The kinetics of milk gel structure formation studies by electron microscopy

The aim of this study is to enhance the comprehension of the mechanism of enzymatic gelation in milk by visualizing the evolution of its microstructure through transmission electron microscopy. In order to minimize the potential for artifacts during the preparation process and eliminate any possible difficulties in interpreting the resulting images, three distinct methods were employed in the research: shading the surface topography with vacuum deposition of heavy metal, negative staining of the specimen with a heavy metal solution and replicating a cleavage of a quick-frozen sample. The selection of time intervals for sampling the gel during its evolution is determined by the most probable significant modifications in the resulting gel. Based on the research, it has been shown that natural milk is a nonequilibrium system from the perspective of statistical thermodynamics. A notable observation is that the glycomacropeptides forming the hair layer on the surface of casein micelles are unevenly distributed, leading to the formation of micelle dimers and trimers. It has been determind that during the initial stage of enzymatic gelation in milk, clusters of loosely bound micelles are formed in areas with the highest concentration. The formation of micelle chains is absent at this stage due to the non-anisometric nature of micelles and the energetic disadvantage of their formation. It has been found that under the influence of enzymatic gelation near the gel point, a hierarchical process involving the transformation of the milk’s protein component is activated. The trigger mechanism for this process is a cooperative conformational transition in clusters of casein micelles, which initiates a chain of more energy-intensive reactions in the following sequence: hydro­phobic interactions → hydrogen bridges → electrostatic interactions → calcium bridges. The result is the conversion of loosely bound micelle clusters into denser aggregates, predominantly contributing to the formation of milk curd. It is worth noting that gelation in milk can be regarded as a process that reduces the free energy of the dispersed system. Understanding the correlation between the decrease in the free energy value during gelation and the physical properties of the finished cheese and other dairy products continues to be a relevant area of research.

1. Lucey, J. A. (2011). Rennet-induced coagulation of milk. Chapter in a book: Encyclopedia of Dairy Sciences. Oxford: Academic Press. 2011.

2. Fox, P. F., Guinee, T. P., Cogan, T. M., McSweeney, P. L. H. (2017). Fundamentals of Cheese Science. Springer, New York. 2017.

3. Amaro-Hernández, J. C., Olivas, G. I., Acosta-Muñiz, C. H., Gutiérrez-Méndez, N., Rios-Velasco, C., Sepulveda, D. R. (2022). Chemical interactions among caseins during rennet coagulation of milk, Journal of Dairy Science, 105(2), 981–989. https://doi.org/10.3168/jds.2021-21071

4. Ptitsyn, O. B. (1995). Molten globule and protein folding. Chapter in a book: Advances in Protein Chemistry. 1995. https://doi.org/10.1016/S0065-3233(08)60546-X

5. Farrell Jr., H. M., Qi, P. X., Brown, E. M., Cooke, P. H., Tunick, M. H., Wickham, E. D. et al. (2002). Molten globule structures in milk proteins: Implications for potential new structure-function relationships. Journal of Dairy Science, 85(3), 459–471. https://doi.org/10.3168/jds.S0022-0302(02)74096-4

6. Fox, P. F., Guinee, T. P. (2013). Cheese science and technology. Chapter in a book: Milk and Dairy Products in Human Nutrition. Oxford, Wiley Blackwell.2013.

7. Smykov, I. T. (2015). Kinetics of Milk Gelation. Part I. Coagulation Mechanism. Chapter in a book: Rheology: Principles, applications and environmental impacts. New York: Nova Science Publ. 2015.

8. Surkov, B. A., Klimovskii, I. I., Krayushkin, V. A. (1982). Turbidimetric study of kinetics and mechanism of milk clotting by rennet. Milchwissenschaft, 37, 393–395.

9. Mosler, A. B., Shaqfeh, E. S. G. (1997). The conformation change of model polymers in stochastic flow fields: Flow through fixed beds. Physics of Fluids, 9, 1222–1234. https://doi.org/10.1063/1.869262

10. Markoska, T., Vasiljevic, T., Huppertz, T. (2020). Unravelling conformational aspects of milk protein structure-contributions from nuclear magnetic resonance studies. Foods, 9(8), Article 1128. https://doi.org/10.3390/foods9081128

11. Horne, D. S., Lucey, J. A. (2017). Rennet-Induced Coagulation of Milk. Chapter in a book: Cheese: Chemistry, Physics and Microbiology, Oxford: Academic Press. 2017. https://doi.org/10.1016/B978-0-12-417012-4.00005-3

12. Fox, P. F., Guinee, T. P., Cogan, T. M., McSweeney, P. L. H. (2016). Enzymatic Coagulation of Milk. pp.185–229. Chapter in a book: Fundamentals of Cheese Science, Springer Nature, New York. 2013. https://doi:10.1007/978-1-4899-7681-9_7

13. Tuszynski, W. B. (1971). A kinetic model of the clotting of casein by rennet.Journal of Dairy Research, 38(2), 115–125. https://doi.org/10.1017/S0022029900019233

14. Witten Jr., T. A., Meakin, P. (1983). Diffusion-limited aggregation at multiple growth sites. Physical Review B, 28(10), 5632–5642. https://doi.org/10.1103/PhysRevB.28.5632

15. De Kruif, C. G., Holt, C. (2003). Casein micelle structure, functions and interactions. Chapter in a book: Advanced Dairy Chemistry — 1 Proteins. Springer, Boston, MA. 2003.

16. Salvador, D., Acosta, Y., Zamora, A., Castillo, M. (2022). Rennet-induced casein micelle aggregation models: A review. Foods. 11(9), Article 1243. https://doi.10.3390/foods11091243

17. Kalab, M. (1979). Microstructure of dairy foods. 1. Milk products based on protein. Journal of Dairy Science, 62(8), 1352–1364.

18. Ong, L., Li, X., Ong, A., Gras, S. L. (2022). New insights into cheese microstructure. Annual Review of Food Science and Technology, 13, 89–115. https://doi.org/10.1146/annurev-food-032519-051812

19. Serna-Hernandez, S. O., Escobedo-Avellaneda, Z., García-García, R., Rostro-Alanis, M. D. de J., Welti-Chanes, J. (2022). Microscopical evaluation of the effects of high-pressure processing on milk casein micelles. Molecules, 27, Article 7179. https://doi.org/10.3390/molecules27217179

20. Panthi, R. R., Kelly, A. L., Sheehan, J. J., Bulbul, K., Vollmer, A.H., McMahon, D. J. (2019). Influence of protein concentration and coagulation temperature on rennet-induced gelation characteristics and curd microstructure. Journal of Dairy Science, 102(1), 177–189. https://doi.org/10.3168/jds.2018-15039

21. Li, R., Ebbesen, M. F., Glover, Z. J., Jæger, T. C., Rovers, T. A. M., Svensson, B. et al. (2023). Discriminating between different proteins in the microstructure of acidified milk gels by super-resolution microscopy. Food Hydrocolloids, 138, Article 108468. https://doi.org/10.1016/j.foodhyd.2023.108468

22. Zhang, Y., Liu, D., Liu, X., Hang, F., Zhou, P., Zhao, J. et al. (2018). Effect of temperature on casein micelle composition and gelation of bovine milk. International Dairy Journal, 78, 20–27. https://doi.org/10.1016/j.idairyj.2017.10.008

23. Foroutanparsa, S., Brüls, M., Tas, R. P., Maljaars, C. E. P., Voets, I. K. (2021). Super resolution microscopy imaging of pH induced changes in the microstructure of casein micelles. Food Structure, 30, Article 100231. https://doi.org/10.1016/j.foostr.2021.100231

24. Glover, Z. J., Ersch, C., Andersen, U., Holmes, M. J., Povey, M. J., Brewer, J. R. et al. (2019). Super-resolution microscopy and empirically validated autocorrelation image analysis discriminates microstructures of dairy derived gels. Food Hydrocolloids, 90, 62–71. https://doi.org/10.1016/j.foodhyd.2018.12.004

25. Karlsson, A. O., Ipsen, R., Ardo, Y. (2007). Observations of casein micelles in skim milk concentrate by transmission electron microscopy. LWT — Food Science and Technology, 40(6), 1102–1107. https://doi.org/10.1016/j.lwt.2006.05.012

26. Matsko, N., Letofsky-Papst, I., Albu, M., Mittal, V. (2013). An analytical technique to extract surface information of negatively stained or heavy-metal shadowed organic materials within the TEM. Microscopy and Microanalysis, 19(3), 642–651. https://doi.org/10.1017/S1431927613000366

27. Kavanagh, G. M., Clark, A. H., Ross-Murphy, S. B. (2000). Heat-induced gelation of globular proteins: 4. Gelation kinetics of low pH β-Lactoglobulin gels. Langmuir, 16, 24, 9584–9594. https://doi.org/10.1021/la0004698

28. Hayat, M. A. (2000). Principles and techniques of electron microscopy: Biological applications. Cambridge University Press. 2000.

29. Dalgleish, D. G., Spagnuolo, P., Goff, H. D. (2004). A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy. International Dairy Journal, 14(12), 1025–1031. https://doi.org/10.1016/j.idairyj.2004.04.008

30. Casanova, H., Chen, J., Dickinson, E., Murray, B. S., Nelson, P. V., Whittle, M. (2000). Dynamic colloidal interactions between protein stabilized particles — experiment and simulation. Physical Chemistry Chemical Physics, 2(17), 3861–3869. https://doi.org/10.1039/b004023l

31. de Kruif, C. G. (1998). Supra-aggregates of casein micelles as a prelude to coagulation. Journal of Dairy Science, 81(11), 3019–3028. https://doi.org/10.3168/jds.S0022-0302(98)75866-7

32. Shamsi, K. (2008). Effects of Pulsed Electric Field Processing on Microbial, Enzymatic and Physical Attributes of Milk and the Rennet-Induced Milk Gel. RMIT University, Melbourne. 2008.