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

Yıl 2025, Cilt: 8 Sayı: 2, 371 - 386, 31.12.2025

Ferhan Yapar , Evrim Güneş Altuntaş , Mehmet Altay Ünal

https://doi.org/10.55930/jonas.1744315

Öz

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.

Anahtar Kelimeler

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

Proje Numarası

FLY-2022-2487

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

Öz

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.

Anahtar Kelimeler

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

Destekleyen Kurum

Ankara Üniversitesi BAP

Proje Numarası

FLY-2022-2487

Teşekkür

Bu çalışma “Ferhan YAPAR” ın Ankara Üniversitesi Biyoteknoloji Enstitüsü’ne sunduğu Yüksek Lisans Tezi’nin bir bölümüdür. Aynı zamanda çalışma Ankara Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından FLY-2022-2487 kodlu proje kapsamında desteklenmiştir.

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