Purification, Structure, and In vitro Antioxidant Activity of Tartary Buckwheat Polypeptides
Buckwheat, also known as black wheat, buckwheat, triangle wheat, etc., belongs to the family Polygonaceae and the genus Buckwheat. It is a dicotyledonous plant that originated in China and is one of the earliest cultivated crops in the world. It is also a unique small grain crop in China that combines food and medicine. According to the Compendium of Materia Medica, bitter buckwheat has a bitter, mild, and cold taste, and has the functions of reducing qi, broadening the intestines, sustaining the spirit, promoting qi circulation, and removing blood stasis. Previous studies have shown that the crude peptide extracts obtained from bitter buckwheat grains and their protein hydrolysis have various biological activities, such as cholesterol lowering, cancer and tumor prevention, hyperlipidemia prevention, and antioxidant effects.
Bioactive peptides have multiple biological functions, which are closely related to their amino acid composition and sequence. Therefore, the key to studying the efficacy of peptides is to identify their structure. Separation and purification is a crucial step in studying the relationship between peptide structure and efficacy. Commonly used peptide separation and purification techniques include chromatography, membrane separation, electrophoresis, etc. Among them, chromatography technology is the most widely used. Due to the complex composition of protein hydrolysates, multiple methods are often used in combination for separation and purification to achieve peptide separation and enrichment. Previous studies have shown that tartary buckwheat protein or enzymatic hydrolysis products have various biological activities, but their biological activity mechanisms are not yet very clear, and there are few reports on the structural analysis and biological activity of tartary buckwheat active peptides. Ma et al. isolated and identified four antioxidant peptides (WPL, VPW, VFPW, and PW) from digested buckwheat protein, which have stronger ability to scavenge ABTS free radicals than buckwheat protein mixtures. Luo used alkaline protease to hydrolyze tartary buckwheat protein to obtain tartary buckwheat protein hydrolysate. After ultrafiltration separation, anion exchange chromatography, and reverse high-performance liquid chromatography, the active peptide component of tartary buckwheat was successfully separated, and its amino acid sequence was obtained as Ala Phe Tyr Arg Trp.
This study used the waste residue of functional extract of tartary buckwheat as raw material to extract crude protein of tartary buckwheat and prepared tartary buckwheat peptides by fermentation with plant lactobacillus. The obtained tartary buckwheat polypeptide fermentation broth was purified by macroporous adsorption resin, dextran gel, and liquid chromatography, and its antioxidant activity was determined. The structure of the selected parts with strong antioxidant activity was identified, and the main source of the antioxidant activity of the crude extract of tartary buckwheat polypeptide was explored, in order to provide a theoretical basis for further research of tartary buckwheat polypeptide and its application in functional food and health products.

 

After Sephadex G-15 glucan gel separation and purification, the ABTS and DPPH free radical scavenging capacity of T-3 component was the best, and the scavenging rates were 96% and 94% respectively. The tartary buckwheat polypeptide purified by macroporous adsorption resin has a strong inhibitory effect on DPPH and ABTS, similar to the T-3 component separated and purified by gel, and shows significant antioxidant activity.
Material identification was carried out on the T-3-1 component separated by liquid chromatography, and a total of 249 proteins and 2324 peptides were identified from the T-3-1 component. The identified peptides mostly consist of 10-15 amino acids and belong to the length range of biologically active peptides. According to the identification results of T-3-1 component mass spectrometry, the amino acid sequences of the main antioxidant peptides are phenylalanine proline tyrosine (Phe Pro Tyr, FPY) and tyrosine leucine proline phenylalanine (Tyr Leu Pro Phe, YLPF).
The antioxidant capacity of tartary buckwheat peptides varies with different purities. After further separation and purification of the T-3 component by liquid chromatography, component T-3-1 was obtained. Although the purity of tartary buckwheat peptides reached 98%, the inhibition rates of DP-PH and ABTS were no longer enhanced, but slightly decreased, with inhibition rates of 89% and 90%, respectively. The antioxidant activity of tartary buckwheat peptides prepared by fermentation method may not be fully positively correlated with peptide purity. When the purity is increased to a certain extent, the antioxidant activity of peptides actually decreases