The Problems With Peroxidases

Peroxidases (POD) are problematic enzymes when it comes to reducing browning or minimising the loss of nutrients in fruits and vegetables.  The enzyme is often used as a quality indicator, especially in the reduction of enzymic browning, because its inactivation can be directly related to an improvement in the shelf life of processed foods (Morales-Blancas et al., 2002). Peroxidase (POD, EC 1.11.1.7) catalyzes the oxidation of many types of compounds by transferring oxygen from peroxides, which act as hydrogen acceptors.

ROOR’ + electron donor (2 e^–) + 2H⁺ → ROH + R’OH

Typical substrates that act as hydrogen donors could be phenols, amines, or other organic compounds and the products generated depend on the source of these substrates (Soysal and Söylemez, 2005). The ideal substrate is hydrogen peroxide but lipid peroxides and other organic hydroperoxides found in fruits are also commonly used.  The undesirable quality losses such as discoloration and browning, flavour changes, and loss of nutrients may occur if POD is not properly inhibited. They form part of a large family of enzymes, the oxidoreductases, all with similar mechanisms by having a haem based active centre. Peroxidases are extensively reviewed by Vámos-Vigyázó (1981). One of the most actively studied from a clinical perspective is that sourced from horse radish.

Assay Of Peroxidase Activity

Peroxidase activity is often assayed spectrophotometrically by mixing an extract of the fruit or vegetable with a buffered solution at pH 6.5 with a 1% (w/v) solution of p-phenylenediamine as the hydrogen donor and hydrogen peroxide proving the oxidant. Oxidation of  p-phenylenediamine is measured in a double beam spectrophotometer at 485nm and 25 °C. The units are recorded as a change in absorbance per minute per unit weight of extract.

Levels Of Peroxidases In Fruits And Vegetables

Levels of the enzyme vary in produce. For example, peroxidase activity in fresh orange juice (3.19 ΔOD/min/g f.w.) is twice that of strawberry puree (1.38 ΔOD/min/g f.w.)  (Cano et al., 1997). Thermal inactivation studies show a biphasic inactivation in the cooking temperature range of 70 to 100 °C.  The level of activity depends on its structure, location in the fruit or vegetable and the milieu in which it resides. Various peroxidase fractions are isolated from the same plant tissues as a mix of different isoenzymes, all with varying thermal stability (Vámos-Vigyázó 1981; Adams 1997). The group led by Professor David Robinson in the Proctor Department of Food Science at the University of Leeds has published a large body of work on this enzyme amongst others of the type. For example, they noted that peroxidases resided in the albedo of orange and could be present in both solubilised or electrostatically linked forms (McLellan & Robinson, 1984). The bound peroxidises were heat stable and could regain some activity at 30 °C having been initially heat processed to denature them. This behaviour has been reported for the enzyme from other produce which implies it is difficult to destroy completely.  Even if the fruit or vegetable is processed, there is the possibility of a return of activity and so verification is needed to check the peroxidise activity will not return. An example of this is implied in an early work on flavour losses in processed orange juice (Bruemmer et al., 1976) and then reported later for asparagus (Rodrigo et al., 1996). Just to reiterate, it is the case that one of the most robust heat processing treatments, HTST (high temperature-short time) will not ‘kill’ the peroxidise (Khan & Robinson,1993; McLellan & Robinson, 1987).

As well as peroxidase to contend with, the enzymes polyphenol oxidase and lipoxygenase  also contribute to nutrient and other quality losses. The study of the enzyme is vast ! A separate post will look at processing methods that will inactivate this enzyme.

References

Adams, J.B. (1997)  Regeneration and the kinetics of peroxidase inactivation. Food Chem. 60(2) pp. 201-206.

Bruemmer, J.H., Roe, B., Bowen, E.R. (2006)  Peroxidase reactions and orange juice quality.  J. Food Sci., 41(1)

Cano, M.P., Hernández, A., De Ancos, B. (1997)  High pressure and temperature effects on enzyme inactivation in strawberry and orange products. J Food Sci. 62 pp. 85–88.

Khan, A. A., & Robinson, D. S. (1993). The thermostability of purified mango isoperoxidases. Food Chem., 47, pp. 53–59.

McLellan, K.M. Robinson, D.S. (1984) Heat stability of peroxidises from orange. Food Chem., 13(2) pp. 139-147

McLellan, K. M., & Robinson, D. S. (1987). The heat stability of purified spring cabbage preoxidase isoenzymes. Food Chem., 26, pp. 97–107.

Morales-Blancas E.F., Chandia, V.E., Cisneros-Zevallos, L. (2002)  Thermal inactivation kinetics of peroxidase and lipoxygenase from broccoli, green asparagus and carrots. J. Food Sci. 67(1) pp. 146–54.

Rodrigo, C., Rodrigo, M., Alvarruiz, A., Frigola, A. (1996) Thermal Inactivation at High Temperatures and Regeneration of Green Asparagus Peroxidase. J. Food Protection. 59 (10) pp. 1056-1107

Soysal, C.¸Söylemez, Z. (2005)  Kinetics and inactivation of carrot peroxidase by heat treatment. J.  Food Engr. 68 pp. 349–56.

Vámos-Vigyázó L. 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Critical Rev Food Sci Nutr 15(1)  pp.49-127.