Elsevier

International Dairy Journal

Volume 67, April 2017, Pages 35-45
International Dairy Journal

β-Lactoglobulin nanofibrils: The long and the short of it

https://doi.org/10.1016/j.idairyj.2016.09.011Get rights and content

Abstract

The bovine whey protein β-lactoglobulin will self-assemble into amyloid-like nanofibrils when heated at pH ≤ 3 and temperatures ≥75 °C. These fibrils have diameters <10 nm and lengths that can exceed 10 μm. The length and stiffness of fibrils depends on ionic strength; heating at low ionic strength produces long semi-flexible fibrils, whereas heating with added salts produces highly flexible ‘worm-like’ fibrils that are an order of magnitude shorter. Over the last two decades there has been a substantial research effort focused on imaging fibrils, manipulating conditions to accelerate fibril formation, and characterising microstructural and rheological properties of fibril dispersions. Here we review the major mechanistic findings, explore potential applications for fibrils as food ingredients, and highlight the major gaps in knowledge surrounding these intriguing self-assembled structures.

Introduction

The term amyloid arises from abnormal tissue deposits in the brain that stain positively with iodine; these deposits were discovered in 1854 and they were found to consist of nanofibrils in 1959 (Sipe & Cohen, 2000). The characteristic stacked β-sheet internal structure of the amyloid nanofibrils is now understood to be a generic folding pattern that almost all proteins are capable of assuming (Goldschmidt et al., 2010, Otzen, 2010). The term ‘amyloid’ usually refers protein structures assembled all instances of in vivo and in vitro should be italicised, and includes a wide variety of non-pathological proteins such as the curli proteins involved in bacterial biofilm formation, chorion proteins reinforcing the egg shells of silk moths, fish and other organisms, and Pmel17 protein involved in human melanosome biogenesis (Berson et al., 2003, Dueholm et al., 2012, Iconomidou and Hamodrakas, 2008).

‘Amyloid-like’ is often used in connection with fibrils assembled in vitro that share the same ‘stacked β-sheet’ structure, and the amount of fibrils can be quantified using fluorescent dyes that bind to this structure, particularly thioflavin T (ThT) and Congo red (Nilsson, 2004). The prefix ‘nano’ refers to the fact that individual fibrils have diameters well below 20 nm. Subsequent reference to fibrils in this paper refers to amyloid-like nanofibrils.

For the milk proteins, amyloid-like fibrils have been shown to form from α-lactalbumin (Goers et al., 2002, Ipsen and Otte, 2007), αS2-casein and κ-casein (Thorn, Ecroyd, Carver, & Holt, 2015), and bovine serum albumin (Usov et al., 2013, Veerman et al., 2003b). However, by far the most common source material for milk protein-derived fibrils is β-lactoglobulin (β-lg), which readily assembles into fibrils when heated at low pH and low ionic strength. The β-lg can be isolated using a simple salt precipitation method (Dave et al., 2013) from whey protein isolate (WPI) – a commercial milk protein fraction that is derived by ultrafiltration of whey, and typically contains >90% protein, most of which is β-lg (Foegeding, Luck, & Vardhanabhuti, 2011).

Fibrils that form in mixed whey protein solutions appear to comprise only β-lg (Bolder, Hendrickx, Sagis, & van der Linden, 2006), and β-lg appears to be the only dairy protein capable of rapidly forming very long fibrils during heating under suitable conditions. These heat-induced β-lg fibrils will be the focus of this review. Amyloid-like aggregation is compared with other protein aggregation patterns in a recent review by Mezzenga and Fischer (2013), and polymer physics aspects of fibril aggregation were discussed by Adamcik and Mezzenga (2012). Fig. 1 shows a typical example of amyloid-like fibrils formed by heating bovine β-lg at low pH and low ionic strength (top). Individual fibrils have a diameter of 5–10 nm and a length that can exceed 10 μm, i.e., an aspect ratio in the order of 1000 or more.

The β-lg amyloid-like fibrils that assemble at low ionic strength and low pH are considered semi-flexible according to biopolymer physics definitions. Fibril flexibility is quantified as persistence length, lp, which typically varies from 1.5 to 4 μm when measured with TEM or AFM images (Loveday et al., 2010, Schleeger et al., 2013). It is the extreme length and flexibility of β-lg fibrils that allows fibril dispersions to form self-supporting physical entanglement networks that gel at much lower protein concentrations than other types of β-lg aggregates (Kavanagh et al., 2000b, Loveday et al., 2009), though with a narrower linear viscoelastic shear rate range (Wu et al., 2016).

In addition to forming water-holding gel networks, β-lg fibrils can also adsorb at interfaces, form fibril–fibril and fibril-polymer superstructures, and they can be surface-functionalised via simple chemical crosslinking reactions. The morphology and functionality of β-lg fibrils can be tuned by manipulating heating conditions. Here we will explore the unique properties of β-lg fibrils and the potential they have as high-value components of food ingredients.

Section snippets

Nanofibril assembly processes

For β-lg and other proteins with a significant degree of tertiary structure, partial denaturation must occur before fibril assembly is possible. Unfolding of the tertiary structure allows β-sheets to come into contact and form hydrogen-bonded stacks. Denaturation with concentrated alcohols or urea will induce β-lg to assemble into fibrils over several days (Gosal et al., 2004a, Hamada and Dobson, 2002), whereas denaturation and assembly occur within hours at pH < 3 and temperatures >75 °C. When

pH, ionic strength and ion-specific effects

The solution pH has a strong influence on protein reactions because it determines the ionisation states of the terminal carboxyl and amino groups as well as the ionisable amino acids – primarily Arg, His, Lys, Asp and Glu. The isoelectric point of β-lg is ∼5.2 (Swaisgood, 1982), and a pH below 2.5 favours dissociation of dimers into monomers (Mercadante et al., 2012). At low ionic strength, long semi-flexible fibrils form in heated β-lg solutions between pH 1.6 and pH 3. Aggregates are much

Fibrils as food ingredients

Relatively extreme process conditions are needed to induce β-lg to form fibrils, and the utility of fibrils as food ingredients depends on their behaviour under conditions more relevant to food systems. This section examines how β-lg fibrils are affected by changing conditions, and how the functionality of fibrils may be enhanced by post-assembly processing such as crosslinking or surface modification. Whey protein fibrils may have applications in biophysical or biotechnological fields (

Conclusions

Over the last two decades there has been a substantial research effort focused on manipulating conditions to accelerate β-lg fibril formation, imaging fibrils and characterising microstructural and rheological properties of fibril dispersions. The vast majority of research has focused on the long semi-flexible fibrils formed at low ionic strength, perhaps because they are amenable to modelling with polymer physics concepts. The shorter wormlike fibrils have received little attention, despite

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