Enzymes in the fruit juice, wine, brewing and distilling industries

One of the major problems in the preparation of fruit juices and wine is cloudiness due primarily to the presence of pectins. These consist primarily of a-1,4-anhydrogalacturonic acid polymers, with varying degrees of methyl esterification. They are associated with other plant polymers and, after homogenisation, with the cell debris. The cloudiness that they cause is difficult to remove except by enzymic hydrolysis. Such treatment also has the additional benefits of reducing the solution viscosity, increasing the volume of juice produced (e.g., the yield of juice from white grapes can be raised by 15%), subtle but generally beneficial changes in the flavour and, in the case of wine-making, shorter fermentation times. Insoluble plant material is easily removed by filtration, or settling and decantation, once the stabilising effect of the pectins on the colloidal haze has been removed.

Commercial pectolytic enzyme preparations are produced from Aspergillus niger and consist of a synergistic mixture of enzymes:

1. polygalacturonase (EC 3.2.1.15), responsible for the random hydrolysis of 1,4-a-D-galactosiduronic linkages;
2. pectinesterase (EC 3.2.1.11), which releases methanol from the pectyl methyl esters, a necessary stage before the polygalacturonase can act fully (the increase in the methanol content of such treated juice is generally less than the natural concentrations and poses no health risk);
3. pectin lyase (EC 4.2.2.10), which cleaves the pectin, by an elimination reaction releasing oligosaccharides with non-reducing terminal 4-deoxymethyl-a-D-galact-4-enuronosyl residues, without the necessity of pectin methyl esterase action; and
4. hemicellulase (a mixture of hydrolytic enzymes including: xylan endo-1,3-b-xylosidase, EC 3.2.1.32; xylan 1,4-b-xylosidase, EC 3.2.1.37; and a-L-arabinofuranosidase, EC 3.2.1.55), strictly not a pectinase but its adventitious presence is encouraged in order to reduce hemicellulose levels.

The optimal activity of these enzymes is at a pH between 4 and 5 and generally below 50°C. They are suitable for direct addition to the fruit pulps at levels around 20 U L⁻¹ (net activity). Enzymes with improved characteristics of greater heat stability and lower pH optimum are currently being sought.

In brewing, barley malt supplies the major proportion of the enzyme needed for saccharification prior to fermentation. Often other starch containing material (adjuncts) are used to increase the fermentable sugar and reduce the relative costs of the fermentation. Although malt enzyme may also be used to hydrolyse these adjuncts, for maximum economic return extra enzymes are added to achieve their rapid saccharification. It not necessary nor desirable to saccharify the starch totally, as non-fermentable dextrins are needed to give the drink 'body' and stabilise its foam 'head'. For this reason the saccharification process is stopped, by boiling the 'wort', after about 75% of the starch has been converted into fermentable sugar.

The enzymes used in brewing are needed for saccharification of starch (bacterial and fungal a-amylases), breakdown of barley b-1,4- and b-1,3- linked glucan (b-glucanase) and hydrolysis of protein (neutral protease) to increase the (later) fermentation rate, particularly in the production of high-gravity beer, where extra protein is added. Cellulases are also occasionally used, particularly where wheat is used as adjunct but also to help breakdown the barley b-glucans. Due to the extreme heat stability of the B. amyloliquefaciens a-amylase, where this is used the wort must be boiled for a much longer period (e.g., 30 min) to inactivate it prior to fermentation. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence of protein- and tannin-containing 'chill-haze' otherwise formed on cooling the beer.

Recently, 'light' beers, of lower calorific content, have become more popular. These require a higher degree of saccharification at lower starch concentrations to reduce the alcohol and total solids contents of the beer. This may be achieved by the use of glucoamylase and/or fungal a-amylase during the fermentation.

A great variety of carbohydrate sources are used world wide to produce distilled alcoholic drinks. Many of these contain sufficient quantities of fermentable sugar (e.g., rum from molasses and brandy from grapes), others contain mainly starch and must be saccharified before use (e.g., whiskey from barley malt, corn or rye). In the distilling industry, saccharification continues throughout the fermentation period. In some cases (e.g., Scotch malt whisky manufacture uses barley malt exclusively) the enzymes are naturally present but in others (e.g., grain spirits production) the more heat-stable bacterial a-amylases may be used in the saccharification.