Skip to main content
SpringerLink
Log in

Milk lactose removal by β-galactosidase immobilized on eggshell membrane

  • Original Paper
  • Published:
European Food Research and Technology Aims and scope Submit manuscript

Abstract

Eggshell membrane (ESM) is used in immobilization studies due to its large surface area, non-toxicity and biodegradability. In this study, β-galactosidase was immobilized on ESM using the adsorption and cross-linking method. The interactions between enzyme and ESM after immobilization were determined by ATR-FTIR (attenuated total reflection fourier transform infrared spectroscopy) and SEM (scanning electron microscope). The optimum temperature of the free enzyme was found to be 35 °C, and this value was 45 °C for the immobilized enzyme. Immobilized enzyme managed to retain more than 50% of its activity after 8 reuses. In the lactose removal experiment from milk, the highest reaction efficiency was found as 55.8% under specified optimization conditions for β-galactosidase immobilized ESM after 3 h. Due to the microfiber protein structure of the ESM, it has improved enzyme stability properties, as a result of the multi protein–protein interactions formed after the immobilization between the proteins in the ESM and the enzyme molecules.

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
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

Not applicable

Code availability

Not applicable.

References

  1. Adhikari K, Dooley LM, Chambers E IV, Bhumiratana N (2010) Sensory characteristics of commercial lactose-free milks manufactured in the United States. LWT - Food Sci Technol 43:113–118. https://doi.org/10.1016/j.lwt.2009.06.017

    Article  CAS  Google Scholar 

  2. Benavente R, Pessela BC, Curiel JA et al (2015) Improving properties of a novel β-galactosidase from Lactobacillus plantarum by covalent immobilization. Molecules 20:7874–7889. https://doi.org/10.3390/molecules20057874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Panesar R, Panesar PS, Singh RS, Kennedy JF (2011) Hydrolysis of milk lactose in a packed bed reactor system using immobilized yeast cells. J Chem Technol Biotechnol 86:42–46. https://doi.org/10.1002/jctb.2481

    Article  CAS  Google Scholar 

  4. Ricardi NC, de Menezes EW, Valmir Benvenutti E et al (2018) Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology. Food Chem 246:343–350. https://doi.org/10.1016/j.foodchem.2017.11.026

    Article  CAS  PubMed  Google Scholar 

  5. Husain Q, Ansari SA, Alam F, Azam A (2011) Immobilization of Aspergillus oryzae β galactosidase on zinc oxide nanoparticles via simple adsorption mechanism. Int J Biol Macromol 49:37–43. https://doi.org/10.1016/j.ijbiomac.2011.03.011

    Article  CAS  PubMed  Google Scholar 

  6. Labus K (2018) Effective detection of biocatalysts with specified activity by using a hydrogel-based colourimetric assay - β-galactosidase case study. PLoS ONE 13:1–11. https://doi.org/10.1371/journal.pone.0205532

    Article  CAS  Google Scholar 

  7. Numanoǧlu Y, Sungur S (2004) β-galactosidase from Kluyveromyces lactis cell disruption and enzyme immobilization using a cellulose-gelatin carrier system. Process Biochem 39:705–711. https://doi.org/10.1016/S0032-9592(03)00183-3

    Article  CAS  Google Scholar 

  8. Klein MP, Fallavena LP, Schöffer JDN et al (2013) High stability of immobilized β-d-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis. Carbohydr Polym 95:465–470. https://doi.org/10.1016/j.carbpol.2013.02.044

    Article  CAS  PubMed  Google Scholar 

  9. Liu W, Wang L, Jiang R (2012) Specific enzyme immobilization approaches and their application with nanomaterials. Top Catal 55:1146–1156. https://doi.org/10.1007/s11244-012-9893-0

    Article  CAS  Google Scholar 

  10. Wang ZG, Wan LS, Liu ZM et al (2009) Enzyme immobilization on electrospun polymer nanofibers: an overview. J Mol Catal B Enzym 56:189–195. https://doi.org/10.1016/j.molcatb.2008.05.005

    Article  CAS  Google Scholar 

  11. Boltachev GS, Nagayev KA, Paranin SN, et al (2011) Theory of the magnetic pulsed compaction of nanosized powders. Nanomater Prop Prep Process 1–58.

  12. Brena B, González-Pombo P, Batista-Viera F (2013) Immobilization of enzymes and cells. Immobil Enzym Cells Third Ed Methods Mol Biol 1051:15–31. https://doi.org/10.1007/978-1-62703-550-7

    Article  CAS  Google Scholar 

  13. Işik C, Arabaci G, Ispirli Doğaç Y et al (2019) Synthesis and characterization of electrospun PVA/Zn2+ metal composite nanofibers for lipase immobilization with effective thermal pH stabilities and reusability. Mater Sci Eng C. https://doi.org/10.1016/j.msec.2019.02.031

    Article  Google Scholar 

  14. Haghju S, Bari MR, Khaled-Abad MA (2018) Affecting parameters on fabrication of β-D-galactosidase immobilized chitosan/poly (vinyl alcohol) electrospun nanofibers. Carbohydr Polym 200:137–143. https://doi.org/10.1016/j.carbpol.2018.07.096

    Article  CAS  PubMed  Google Scholar 

  15. Kessi E, Arias JL (2019) Using natural waste material as a matrix for the immobilization of enzymes: chicken eggshell membrane powder for β-galactosidase immobilization. Appl Biochem Biotechnol 187:101–115. https://doi.org/10.1007/s12010-018-2805-4

    Article  CAS  PubMed  Google Scholar 

  16. Klein MP, Hackenhaar CR, Lorenzoni ASG et al (2016) Chitosan crosslinked with genipin as support matrix for application in food process: Support characterization and β-d-galactosidase immobilization. Carbohydr Polym 137:184–190. https://doi.org/10.1016/j.carbpol.2015.10.069

    Article  CAS  PubMed  Google Scholar 

  17. Pan C, Hu B, Li W et al (2009) Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4-chitosan nanoparticles. J Mol Catal B Enzym 61:208–215. https://doi.org/10.1016/j.molcatb.2009.07.003

    Article  CAS  Google Scholar 

  18. Verma ML, Barrow CJ, Kennedy JF, Puri M (2012) Immobilization of β-d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: characterization and lactose hydrolysis. Int J Biol Macromol 50:432–437. https://doi.org/10.1016/j.ijbiomac.2011.12.029

    Article  CAS  PubMed  Google Scholar 

  19. Wahba MI (2016) Treated calcium pectinate beads for the covalent immobilization of β-D-galactosidase. Int J Biol Macromol 91:877–886. https://doi.org/10.1016/j.ijbiomac.2016.06.044

    Article  CAS  PubMed  Google Scholar 

  20. Wolf M, Gasparin BC, Paulino AT (2018) Hydrolysis of lactose using β-D-galactosidase immobilized in a modified Arabic gum-based hydrogel for the production of lactose-free/low-lactose milk. Int J Biol Macromol 115:157–164. https://doi.org/10.1016/j.ijbiomac.2018.04.058

    Article  CAS  PubMed  Google Scholar 

  21. Hernandez K, Fernandez-Lafuente R (2011) Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzyme Microb Technol 48:107–122. https://doi.org/10.1016/j.enzmictec.2010.10.003

    Article  CAS  PubMed  Google Scholar 

  22. Rueda N, dos Santos JCS, Ortiz C et al (2016) Chemical modification in the design of immobilized enzyme biocatalysts: drawbacks and opportunities. Chem Rec 16:1436–1455. https://doi.org/10.1002/tcr.201600007

    Article  CAS  PubMed  Google Scholar 

  23. Bedade DK, Sutar YB, Singhal RS (2019) Chitosan coated calcium alginate beads for covalent immobilization of acrylamidase: process parameters and removal of acrylamide from coffee. Food Chem 275:95–104. https://doi.org/10.1016/j.foodchem.2018.09.090

    Article  CAS  PubMed  Google Scholar 

  24. de Lima JS, Cabrera MP, Casazza AA et al (2018) Immobilization of Aspergillus ficuum tannase in calcium alginate beads and its application in the treatment of boldo (Peumus boldus) tea. Int J Biol Macromol 118:1989–1994. https://doi.org/10.1016/j.ijbiomac.2018.07.084

    Article  CAS  PubMed  Google Scholar 

  25. Sondhi S, Kaur R, Kaur S, Kaur PS (2018) Immobilization of laccase-ABTS system for the development of a continuous flow packed bed bioreactor for decolorization of textile effluent. Int J Biol Macromol 117:1093–1100. https://doi.org/10.1016/j.ijbiomac.2018.06.007

    Article  CAS  PubMed  Google Scholar 

  26. Chattopadhyay S, Sen R (2012) A comparative performance evaluation of jute and eggshell matrices to immobilize pancreatic lipase. Process Biochem 47:749–757. https://doi.org/10.1016/j.procbio.2012.02.003

    Article  CAS  Google Scholar 

  27. Jiang C, Cheng C, Hao M et al (2017) Enhanced catalytic stability of lipase immobilized on oxidized and disulfide-rich eggshell membrane for esters hydrolysis and transesterification. Int J Biol Macromol 105:1328–1336. https://doi.org/10.1016/j.ijbiomac.2017.07.166

    Article  CAS  PubMed  Google Scholar 

  28. Du L, Huang M, Feng JX (2017) Immobilization of α-amylase on eggshell membrane and Ag-nanoparticle-decorated eggshell membrane for the biotransformation of starch. Starch/Staerke 69:1–7. https://doi.org/10.1002/star.201600352

    Article  CAS  Google Scholar 

  29. Al-Ghouti MA, Khan M (2018) Eggshell membrane as a novel bio sorbent for remediation of boron from desalinated water. J Environ Manage 207:405–416. https://doi.org/10.1016/j.jenvman.2017.11.062

    Article  CAS  PubMed  Google Scholar 

  30. Cordeiro CMM, Hincke MT (2015) Quantitative proteomics analysis of eggshell membrane proteins during chick embryonic development. J Proteomics 130:11–25. https://doi.org/10.1016/j.jprot.2015.08.014

    Article  CAS  PubMed  Google Scholar 

  31. Du J, Hincke MT, Rose-Martel M et al (2015) Identifying specific proteins involved in eggshell membrane formation using gene expression analysis and bioinformatics. BMC Genomics 16:1–14. https://doi.org/10.1186/s12864-015-2013-3

    Article  CAS  Google Scholar 

  32. Rao A, Arias JL, Cölfen H (2017) On mineral retrosynthesis of a complex biogenic scaffold. Inorganics 5:16. https://doi.org/10.3390/inorganics5010016

    Article  CAS  Google Scholar 

  33. Baláž M (2014) Eggshell membrane biomaterial as a platform for applications in materials science. Acta Biomater 10:3827–3843. https://doi.org/10.1016/j.actbio.2014.03.020

    Article  CAS  PubMed  Google Scholar 

  34. Park S, Choi KS, Lee D et al (2016) Eggshell membrane: review and impact on engineering. Biosyst Eng 151:446–463. https://doi.org/10.1016/j.biosystemseng.2016.10.014

    Article  Google Scholar 

  35. Bahl OP, Agrawal KML (1969) Glycosidases of Aspergillus niger. J Biol Chem 244:2970–2978. https://doi.org/10.1016/S0021-9258(18)91719-9

    Article  CAS  PubMed  Google Scholar 

  36. 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  PubMed  Google Scholar 

  37. Işık C, Saraç N, Teke M, Uğur A (2021) A new bioremediation method for removal of wastewater containing oils with high oleic acid composition: Acinetobacter haemolyticuslipase immobilized on eggshell membrane with improved stabilities. New J Chem 45:1984–1992. https://doi.org/10.1039/D0NJ05175F

    Article  Google Scholar 

  38. Pundir CS, Chauhan NS, Bhambi M (2008) Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination. Anal Biochem 374:272–277. https://doi.org/10.1016/j.ab.2007.11.008

    Article  CAS  PubMed  Google Scholar 

  39. Bayramoglu G, Tunali Y, Arica MY (2007) Immobilization of β-galactosidase onto magnetic poly(GMA-MMA) beads for hydrolysis of lactose in bed reactor. Catal Commun 8:1094–1101. https://doi.org/10.1016/j.catcom.2006.10.029

    Article  CAS  Google Scholar 

  40. Zhang S, Gao S, Gao G (2010) Immobilization of β-galactosidase onto magnetic beads. Appl Biochem Biotechnol 160:1386–1393. https://doi.org/10.1007/s12010-009-8600-5

    Article  CAS  PubMed  Google Scholar 

  41. Zhang Z, Zhang R, Chen L, McClements DJ (2016) Encapsulation of lactase (β-galactosidase) into κ-carrageenan-based hydrogel beads: Impact of environmental conditions on enzyme activity. Food Chem 200:69–75. https://doi.org/10.1016/j.foodchem.2016.01.014

    Article  CAS  PubMed  Google Scholar 

  42. Das B, Roy AP, Bhattacharjee S et al (2015) Lactose hydrolysis by β-galactosidase enzyme: optimization using response surface methodology. Ecotoxicol Environ Saf 121:244–252. https://doi.org/10.1016/j.ecoenv.2015.03.024

    Article  CAS  PubMed  Google Scholar 

  43. Estevinho BN, Damas AM, Martins P, Rocha F (2014) Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process. Food Res Int 64:134–140. https://doi.org/10.1016/j.foodres.2014.05.057

    Article  CAS  PubMed  Google Scholar 

  44. Weymuth T, Jacob CR, Reiher M (2010) A local-mode model for understanding the dependence of the extended amide III vibrations on protein secondary structure. J Phys Chem B 114:10649–10660. https://doi.org/10.1021/jp104542w

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study is a part of Selen Kızıldağ’s MSc thesis.

Author information

Authors and Affiliations

  1. Faculty of Science, Chemistry Department, Muğla Sıtkı Koçman University, Muğla, Türkiye

    Selen Kızıldağ, Ceyhun Işık & Mustafa Teke

Authors
  1. Selen Kızıldağ
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ceyhun Işık
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mustafa Teke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Not applicable.

Corresponding author

Correspondence to Ceyhun Işık.

Ethics declarations

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kızıldağ, S., Işık, C. & Teke, M. Milk lactose removal by β-galactosidase immobilized on eggshell membrane. Eur Food Res Technol 249, 2125–2136 (2023). https://doi.org/10.1007/s00217-023-04280-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00217-023-04280-3

Keywords

Search

Navigation