results and Discussion

Enzymatic Preparation of Pea Peptides

The enzymatic hydrolysis process was optimized to enhance the yield and activity of peptides. The optimized conditions were determined to effectively release peptides with potential antitumor properties.

Antitumor Activity Analysis

The peptides exhibited significant antitumor activity against the tested cancer cell lines. The MTT assay results demonstrated the peptides’ effectiveness in inhibiting cancer cell proliferation, with varying degrees of potency across different cell lines.

Peptide Isolation and Purification

Peptides were separated using ultrafiltration membranes and further purified by gel chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), resulting in fractions with high antitumor activity.

Structural Identification of Antitumor Peptides

The structures of active peptides were identified using LC-ESI-MS/MS analysis, and potential active peptide sequences were synthesized for further validation of antitumor activity.

Conclusion

This study successfully demonstrates the feasibility of producing antitumor peptides from pea protein through enzymatic hydrolysis. The identified peptides show promising antitumor activities, providing a foundation for future research into their therapeutic applications. Further studies on the mechanisms of action and in vivo efficacy of these peptides are warranted to explore their potential as cancer therapeutics.

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Original authors: Pan Fen, Wang Yanping, Mo Zhaohui (Food Engineering, Tianjin University of Science and Technology, Tianjin, 300457)

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