Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Evanthia Monogioudi

Research output: ThesisDissertationCollection of Articles

1 Citation (Scopus)

Abstract

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of Beta-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible - multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of Beta-casein resulted in the formation of high molecular mass (MM ca. 1350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial Beta-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91% of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of Beta-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked Beta-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked Beta-casein was more resistant to pepsin digestion when compared to that of non modified Beta-casein. The effects of enzymatic cross-linking of Beta-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native Beta-casein by 14% when cross-linked by TrTyr and by 6% after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Mattinen, Maija-Liisa, Supervisor, External person
  • Buchert, Johanna, Supervisor, External person
Award date28 Jan 2011
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-7421-6
Electronic ISBNs978-951-38-7422-3
Publication statusPublished - 2010
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Caseins
Monophenol Monooxygenase
Enzymes
Transglutaminases
Pepsin A
Proteins
Nuclear magnetic resonance
Size exclusion chromatography
Cosmetics
Milk Proteins
Molecular mass
Light scattering
Phosphorus
Chemical properties
Nuclear magnetic resonance spectroscopy
Ionization
Mass spectrometry
Tyrosine
Desorption
Textiles

Keywords

  • Tyrosinase
  • Transglutaminase
  • Protein
  • ?-casein
  • cross-linking
  • digestibility
  • allergenicity
  • SEC-UV/Vis-MALLS
  • MALDI-TOF MS
  • 31P-NMR
  • AFM

Cite this

Monogioudi, E. (2010). Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity. Espoo: VTT Technical Research Centre of Finland.
Monogioudi, Evanthia. / Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity. Espoo : VTT Technical Research Centre of Finland, 2010. 86 p.
@phdthesis{76f58a16d39b4a1d8e2deb20a0f35820,
title = "Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity",
abstract = "Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of Beta-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible - multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of Beta-casein resulted in the formation of high molecular mass (MM ca. 1350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial Beta-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91{\%} of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of Beta-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked Beta-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked Beta-casein was more resistant to pepsin digestion when compared to that of non modified Beta-casein. The effects of enzymatic cross-linking of Beta-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native Beta-casein by 14{\%} when cross-linked by TrTyr and by 6{\%} after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.",
keywords = "Tyrosinase, Transglutaminase, Protein, ?-casein, cross-linking, digestibility, allergenicity, SEC-UV/Vis-MALLS, MALDI-TOF MS, 31P-NMR, AFM",
author = "Evanthia Monogioudi",
year = "2010",
language = "English",
isbn = "978-951-38-7421-6",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "752",
address = "Finland",
school = "University of Helsinki",

}

Monogioudi, E 2010, 'Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity', Doctor Degree, University of Helsinki, Espoo.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity. / Monogioudi, Evanthia.

Espoo : VTT Technical Research Centre of Finland, 2010. 86 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

AU - Monogioudi, Evanthia

PY - 2010

Y1 - 2010

N2 - Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of Beta-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible - multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of Beta-casein resulted in the formation of high molecular mass (MM ca. 1350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial Beta-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91% of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of Beta-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked Beta-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked Beta-casein was more resistant to pepsin digestion when compared to that of non modified Beta-casein. The effects of enzymatic cross-linking of Beta-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native Beta-casein by 14% when cross-linked by TrTyr and by 6% after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

AB - Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of Beta-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible - multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of Beta-casein resulted in the formation of high molecular mass (MM ca. 1350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial Beta-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91% of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of Beta-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked Beta-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked Beta-casein was more resistant to pepsin digestion when compared to that of non modified Beta-casein. The effects of enzymatic cross-linking of Beta-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native Beta-casein by 14% when cross-linked by TrTyr and by 6% after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

KW - Tyrosinase

KW - Transglutaminase

KW - Protein

KW - ?-casein

KW - cross-linking

KW - digestibility

KW - allergenicity

KW - SEC-UV/Vis-MALLS

KW - MALDI-TOF MS

KW - 31P-NMR

KW - AFM

M3 - Dissertation

SN - 978-951-38-7421-6

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Monogioudi E. Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity. Espoo: VTT Technical Research Centre of Finland, 2010. 86 p.