Periodic Reporting for period 1 - CASMOX (Analysis of the oxidative modifications on casein micelles exposed to different oxidative sources: determining the impact of these modifications at a structural and nutritional level)
Période du rapport: 2020-08-01 au 2022-07-31
Milk and dairy products are one of the most consumed drinks and foods worldwide representing a global market of ~6 billion EUR. It is one of the most relevant foods for humans due to its affordability and complete nutritional composition including proteins, lipids, carbohydrates, vitamins and minerals. However, this complex composition favors the formation of oxidants (e.g. free radicals and singlet oxygen) during milk processing and storage. Some of the modifications triggered by the reaction between oxidants with proteins can lead to the loss of essential amino acids for human nutrition, and to the formation of products that can alter protein structure and function. Caseins are the most abundant proteins in bovine milk (80% of the total protein content), and play a key role in stabilizing the lipid-water interface in this food thanks to their amphiphilic nature. However, these same hallmarks make them prone to suffering oxidative damage. Despite of this knowledge, little is known about the pathways contributing to these reactions and the consequences of consuming such modified proteins. This knowledge is key to creating better processing and storage policies in the food industry in order to assess food quality. In this context, this project aimed: To gain an improved understanding of degradation pathways of the most abundant milk proteins (caseins), the micelle structures that these proteins form, and the key drivers of these reactions. To address this, this project investigated the oxidative modifications on casein micelles, and the related consequences to their nutritional and health value, after their reaction with different oxidative sources. This project aimed to assess whether and how the association of casein proteins (favored at high protein concentrations) modified their reactivity towards oxidants, making these more prone to changes in structure, molecular mass, and function, with consequential modification on their nutritional and health value.
This project has shed light on the key factors contributing to the oxidative modification of casein proteins on exposure to oxidative insults that are commonly generated during milk processing and storage. One of the major findings of this work was that concentrated solutions of casein proteins exacerbate radical chain reactions and increase the extent of oxidative damage (DOI: 10.1016/j.foodhyd.2021.107060). Thus, the modification of the intrinsically-disordered casein proteins is shown to be highly dependent on the protein concentration. Crowded environments arising from high casein concentrations (at concentrations found in milk), and the close association between protein chains (due to formation of micelle-like structures), favors radical chain propagation reactions with this enhancing damage. In addition, oxidation of cysteine, tyrosine, methionine and tryptophan residues in κ-casein modulated the formation of amyloid-like fibrils. Additionally, the rates of oxidation, the total extent of modification, and the nature of the products formed are modulated by crowded environments (DOI: 10.1016/j.redox.2021.102202) as demonstrated for tryptophan residues (an essential amino acid for human nutrition). This work revealed the significance of volume effects on oxidative processes and the importance of developing approaches to minimize tryptophan loss and modification in crowded environments such as food systems including milk and dairy products.
Finally, this project revealed that photo-oxidation of casein proteins as consequence of illumination with visible light in the presence of the photosensitizer riboflavin (i.e. vitamin B2) can lead to the oxidation of tyrosine residues triggering the formation of quinone products. These quinone species react efficiently with Cys-containing peptides and proteins (e.g. κ-casein and β-lactoglobulin) mediating the formation of stable covalent cross-links that mediate the formation of high molecular mass aggregates (DOI: 10.1016/j.foodchem.2022.132667). These species have been shown to be long-lived and have the potential of being used by industry as biomarkers to determine the quality of milk proteins after processing to assure quality.
Exploitation and dissemination of results:
Conferences:
-Invited speaker, Redox Biology Congress: Oxidative stress, Redox biology and antioxidants from plants to humans, August 2022, Ghent, Belgium.
-Invited speaker, Future of Redox Biology meeting, June 2022, Siena, Italy.
-Invited speaker, “Pathways and consequences of the oxidation of macromolecules”. Pontifical Catholic University of Chile, Chile and University of Buenos Aires, Argentina, 2021.
-Short oral communication, Society for Free Radical Research Europe (SFRR-E), Belgrade 2021.
-Short oral communication at 20th biennial meeting of the Society for Free Radical Research International (SFRR-I), Virtual meeting.
Scientific publications:
1) C. Rossi et al., Food Chemistry, 2022, 385, 132667. DOI: 10.1016/j.foodchem.2022.132667.
2) E. Fuentes-Lemus et al., Molecules, 2022, 27, 15. DOI: 10.3390/molecules27010015.
3) E. Fuentes-Lemus et al., Redox Biology, 2021, 48, 102202. DOI: 10.1016/j.redox.2021.102202.
4) E. Fuentes-Lemus et al., Food Hydrocolloids, 2021, 121, 107060. DOI: 10.1016/j.foodhyd.2021.107060.
Dissemination and communication by other means than scientific publications:
The results have been posted on social media (e.g. LinkedIn) and presented in local meetings (primarily via zoom). Moreover, in October 2021 part of the results were presented to an audience from South America in a (virtual) workshop organized by the University of Buenos Aires (Argentine) and the Pontifical Catholic University of Chile (Chile). This activity was an excellent opportunity to reach bachelor’s students, young researchers and academics working in countries outside the EU, and introduce to them a current scientific problem, and the necessity of tackling this to improve the quality of food products. In all these activities, the acknowledgements to the funding agency were included.
Finally, this project revealed that photo-oxidation of casein proteins as consequence of illumination with visible light in the presence of the photosensitizer riboflavin (i.e. vitamin B2) can lead to the oxidation of tyrosine residues triggering the formation of quinone products. These quinone species react efficiently with Cys-containing peptides and proteins (e.g. κ-casein and β-lactoglobulin) mediating the formation of stable covalent cross-links that mediate the formation of high molecular mass aggregates (DOI: 10.1016/j.foodchem.2022.132667). These species have been shown to be long-lived and have the potential of being used by industry as biomarkers to determine the quality of milk proteins after processing to assure quality.
Exploitation and dissemination of results:
Conferences:
-Invited speaker, Redox Biology Congress: Oxidative stress, Redox biology and antioxidants from plants to humans, August 2022, Ghent, Belgium.
-Invited speaker, Future of Redox Biology meeting, June 2022, Siena, Italy.
-Invited speaker, “Pathways and consequences of the oxidation of macromolecules”. Pontifical Catholic University of Chile, Chile and University of Buenos Aires, Argentina, 2021.
-Short oral communication, Society for Free Radical Research Europe (SFRR-E), Belgrade 2021.
-Short oral communication at 20th biennial meeting of the Society for Free Radical Research International (SFRR-I), Virtual meeting.
Scientific publications:
1) C. Rossi et al., Food Chemistry, 2022, 385, 132667. DOI: 10.1016/j.foodchem.2022.132667.
2) E. Fuentes-Lemus et al., Molecules, 2022, 27, 15. DOI: 10.3390/molecules27010015.
3) E. Fuentes-Lemus et al., Redox Biology, 2021, 48, 102202. DOI: 10.1016/j.redox.2021.102202.
4) E. Fuentes-Lemus et al., Food Hydrocolloids, 2021, 121, 107060. DOI: 10.1016/j.foodhyd.2021.107060.
Dissemination and communication by other means than scientific publications:
The results have been posted on social media (e.g. LinkedIn) and presented in local meetings (primarily via zoom). Moreover, in October 2021 part of the results were presented to an audience from South America in a (virtual) workshop organized by the University of Buenos Aires (Argentine) and the Pontifical Catholic University of Chile (Chile). This activity was an excellent opportunity to reach bachelor’s students, young researchers and academics working in countries outside the EU, and introduce to them a current scientific problem, and the necessity of tackling this to improve the quality of food products. In all these activities, the acknowledgements to the funding agency were included.
This project has contributed to understanding the pathways leading to the modification of casein proteins, the most abundant proteins in bovine milk. This knowledge is essential to create better policies regarding milk processing and storage to avoid these processes and modifications. Moreover, it has reported different methodologies that have the potential of being used by industry to determine food quality. This project has therefore generated results that are of relevance for both the industry and society.
The major findings obtained have been published in different scientific journals as open access articles and are therefore available to the scientific community, industrial parties and to the non-academic sector of society. The researcher and his supervisor are currently working on submitting a new grant proposal to a funding agency belonging to a Danish dairy company – Arla Food for Health program – and are therefore committed to continue investigating the topic of this project. The results of this project have been presented at different international (European) conferences.
The major findings obtained have been published in different scientific journals as open access articles and are therefore available to the scientific community, industrial parties and to the non-academic sector of society. The researcher and his supervisor are currently working on submitting a new grant proposal to a funding agency belonging to a Danish dairy company – Arla Food for Health program – and are therefore committed to continue investigating the topic of this project. The results of this project have been presented at different international (European) conferences.