The causes of protein instability in white wine

Karien O'Kennedy - 29 Apr 2016

Protein deposits in white wine are an aesthetic problem and unacceptable to the consumer. The use of bentonite to prevent this problem has disadvantages, but research indicates there may be promising alternatives. Winemakers prevent the problem of protein deposits in white wine by removing proteins from the juice or wine by means of bentonite. The use of bentonite has several disadvantages, however, and over the past 20 years there has been ongoing research to find alternatives. Recently various alternatives were identified, the most promising of which appears to be the protease, Proctase®. This article provides a short overview of the proteins responsible for instability, as well as the factors that impact on their stability.

 

The proteins

The two principal groups of proteins responsible for instability are chitinases and thaumatin-like proteins (TLPs). Beta-glucanases may also cause instability, but occur in much smaller quantities in wine and therefore they do not make a significant contribution. The amount of chitinases and TLPs that occur in wine depends on the cultivar, vintage, grapevine disease and pressing conditions. Chitinases and TLPs are known as pathogen related proteins, in other words, proteins produced by the grapevine following an attack by fungi or viruses. Over the centuries the grapevine has adapted to such an extent however that these proteins may be formed even if there is no pathogen that stimulates it (constitutive expression).

Chitinases and TLPs are resistant to fungal infections as a result of their globular structure and multiple disulphide bridges and consequently proteases deriving from fungi such as Aspergillus niger cannot effectively break down these proteins in their natural form. Over the past 20 years in depth research has been conducted into the structures of these proteins, leading to a better grasp of white wine protein instability and the prevention thereof.

 

The role of temperature

Temperature plays a very important role in the structure of proteins and proteins differ in respect of their sensitivity to temperature. Chitinases are more sensitive than TLPs and may denaturate within minutes at temperatures above 40°C, compared to TLPs where this may take weeks. Denaturation basically means that the protein unfolds and loses its globular appearance, as well as its specific action (for example enzymatic action). Denaturation also causes the exposure of parts of the protein that would otherwise be sheltered against the environment. If these recently exposed parts of the protein are hydrophobic, they attract adjacent proteins that also have exposed hydrophobic parts. Their objective is to move away from the aqueous environment by forming aggregates. The bigger these aggregates, the more visible they become to the naked eye. Only proteins that denaturate therefore result in a protein deposit. Not all TLPs result in a deposit, because some of them may fold up again should conditions become favourable.

 

Ionic strength

Proteins in wine pH are positively charged. They repel each other in a process known as electrostatic repulsion. The smaller they are, the more readily they remain in suspension. Electrostatic repulsion also counters hydrophobic attraction. There are many components with negative charges in wine that may bind to proteins and by so doing reduce electrostatic repulsion. Examples of wine components that may bind to proteins are sulphate (SO4), polyphenols, polysaccharides and organic acids. Sulphate plays the biggest role in the formation of aggregates and in more than one way (not just the influence on ionic strength).

In the course of time protein instability may also take place without exposure to high temperatures. This indicates that other factors also impact on protein structure. Research has found that a change in wine pH may result in protein unfolding. This is the case with chitinases especially. TLPs are more stable under altered pH conditions.

 

Three phases of protein instability

Protein aggregates that are visible to the naked eye and that may be deposited due to gravity are formed by a mechanism that consists of three phases:

1. Proteins in young wines are stable and not visible to the naked eye. Increased storage temperature or wine pH may result in changes in protein structure (unfolding). Such unfolding may then expose the hydrophobic (water averse) or hydrophylic (water loving) interior parts of the protein.

2. Only proteins with hydrophobic interior parts are able to attract each other and form aggregates. Wine salts and sulphates may aid the process. Hydrophylic proteins may fold up again with cooling.

3. Protein aggregates may also bind to other wine components, such as for example polyphenols, to form even bigger compounds which eventually become visible to the naked eye and are deposited.

More thorough knowledge of the specific proteins involved in instability, their structure, as well as the mechanism by which they unfold and aggregate, will eventually result in more effective strategies to measure and prevent instability. The protease enzyme, Proctase®, which derives from the fungus Aspergillus niger, is able to prevent instability by further breaking down of the proteins that have already unfolded due to heat, and by so doing prevent hydrophobic interactions and aggregate formation. This nevertheless has a minimal influence on proteins that are still in a natural globular form.

 

Reference:

Sluyter, S.C., McRae, J.M., Falconer, R.J., Smith, O.A., Bacic, A., Waters, E.J. and M. Marangon (2015). Wine protein haze: Mechanisms of formation and advances in prevention. J. Agric. Food Chem. 63, 4020-4030.

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