THE USE OF GELATINE IN WINE FINING C.G.B. Cole





Published in the Proceedings of the 1st SAAFoST Technical Symposium, Emulsifiers, Stabilisers and Thickeners in the Food Industry 1, held in Durban, RSA, in April 1986. Natal Technikon Printers. PO Box 953 Durban 4000

SYNOPSIS

The basic chemistry of protein is developed to enable an understanding of its fining reactions in beverage clarification. Results of various European and South African wine fining experiments are presented. The efficiency of both very high and very low molecular weight gelatins are discussed and it is proposed that protein isoelectric point is a more important attribute in determining fining performance than is the Bloom strength of the gelatine.

Introduction.

Beverages like wine, cider and unfermented fruit juices contain insoluble matter which imparts a haze to the beverage which is often not practical to remove by filtration.

The process of haze removal is known as fining. It involves the formation of a floccular precipitate in the beverage which will absorb the natural haze forming constituents while settling. After a settling period, the supernatant can be withdrawn and given a polishing filtration prior to sale.

It is important that the settling process be efficient in the removal of natural haze. It must also be reasonably rapid, and the loss of salable product in the sediment or lees should be minimal. Finally, the beverage, once clarified, should remain clear and the clarification or fining process should not have any undesirable effect like the removal of wanted flavourants or the addition of unwanted flavour components.

Gelatine has been used for the clarification or fining of wine since the Roman civilization (Ringland 1983) and probably before that as well. At that time the chemistry of the process was certainly very poorly understood, and hence it is not surprising that the process is often considered to be an art rather than a science, and like all arts, the process can be surrounded by misconceptions which can result in inefficiency. Hence, it is the intention to review the chemistry of fining and to present some results of South African investigations and compare these with results obtained in Europe and to attempt to account for the discrepancies.

Fining Reactions.

The primary reaction occurring with gelatine is a complex formation between polyphenols in the wine and the protein of gelatine to give the desired floccular precipitate.

The second reaction, less well understood, but equally important, is the complex formation between the natural proteins of the wine and the added protein, gelatin.

The third reaction is between bentonite or silica sol (which should be added after the gelatin) which absorbs or complexes with any residual dissolved protein, be it gelatin or natural protein in the beverage.

The Nature of Gelatin.

Gelatin is a protein, that is, it is a polymer of amino acids joined together by peptide bonds as shown in Figure 1a. Hence, proteins can be depicted as long molecules with many different side chains (Figure 1b), which accounts for their varying properties.

Figure 1. Developing concepts of protein structure, a) the formation of the peptide bond and the polypeptide, b) peptide side chains, c) peptides as amphoteric compounds and d) charge on the peptide chain.



The side chains can be, for example:



Neutral Side Chains



R = -H Glycine

R = -CH3 Alanine

R =

Tyrosine

Cationic Side Chains



R = -CH2 - CH2 - CH2 - NH2 Lysine



Anionic Side Chains



R = -CH2 - COOH Aspartic Acid



Cyclic Side Chains



Proline



Proline is very important in that it imparts a twist to the chain and affects the shape of the protein molecule and its rigidity.

The protein chain is more accurately depicted in Figure 1c, that is the molecule is amphoteric and can carry either a positive or negative charge depending on the pH of the medium. In wine and beverages at a pH of 3.6, one would expect most of the amino groups to be positively charged and most of the acidic groups to be uncharged as in Figure 1d.

The molecule would then behave as a cation provided the pH was below the isoelectric point, i.e. It would attract and form polar associates with anions in solution. In addition proteins form associations due to hydrogen bonding using the negatively charged oxygen and nitrogen atoms in the molecules.

The isoelectric point (pI) of a protein is that pH at which the protein will not migrate in an electric field. This is due to the fact that at that pH the molecule carries an equality of positive and negative charges, i.e. the molecule is isoionic, in the absence of added ions other than hydrogen and hydroxyl ions in solution. Gelatin, is rather unique in that it can have an isoelectric point anywhere between pH 9 and pH 5, depending upon the source and method of production.

Type A gelatins are usually derived from acid pretreated pigskin and have isoelectric points between 6 and 9, with the high gel strength (Bloom strength) gelatins having the higher pI and the low Bloom strength gelatins having a pI closer to 6. Gelatins derived from limed hide or limed ossein are known as Type B gelatins and all of them have a pI close to 5. The significance of pI is, of course, that the higher the pI, the greater the cationic charge on the molecule at a beverage pH of say 3.6. In other words, at pH 3.6, all gelatins would be positively charged, but the charge density would be far higher for high pI gelatins.

Phenol - Protein reaction.

Both tannins and anthocyanins in beverages are molecules containing benzene rings with adjacent hydroxyl groups as shown by the gallic group (Figure 2) which are proposed as the major source of the hydrogen bonds which are the basis of complex formation between gelatin and tannins or anthocyanins in beverages (Figure 3):

Figure 2. Galloyl group, a major constituent of tannins.

Figure 3. Polyphenol - peptide hydrogen bonding (Ringland 1983).

Gelatin is held to be particularly suited to hydrogen bonding because one third of the amino acids are glycine, where R = H , and hence steric hindrance to hydrogen bonding would be far less than with proteins containing less glycine. However, the tannin/gelatin complex is also very pH dependent and disappears at approximately pH 8, which would be due to both molecules becoming negatively charged and hence mutually repulsive. Hence, the role of polar bonding between molecules of dissimilar charge must not be overlooked.

Protein - Protein Interaction.

Beverage proteins would be derived from the enzymes which are responsible for the diversity of the biological processes occurring prior to and during conversion of the substrate into a beverage. In beer the proteins would be in the form of wort enzymes needed to convert starch into glucose and then alcohol. In wine the growth and ripening enzymes of the grape and the fermentation enzymes would provide the protein. Both beer and wine makers know that, with time, these proteins associate to form insoluble precipitates, i.e. they are responsible for "protein instability". It is worth noting that for beer fining, Isinglass, a close relative of gelatin derived from fish swim bladder is most effective. This protein has an extremely high molecular weight and a pI of 4.5 to 4.8 (i.e. higher than the pH of beer). (Vickers & Bracher 1966).

For protein-protein interaction it is necessary that the two proteins be of opposite charge at the beverage pH for polar association to occur. This association leads to a reduction of hydrophilic sites and hence precipitation. Also, further hydrophobic bonding due to association of hydrophobic sites in aqueous medium can lead to an increase in effective molecular weight and precipitation.

It would be wrong to neglect to mention the two other proteins that have received a fair amount of use in fining, namely egg albumin and casein. However in both these cases the floc formation is due to the insolubility of the fining protein at pHs below their pIs, hence the fining action is not the same as in the case of gelatin which is soluble at all pHs, even at its isoelectric pH.

Gelatin Fining.

In Europe, from where we inherited the art of wine making, most of the available gelatin is Type A pigskin gelatin. Ossein gelatin, having a higher viscosity, is mainly employed in film forming applications, and in Europe there has been a tendency to think that cattle hide gelatin was only suitable for use as glue. In RSA however, gelatin is only made from cattle hide and is thus Type B, and we would contend that it is in no way inferior to Type A, especially in fining applications!

There is a wealth of European data which appears to show quite conclusively that low Bloom strength gelatin is optimum for fining, as shown in Table 1. This will come as no surprise to the technologist trained in Europe, and this is so much a dictate of the art that it requires a real "Thomas" to consider the possibility of reinventing the wheel. Table 2, however from the study of W. Bestbier of KWV, shows that there really is no detectable difference in performance between the use of gelatins of between 100g and 275g Bloom strength. This applied to all parameters tested, i.e. sediment volume, clarity of supernatant and protein stability. Hence the gelatin to use is determined by economics alone and once again it looks as though the Europeans are correct, because traditionally, the lowest Bloom strength gelatins command the lowest price. However, there is one vintner who will not be swayed from the use of "Superfine" 250-270 Bloom strength gelatin. Such a gelatin, of very high molecular weight, is partly insoluble and forms a coacervate in 10 % alcohol solution. Hence, this user of gelatin is managing to add the insoluble floc fining action of egg albumin or casein to the normal fining action of gelatin and it is not considered to be a trade secret! Thus the advantage of superior hydrogen bonding reactivity of gelatin protein, as well as the apparently ideal pI of gelatin protein and additionally, the unusual induced insolubility of gelatin protein, is used and the result is a vastly superior rate of settling. Overnight settling and a compact sediment is said to outweigh completely the higher cost of Superfine gelatin. The price differential between 275g and 100g Bloom Strength gelatin would be 27 c/g and a usage rate of 4 g/hl would therefore equate to an increased cost of some 1.08 c/hl!

TABLE 1. The range of quantities of individual types of gelatin which achieve optimum fining.

Gelatin Type - Bloom Strength. Optimum usage rate.

A-267 90-100 g/hl

A-210 80-90 g/hl

A-195 80 g/hl

A-141 50-70 g/hl

A-120 40-60 g/hl

A-100 30-60 g/hl

A-80 30-90 g/hl

A-60 25-100 g/hl

From: Wucherpfennig, K., Ph. Possmann, Kettern, W. and Scherpe, W.

TABLE 2. Gelatin Fining Tests on 1982 Stein, using Type B gelatin at 3 g/hl.





Gelatin Bloom Str. g Control 100 125 150 175 200 225 250 275 Determination Bentonite used g/hl Sediment % 20

40

60

80 3.2

4.4

5.4

6.2 4.2

5.4

6.4

7.4 4.4

5.3

6.5

7.3 4.4

5.4

6.4

7.4 4.6

5.5

6.5

7.5 4.8

5.2

6.6

7.4 4.6

5.4

6.7

7.5 4.7

5.3

6.5

7.4 4.6

5.6

8.8

7.6 Clarity (%T @ 520 nm) 20

40

60

80 88

89

89

88 91

92

92

91 92

91

91

91 91

92

91

91 92

91

91

92 92

91

92

91 91

91

91

92 91

92

91

92 91

91

92

91 Protein Stability

O= Unstable

S= Stable 20

40

60

80 O

S

S

S O

S

S

S O

S

S

S O

S

S

S O

S

S

S O

S

S

S O

S

S

S O

S

S

S O

S

S

S

From: Bestbier, W.

In taking the liberty of introducing the advantages of high Bloom Strength gelatin in fining, it is necessary to emphasize that, if one does not use a very dilute solution of gelatin, then one must remember that, on cooling, the solution will gel. Hence it is of prime importance to ensure that when warm gelatin solution is added, it is added at a point of very intense agitation such that the small amount of gelatin is intimately mixed into a large bulk of beverage before any gelling can occur. One point of application that can be recommended is into the suction of a centrifugal transfer pump as depicted in Figure 4.



Figure 4. Techniques for gelatin addition under large scale conditions. (Troost 1980)



Liquid Gelatin Fining.

A disadvantage of gelatin fining is the difficulty of gelatin dissolution. It requires both heat and time, and in addition, gelatin solutions gel on cooling, and further more, they should not be stored for more than a few hours at a time because gelatin is an excellent nutrient for most forms of microbiological life. Hence, in line with European dictate that lower Bloom strength is better for fining, a number of manufacturers have produced a highly concentrated solution of non-gelling hydrolyzed gelatin preserved with SO 2 which is allowed in wine. This concept has a lot of convenience advantages and has received support from the wine industry in New Zealand in particular. Researchers at KWV were very interested in the concept and have agreed to the use of previously unpublished results in Tables 3 to 5.

TABLE 3. Influence of gelatin fining on the polyphenol content of a South African 1984 Riesling.

Fining Polyphenol Content

Control + 40 g/hl bentonite 225 ppm*

Liquifine 3 g/hl + 40 g/hl bentonite 215 ppm

Superfine 3 g/hl + 40 g/hl " 210 ppm

* Method of Slinkard and Singleton (1987)

From: Baumgarten, G. (1984)

TABLE 4. The influence of gelatin fining on the filter-ability of 1984 Pinotage.

Fining Filtrate ml*

Control 13

Liquifine 2 g/hl 20

3 g/hl 21

4 g/hl 21

Superfine 2 g/hl 21

3 g/hl 21

4 g/hl 20

* ml clear supernatant wine filtering through Whatman No. 1 filter paper in 120 sec.

From: Baumgarten, G. (1984).

TABLE 5. The influence of gelatin fining on the protein stability of a protein stable 1980 Cabernet.

Fining Protein Stability *

Control Stable

Liquifine 2 g/hl Unstable

3 g/hl "

4 g/hl "

Superfine 2 g/hl Stable

3 g/hl "

4 g/hl "

* Bento test and heating to 85°C for 5 hours.

Table 3 shows that the high molecular weight gelatin "Superfine" removed slightly more polyphenol from the Riesling than did the low molecular weight "Liquifine". However, both gelatins removed significant amounts of polyphenol.

Table 4 indicated that the gelatin molecular weight did not affect the filter-ability of the wine after settling.

Table 5 showed that without adequate precautions by way of bentonite usage, the convenience of using low molecular weight hydrolyzed gelatin, "Liquifine", can be offset by inducing protein instability into the wine.

The use of finings at the pressing stage has not received much acceptance, largely it is said, because the vintner feels he should see what the grapes are providing before he modifies this gift of God in any way. However, some vintners in Australia have reported greatly increased yields due to the formation of very compact sediment, by the use of Liquifine hydrolyzed gelatin in the pre-fermentation stage of wine making. The addition of gelatin to cold grapes would generally lead to waste due to gelling of the solution and it is here that Liquifine has an undisputed advantage over gelatin, as a fining agent.

Conclusions.



In contrast to the European findings with Type A gelatins, that low Bloom strength gelatin has optimum performance characteristics, it has been shown with South African wines and Type B gelatins, that Bloom strength has no influence on fining performance. When it is realised that high Bloom strength Type A gelatins have a pI close to 9, whereas low Bloom strength Type A gelatins have a pI close to 6 and Type B gelatins have pI close to 5, it appears probable that the superior fining action of low Bloom strength Type A gelatins when compared to high Bloom strength Type A gelatins, is more a function of pI than of Bloom strength, and as has been found with beer, the closer the beverage pH to the pI of the fining agent, the better is the fining performance. The use of hydrolyzed gelatin as a fining agent has convenience advantages but has been shown possibly to lead to protein instability. The use of high (250-270) Bloom strength (250-270) Type B gelatine for wine fining has been reported to reduce greatly the settling time required for the fining of South African wines.

Acknowledgments.

The author wishes to thank Davis Gelatin Industries (Pty) Ltd. and KWV (Suider-Paarl) for permission to use previously unpublished data.

References.

Baumgarten, G. 1984. The use of Liquifine Gelatine as a substitute for Gelatine powder in wine preparation. (Private communication.)

Bestbier, W. 1983. Ondersoek na die brei-effekt van gelastien met verskillende Bloom-getalle. Die Wynboer 621, 61-62.

Ringland, C. & Eschenbruch, R. 1983. Gelatine for juice and wine fining. Food Technology in New Zealand. August 1983.

Slinkard, K & Singleton, V.L. 1977. Total phenol analysis. Am. J. Enol. Vitic. 28. 49-58.

Troost, G. 1980. Technologie des weines. Verlag Eugen Ulmer, Stuttgart, Germany.

Vickers, J. & Ballard, G. 1966. Fining in perspective. Brewers Guild Journal. June 1966.

Vickers, J. & Bracher, C. 1961. Isinglass & Finings. Brewers Guild Journal. June 1961.

Wucherpfennig, K., Ph. Possmann, Kettern, W. & Scherpe, W. The effect of gelatine type on fining in white wines. Wein und Rebe, Wissenschaft, Forschung, Praxis. 55, 92-937.