Food and Bioprocess Technology

, Volume 8, Issue 2, pp 287–300 | Cite as

Production of Antioxidant Egg White Hydrolysates in a Continuous Stirred Tank Enzyme Reactor Coupled with Membrane Separation Unit

  • Sonja Jakovetić
  • Nevena Luković
  • Branimir Jugović
  • Milica Gvozdenović
  • Sanja Grbavčić
  • Jelena Jovanović
  • Zorica Knežević-Jugović
Original Paper


The objective of this research was to design an efficient continuously operated membrane reactor with a separation unit for egg white protein (EWP) hydrolysis and production of hydrolysates with improved antioxidant properties. For this purpose, a mechanically stirred tank reactor coupled with the polyethersulfone ultrafiltration module with a molecular weight cutoff of 10 kDa was employed. Several proteolytic enzymes have been tested in order to obtain the best quality of peptide-based formulations intended for human consumption. Among protease from Bacillus licheniformis (Alcalase), protease from Bacillus amyloliquefaciens (Neutrase), and protease from papaya latex (papain), the highest degree of hydrolysis (DH), as well as the best antioxidant properties of obtained hydrolysates, was achieved with Alcalase. The effects of operating variables such as enzyme/substrate ([E]/[S]) ratio, impeller speed, and permeate flow rate were further studied using response surface methodology (RSM) and Box–Behnken experimental design. Results obtained in RSM analysis confirmed that over the studied range [E]/[S] ratio, impeller speed and permeate flow rate had the significant effect on the DH and reactor capacity. The effects of different impeller geometries were also studied and four-bladed propeller stirrer enabled the highest DH. Antioxidant properties were analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), by the 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical scavenging activity, and by the linear voltammetry methods. Results show that the use of Alcalase in the membrane reactor system is of potential interest for the EWP hydrolysis and obtaining value-added egg products.


Egg white protein hydrolysis Proteases Continuous membrane reactor Polyethersulfone ultrafiltration module Response surface methodology Antioxidant properties 



This work was supported by Grant numbers E!6750 and III 46010 from the Ministry of the Education, Science and Technological Development, Republic of Serbia. The authors would like to thank the project partner Jata Emona d.o.o. (Ljubljana, Slovenia) for providing us with pasteurized egg white samples.


  1. Cheison, S. C., Wang, Z., & Xu, S.-Y. (2007). Use of response surface methodology to optimise the hydrolysis of whey protein isolate in a tangential flow filter membrane reactor. Journal of Food Engineering, 80(4), 1134–1145.CrossRefGoogle Scholar
  2. Chen, J., Gorton, L., & Åkesson, B. (2002). Electrochemical studies on antioxidants in bovine milk. Analytica Chimica Acta, 474(1–2), 137–146.CrossRefGoogle Scholar
  3. Chen, C., Chi, Y.-J., & Xu, W. (2012). Comparisons on the functional properties and antioxidant activity of spray-dried and freeze-dried egg white protein hydrolysate. Food and Bioprocess Technology, 5(6), 2342–2352.CrossRefGoogle Scholar
  4. Chiang, W.-D., Shih, C.-J., & Chu, Y.-H. (1999). Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system. Food Chemistry, 65(2), 189–194.CrossRefGoogle Scholar
  5. Chiang, W.-D., Tsou, M.-J., Tsai, Z.-Y., & Tsai, T.-C. (2006). Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 98(4), 725–732.CrossRefGoogle Scholar
  6. Cigić, B., & Zelenik-Blatnik, M. (2004). Preparation and characterization of chicken egg white hydrolysate. Acta Chimica Slovenica, 51(1), 177–188.Google Scholar
  7. Damrongsakkul, S., Ratanathammapan, K., Komolpis, K., & Tanthapanichakoon, W. (2008). Enzymatic hydrolysis of rawhide using papain and neutrase. Journal of Industrial and Engineering Chemistry, 14(2), 202–206.CrossRefGoogle Scholar
  8. de Oliveira, C. F., Corrêa, A. P. F., Coletto, D., Daroit, D. J., Cladera-Olivera, F., & Brandelli, A. (2014). Soy protein hydrolysis with microbial protease to improve antioxidant and functional properties. Journal of Food Science and Technology. doi: 10.1007/s13197-014-1317-7.Google Scholar
  9. Demirhan, E., Apar, D. K., & Özbek, B. (2010). Sesame cake protein hydrolysis by alcalase: effects of process parameters on hydrolysis, solubilisation, and enzyme inactivation. Korean Journal of Chemical Engineering, 28(1), 195–202.CrossRefGoogle Scholar
  10. Gorjanović, S. Z., Novaković, M. M., Vukosavljević, P. V., Pastor, F. T., Tešević, V. V., & Sužnjević, D. Z. (2010). Polarographic assay based on hydrogen peroxide scavenging in determination of antioxidant activity of strong alcohol beverages. Journal of Agricultural and Food Chemistry, 58(14), 8400–8406.CrossRefGoogle Scholar
  11. Goswami, D., De, S., & Basu, J. K. (2012). Effects of process variables and additives on mustard oil hydrolysis by porcine pancreas lipase. Brazilian Journal of Chemical Engineering, 29, 449–460.CrossRefGoogle Scholar
  12. Guadix, A., Camacho, F., & Guadix, E. M. (2006). Production of whey protein hydrolysates with reduced allergenicity in a stable membrane reactor. Journal of Food Engineering, 72(4), 398–405.CrossRefGoogle Scholar
  13. Jakovetić, S. M., Jugović, B. Z., Gvozdenović, M. M., Bezbradica, D. I., Antov, M. G., Mijin, D. Z., & Knezevic-Jugovic, Z. D. (2013a). Synthesis of aliphatic esters of cinnamic acid as potential lipophilic antioxidants catalyzed by lipase B from Candida antarctica. Applied Biochemistry and Biotechnology, 170(7), 1560–1573.CrossRefGoogle Scholar
  14. Jakovetić, S. M., Luković, N. D., Bošković-Vragolović, N. M., Bezbradica, D. I., Picazo-Espinosa, R., & Knezevic-Jugovic, Z. D. (2013b). Comparative study of batch and fluidized bed bioreactors for lipase-catalyzed ethyl cinnamate synthesis. Industrial and Engineering Chemistry Research, 52(47), 16689–16697.CrossRefGoogle Scholar
  15. Jin, T., Li, W., & Wu, Y. (2012). Production and characteristics of protein hydrolysates from little hairtail (Trichiurus haumela) of East China Sea. Journal of Food, Agriculture & Environment, 10(3&4), 81–85.Google Scholar
  16. Jing, H., Yap, M., Wong, P. Y., & Kitts, D. (2011). Comparison of physicochemical and antioxidant properties of egg-white proteins and fructose and inulin Maillard reaction products. Food and Bioprocess Technology, 4(8), 1489–1496.CrossRefGoogle Scholar
  17. Kedare, S., & Singh, R. P. (2011). Genesis and development of DPPH method of antioxidant assay. Journal of Food Science and Technology, 48(4), 412–422.CrossRefGoogle Scholar
  18. Kim, S. B., Seo, I. S., Khan, M. A., Ki, K. S., Nam, M. S., & Kim, H. S. (2007). Separation of iron-binding protein from whey through enzymatic hydrolysis. International Dairy Journal, 17(6), 625–631.CrossRefGoogle Scholar
  19. Knežević-Jugović, Z. D., Stefanović, A. B., Žuža, M. G., Milovanović, S. L., Jakovetić, S. M., Manojlović, V. B., & Bugarski, B. M. (2012). Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins. Acta Periodica Technologica, 43, 33–41.CrossRefGoogle Scholar
  20. Laohakunjit, N., Selamassakul, O., & Kerdchoechuen, O. (2014). Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria sp.). Food Chemistry, 158, 162–170.CrossRefGoogle Scholar
  21. Li, X., Lin, J., Gao, Y., Han, W., & Chen, D. (2012). Antioxidant activity and mechanism of Rhizoma cimicifugae. Chemistry Central Journal, 6(1), 140.CrossRefGoogle Scholar
  22. Li-Chan, E., & Nakai, S. (1989). Biochemical basis for the properties of egg white. Critical Reviews in Poultry Biology, 2, 21–58.Google Scholar
  23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265–275.Google Scholar
  24. Martos, G., López-Fandiño, R., & Molina, E. (2013). Immunoreactivity of hen egg allergens: influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk. Food Chemistry, 136(2), 775–781.CrossRefGoogle Scholar
  25. Nouri, L., Legrand, J., Popineau, Y., & Belleville, P. (1997). Enzymatic hydrolysis of wheat proteins. Part I. Enzymatic kinetics and study of limited hydrolysis in a batch stirred reactor. Chemical Engineering Journal, 65(3), 187–194.CrossRefGoogle Scholar
  26. Ou, K., Liu, Y., Zhang, L., Yang, X., Huang, Z., Nout, M. J. R., & Liang, J. (2010). Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells. Journal of Agricultural and Food Chemistry, 58(8), 4894–4900.CrossRefGoogle Scholar
  27. Prieto, C. A., Guadix, A., González-Tello, P., & Guadix, E. M. (2007). A cyclic batch membrane reactor for the hydrolysis of whey protein. Journal of Food Engineering, 78(1), 257–265.CrossRefGoogle Scholar
  28. Prieto, C. A., Guadix, E. M., & Guadix, A. (2008). Influence of temperature on protein hydrolysis in a cyclic batch enzyme membrane reactor. Biochemical Engineering Journal, 42(3), 217–223.CrossRefGoogle Scholar
  29. Prieto, C. A., Guadix, E. M., & Guadix, A. (2010). Optimal operation of a protein hydrolysis reactor with enzyme recycle. Journal of Food Engineering, 97(1), 24–30.CrossRefGoogle Scholar
  30. Qu, W., Ma, H., Zhao, W., & Pan, Z. (2013). ACE-inhibitory peptides production from defatted wheat germ protein by continuous coupling of enzymatic hydrolysis and membrane separation: modeling and experimental studies. Chemical Engineering Journal, 226, 139–145.CrossRefGoogle Scholar
  31. Radzi S. M., Mohamad R., Basri M., Salleh A. B., Ariff A., Rahman M. B. A., & Rahman R. N. Z. R. A. (2010) Kinetics of Enzymatic Synthesis of Liquid Wax Ester from Oleic Acid and Oleyl Alcohol. Journal of Oleo Science, 59(3), 127–134.Google Scholar
  32. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9–10), 1231–1237.CrossRefGoogle Scholar
  33. Ren, Y., Wu, H., Li, X., Lai, F., Zhao, G., & Xiao, X. (2014). A two-step, one-pot enzymatic method for preparation of duck egg white protein hydrolysates with high antioxidant activity. Applied Biochemistry and Biotechnology, 172(3), 1227–1240.CrossRefGoogle Scholar
  34. Ruan, C.-Q., Chi, Y.-J., & Zhang, R.-D. (2010). Kinetics of hydrolysis of egg white protein by pepsin. Czech Journal of Food Sciences, 28, 355–363.Google Scholar
  35. Sáez-Plaza, P., Michałowski, T., Navas, M. J., Asuero, A. G., & Wybraniec, S. (2013). An overview of the Kjeldahl method of nitrogen determination. Part I. Early history, chemistry of the procedure, and titrimetric finish. Critical Reviews in Analytical Chemistry, 43(4), 178–223.CrossRefGoogle Scholar
  36. Sanhong, F., Yanan, H., Chen, L., & Yanrong, L. (2014). Optimization of preparation of antioxidative peptides from pumpkin seeds using response surface method. PLoS One, 9(3).Google Scholar
  37. Shi, L.-E., Ying, G.-Q., Tang, Z.-X., Chen, J.-S., Xiong, W.-Y., & Wang, H. (2009). Continuous enzymatic production of 5′-nucleotides using free nuclease P1 in ultrafiltration membrane reactor. Journal of Membrane Science, 345(1–2), 217–222.CrossRefGoogle Scholar
  38. Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. The Journal of Nutritional Biochemistry, 11(3), 128–131.CrossRefGoogle Scholar
  39. Van der Plancken, I., Van Loey, A., & Hendrickx, M. E. (2005). Combined effect of high pressure and temperature on selected properties of egg white proteins. Innovative Food Science & Emerging Technologies, 6(1), 11–20.CrossRefGoogle Scholar
  40. Van der Plancken, I., Van Loey, A., & Hendrickx, M. E. (2007). Foaming properties of egg white proteins affected by heat or high pressure treatment. Journal of Food Engineering, 78(4), 1410–1426.CrossRefGoogle Scholar
  41. Zheleva-Dimitrova, D., Nedialkov, P., & Kitanov, G. (2010). Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria. Pharmacognosy Magazine, 6(22), 74–78.CrossRefGoogle Scholar
  42. Zhu, L., Chen, J., Tang, X., & Xiong, Y. L. (2008). Reducing, radical scavenging, and chelation properties of in vitro digests of alcalase-treated zein hydrolysate. Journal of Agricultural and Food Chemistry, 56(8), 2714–2721.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sonja Jakovetić
    • 1
  • Nevena Luković
    • 1
  • Branimir Jugović
    • 2
  • Milica Gvozdenović
    • 1
  • Sanja Grbavčić
    • 3
  • Jelena Jovanović
    • 1
  • Zorica Knežević-Jugović
    • 1
  1. 1.Faculty of Technology and MetallurgyUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of Technical ScienceSerbian Academy of Science and ArtsBelgradeSerbia
  3. 3.Innovation Center of Faculty of Technology and MetallurgyUniversity of BelgradeBelgradeSerbia

Personalised recommendations