Proteins are produced in the grape during the ripening process, and in response to diseases and injuries1. These proteins end up in the wine and are poorly soluble so that they can cause an undesired cloudiness – a protein haze. Bentonite is one of the most used products to remove the proteins from the wine and to prevent a protein haze. Unfortunately, there are also a number of disadvantages to the use of bentonite.
This article will discuss the (molecular) action of bentonite, the different types of bentonite, the disadvantages of the use of bentonite and the optimal timing and conditions for its use during vinification. Finally, the (future) alternatives to bentonite are shortly discussed.
Bentonite is a clay that originates from volcanic ash that settled on the land millions of years ago. It was named by Wilbur C. Knight in 1898 after the Benton formation (Wyoming, USA) where the bentonite was found. Bentonite is a mixture of minerals that for the most part consists of the mineral montmorillonite, supplemented with minerals such as plagioclase, biotite, quartz, gypsum and calcite, and traces of heavy metals. Today, bentonite is mined in many places in the world and the composition of bentonite is therefore always slightly different2.
The function of bentonite is dependent on the mineral montmorillonite. Montmorillonite is a 2:1 clay, two parts silicon and one part aluminum. In practise, this means that the crystal structure of montmorillonite is made up of two layers of silicon atoms (Si) bound to oxygen (O), between which there is one layer of aluminum atoms (Al) that are also bound to oxygen. These silicon and aluminum ‘sandwiches’ make up the montmorillonite layers which are 1 nanometer thick, and can be up to 1000 nanometers wide. The montmorillonite layers are in turn also stacked on top of each other3.
The molecular structure of montmorillonite. Each montmorillonite particle consists of two layers of silicon atoms bound to oxygen with a layer of aluminum atoms bound to oxygen in between.
The silicon and aluminum atoms in the montmorillonite have a positive charge that is canceled by the negative charge of the oxygen atoms. Only in the most ideal case are the layers of montmorillonite made of only silicon and aluminum. Often the silicon and aluminum atoms are partly replaced by iron and magnesium atoms with a smaller charge. As a result, there are fewer positive charges in the structure and a negative charge is created on the surface of the montmorillonite layers. This charge difference must be eliminated, and therefore the montmorillonite layers bind cations – these are particles with a positive charge – such as sodium (Na+), magnesium (Mg2+) and calcium (Ca2+) REF4.
The more silicon and aluminum atoms that are replaced in the montmorillonite structure, the greater the negative charge of the surface becomes, and the more cations are bound between the montmorillonite layers. In a solution (such as water or wine), these cations can be exchanged for other positively charged molecules, such as positively charged proteins. The montmorillonite adsorbs the positive proteins and in return the cations are released in the wine5, 6. A larger negative surface charge ensures a greater cation exchange capacity. This means that there are more positively charged particles bound to the montmorillonite that can be exchanged, which therefore results in a better protein adsorption.
Montmorillonite properties. The negative surface charge of the montmorillonite to which cations bind (left). The absorption of water between the layers and the adsorption of proteins on the montmorillonite layers (middle). In low pH solutions (below 7) montmorillonite layers form a “house of cards” structure due to the positive charge at the edges (right).
HOUSE OF CARDS
The pH of the liquid, must or wine in which the montmorillonite is used determines how the montmorillonite layers are stacked. In addition to the negative surface charge, montmorillonite also has a positive charge on the edges of its layers. This positive charge becomes stronger at a lower pH. The positively charged edges are attracted to the negative surface charges of other montmorillonite layers, which makes that the layers interact. In low pH solutions (below 7 pH), the montmorillonite layers are therefore not neatly stacked, but form a house of cards structure7-9. Although much is unknown about the precise effect of this structure, it is likely to cause a more negative surface charge, which contributes to the adsorption of (the positively charged) proteins from the liquid10. However, the formation of the ‘house of cards’ structure is at its optimum between a pH of 4.5 to 5.5. A very acidic solution (like must or wine) negatively affects the formation of this structure. At pH below 4.5 the surface-to-surface stacking is likely dominant due to the high number of ions in the solution that compete to bind to the montmorillonite and disturb the edge-surface interactions8, 10.
SODIUM vs. CALCIUM MONTMORILLONITE
The naming of the montmorillonite type – and also bentonite type – depends on the cation that is most present between the layers of montmorillonite. Montmorillonite with mainly Na+ ions is called sodium montmorillonite and calcium montmorillonite mainly has Ca2+ bound11.
The extent to which the montmorillonite absorbs water and can adsorb proteins depends on the type of cation between the layers, and on the pH of the liquid. In practice there are two types of montmorillonite (and bentonite) that are used; sodium montmorillonite and calcium montmorillonite. Na+ dissolves easily in water and therefore ensures that sodium montmorillonite easily absorbs water and can exchange many cations. Ca2+ dissolves less easily and therefore remains “sticking” between the montmorillonite layers. This keeps the montmorillonite more compact and less water is absorbed between the layers. In comparison to Na+, Ca2+ ensures that the edges of the montmorillonite have a larger positive charge, making it easier to create a house of cards structure. However, this property also makes that calcium bentonite tends to clump more easily on swelling (with low mineral water). On the upside, calcium bentonite produces a heavier sediment that is easier to remove. Nevertheless, sodium montmorillonite adsorbs proteins a lot more efficiently than calcium montmorillonite5, 12.
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The ratio of the cations in the montmorillonite structure is therefore very decisive for the ab- and adsorbent effect when clarifying the wine. Where Na+ in particular ensures a high cation exchange and swelling of the montmorillonite (due to absorption of water), Ca2+ ensures that the montmorillonite assumes a house of cards structure and remains more compact.
Although sodium bentonite adsorbs proteins best, calcium bentonite or a mixture of sodium and calcium bentonite is almost always used during vinification. The use of sodium bentonite has the disadvantage that it causes a greater volume loss and large amounts of sodium that enter the wine7. High sodium intake is associated with cardiovascular diseases13, and the use of “pure” sodium bentonite in wine is therefore prohibited in, among others, Germany14. In addition, international guidelines have been drawn up for the maximum amount of interchangeable cations such as Na+ and Ca2+ that may enter the wine11. Ca2+ is a lot safer for human consumption, but can cause the crystallization and precipitation of tartar15 if concentrations are too high.
Through the use of bentonite, in addition to sodium and calcium, there is a list of other elements such as Li, Be, Na, Mg, Al, Ca, Sc, V, Mn, Fe, Co, Ni, Ga, Ge, As, Sr, Y , Zr, Nb, Mo, Cd, Sn, Sb, Ba, W, Tl, Bi, and W that are released in the wine16. To prevent extensive pollution, the International Oenological Codex stipulates that only bentonite consisting of at least 80% montmorillonite should be used11. In practice, however, it is often difficult to find out the precise composition of the bentonite. As a result, the bentonite may consist of a low percentage of montmorillonite, it may be unclear what the ratio of calcium and sodium montmorillonite is, the exchange capacity of cations may be limited, and it may contain heavy metal impurities. It is therefore important not only to use as little bentonite as possible, but also to use the purest possible quality of bentonite for wine fining.
OPTIMAL CONDITIONS FOR BENTONITE
To minimize the use of bentonite, it must be used under conditions where it works as efficiently as possible. The optimal effect of bentonite depends on the pH, the temperature and the alcohol percentage of the must / wine. The pH is the most important variable for the adsorption of proteins. This not only determines the charge of bentonite (montmorillonite, see above), but also the ionization of the proteins17. At a low pH, more proteins are positively charged (their isoelectric point is higher) and will bind to the negatively charged surface of the montmorillonite. When the pH of the must / wine is too high, more proteins do not get a positive charge and are therefore not adsorbed by the montmorillonite. The wine will remain to have a protein haze. The conditions can be optimized to let the bentonite work better. For example, the must can be acidified, or the bentonite can be used before fermentation when the pH is lower. The minimum pH required for all proteins to be bound by bentonite is different for each grape variety18, and ranges roughly between a pH of 2.5 to 3.5. This is because the composition of the proteins differs per grape variety19 and is influenced each year by the climatic conditions in the vineyard20.
In addition to the pH, both a higher temperature and a higher alcohol percentage – in particular at >10% – ensure improved adsorption of the proteins5, 21. Alcohol, just like water, nestles between the layers of the montmorillonite and makes the space between the layers slightly larger, making it easier for the proteins to reach the deeper interchangeable cations5. A higher alcohol percentage therefore ensures better adsorption of large proteins, but has no effect on the proteins that are already adsorbed anyway17, 21.
With the optimal conditions for the use of bentonite in mind, one would prefer to use bentonite in a must / wine stage in which the pH is low, and the temperature and the alcohol percentage are high. During the alcoholic fermentation the pH rises, but the temperature and the alcohol percentage are higher. The optimum condition is therefore always a consideration of these three factors.
OPTIMAL CONDITIONS FOR THE WINE
Bentonite causes a loss of colorants and aromas in the wine because they are adsorbed by montmorillonite, or bind to the proteins extracted from the wine by bentonite4, 22. To limit this loss, it is desirable to use bentonite as little and as efficiently as possible. A heat test or a bento test can be used to determine the amount of bentonite required. However, it is more difficult to determine the best time to use bentonite during vinification. The moment of addition (before, during or after fermentation) is not only important for the efficiency of bentonite (see paragraph above), but can also have a different effect on the loss of aromas and colorants. In addition, bentonite also binds nitrogen compounds, which has an effect on the amount of yeast food present23.
The use of bentonite in the middle and towards the end of the alcoholic fermentation is the most efficient, and also has the least effect on the aromas of the wine24. As such, it seems that the most efficient time point during the vinification to use bentonite fortunately coincides with the moment that bentonite has the least effect on the wine’s aroma profile. Further, the use of bentonite after fermentation and just before bottling is not recommended25. The wine can then hardly recover from the loss of aromas and colorants and becomes unbalanced.
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The amount of research into the timing of bentonite use during the vinification and its effects on the wine’s aroma profile is limited. The above referred research gives a good indication of the effects of bentonite, but has only been performed on still Spanish Albarino wines. It is likely that the ideal moment to use bentonite differs per grape variety and per wine type. After all, every grape and type of wine (sparkling, white, orange) has a unique composition of aromas and (undissolved) proteins.
THE (FUTURE) ALTERNATIVES
The disadvantages of using bentonite in vinification – loss of volume, aroma and color – are widely recognized, and therefore better alternatives are being sought. Research is being done on genetically modified yeasts that produce more mannoproteins26, on enzymes such as proctase that specifically break down the proteins responsible for the protein haze27, and there is plenty of experimentation with replacement filter substances such as the polysaccharide chitosan28 and porous nanoparticles29, 30. The use of genetically modified yeast is however often not (yet) desirable, and proctase requires flash pasteurization (rapid heating to 70℃) to be effective. Apart from the effects this has on wine, expensive equipment is also required for this rapid heating. Fortunately, the results with chitosan and especially with the porous nanoparticles look promising. Just like montmorillonite, these nanoparticles are composed of silicon and aluminum atoms and form a porous structure in which proteins can bind. In comparison to bentonite, however, they have a much smaller effect on the aroma profile of the wine29. But, until this is properly investigated and approved for use in wine making, bentonite – despite its disadvantages – will remain the standard for removing proteins from wine.
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