Plant Foods for Human Nutrition

, Volume 73, Issue 3, pp 241–246 | Cite as

Antithrombotic Activity of Brewers’ Spent Grain Peptides and their Effects on Blood Coagulation Pathways

  • Raúl E. CianEmail author
  • Antonela G. Garzón
  • Olga Martínez-Augustin
  • Cecilia C. Botto
  • Silvina R. Drago
Original Paper


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 



Angiotensin-converting enzyme


Hydrolysate obtained with alkaline protease during 2 h + Flavourzyme 2 h


Gastrointestinal digested sample from AF


Brewers’ spent grain


Electronic supplementary material


Hydrolysate obtained with neutral protease during 2 h + Flavourzyme 2 h


Gastrointestinal digested sample from PF



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.

Compliance with Ethical Standards

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.

Supplementary material

11130_2018_682_MOESM1_ESM.docx (115 kb)
ESM 1 (DOCX 115 kb)


  1. 1.
    Vieira E, Angélica M, Rocha M, Coelho E, Pinho O, Saraivab J, Ferreira I, Coimbra M (2014) Valuation of brewer’s spent grain using a fully recyclable integrated process for extraction of proteins and arabinoxylans. Ind Crop Prod 52:136–143CrossRefGoogle Scholar
  2. 2.
    McCarthy A, O’Callaghan Y, Connolly A, Piggott C, FitzGerald R, O’Brien N (2013) In vitro antioxidant and anti-inflammatory effects of brewers' spent grain protein rich isolate and its associated hydrolysates. Food Res Int 50:205–212CrossRefGoogle Scholar
  3. 3.
    Vieira E, Teixeira J, Ferreira I (2016) Valorization of brewers’ spent grain and spent yeast through protein hydrolysates with antioxidant properties. Eur Food Res Technol 242:1975–1984CrossRefGoogle Scholar
  4. 4.
    Mussatto S, Dragone G, Roberto I (2006) Brewers’ spent grain: generation, characteristics and potential applications. J Cereal Sci 43:1–14CrossRefGoogle Scholar
  5. 5.
    Clemente A (2000) Enzymatic protein hydrolysates in human nutrition. Trends Food Sci Technol 11:254–262CrossRefGoogle Scholar
  6. 6.
    Connolly A, Piggott C, FitzGerald R (2014) In vitro α-glucosidase, angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory properties of brewers' spent grain protein hydrolysates. Food Res Int 56:100–107CrossRefGoogle Scholar
  7. 7.
    Connolly A, O’Keeffe M, Nongonierma A, Piggott C, FitzGerald R (2017) Isolation of peptides from a novel brewers spent grain protein isolate with potential to modulate glycaemic response. Int J Food Sci Technol 52:146–153CrossRefGoogle Scholar
  8. 8.
    Kotlar C, Ponce A, Roura S (2013) Improvement of functional and antimicrobial properties of brewery byproduct hydrolysed enzymatically. LWT-Food Sci Technol 50:378–385CrossRefGoogle Scholar
  9. 9.
    Connolly A, O'Keeffe M, Piggott C, Nongonierma A, FitzGerald R (2015) Generation and identification of angiotensin converting enzyme (ACE) inhibitory peptides from a brewers' spent grain protein isolate. Food Chem 176:64–71CrossRefPubMedGoogle Scholar
  10. 10.
    Crowley D, O'Callaghan Y, McCarthy A, Connolly A, Piggott C, FitzGerald R, O'Brien N (2015) Immunomodulatory potential of a brewers' spent grain protein hydrolysate incorporated into low-fat milk following in vitro gastrointestinal digestion. Int J Food Sci Nutr 66:672–676CrossRefPubMedGoogle Scholar
  11. 11.
    Vieira E, das Neves J, Vitorino R, Dias da Silva D, Carmo H, Ferreira I (2016) Impact of in vitro gastrointestinal digestion and transepithelial transport on antioxidant and ACE-inhibitory activities of brewer's spent yeast autolysate. J Agric Food Chem 64:7335–7341CrossRefPubMedGoogle Scholar
  12. 12.
    Connolly A, Piggott C, FitzGerald R (2014) Technofunctional properties of a brewers' spent grain protein-enriched isolate and its associated enzymatic hydrolysates. LWT-Food Sci Technol 59:1061–1067CrossRefGoogle Scholar
  13. 13.
    Niemi P, Martins D, Buchert J, Faulds C (2013) Pre-hydrolysis with carbohydrases facilitates the release of protein from brewer’s spent grain. Bioresour Technol 136:529–534CrossRefPubMedGoogle Scholar
  14. 14.
    Sabbione A, Scilingo A, Añón M (2015) Potential antithrombotic activity detected in amaranth proteins and its hydrolysates. LWT-Food Sci Technol 60:171–177CrossRefGoogle Scholar
  15. 15.
    Rojas-Ronquillo R, Cruz-Guerrero A, Flores-Nájera A, Rodríguez-Serrano G, Gómez-Ruiz L, Reyes-Grajeda J, Jiménez-Guzmán J, García-Garibay M (2012) Antithrombotic and angiotensin-converting enzyme inhibitory properties of peptides released from bovine casein by Lactobacillus casei Shirota. Int Dairy J 26:147–154CrossRefGoogle Scholar
  16. 16.
    Jung W, Kim S (2009) Isolation and characterisation of an anticoagulant oligopeptide from blue mussel, Mytilus edulis. Food Chem 117:687–692CrossRefGoogle Scholar
  17. 17.
    Kong Y, Li S, Shao Y, He Z, Chen M, Ming X, Wei J (2014) Antithrombotic peptides from Scolopendra subspinipes mutilans hydrolysates. Int J Pept Res Ther 20:245–252CrossRefGoogle Scholar
  18. 18.
    Sabbione A, Ibañez S, Martínez E, Añón A, Scilingo A (2016) Antithrombotic and antioxidant activity of amaranth hydrolysate obtained by activation of an endogenous protease. Plant Foods Hum Nutr 71:174–182CrossRefPubMedGoogle Scholar
  19. 19.
    Jo HY, Jung WK, Kim SK (2008) Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm Urechis unicinctus. Process Biochem 43:179–184CrossRefGoogle Scholar
  20. 20.
    Roškar I, Štrukelj B, Lunder M (2016) Screening of phenolic compounds reveals inhibitory activity of nordihydroguaiaretic acid against three enzymes involved in the regulation of blood glucose level. Plant Foods Hum Nutr 71:88-89CrossRefPubMedGoogle Scholar
  21. 21.
    Mullally M, O'Callaghan D, FitzGerald R, Donnelly W, Dalton J (1994) Proteolytic and peptidolytic activities in commercial pancreatic protease preparations and their relationship to some whey protein hydrolyzate characteristics. J Agric Food Chem 42:2973–2981CrossRefGoogle Scholar
  22. 22.
    Jao C, Huang S, Hsu K (2012) Angiotensin I-converting enzyme inhibitory peptides: inhibition mode, bioavailability, and antihypertensive effects. Biomed 2:130–136CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Raúl E. Cian
    • 1
    Email author
  • Antonela G. Garzón
    • 1
  • Olga Martínez-Augustin
    • 2
  • Cecilia C. Botto
    • 3
  • Silvina R. Drago
    • 1
  1. 1.Instituto de Tecnología de Alimentos, CONICET, FIQ – UNLSanta FeArgentina
  2. 2.Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibsUniversity of GranadaGranadaSpain
  3. 3.Facultad de Bioquímica y Ciencias Biológicas – UNLSanta FeArgentina

Personalised recommendations