Background information enzymes in fruit juice production Jam-makers have valued it for hundreds of years, wine-makers endeavour to destroy it and food technologists are variously trying to block or improve the enzymes that attack it. After cellulose, pectin is one of the most abundant carbohydrates on Earth. Yet despite its importance in the production of food and drink, pectin and the enzymes that break it down seldom receive the attention they deserve. This article describes the rôle of pectin, pectinases and other enzymes in fruit juice production. Structure of fruit microfibrils are arranged at random rather than in the stronger regular pattern seen in the secondary The structure of plant cells and the way they are wall. Degradation of the cellulose microfibrils leads built into tissues is complex. Only a simplified to breakdown of the cell walls and their eventual description will be given here, including sufficient disintegration. Table 1 shows the proportions of detail for the reader to appreciate the action of various materials found in the cell walls of some enzymes in fruit juice production. important fruit crops. Fruits are comprised mostly of parenchyma cells. These are relatively simple ‘general purpose’ plant Changes to fruit during ripening cells, with thin walls made of two layers. The outermost of these, the primary cell wall, is made There are two main stages in the formation of fruit. from cellulose fibres called microfibrils surrounded Soon after fertilisation the cells of the ovary (and by a matrix of pectins, hemicelluloses and proteins. in fruit like apples, those of the surrounding tissue The inner, or secondary, cell wall is similar but as well) divide rapidly. In later weeks the young contains less pectin (Figure 1). fruit swells as both these cells and the spaces between them enlarge. As the fruit ripens it Between the cellular ‘bricks’ is a ‘mortar’ of pectin. becomes progressively softer. Complete removal of this binding layer causes the tissue to fall apart. This is what has happened to Much research has been done to discover exactly fruits or vegetables which have gone soft. In such which enzymes are responsible for these and cases digestive enzymes, which have broken down other changes in maturing fruit. One hope is that the long chains of pectin molecules, have been by monitoring the levels of these enzymes it produced by microbes such as the ‘soft-rot’ might be possible for growers to harvest their bacterium Erwinia carotovora. crops at just the right time or even to select varieties with desirable characteristics, such as Cellulose is susceptible to enzymatic attack too, better storage properties or greater yields and especially in the primary cell wall where its improved quality of juice. Figure 1 Outer, primary cell wall The general structure of with random cellulose Middle lamella between plant cell walls. microfibrils in pectin cells — mostly pectin The outer cell wall is made of cellulose microfibrils embedded in a matrix of pectins, hemicelluloses and proteins. In woody Inner, secondary cell wall tissue, this wall also contains lignin. The inner, secondary cell with regular cellulose wall is composed of pectin and some lignin. The middle lamella, microfibrils embedded in which binds the cells together, is made mainly of pectin. pectin and hemicellulose 4| IN A JAM AND OUT OF JUICE | Version 1.0 | December 2000 Background information For many years it was (not unreasonably) assumed Percentage of fresh mass of cell wall that the softening associated with ripening was caused by cellulases acting on the cell walls. For Pectin Hemicellulose Cellulose Glycoprotein apples at least this has been shown not to be so, although cellulases do play a part in the ripening Cherry 0.51 0.06 0.17 0.32 Pineapple 0.21 0.35 0.27 0.12 of softer fruits such as peaches and tomatoes. Mango 1.02 0.23 0.59 0.32 Apple 0.54 0.34 0.70 0.15 The other candidate for change causing fruit to Pear 0.42 0.22 0.40 0.12 soften is pectin, the inter-cellular ‘cement’. In unripe fruit, the pectin is bound to cellulose microfibrils Table 1 in the cell walls. Such pectin is insoluble and the Waxy The major components liquid within the cells remains fluid, conferring cuticle of the cell walls of rigidity on them (by pushing on the cell wall). Flavedo containing oil glands some important fruits. During ripening, the pectin is altered by naturally- and pigments occurring enzymes in the fruit. These ‘alterations’ From Voragen and van den Broek, may involve the breakdown of the pectin chains 1991. or of side chains attached to the units from which they are made (see diagram of pectin structure on Albedo page 9). In either case, the result is that the pectin Figure 2 becomes more soluble and its grip on the The structure of citrus surrounding cell walls is loosened, so that the plant Lamella fruit and apples. tissue softens. Seed The bulk of both of these fruits is formed Intuitively, this suggests that it should be easier to Carpel (segment) from unspecialised press the juice from ripe fruits than unripe ones. containing juice sacs tissue known as Surprisingly, the reverse is true. One consequence parenchyma, which of the partial breakdown of insoluble pectin is that holds most of the juice. it becomes soluble in water. Some of the pectin Vascular bundles in the outer portion of the ovary molecules are released into the juice, so that it becomes more viscous, and therefore more difficult to squeeze from the fruit during processing. Important colour and flavour compounds may also Enlarged receptacle Seed be retained within the fruit so that juice pressed from it is of inferior quality. Furthermore, the juice may be difficult to clarify and filter due to suspended pectin particles. In the fruit juice industry pectinases, obtained from the fungus Aspergillus, are used to help overcome these Remmants of problems. petals and sepals Use of pectinases and other throughout the world. Some 4 000 million litres of enzymes in fruit juice production orange juice and over 450 million litres of grapefruit juice are also produced each year. Home winemakers will be familiar with pectinase or pectolytic enzyme. Pectinases were some of the Pectinases and other enzymes are an essential part first enzymes to be used at home and they were of fruit juice technology (see Table 2, page 7). They utilised in the commercial preparation of wines are used to help extract, clarify and modify juices and fruit juices during the 1930s. Before this juices from many crops including berries, stone and citrus had been prepared by mechanical means — simply fruits, grapes, apples, pears and even vegetables. by pressing fruit and filtering the liquid which Where a cloudy juice or ‘nectar’ is preferred (for emerged. Even so the early use of pectinase to example, with oranges, pineapples or apricots) clarify apple juice was developed by trial and error. there is no need to clarify the liquid, though Only in the 1960s did the chemical structure of enzymes are used here to enhance extraction or plant tissues become apparent, and with this perform other modifications. knowledge food technologists began to use a greater range of enzymes more effectively. The methods used for apple juice production are the same as those for many other fruits, and Enzymatic juice extraction from apples was therefore serve as a useful example. Citrus fruits An apple crusher introduced 30 years ago and now more than 5 million present special requirements of their own, so these and juice press, for tons of apples are processed into juice annually are dealt with separately. ➔ home use, circa 1890. www.ncbe.reading.ac.uk |5 Background information Apple juice production Prepress treatment with pectinases takes anything from 15 minutes to 2 hours depending upon the Several types of apple juice are available exact nature of the enzyme and how much is used, commercially: the most familiar are hazy unfiltered the reaction temperature and the variety of apple and unclarified juice (usually a premium product); chosen. Some apple varieties such as Golden and a clear, filtered, amber-coloured juice. Both Delicious are very difficult to break down. can be prepared using enzymes — the main steps involved are shown in Figure 3. During incubation, the pectinase degrades soluble pectin in the pulp, making the juice flow more Pre-press treatment freely. The enzyme also helps to break down After the apples have been washed and sorted, they insoluble pectin, which hampers juice extraction are crushed in a mill. Peels and cores from apple in two ways. First, if they are left, these slimy pectin slice or sauce production may also be used together particles become saturated with juice which is then with whole apples. Although pectinases are often difficult to extract from the pulp and second, they added at this stage, better results are achieved if block drainage channels in the pulp through which the apple pulp is first stirred in a holding tank for the juice must drain. Enzyme treatment is 15–20 minutes so that enzyme inhibitors considered to be complete once the viscosity of (polyphenols) are oxidised (by naturally-occurring the juice has returned to its original level or less. polyphenol oxidase in the fruit). The pulp is then (Remember, the degradation of insoluble pectin heated to an appropriate temperature before causes the juice to thicken at the start.) It is enzyme treatment. For apples, 30 °C is the optimal important that the pulp is not broken down too temperature, whereas stone fruits and berries much as it would then be difficult to press. ▼ Figure 3 generally require higher temperatures — around The main stages in the 50 °C.