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Bentonite - more than just dirt


Paul Bowyer

Paul K. Bowyer1 & Virginie Moine-Ledoux2

1 Technical Manager – Australasia, Laffort Oenologie Australia, 5 Williams Circuit, Pooraka SA 5095, Australia

2 Product Manager – Fining Agents, Laffort Oenologie, BP 40, 33015 Bordeaux Cedex, France


Stability is an aspect of winemaking that all winemakers think about throughout the year, whether it’s the stability of grape aromatics at the crusher during vintage, the stability of colour during maturation or micro oxygenation, or simply the stability of potassium bitartrate (Bowyer, 2001) when Joe Public puts a bottle of white into the fridge. Some of these stability problems are more easily dealt with than others, such as using inert gases during winemaking or inhibiting bitartrate precipitation through cold stabilisation (or with greater technological finesse and efficiency by using a purified mannoprotein such as Mannostab™ more on that one in a future article). The one stability issue that we have not yet found an alternative treatment for is protein instability.



FIG 1: Graph showing the variation in protein solubility with changes in pH.

Proteins

The structure of proteins governs many of their characteristics. For starters, they can have up to four distinct modes of structural variation (referred to as their primary, secondary, tertiary and quaternary structures). Proteins, like many organic molecules, are stable only under certain conditions. They are affected by changes in pH, the polarity of the solution they are in (Bowyer, 2003) and are also at the mercy of exposure to heat. When some proteins are heated to levels that exceed normal biological conditions (~37ºC) the hydrogen bonds that help the molecule retain its secondary, tertiary and quaternary structures are disrupted. Once these bonds are destroyed it is practically impossible for them to regain their original form, meaning that the protein has been irrevocably altered. We call this process “denaturation”, and there are many common examples of this process. Albumin, the major protein of egg white, is soluble at room temperature but denatures and becomes insoluble when heated, hence the term egg “white”. Crustacean shells change colour when they are cooked, due to the denaturing of a shell protein bound to a pigment. In the combined state the pigment appears blue, but when the binding protein is denatured the pigment reverts to its free molecular colouration of orange-pink.



FIG 2: The crystal lattice structure of kaolinite.

A protein’s primary structure (i.e. the amino acid sequence) can have a major impact on the physical characteristics of the molecule. For example, some amino acids have functionality that responds to a change in pH, such as a wine during fermentation. Any change in solution pH therefore can impact upon a protein’s primary structure, which in turn can impact on higher protein structures and also on the solubility of the protein. As with all weak acid and base interactions, equilibrium can be reached (Bowyer, 2001) and an overall or net charge on the molecule exists at any given pH. If the charge is high, either positive or negative, the protein will be quite soluble, since charged species dissolve in water fairly easily (remember that wine is typically >85% water). However, if these charges cancel each other out, the overall charge on the protein will be zero. The solution pH at which this occurs is defined as the protein’s isoelectric point (pI), and the protein’s solubility will be lowest (Figure 1).



FIG 3: The crystal lattice structure of mont­mo­rillonite.

For an excellent and recent overview on wine proteins see Waters et al (2005). Proteins are present naturally in grapes, and are found in the grape skin, flesh and seed (Fukui and Yokotsuka, 2003). Protein is, in the case of both white and red wines, associated with the formation of hazes and is typically removed before bottling. If left in the wine the possibility of haze formation remains, which is poorly regarded by consumers. The conditions under which a protein-induced haze will form are known, with the most prominent factor involved being the elevation of a wine’s temperature. This can take place during transportation (it can get quite hot on the back of a truck parked in the sun in South Africa for a few hours), in the boot of a car driving away from a cellar door (likewise) or even in that bottle you’ve got in the small wine rack above the fridge, just above the heat exchanging element.


Most wine proteins have an isoelectric point that lies above typical wine pH (pH 3.0 - 3.5), thus in wine they will bear a net positive charge (in contrast, laccase has a pI of around 2.5, meaning that in wine it bears a negative charge, hence its lack of removal by treatment with bentonite, and it remains quite soluble), (Ribéreau-Gayon et al, 2006b). This is the fundamental principle on which the action of bentonite towards wine protein is based, yet it is only half of the equation. To understand the other half, we need to examine the structure of bentonite in a likewise manner.


Bentonite

Bentonite is a very interesting mineral, finding application in many diverse areas such as sand casting, catalysis, dam sealing, intestinal evacuation, cat litter, sandcastle building and of course, wine processing. It even plays a role in the removal of contaminants found in wine (Ruediger et al, 2004). Bentonite is quite topical, making regular appearances in both the scientific and industrial literature (for a recent article focusing on efficiency of the usage of bentonite, see Colby et al, 2006). Bentonite is a hydrated aluminium silicate and a member of the smectite class of clays. It commonly contains two forms of mineral, kaolinite and montmorillonite, with the latter being the major form found in the bentonite that is active in wine processing. Both minerals have crystal structures that exist as sheets or layers. Montmorillonite can be subdivided again into two forms, depending on the predominant cation (positively charged ion) found in the inter-laminar regions of the sub-crystalline structure. If this seems a little confusing it will become clearer with a little more explanation.



FIG 4: The relative settling abilities of some sodium, calcium and sodium/calcium bentonite samples.


FIG 5: The different protein removal capacities of various bentonite samples.


FIG 6: A plot of wine pH versus turbidity showing the change in bentonite effectiveness as a wine's pH is altered. The hashed line at 2 NTU indicates protein stability.

Bentonite (both kaolinite and montmorillonite) is comprised mainly of oxides of aluminium and silicon. The difference between kaolinite and montmorillonite structures lies in the type of layering and the ratio of silicon to aluminium oxides. In kaolinite the layering is quite simple, with a layer each of silicon and aluminium oxides, as shown in Figure 2. In the case of kaolinite there is one very important difference between the outer oxygen layers: the outer layer of oxygen atoms closest to silicon is present as oxygen, whilst the oxygen atoms in the outer layer adjacent to aluminium is present in the form of hydroxyl (OH) groups in order to balance the charge. This means that the kaolinite crystal lattice sheet structure is different on the top and bottom. More importantly, the difference means that the upper surface of one layer is able to hydrogen bond to the lower surface of the other, so the overall structure resembles a layer cake.


The hydrogen bonding between the layers of kaolinite imparts two important properties to the mineral. Firstly, the layers are held quite tightly together. Secondly, as a result of the difficulty of separating the kaolinite layers due to the hydrogen bonding, it is not easy for water molecules to enter the inter-laminar region. The importance of this will become clear in a moment.


In montmorillonite the same structural components as kaolinite are present, with two important exceptions. In the first exception, a second layer of silicon oxide is present, such that the aluminium oxide layer sits in between the two silicon oxide layers. This forms a symmetrical structure similar to a Jersey caramel lolly (Figure 3). This means that on both upper and lower faces the lattice projects only oxygen atoms, hence the possibility of hydrogen bonding is removed. The second exception is that occasionally in the montmorillonite structure aluminium is replaced with a different metal such as iron, manganese or magnesium. This may seem like a small change, but it is fact the crux of bentonite’s ability to remove wine proteins.



Table 1: Comparative characteristics of the Laffort Oenologie Microcol bentonites.

When aluminium ionises it forms trivalent Al3+. Magnesium, manganese and iron tend to form divalent cations, hence the substitution of a trivalent cation for a divalent cation generates a deficiency in positive charge. Overall, therefore, the lattice takes on a net negative charge.


In practical terms in montmorillonite we have flat crystalline sheets bearing negative charges. Electrostatically, like charges repel one another in a similar manner to identical poles of magnets. In montmorillonite this translates into the lattice sheets repelling one another quite fiercely, yet bentonite does not simply explode into a fine dust of individual lattice sheets. Why does this not happen?


In nature structures such as minerals do not exist in totality as charged species. The charge must always be balanced by other species. In the case of montmorillonite, this charge excess is balanced by the presence of cations that reside in the inter-laminar regions, between the lattice sheets. When these cations are incorporated, the overall charge of the mineral is balanced and the material as a whole bears no net charge. The type of cation that fulfils this charge-balancing role impacts markedly on the performance of the bentonite in terms of its capacity for swelling and protein removal.


For an excellent website to help you visualise kaolinite and montmorillonite (listed therein as smectite) using movable three-dimensional images structures, visit: http://www.soils.wisc.edu/virtual_museum/displays.html. The images in Figures 2 and 3 were generated using these models.


Sodium bentonite

In sodium bentonite the inter-laminar region is occupied mainly by sodium ions and water molecules. The high polarity of water molecules makes them excellent stabilisers for cations and anions (negatively charged ions) alike. Sodium forms monovalent cations (i.e. Na+). The ionic radius of Na+ is around 100 pm (1 x 10-10 m, or 1 Å) (Aylward and Findlay, 1987), which makes Na+ reasonably large in terms of the volume each cation displaces. In filling the inter-laminar region of montmorillonite with a complement of water molecules, Na+ is acting as a sort of ionic glue, holding the lattice plates together. It is not particularly efficient glue due to its small charge density, a result of its monovalent charge and portly ionic radius, so the overall structure is relatively loosely constrained.


When sodium bentonite is added to water, the sheets begin to break apart, since the Na+ ions do not possess sufficient charge density to hinder the process to any great extent. This process is expedited by using hot (higher energy) water. Effectively, the absorption of water is very high because the plates separate easily, meaning that the swelling capacity of sodium bentonite is very high. This is the reason that the hydration of sodium bentonite can take some time, requiring continual stirring, often overnight.


The high dispersion rate of sodium bentonite has some interesting ramifications. Firstly, the surface area of sodium bentonite is very high. Secondly, the high dispersion rate means that flocculation and settling are both slower, and the formation of a relatively stable colloidal suspension can occur. This can impact dramatically on wine or juice processing efficiency. Thirdly, the protein removal capacity is very high, since the activity for protein removal is governed primarily by the charge density of the lattice and the surface area the mineral occupies: as each increases, so does the protein binding capacity.


Calcium bentonite

The only important difference between sodium and calcium bentonites is the relative proportions of the two cations in the inter-laminar region. In calcium bentonite, the divalent calcium ion predominates. The ionic radius of Ca2+ is 99 pm (Aylward and Findlay, 1987), which is almost identical to that of Na+. Although the ions occupy the same volume in space the charge density of the Ca2+ ion is twice that of the Na+ ion. In physical terms this impacts greatly on physiochemical characteristics of the two minerals, since the negatively charged montmorillonite lattice sheets are more tightly held together by the calcium ions.



FIG 7: The observed reduction in Sauvignon blanc varietal character (represented by 4MMP) as the dosage of bentonite is increased in a wine. Columns show the concentration of 4MMP remaining in the wine after treatment.


FIG 8: An example of some of the testing undertaken by Laffort Oenologie when developing new bentonite products: lees compaction. Note that the samples in the graph do not correspond to those in the measuring cylinders.


FIG 9: A comparison of a sodium bentonite and Microcol CL with respect to lees compaction.

Calcium bentonite, having a better “glue” in between the lattice sheets in the form of calcium ions, does not disperse in water as readily as sodium bentonite, meaning that hydration (swelling) does not occur to the same extent. Flocculation and settling for calcium bentonite are much more rapid than for sodium bentonite, since the calcium ions cause faster re-association of the lattice sheets (Figure 4). In winemaking terms this means that calcium bentonites settle more rapidly and to a more compact state than sodium bentonites. The surface area presented by a suspension of calcium bentonite is not as great as for sodium bentonite, even though the negative charge on the lattice plates is equivalent, hence the protein removal capacity of calcium bentonite is lower than for sodium bentonite. A graph illustrating the differing protein removal capacities of some bentonite samples with differing inter-laminar compositions is given in Figure 5.


Sodium-activated calcium bentonite

Some bentonites naturally contain a close ratio of sodium and calcium in the inter-laminar region of the structure. It is also possible to alter the cationic balance in the inter-laminar region of bentonite artificially through chemical treatment. This is achieved by heating a calcium-rich bentonite in water at approximately 80ºC with sodium carbonate. The purpose of this process is to enrich the naturally calcium-dominant bentonite with sodium, thereby increasing its protein removal capacity. The results of this process of activation can vary depending on the type (origin) of the bentonite used.


For an example of a scientific evaluation of this process, see Gougeon et al (2003). In this study the authors examined two calcium bentonites before and after sodium “activation” to elucidate the relative amounts of protein adsorption and the extent of Ca2+/Na+ exchange. The findings of the study indicated that cation exchange occurs mainly only at the edges of the inter-laminar region, which constitutes a relatively low proportion of the total exchangeable cation content. Activation did lead to an increase in the amount of protein adsorption, although this was not quantified.


Bentonite mode of action – ion exchange

Now that we have an understanding of the structures of both proteins and bentonite, we can consider more closely what actually happens when the two are combined in wine. Recall that most wine proteins possess a positive charge in wine, and that the lattice sheets of bentonite have negative charges. When they are combined in wine, the proteins are electrostatically attracted to and adsorb onto the surface of the bentonite sheets. This process upsets the charge balance of the wine medium, as positively charged ions (i.e. the wine proteins) are being removed from the wine. This charge imbalance is corrected by the concerted migration of cations (either Na+ or Ca2+, or other trace cations) from the inter-laminar region of the bentonite into the wine. The action of bentonite, therefore, is an ion-exchange process. In effect, when discussing the protein removal capacity of bentonite we are really discussing its ion-exchange capacity.



FIG 10: The levels of 3MH remaining in a Sauvignon blanc wine after treatment at the designated levels with different types of bentonite. Columns show the concentration of 4MH remaining in the wine after treatment.


FIG 11: The reduction in Sauvignon blanc varietal aroma (represented by 4MMP) with increasing levels of bentonite treatment. Columns show the concentration of 4MMP remaining in the wine after treatment.


FIG 12: The reduction in Sauvignon blanc varietal aroma (represented by 4MMP) with increasing levels of bentonite treatment. Columns show the concentration of 4MMP remaining in the wine after treatment.

In consideration of this, wines for export to markets where maximum sodium levels apply (such as the EU) might require protein stabilisation with calcium rather than sodium bentonite.


Parameters to consider when choosing bentonite

Knowing something about the structures of proteins and bentonite allows us to better predict our fining needs. Firstly, if protein removal is your priority then sodium bentonite is more effective. If you are concerned about introducing extra sodium into your wine, then you should consider using a calcium bentonite at the expense of protein removal. If processing time is at a minimum then calcium bentonite will sediment more rapidly. Variations in varietal protein contents and compositions will have an effect, as well as climate differences between growing regions and seasonal variations from year to year. Wine pH also plays a role (Figure 6), since at lower pH wine proteins will carry greater positive charge density, which will reduce the bentonite requirement to achieve stability. In effect, the correct product you should use depends on your priorities with respect to the wine. It is a balancing act that all winemakers must perform.


Speciality bentonite – aroma retention

Bentonite is well known for causing the loss of aroma compounds at higher rates of application (Figure 7) (Ribéreau-Gayon et al, 2006a), which reinforces the absolute requirement for bench trials to be conducted on every wine before any additions are made. Since bentonite is a natural product, being a mineral that is obtained from mining operations in various locations throughout the world, there can be significant variation between products. Some bentonites will display greater protein removal capacity, whilst others will have better lees compaction. In order to make the best products available to its customers, Laffort Oenologie, through its SARCO research facility, recently invested heavily in an exhaustive examination of over 50 natural bentonites (all Laffort Oenologie products are certified GMO-free and we use natural products wherever possible). The samples were investigated for all parameters that impact on wine quality and protein efficiency, such as protein removal capacity, lees compaction and wine varietal aroma retention. Some examples of the results are given in Figure 8.


The result of this investigation was the development of two new products in the Laffort Oenologie range: Microcol Alpha™ and Microcol CL™ (Microcol™ is an existing product sold in the EU). Microcol CL™ and Microcol Alpha™ display the characteristics indicated in Table 1. Figure 9 shows, for example, the excellent lees compaction of Microcol CL™ in comparison with a sodium bentonite. Additional data are presented below.


Aroma retention

Two of the characteristic aroma molecules found in Sauvignon blanc wine are 3-mercaptohexanol (3MH) and 4-mercapto-4-methylpentan-2-one (4MMP), contributing passion fruit/grapefruit and boxwood aromas respectively. Such compounds are known to be removed by bentonite treatments. Microcol CL™ and Microcol Alpha™ were compared with other commercial bentonites (brands not disclosed) for their respective abilities to permit the retention of these compounds in wines containing this varietal.


In Figure 10 the levels of 3MH remaining such a wine are illustrated, after treatment with increasing levels of the designated bentonites. It can be seen that Microcol Alpha and bentonite X perform very well in this test, whilst Microcol CL removes slightly more 3MH. Bentonite Z, performs relatively poorly, with a significant reduction in 3MH levels in the wine at dosages above 50 g/hL.


Microcol CL™ was also compared with another bentonite (E) and Microcol™ for the effect on 4MMP levels in a Sauvignon blanc wine (Figure 11). Above dosages of 10 g/hL some reduction in 4MMP levels remaining in the wine is noted for all samples, with bentonite E performing relatively poorly. At a high dosage rate (100 g/hL) Microcol CL still allows the retention of more than 80% of the original 4MMP concentration in the wine, making it particularly effective for retaining grapefruit/passion fruit characters in this varietal.


Dosage requirement and lees volume

In addition to improvements in wine quality, winemakers are concerned with increases in logistics and efficiency. For these reasons Microcol CL™ and Microcol Alpha™ were compared with some other commercial bentonites regarding dosages required to achieve protein stability and lees compaction. As can be seen in Figure 12, Microcol Alpha™, being a natural sodium bentonite, performs exceptionally well at protein removal with a correspondingly higher volume of lees. In comparison, Microcol CL™, being a natural calcium bentonite, requires a higher dosage to effect protein stability, yet displays excellent lees compaction.


Laffort South Africa is represented by Grettchen Visagie and Morne Kempthey can be contacted at grettchen.visagie@laffort.com and morne.kemp@laffort.com respectively.


Summary

A working understanding of protein and bentonite structures allows winemakers to make informed choices on the products and methodologies that are most likely to give the highest performance under a given set of conditions. Laffort Oenologie caters for variation in these parameters by providing natural bentonite products that offer high performance in different areas, such as the capacity for protein removal (i.e. ion exchange), lees volume and, importantly for producing high quality wines, wine varietal aroma retention. Winemakers, working in combination with Laffort Oenologie, are therefore provided with understanding of the technical issues, in addition to the products best suited to perform the task required.


References

Bowyer, P.K. (2001). Bitartrate instability and the pH see-saw, The Australian Grapegrower and Winemaker, September, 100 - 101.

Bowyer, P.K. (2003). Molecular polarity – it’s behind more than you think, The Australian Grapegrower and Winemaker, November, 89 - 91.

Colby, C.B., Waters, E., Nordestgaard, S. & O’Neill, B.K. (2006). Bentonite fining: can we improve performance and efficiency and decrease value losses? The Australian and New Zealand Grapegrower and Winemaker – Annual Technical Issue 2006, 82 - 88.

Gougeon, R.D., Soulard, M., Miehe-Brendle, J., Chezeau, J-M., Le Dred, R., Jeandet, P. & Marchal, R. (2003). Analysis of two bentonites of enological interest before and after commercial activation by solid Na2CO3. Journal of Agricultural and Food Chemistry, 51, 4096 - 4100.

Fukui, M. & Yokoysuka, K. (2003). Content and origin of protein in white and red wines: changes during fermentation and maturation. A.J. Enol. Vitic. 54: 178 - 188.

Ribéreau-Gayon, P., Glories, Y., Maujean, A. & Dubourdieu, D. (2006a). Handbook of Enology Volume 2 – The Chemistry of Wine Stabilization and Treatment. Wiley: Chichester, pp 132.

Ribéreau-Gayon, P., Glories, Y., Maujean, A. & Dubourdieu, D. (2006a). Handbook of Enology Volume 2 – The Chemistry of Wine Stabilization and Treatment. Wiley: Chichester, pp 290.

Ruediger, G.A., Pardon, K.H., Sas, A.N., Godden, P.W. & Pollnitz, A.P. (2004). Removal of pesticides from red and white wine by the sue of fining and filter agents. Australian Journal of Grape and Wine Research, 10, 8 - 16.

Waters, E.J., Alexander, G., Muhlack, R., Pocock, K.F., Colby, C., O’Neill, B.K., Høj, P.B. & Jones, P. (2005). Preventing protein haze in bottled wine. Australian Journal of Grape and Wine Research, 11, 215 - 225.

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