Duerasch Anja, Herrmann Pia, Hogh Konstantin, Henle Thomas
Chair of Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany.
J Agric Food Chem. 2020 Nov 25;68(47):13940-13949. doi: 10.1021/acs.jafc.0c00904. Epub 2020 Nov 17.
β-Casein is an amphiphilic protein and thus considered as multilaterally bound in casein micelles. Its polar molecule part, in particular the phosphoserine residues, can interact electrostatically with colloidal calcium phosphate (CCP) to form nanoclusters and its nonpolar molecule part enhances micellar stability by forming hydrophobic bonds to other caseins. Because cooling weakens hydrophobic interactions, a substantial portion of β-casein can be irreversibly removed from the casein micelle by repeated depletion steps, including cooling and subsequent ultracentrifugation. Although this effect of cooling on the micellar β-casein concentration has been well known for decades, the influence of depletion on the main characteristics of casein micelles has been less investigated yet. Therefore, we aimed to analyze the consequences of β-casein depletion on the stability as well as the functionality of casein micelles to evaluate the suitability of depleted compared to native casein micelles as nanocarriers. Up to 43.2% of the total β-casein was irreversibly sequestered from native casein micelles by repeated cooling and ultracentrifugation steps. Depletion showed no effect on size distribution as well as polydispersity and particle concentration of micelle suspensions as measured via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), respectively. Furthermore, the stability of the micelles against ethanol or the chelating agent ethylene glycol-bis(β-aminoethyl ether)-,,','-tetraacetic acid (EGTA) was not influenced by β-casein depletion. Notwithstanding, depleted micelles were less susceptible to enzymatic cross-linking by microbial transglutaminase (mTG), indicating narrowed water channels due to depletion. Additionally, loading experiments showed that depleted micelles could be loaded with linoleic acid (LA) as intensively as native micelles, whereupon LA displaces up to 81.3% of β-casein from native micelles. Our results confirm that depletion does not enhance the ability of the casein micelle to act as a nanocarrier for hydrophobic substances but could support the understanding of the casein micelle structure. Based on the observed unchanged stability against EGTA, the hindered enzymatical cross-linking, and the efficient displacing of β-casein by LA, we suggest that the major portion of micellar β-casein is hydrophobically incorporated into the micelle structure without impact on the formation of calcium phosphate nanoclusters. The main role of β-casein for the casein micelle structure, therefore, might be to facilitate the high hydration of the interior and thus the high permeability of casein micelles.
β-酪蛋白是一种两亲性蛋白质，因此被认为在酪蛋白胶束中以多面结合的形式存在。其极性分子部分，特别是磷酸丝氨酸残基，可与胶体磷酸钙（CCP）发生静电相互作用形成纳米簇，而非极性分子部分则通过与其他酪蛋白形成疏水键来增强胶束稳定性。由于冷却会削弱疏水相互作用，通过包括冷却和随后的超速离心在内的反复耗尽步骤，相当一部分β-酪蛋白可从酪蛋白胶束中不可逆地去除。尽管冷却对胶束β-酪蛋白浓度的这种影响已为人所知数十年，但耗尽对酪蛋白胶束主要特性的影响尚未得到充分研究。因此，我们旨在分析β-酪蛋白耗尽对酪蛋白胶束稳定性和功能的影响，以评估与天然酪蛋白胶束相比，耗尽的酪蛋白胶束作为纳米载体的适用性。通过反复冷却和超速离心步骤，高达43.2%的总β-酪蛋白从天然酪蛋白胶束中被不可逆地分离出来。分别通过动态光散射（DLS）和纳米颗粒跟踪分析（NTA）测量发现，耗尽对胶束悬浮液的尺寸分布、多分散性和颗粒浓度没有影响。此外，胶束对乙醇或螯合剂乙二醇双（β-氨基乙醚）-N,N,N',N'-四乙酸（EGTA）的稳定性不受β-酪蛋白耗尽的影响。尽管如此，耗尽的胶束对微生物转谷氨酰胺酶（mTG）的酶促交联不太敏感，这表明由于耗尽导致水通道变窄。此外，负载实验表明，耗尽的胶束与天然胶束一样能够大量负载亚油酸（LA），随后LA可从天然胶束中取代高达81.3%的β-酪蛋白。我们的结果证实，耗尽不会增强酪蛋白胶束作为疏水性物质纳米载体的能力，但有助于理解酪蛋白胶束的结构。基于观察到的对EGTA稳定性不变、酶促交联受阻以及LA对β-酪蛋白的有效取代，我们认为胶束β-酪蛋白的大部分通过疏水作用融入胶束结构，而不会影响磷酸钙纳米簇的形成。因此，β-酪蛋白对酪蛋白胶束结构的主要作用可能是促进内部的高水合作用，从而提高酪蛋白胶束的高渗透性。
