Antithrombotic activity of brewers’ spent grain peptides before and after simulated gastrointestinal digestion and their effects on blood coagulation pathways were evaluated. Two hydrolysates were produced using sequential enzymatic systems: alkaline protease + Flavourzyme (AF) and neutral protease + Flavourzyme (PF). Simulation of gastrointestinal digestion of AF and PF hydrolysates was made using porcine pepsin and pancreatin enzymes, obtaining the corresponding digested samples: AFD and PFD, respectively. Peptides were fractionated by ultrafiltration using a 1 kDa cut-off membrane. Hydrolysates had peptides with medium and low molecular weights (2100 and 500 Da, respectively), and Glu, Asp, Leu, Ala, and Phe were the most abundant amino acids. Gastrointestinal digested hydrolysates presented high proportion of small peptides (~500 Da), and higher amount of Val, Tyr, and Phe than hydrolysates. Mass spectrum (HDMS Q-TOF) of AFD-ultrafiltered fraction <1 kDa exhibited peptides from 500 to 1000 Da, which are not present in AF. PFD showed the generation of new peptides from 430 to 1070 Da. All samples showed thrombin inhibitory activity. However, no effect was observed on prothrombin time. Peptides <1 kDa from hydrolysates and digested samples delayed thrombin and thromboplastin time respect to the control (~63%). Also the samples showed thrombin inhibitory activity at common pathway level. Thus, brewers’ spent grain peptides exerted their antithrombotic activity by inhibiting the intrinsic and common pathways of blood coagulation. This is the first report to demonstrate that brewers’ spent grain peptides are able to delay clotting time after simulated gastrointestinal digestion.
Multi-enzyme bioactive peptide extraction Antithrombotic peptides ACE I and α-amylase inhibition mechanism In vitro gastrointestinal digestion Brewers’ spent grain
Hydrolysate obtained with alkaline protease during 2 h + Flavourzyme 2 h
Gastrointestinal digested sample from AF
Brewers’ spent grain
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Hydrolysate obtained with neutral protease during 2 h + Flavourzyme 2 h
Gastrointestinal digested sample from PF
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REC, AGG, OMA and CCB carried out the experiment. REC and SRD analyzed the data and wrote the paper, and had primary responsibility for final content. All authors read and approved the final manuscript. The authors are thankful to PICT-2016-2716 and PICT-2016-2879 from ANPCyT for the financial support.
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Conflict of Interest
The authors declare that there are no conflicts of interest.
Human and Animal Studies
This article does not contain any studies with human or animal subjects.
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