Skip to main content
SpringerLink
Log in

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

  • Original Paper
  • Published:
Journal of the American Oil Chemists' Society

Abstract

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Zou X-Q, Huang J-H, Jin Q-Z, Liu Y-F, Tao G-J, Cheong L-Z, Wang X-G (2012) Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid: combination of enzymatic and physical methods. J Agric Food Chem 60:9415–9423

    Article  CAS  Google Scholar 

  2. Turan D, Yeşilçubuk NS, Akoh CC (2012) Production of human milk fat analogue containing docosahexaenoic and arachidonic acids. J Agric Food Chem 60:4402–4407

    Article  CAS  Google Scholar 

  3. Jensen RG (2001) Infant formulas. In: Gunstone FD (ed) Structured and modified lipids. Dekker, New York, pp 455–464

    Google Scholar 

  4. Soumanou MM, Pérignon M, Villeneuve P (2013) Lipase-catalyzed interesterification reactions for human milk fat substitutes production: a review. Eur J Lipid Sci Technol 115:270–285

    Article  CAS  Google Scholar 

  5. Sahín N, Akoh CC, Karaali A (2005) Lipase-catalyzed acidolysis of tripalmitin with hazelnut oil fatty acids and stearic acid to produce human milk fat substitutes. J Agric Food Chem 53:5779–5783

    Article  Google Scholar 

  6. Sahín N, Akoh CC, Karaali A (2005) Enzymatic production of human milk fat substitutes containing γ-linolenic acid: optimization of reactions by response surface methodology. J Am Oil Chem Soc 82:549–557

    Article  Google Scholar 

  7. Sahín N, Akoh CC, Karaali A (2006) Human milk fat substitutes containing omega-3 fatty acids. J Agric Food Chem 54:3717–3722

    Article  Google Scholar 

  8. Tecelão C, Silva J, Dubreucq E, Ribeiro MH, Ferreira-Dias S (2010) Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase. J Mol Catal B Enzym 65:122–127

    Article  Google Scholar 

  9. Tecelão C, Guillén M, Valero F, Ferreira-Dias S (2012) Immobilized heterologous Rhizopus oryzae lipase: a feasible biocatalyst for the production of human milk fat substitutes. Biochem Eng J 67:104–110

    Article  Google Scholar 

  10. Tecelão C, Rivera I, Sandoval G, Ferreira-Dias S (2012) Carica papaya latex: a low-cost biocatalyst for human milk fat substitutes production. Eur J Lipid Sci Technol 114:266–276

    Article  Google Scholar 

  11. Li Y, Mu H, Andersen JET, Xu X, Meyer O, Ørngreen A (2010) New human milk fat substitutes from butterfat to improve absorption. Food Res Int 43:739–744

    Article  Google Scholar 

  12. Jiménez MJ, Esteban L, Robles A, Hita E, González PA (2010) Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis. Process Biochem 45:407–414

    Article  Google Scholar 

  13. Nielsen NS, Yang T, Xu X, Jacobsen C (2006) Production and oxidative stability of a human milk fat substitute produced from lard by enzyme technology in a pilot packed-bed reactor. Food Chem 94:53–60

    Article  CAS  Google Scholar 

  14. Wang YH, Qin XL, Zhu Q, Zhou R, Yang B, Li L (2010) Lipase-catalyzed acidolysis of lard for the production of human milk fat substitutes. Eur Food Res Technol 230:769–777

    Article  CAS  Google Scholar 

  15. Xu X, Fomuso LB, Akoh CC (2000) Modification of menhaden oil by enzymatic acidolysis to produce structured lipids: optimization by response surface design in a packed bed reactor. J Am Oil Chem Soc 77:171–176

    Article  CAS  Google Scholar 

  16. Xu X, Skands ARH, Høy C-E, Mu H, Balchen S, Adler-Nissen J (1998) Production of specific-structured lipids by enzymatic interesterification: elucidation of acyl migration by response surface design. J Am Oil Chem Soc 75:1179–1186

    Article  CAS  Google Scholar 

  17. Nunes PA, Pires-Cabral P, Guillén M, Valero F, Luna D, Ferreira-Dias S (2011) Production of MLM-type structured lipids catalyzed by immobilized heterologous Rhizopus oryzae lipase. J Am Oil Chem Soc 88:473–480

    Article  CAS  Google Scholar 

  18. Nunes PA, Pires-Cabral P, Guillén M, Valero F, Ferreira-Dias S (2012) Optimized production of MLM triacylglycerols catalyzed by immobilized heterologous Rhizopus oryzae lipase. J Am Oil Chem Soc 89:1287–1295

    CAS  Google Scholar 

  19. Nunes PA, Pires-Cabral P, Guillén M, Valero F, Ferreira-Dias S (2012) Batch operational stability of immobilized heterologous Rhizopus oryzae lipase during acidolysis of virgin olive oil with medium-chain fatty acids. Biochem Eng J 67:265–268

    Article  CAS  Google Scholar 

  20. Guillén M, Benaiges MD, Valero F (2011) Comparison of the biochemical properties of a recombinant lipase extract from Rhizopus oryzae expressed in Pichia pastoris with a native extract. Biochem Eng J 54:117–123

    Article  Google Scholar 

  21. Arnau C, Ramón R, Casas C, Valero F (2010) Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess. Enzym Microb Technol 46:494–500

    Article  CAS  Google Scholar 

  22. Osório NM, Dubreucq E, da Fonseca MM, Ferreira-Dias S (2009) Lipase/acyltransferase-catalysed interesterification of fat blends containing omega-3 polyunsaturated fatty acids. Eur J Lipid Sci Technol 111:120–134

    Article  Google Scholar 

  23. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  24. Haaland PD (1989) Experimental design in biotechnology, statistics: textbooks and monographs. Marcel Dekker Inc., New York

    Google Scholar 

  25. Jennings BH, Akoh CC (2000) Lipase-catalyzed modification of rice bran oil to incorporate capric acid. J Agric Food Chem 48:4439–4443

    Article  CAS  Google Scholar 

  26. Esteban L, Jiménez MJ, Hita E, González PA, Martín L, Robles A (2011) Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis. Biochem Eng J 54:62–69

    Article  CAS  Google Scholar 

  27. Zhao H, Lu Z, Bie X, Lu F, Liu Z (2007) Lipase catalyzed acidolysis of lard with capric acid in organic solvent. J Food Eng 78:41–46

    Article  CAS  Google Scholar 

  28. Nascimento AC, Tecelão CSR, Gusmão JH, da Fonseca MMR, Ferreira-Dias S (2004) Modelling lipase-catalysed transesterification of fats containing n-3 fatty acids monitored by their solid fat content. Eur J Lipid Sci Technol 106:599–612

    Article  CAS  Google Scholar 

  29. Osório NM, da Fonseca MM, Ferreira-Dias S (2006) Operational stability of Thermomyces lanuginosa lipase during fats interesterification in continuous-packed-bed reactors. Eur J Lipid Sci Technol 108:545–553

    Article  Google Scholar 

  30. Haas MJ, Allen J, Berka TR (1991) Cloning, expression and characterization of a cDNA encoding a lipase from Rhizopus delemar. Gene 109:107–113

    Article  CAS  Google Scholar 

  31. Haas MJ, Bailey DG, Baker W, Berka TR, Cichowicz DJ, Derewenda ZS, Genuario RR, Joerger RD, Klein RR, Scott K, Woolf DJ (1999) Biochemical and molecular biological characterization of a lipase produced by the fungus Rhizopus delemar. Fett/Lipid 101:364–370

    Article  CAS  Google Scholar 

  32. Ghamgui H, Miled N, Karrachaabouni M, Gargouri Y (2007) Immobilization studies and biochemical properties of free and immobilized Rhizopus oryzae lipase onto CaCO3: a comparative study. Biochem Eng J 37:34–41

    Article  CAS  Google Scholar 

  33. Kharrat N, Ali YB, Marzouk S, Gargouri Y-T, Karra-Châabouni M (2011) Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: comparison with the free enzyme. Process Biochem 46:1083–1089

    Article  CAS  Google Scholar 

  34. Casas-Godoy L, Marty A, Sandoval G, Ferreira-Dias S (2013) Optimization of medium chain length fatty acid incorporation into olive oil catalyzed by immobilized Lip2 from Yarrowia lipolytica. Biochem Eng J 77:20–27

    Article  CAS  Google Scholar 

  35. Guillén M, Benaiges MD, Valero F (2011) Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: comparison between native and recombinant variants. Biotechnol Prog 27:1232–1241

    Article  Google Scholar 

Download references

Acknowledgments

This study was supported by the: (1) Program CYTED, under the scope of the “Ibero-American Network for the Extraction and Enzymatic Transformation of Functional Ingredients and Nutraceuticals from Regional Plants and Agro-Residues (ENZNUT)”, (2) by the project CTQ2010-15131—of the Spanish Ministry of Science and Innovation, (3) by 2009-SGR-281 and Reference Network in Biotechnology (XRB) (Generalitat de Catalunya), (4) by the Strategic Project PEst-OE/AGR/UI0245/2011 of CEER, Biosystems Engineering, a research unit supported by the national funding of Fundação para a Ciência e a Tecnologia, Portugal, and by (5) the Integrated Action Portugal-Spain E-29/11.

Author information

Authors and Affiliations

  1. Marine Resources Research Group, School of Tourism and Maritime Technology, Polytechnic Institute of Leiria, 2520-641, Peniche, Portugal

    Tiago Simões & Carla Tecelão

  2. Departament d’Enginyeria Quimica (EE), Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain

    Francisco Valero

  3. CEER, Biosystems Engineering, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Lisbon, Portugal

    Carla Tecelão & Suzana Ferreira-Dias

Authors
  1. Tiago Simões
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francisco Valero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carla Tecelão
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Suzana Ferreira-Dias
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Suzana Ferreira-Dias.

About this article

Cite this article

Simões, T., Valero, F., Tecelão, C. et al. Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases. J Am Oil Chem Soc 91, 411–419 (2014). https://doi.org/10.1007/s11746-013-2379-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11746-013-2379-9

Keywords

Search

Navigation