What Are Polyphenol Oxidases?

Polyphenol oxidases (PPOs) are widely distributed in both plants and animals. They are a major cause of enzymic browning in fruits and vegetables. Browning reactions generally reduces the value of fruit and vegetables both nutritionally and commercially.

PPO is a copper-containing enzyme, which catalyses the o-hydroxylation of monophenols to o-diphenols and the oxidation of o-dihydroxyphenols to o-quinones. The enzyme uses molecular oxygen.

The first type of reaction illustrates its monophenolase activity whilst the second reaction illustrates its diphenolase activity (Chararra et al., 2001).

The enzyme has two numbers: (EC 1.14.18.1 and EC 1.10.3.1). 

The enzyme has two conserved copper‐binding domains, CuA and CuB, responsible for copper coordination and interaction with molecular oxygen and phenolic substrates. The enzyme also possesses a transit peptide targeted to interact with the thylakoid lumen (Steffens et al., 1994). 

The corresponding o-quinones subsequently polymerise to brown pigments. The enzyme is related to lipoxygenase and peroxidase which are also found in the same animals and plants.

The action of polyphenol oxidases leads to the change in the colour and flavour of fruits and vegetables during harvest, storage and processing, which reduces the commercial value of the fruits, vegetables and their products.

Sources Of PPO

PPO has been widely studied in various plants such as pear (Halim & Montgomery, 1978), blueberry (Farid et al., 1997), plum (Siddig et al., 1992), grape (Cash et al., 1976), medlar (Barbaros et al., 2002), banana (Yang et al., 2000), tea leaf (Halder et al., 1998) and apple (Shannon & Pratt, 1967).

The enzyme is mainly found in the pericarp of fruits although it can be extracted from the peel and skin. There is a tendency for the enzyme to be found in higher concentrations in the skin than the pulp as has been reported a number of fruits especially mango (Cheema et al., 2015). the extraction and purification of the enzyme for research purposes was recently reviewed (Panadare and Rathod, 2018).

The pH optimum is around 6 with a temperature optimum at 20 °C for the PPO from plum (Siddig et al., 1992).  A grape PPO had optimal activity at pH 3.4 but there was at least 90% activity between pH3 and 4 and then a very sharp drop above pH 4 (Ünal et al., 2007). The PPO from blueberry has a temperature optima at 35 C and pH optima between 6.12 and 6.3 (Siddiq & Dolan, 2017).

The preferred substrates are polyphenols. Among substrates, 4‐methylcatechol was oxidized more rapidly, although catechol, 3-methylcatechol, dopamine, pyrogallol and caffeic acid were also good substrates.

The enzyme from plum for example has a K_m of 20 raM of catechol and V_maxof 5.41 × 10⁻¹ O.D./ min at pH 6.0 (Siddig et al., 1992).

The enzyme is effectively inhibited by sodium metabisulphite (Na₂S₂O₅), sodium ascorbate, kojic acid, L‐cysteine, L-glutathione and ascorbic acid. Inhibition is usually of a non-competitive type. L-cysteine is claimed to completely inhibit the PPO from Whangkeumbae pear (Zhou et al., 2018).

Purification Of Polyphenol Oxidases

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE−Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. More sophisticated methods use three phase partitioning (TPP), aqueous two phase extraction (ATPE), reverse micellar extraction (RME) and chromatographic techniques (Panadare & Rathod, 2018). 

Assay Of PPO Activity

The activity of PPO is often measured by spectrophotometry using catechol as the substrate. The increase in absorbance at 410nm is recorded. A sample cuvette containing 2.0 mL of 20 mm catechol (prepared in 0.2 m sodium acetate buffer, pH 4.0), 0.9 mL of 0.2 m sodium acetate buffer pH 4.0 and 0.1 mL of enzyme solution. Guiacol is also another suitable substrate for this assay.

Enzyme Behaviour

PPO has an unusual and interesting characteristic not usually found with other enzymes. It can form an inactive or latent state (Mayer & Harel, 1979). A number of agents  and processing treatments can convert PPO from its latent form to a more active type. The PPO exists in its latent form when bound to cell membranes but is often activated when fruit is processed (Yoruk & Marshall, 2003).

The PPO in strawberry puree is highly thermostable which makes it difficult to produce a non-browned product. Even treatment with steam for 30 minutes has little impact (Terefe et al., 2010). A related enzyme peroxidase is however thermosensitive with inactivation in less than 5 minutes at 70 °C . The PPO in blueberry is completely inactivated in 20 minutes at 85°C.

Inactivation Of Polyphenol oxidases

Blanching has long been the accepted method for inactivating any enzymes in food. Hot water blanching is probably the most commercially acceptable because it is so simple and has considerable economic advantages. The disadvantage however is the loss of nutrients as a result of such processing – sugars, vitamins, proteins and water-soluble minerals can all be degraded or leached away when bidding to destroy enzyme activity (Lee, 1958). Some have speculated on whether removing copper from the seed for example might help suppress PPO activity.

High temperature -short time (HTST) blanching of apple pieces inactivates PPO. The hotter the water the more effective the treatment. A temperature above 55 ºC is required as below this there is very little deactivation (del Valle et al., 1998).

Another possible treatment is pressure assisted thermal processing (PATP) which was tried on coconut water (Chourio et al., 2018). No enzymatic activity was detected for both enzymes within 300 s at 90 °C/400–600 MPa.

Supercritical carbon dioxide has also been accepted as a process (Gui et al., 2007; Xu et al., 2011) for apple juice. One study found the maximum reduction of PPO activity was 60% when a high carbon dioxide pressure of 30 MPa at 55 °C for 60 min was applied. The apple juice browned but at a slower rate (Gui et al., 2007).

Ultrasound shows possibilities. The inactivation of PPO and peroxidase follows a 1st-order model when ultrasound is applied. It is particularly effective for treating puree and juice (Cao et al., 2018) where the cavitation helps to release PPO from its membrane form which is subsequently denatured by heat.

References

Siddiq, M & Dolan, K.D. (2017) Charcterization of polyphenol oxidase from blueberry (Vaccinium corybosum L.). Food Chem., 218 March pp. 216-220 https://doi.org/10.1016/j.foodchem.2016.09.061

Valle, J.M., Aranguiz, V. & Leon, H. (1998). Effects of blanching and calcium infiltration on PPO activity, texture, microstructure and kinetics of osmotic dehydration of apple tissue. Food Research International, 31, pp. 557–569 https://doi.org/10.1016/S0963-9969(99)00029-0