Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation

Jing Che; Zekun Fan; Etske Bijl; Julia Prangchat Stub Thomsen; Ivan Mijakovic; Kasper Hettinga; Nina Aagaard Poulsen; Lotte Bach Larsen

doi:10.1016/j.foodhyd.2024.110615

Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation

Jing Che^*, Zekun Fan, Etske Bijl, Julia Prangchat Stub Thomsen, Ivan Mijakovic, Kasper Hettinga, Nina Aagaard Poulsen, Lotte Bach Larsen

^*Corresponding author for this work

Department of Food Science - Food Chemistry

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

This study investigated the effect of bovine casein (CN) phosphorylation degree on micelle reassembly, stability, calcium-binding, and acid-induced gelation or precipitation, with the aim of assessing the feasibility of utilising microbial CNs in future dairy production. The four CNs (αS1-, αS2-, β-, and κ-CN) were purified from bovine milk and subjected to enzymatic dephosphorylation, resulting in three pools – fully and partially phosphorylated and fully dephosphorylated. Nine reassembled CN micelle solutions (RCMS) were investigated, consisting of three systems (two of four-CN, and one of β/κ-CN) and three phosphorylation degrees of the corresponding CNs. The distribution of different components (protein, calcium, and phosphorus) into micellar, serum, and insoluble fractions after two centrifugation steps was studied. The pH stability relative to acid-gelation or precipitation of the formed RCMS was further investigated. Results showed that the micelle reassembly ability of RCMS containing all four CNs was proportional to the phosphorylation degree; CNs with higher phosphorylation degrees contained a higher proportion of micelles and exhibited greater calcium-binding ability, whereas fully dephosphorylated CNs hardly formed CN micelle structures. The gelation pH of RCMS increased with decreasing phosphorylation degree, whereas the fully dephosphorylated CNs completely failed in gelation but precipitated when reaching their isoelectric point at pH 5.5. Moreover, RCMS containing only β/κ-CN at their native full phosphorylation were unstable at the salt concentrations applied, with more than half of the CNs self-associating to flocculate. Our study confirms the essential role of phosphorylation degree in micelle reassembly and stability, providing important information for potential future applications of microbial CNs that are expected to exhibit non-native phosphorylation levels.

Original language	English
Article number	110615
Journal	Food Hydrocolloids
Volume	159
Number of pages	10
ISSN	0268-005X
DOIs	https://doi.org/10.1016/j.foodhyd.2024.110615
Publication status	Published - Feb 2025

Keywords

Acid-induced gelation
Calcium-binding ability
Dephosphorylation
Reassembled casein micelles
TEM

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Che, J., Fan, Z., Bijl, E., Thomsen, J. P. S., Mijakovic, I., Hettinga, K., Poulsen, N. A., & Larsen, L. B. (2025). Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation. Food Hydrocolloids, 159, Article 110615. https://doi.org/10.1016/j.foodhyd.2024.110615

@article{d13226cecc2145598ec93c9cb1003b25,

title = "Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation",

abstract = "This study investigated the effect of bovine casein (CN) phosphorylation degree on micelle reassembly, stability, calcium-binding, and acid-induced gelation or precipitation, with the aim of assessing the feasibility of utilising microbial CNs in future dairy production. The four CNs (αS1-, αS2-, β-, and κ-CN) were purified from bovine milk and subjected to enzymatic dephosphorylation, resulting in three pools – fully and partially phosphorylated and fully dephosphorylated. Nine reassembled CN micelle solutions (RCMS) were investigated, consisting of three systems (two of four-CN, and one of β/κ-CN) and three phosphorylation degrees of the corresponding CNs. The distribution of different components (protein, calcium, and phosphorus) into micellar, serum, and insoluble fractions after two centrifugation steps was studied. The pH stability relative to acid-gelation or precipitation of the formed RCMS was further investigated. Results showed that the micelle reassembly ability of RCMS containing all four CNs was proportional to the phosphorylation degree; CNs with higher phosphorylation degrees contained a higher proportion of micelles and exhibited greater calcium-binding ability, whereas fully dephosphorylated CNs hardly formed CN micelle structures. The gelation pH of RCMS increased with decreasing phosphorylation degree, whereas the fully dephosphorylated CNs completely failed in gelation but precipitated when reaching their isoelectric point at pH 5.5. Moreover, RCMS containing only β/κ-CN at their native full phosphorylation were unstable at the salt concentrations applied, with more than half of the CNs self-associating to flocculate. Our study confirms the essential role of phosphorylation degree in micelle reassembly and stability, providing important information for potential future applications of microbial CNs that are expected to exhibit non-native phosphorylation levels.",

keywords = "Acid-induced gelation, Calcium-binding ability, Dephosphorylation, Reassembled casein micelles, TEM",

author = "Jing Che and Zekun Fan and Etske Bijl and Thomsen, {Julia Prangchat Stub} and Ivan Mijakovic and Kasper Hettinga and Poulsen, {Nina Aagaard} and Larsen, {Lotte Bach}",

year = "2025",

month = feb,

doi = "10.1016/j.foodhyd.2024.110615",

language = "English",

volume = "159",

journal = "Food Hydrocolloids",

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Che, J, Fan, Z, Bijl, E, Thomsen, JPS, Mijakovic, I, Hettinga, K, Poulsen, NA & Larsen, LB 2025, 'Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation', Food Hydrocolloids, vol. 159, 110615. https://doi.org/10.1016/j.foodhyd.2024.110615

Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation. / Che, Jing; Fan, Zekun; Bijl, Etske et al.
In: Food Hydrocolloids, Vol. 159, 110615, 02.2025.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation

AU - Che, Jing

AU - Fan, Zekun

AU - Bijl, Etske

AU - Thomsen, Julia Prangchat Stub

AU - Mijakovic, Ivan

AU - Hettinga, Kasper

AU - Poulsen, Nina Aagaard

AU - Larsen, Lotte Bach

PY - 2025/2

Y1 - 2025/2

N2 - This study investigated the effect of bovine casein (CN) phosphorylation degree on micelle reassembly, stability, calcium-binding, and acid-induced gelation or precipitation, with the aim of assessing the feasibility of utilising microbial CNs in future dairy production. The four CNs (αS1-, αS2-, β-, and κ-CN) were purified from bovine milk and subjected to enzymatic dephosphorylation, resulting in three pools – fully and partially phosphorylated and fully dephosphorylated. Nine reassembled CN micelle solutions (RCMS) were investigated, consisting of three systems (two of four-CN, and one of β/κ-CN) and three phosphorylation degrees of the corresponding CNs. The distribution of different components (protein, calcium, and phosphorus) into micellar, serum, and insoluble fractions after two centrifugation steps was studied. The pH stability relative to acid-gelation or precipitation of the formed RCMS was further investigated. Results showed that the micelle reassembly ability of RCMS containing all four CNs was proportional to the phosphorylation degree; CNs with higher phosphorylation degrees contained a higher proportion of micelles and exhibited greater calcium-binding ability, whereas fully dephosphorylated CNs hardly formed CN micelle structures. The gelation pH of RCMS increased with decreasing phosphorylation degree, whereas the fully dephosphorylated CNs completely failed in gelation but precipitated when reaching their isoelectric point at pH 5.5. Moreover, RCMS containing only β/κ-CN at their native full phosphorylation were unstable at the salt concentrations applied, with more than half of the CNs self-associating to flocculate. Our study confirms the essential role of phosphorylation degree in micelle reassembly and stability, providing important information for potential future applications of microbial CNs that are expected to exhibit non-native phosphorylation levels.

AB - This study investigated the effect of bovine casein (CN) phosphorylation degree on micelle reassembly, stability, calcium-binding, and acid-induced gelation or precipitation, with the aim of assessing the feasibility of utilising microbial CNs in future dairy production. The four CNs (αS1-, αS2-, β-, and κ-CN) were purified from bovine milk and subjected to enzymatic dephosphorylation, resulting in three pools – fully and partially phosphorylated and fully dephosphorylated. Nine reassembled CN micelle solutions (RCMS) were investigated, consisting of three systems (two of four-CN, and one of β/κ-CN) and three phosphorylation degrees of the corresponding CNs. The distribution of different components (protein, calcium, and phosphorus) into micellar, serum, and insoluble fractions after two centrifugation steps was studied. The pH stability relative to acid-gelation or precipitation of the formed RCMS was further investigated. Results showed that the micelle reassembly ability of RCMS containing all four CNs was proportional to the phosphorylation degree; CNs with higher phosphorylation degrees contained a higher proportion of micelles and exhibited greater calcium-binding ability, whereas fully dephosphorylated CNs hardly formed CN micelle structures. The gelation pH of RCMS increased with decreasing phosphorylation degree, whereas the fully dephosphorylated CNs completely failed in gelation but precipitated when reaching their isoelectric point at pH 5.5. Moreover, RCMS containing only β/κ-CN at their native full phosphorylation were unstable at the salt concentrations applied, with more than half of the CNs self-associating to flocculate. Our study confirms the essential role of phosphorylation degree in micelle reassembly and stability, providing important information for potential future applications of microbial CNs that are expected to exhibit non-native phosphorylation levels.

KW - Acid-induced gelation

KW - Calcium-binding ability

KW - Dephosphorylation

KW - Reassembled casein micelles

KW - TEM

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U2 - 10.1016/j.foodhyd.2024.110615

DO - 10.1016/j.foodhyd.2024.110615

M3 - Journal article

SN - 0268-005X

VL - 159

JO - Food Hydrocolloids

JF - Food Hydrocolloids

M1 - 110615

ER -

Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation

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Keywords

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