THE EFFECT OF HEAT TREATMENT AND STORAGE CONDITIONS ON THE STRUCTURAL INTEGRITY F HUMAN MILK BIOACTIVE PROTEINS: AN IN VITRO AND IN SILICO STUDY

Abstract

Human milk is a complex biological food rich in bioactive proteins like lactoferrin and osteopontin, crucial for infant nutrition, growth, immune regulation, and protection against infections. Despite their benefits, these proteins are highly susceptible to degradation during common practices, including various storage conditions (room temperature, refrigeration, freezing) and heat treatments such as pasteurization. Previous studies primarily focused on functional effects (e.g., enzyme activity, iron/vitamin binding, antimicrobial activity), while the subtle structural changes of these critical proteins remained largely unexplored. This knowledge gap hinders the development of more effective preservation strategies for human milk. This study aimed to elucidate the structural changes occurring in lactoferrin and osteopontin proteins in human milk due to different storage conditions and heat treatment applications. Our approach combined in vitro experimental methods and in silico molecular dynamics simulations to provide a detailed understanding of how these proteins are affected. For in vitro experiments, commercially obtained lactoferrin and osteopontin were prepared at concentrations mimicking colostrum. Proteins were subjected to various conditions including freeze-thawing (-80 °C and -20 °C), freeze-heating (37 °C), Holder pasteurization (62.5 °C), refrigeration (+2-8 °C), room temperature (+20-25 °C), and body temperature (37 °C). Protein concentrations were measured spectrophotometrically at 280 nm, and SDS-PAGE analysis was performed to assess molecular weight and purity changes. For in silico analysis, molecular dynamics simulations were conducted for lactoferrin (PDB ID: 1LFG) and a predicted osteopontin structure, using the CHARMM36 force field at room temperature (300 K) and Holder pasteurization (336 K) conditions. The results from both in vitro and in silico analyses indicate that Holder pasteurization significantly impacts the structural integrity of both lactoferrin and osteopontin. Spectrophotometric analysis showed a significant increase in lactoferrin concentration after pasteurization, attributed to protein aggregation. Similarly, osteopontin concentration increased after pasteurization, and SDS-PAGE confirmed a significant decrease in its band intensity, suggesting structural changes and potential aggregation. Molecular dynamics simulations further supported these findings, demonstrating a clear disruption in the stability of lactoferrin at pasteurization temperature, and some oscillation in osteopontin. While other conditions like freezing and thawing also showed some effects, pasteurization consistently emerged as the most impactful. These observed structural alterations and energy changes suggest a potential reduction in the biological activity of the proteins. The insights gained from this research are expected to contribute to future strategies for developing improved preservation methods for human milk, optimizing processing protocols, and designing beneficial nutritional supplements that maintain the integrity of these vital bioactive components.

Keywords

• Human milk
• Lactoferrin
• Osteopontin
• Heat treatment
• Storage
• Structural integrity

ANNE SÜTÜ BİYOAKTİF PROTEİNLERİNİN YAPISAL BÜTÜNLÜĞÜ ÜZERİNE ISIL İŞLEM VE DEPOLAMA KOŞULLARININ ETKİSİ: BİR IN VITRO VE IN SILICO ARAŞTIRMA

Abstract

Anne sütü, bebek beslenmesi, büyümesi, bağışıklık düzenlemesi ve enfeksiyonlara karşı korunması için kritik roller oynayan laktoferrin ve osteopontin gibi biyoaktif proteinler açısından zengin, karmaşık bir biyolojik besindir. Bu proteinlerin faydalarına rağmen, oda sıcaklığı, buzdolabı ve derin dondurucu depolama gibi çeşitli depolama koşulları ve pastörizasyon gibi ısıl işlemler dahil olmak üzere yaygın uygulamalar sırasında bozulmaya karşı oldukça hassastır. Önceki çalışmalar ağırlıklı olarak enzim aktivitesi, demir/vitamin bağlama kapasitesi ve antimikrobiyal aktivite gibi fonksiyonel etkilerine odaklanırken, bu kritik proteinlerin hassas yapısal değişimleri büyük ölçüde keşfedilmemiştir. Bu çalışma, anne sütündeki laktoferrin ve osteopontin proteinlerinin farklı depolama koşulları ve ısıl işlem uygulamaları sonucunda meydana gelen yapısal değişimlerini aydınlatmayı amaçlamıştır. In vitro deneysel yöntemler ve in silico moleküler dinamik simülasyonlarını birleştiren yaklaşımımız, bu proteinlerdeki önemli yapısal modifikasyonları ortaya koymuş ve bu modifikasyonların işlevsel bozulmaların temelini oluşturduğunu göstermiştir. Bu araştırmadan elde edilen bilgilerin, anne sütünün daha iyi muhafaza yöntemlerinin geliştirilmesine, işleme protokollerinin optimize edilmesine ve bu hayati biyoaktif bileşenlerin bütünlüğünü koruyan faydalı besin takviyelerinin tasarımına yönelik gelecekteki stratejilere katkı sağlaması beklenmektedir.

Keywords

• Anne sütü
• Laktoferrin
• Osteopontin
• Isıl işlem
• Depolama
• Yapısal bütünlük

References

1. Andreas, N. J., Kampmann, B., & Le Doare, K. (2015). Human milk: A source of more than just nutrition. Pharmacological Research, 99, 137-148.
2. Brisson G, Britten M, Pouliot Y (2007). Heat-induced aggregation of bovine lactoferrin at neutral pH: Effect of iron saturation. Int Dairy J., 17(6):617–24
3. Bhatnagar BS, Bogner RH, Pikal MJ (2008). Protein Stability During Freezing: Separation of Stresses and Mechanisms of Protein Stabilization, 12(5):505–23
4. Chang BS, Kendrick BS, Carpenter JF (1996). Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci, 85(12):1325–305
5. Chung TDY, Raymond KN (1993). Lactoferrin: The Role of Conformational Changes in Its Iron Binding and Release. J Am Chem Soc , 115(15):6765–8.
6. Çiçek, N., & Ünal, M. A. (2021). The effects of pasteurization on bioactive compounds in human milk: A review. Current Nutrition & Food Science, 17(5), 522-530.
7. Drouin, E. M., & Lönnerdal, B. (2020). Human milk osteopontin: A review of its role in infant nutrition and health. Nutrients, 12(4), 939.
8. Elwakiel M, Boeren S, Wang W, Schols HA, Hettinga KA (2020). Degradation of Proteins From Colostrum and Mature Milk From Chinese Mothers Using an in vitro Infant Digestion Model. Front Nutr, 16;0:162

9. Haridas M, Anderson BF, Baker EN (1995). Structure of human diferrric lactoferrin refined at 2.2 Angstrom resolution. Acta Crystallogr - Sect D Biol Crystallogr. 51(5):629–46
10. Hawe A, Kasper JC, Friess W, Jiskoot W (2009). Structural properties of monoclonal antibody aggregates induced by freeze–thawing and thermal stress. Eur J Pharm Sci, 38(2):79–87
11. Hinde, K., & Milligan, J. (2018). Human milk: An evolutionary resource for health. Maternal & Child Nutrition, 14(S1), e12595.
12. Kueltzo LA, Wang W, Randolph TW, Carpenter JF (2008). Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing. J Pharm Sci ,97(5):1801–12
13. Lam XM, Costantino HR, Overcashier DE, Nguyen TH, Hsu CC (1996). Replacing succinate with glycolate buffer improves the stability of lyophilized interferon-γ. Int J Pharm, 142(1):85–95
14. Lönnerdal, B. (2009). Lactoferrin: A bioactive protein in human milk. Annals of Nutrition and Metabolism, 54(S1), 31-35
15. MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, et al (1998). All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B, 102(18):3586– 616
16. Peila, C., Moro, G. E., & Cavallarin, L. (2016). Effects of pasteurization and freezing on the microbiological and immunological quality of human milk. Journal of Pediatric Gastroenterology and Nutrition, 63(6), 668-675
17. Picaud, J. C., Rigo, J., & De la Mettrie, J. (2007). Effect of thermal processing on human milk quality. Journal of Pediatric Gastroenterology and Nutrition, 44(S1), S19-S23
18. Privalov PL (1990). Cold Denaturation of Protein. Crit Rev Biochem Mol Biol, 25(4):281-305
19. Roterman I, Marchewka D, Strus M, Śpiewak K, Majka G (2012). Structural Analysis of the Lactoferrin Iron Binding Pockets. Bio-Algorithms and Med-Systems 8(4):351–9.
20. Xiong L, Boeren S, Vervoort J, Hettinga K (2021). Effect of milk serum proteins on aggregation, bacteriostatic activity and digestion of lactoferrin after heat treatment. Food Chem. 1;337:127973.
21. Wang B, Timilsena YP, Blanch E, Adhikari B (2019). Lactoferrin: Structure, function, denaturation and digestion. Crit Rev Food Sci Nutr, 59(4):580–96