Food background notesEnzymes and carrot juicing

Carrots are a nutritious vegetable. They contain a good supply of the precursors of vitamin A (retinol), with the main ones being α-carotene and β-carotene. Carrots also supply dietary fibre, minerals and a range of other vitamins. However there are challenges in the production of juice related to the enzymes naturally present within the carrot after harvesting. [1]

The processing of carrot juice The primary problem is that, as soon as we disrupt the cellular structure of the plant tissue, compartmentalisation is lost. This affects cell walls, cell membranes and well as the membranes enclosing sub-cellular structures including vacuoles, nucleii and chloroplasts. This important as these barriers usually are able to keep various components so that these are unable to interact or undergo chemical reactions

As with many living tissues, as long as there is no juicing or grinding, the enzymes present are effectively idle, because they do not have access to their substrates. However upon juicing, as a result of the loss of compartmentalisation, various enzymes naturally present in the carrots will be able to mix freely with their substrates which are also commonly present within the cells of carrots. The resultant reactions are undesirable as they influence quality of food products and so the enzymes are referred to as being deteriorative.

Deteriorative enzymes in carrots There are three deteriorative enzymes found to be a serious problem in the processing of carrots:

1. Polyphenol oxidase (PPO) which is responsible for browning reactions. There are a number of distinct enzymes which are included as PPO and have similar effects during processing. These vary in their specificity for particular substrates [2].

2. Peroxidase (PO, EC 1.11.1.7) which is involved with oxidation reactions and the main substrate is hydrogen peroxide (H2O2) [3]. This enzyme is known to cause off-flavours in various foods

3. Pectin methyl esterase (PME, EC 3.1.1.11) which is one of the enzymes needed for the hydrolysis of pectin molecules [3]. These polysaccharides are a major component form the basic structure of the cell walls of carrots.

Each of these enzymes impacts directly on the quality and shelf-life of carrot juice. In order to overcome these deteriorative effects we will need to blanch the carrots before any disruption of the carrot tissues.

The term blanching refers to any brief treatment applied in order to minimise deteriorative changes. These might be applied immediately post-harvest or alternatively just prior to the commencement of processing of a vegetable or other ingredient.

Blanching of carrots Some of the options which have been studied are the use of steam, boiling water and microwave treatment [4]. The results of comparative trials showed that microwave was not effective for the uniform heating of the carrots, partly due to their tapered shape. Steam and boiling treatments were quite effective approaches to blanching. The study was extended to a comparison of four common genetic varieties of carrots [4].

Blanching and the inactivation of enzymes When the three enzymes were assayed during steam blanching, in all cases it was confirmed that higher temperatures resulted in more rapid inactivation. As an example of the pattern, the results for PPO for one particular variety of carrots (RHC 100) are presented in Figure 1.

Figure 1 The inactivation of PPO in carrots during steam blanching at various temperatures(RHC is variety Red Hot Carotene 100)

Thermal inactivation was assessed over a range of temperatures and our research showed that the three enzymes are inactivated at different temperatures. PPO was found to be the least stable and pectinesterase the most stable for each of the four varieties studied. In most cases effective inactivation was achieved within 2 minutes at 85C (as in Figure 1) [4].

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Food background notes: enzymes and carrot juice page 2 of 2

In addition there was some variation between the results for when each of the enzymes was compared for the four varieties. The enzymes in one (Top Pak) showed greater thermal stability. In this case, pectinesterase required treatment at 90C to ensure rapid inactivation.

Further analyses showed that there was uneven distribution of the enzymes in the core and outermost layers of the carrot root (Table 1). On the other hand no variation was found between the two ends of the root (tip and stem end, data not shown) [4].

Selection of treatment conditions In processing situations it is generally necessary to blanch using conditions which result in inactivation of the most stable of the enzymes. At the same time it is important to minimise the treatment so that the texture and flavour of the resultant product is not modified too much. For carrots this means that shorter periods of heating reduce the likelihood that there are caramelisation reactions influencing flavour. Consumers do not want a juice product to have a “cooked” taste. Our studies also indicated that there were differences in the effects of blanching for a series of different genetic varieties of carrots

Indicator enzymes In processing it is common to use one enzyme as an indicator. Thus, if we measure that enzyme and find that it has no activity, we can be sure that other deteriorative enzymes have also been inactivated. For carrots, it was concluded that pectinesterase should be used as the indicator enzyme in the assessment of blanching sufficiency.

Post blanching treatment of carrots Once the carrots are blanched, cooled and passed through a juicer we have a thick solution which is quite cloudy. Some form of filtration/centrifugation step is needed in processing so that the final product is relatively uniform in appearance and there is as little sediment as possible. In the research on carrots it was found that the addition of two commercial enzyme preparations to the juice had a number of benefits.

The two enzymes had commercial names of Rohament (given by the manufacturer) but the actual active enzymes present in these were PME (the same as referred to above as a deteriorative enzyme) and another called polygalacturonase. This latter enzyme also acts to break down pectin molecules, in this case by breaking the links between adjacent monosaccharide units in the backbone of the pectin molecule.

This provides two advantages, the first being greatly enhanced yields of juice because the cell contents are more readily released from within the cell structures. The second was higher quality juice because there is more sugar (enhancing flavour) and carotenes (good nutritionally but more importantly giving a brighter colour and hence visual appeal to the product.

Table 1 The relative distribution of deteriorative enzymes in carrots (expressed as a percentage of total activity, for variety RHC100) [4]

Enzyme Superficial Core

Peroxidase 72.8 27.2

PPO 100 0

Pectinesterase 53.4 46.6

So the combined commercial enzymes are added at lower levels and allowed to act on the juice prior to centrifugation/clarification filtration treatments. Note that the commercial enzymes are probably inactivated by subsequent steps in the procedure because there is a concentration step applied to reduce the bulk and hence the cost of transportation of the juice.

Conclusion The research showed that the one particular enzyme can be both deteriorative as well as beneficial. The fundament principle here is that the processor must be in control of what is happening at all times. This includes, for example, temperatures, how times, as well as the relative amounts of the two commercial enzyme preparations .

References and further reading

[1] Vora HM, Kyle WSA, Small DM. 1998. Composition and juicing potential of Australian varieties of carrots. Food Aust 50(1):24-26.

[2] Ramı´rez EC, Whitaker JR, Virador VM. 2002. Polyphenol Oxidase. In: Whitaker JR, Voragen AGJ, Wong DWS, editors. Handbook of food enzymology. New York: Marcel Dekker. 39 p1-15.

[3] Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. 2011. Enzyme nomenclature. [Online. Internet.] Available from: http://www.chem.qmul.ac.uk/iubmb/enzyme/.

[4] Vora HM, Kyle WSA, Small DM. 1999. The activity, location and thermal inactivation of three deteriorative enzymes from Australian carrot (Daucus carota L) varieties. J Sci Food Agric 79(8):1129-35.

[5] Vora HM, Kyle WSA, Small DM. 1999. The application of commercial enzyme preparations in the production of juice from Australian carrots. Food Aust 51(6):122-23.

Prepared by Assoc Prof Darryl M Small

send feedback or questions to <darryl.small@rmit.edu.au >