Heat inactivation and pH optima of peroxidase and catalase in carrot, swede and Brussels sprouts

FoodChemistr) 5 (1980)169 174

HEAT INACTIVATION A N D pH OPTIMA OF PEROXIDASE A N D CATALASE IN CARROT, SWEDE A N D BRUSSELS SPROUTS

PERNILLE BAARDSETH& ERIK SLINDE

Norwegian Food Research Institute, Postbox 50, 3[-1432 Aas-NLH, Norway (Received: 3 January 1979)

ABSTRACT

Heat inactivation and p H optima of the enzymes per oxidase and catalase were studied in carrot, swede and Brussels sprouts. There were differences in the heat stabilities of the peroxidases from different vegetables, but all peroxidases were more heat stable than the catalases. From the pH profileS and the heat stability curves it was concluded that both the peroxidases and catalases in the three vegetable species are somewhat different. Lipoxygenase and phenolase activities were not detected by the methods used.

INTRODUCTION

It has been reported recently (Baardseth, 1978) that unblanched leek, onion and swede did not develop detectable off-flavour or off-odour during storage at - 20 °C and - 30 °C for 15 months. Carrot, cauliflower and French bean, on the other hand, have to be blanched, but a 5 ~ residual activity of peroxidase did not affect quality during storage. Some vegetables can be stored as such for months, for example, carrot, swede, onion (8 months at 0 °C) and leek (5 months at 0 °C). Brussels sprouts (6 weeks at 0 °C), beans and cauliflower (2 weeks at 0 °C), however, deteriorate within a relatively short time (Berg, van der & Lentz, 1977), Carrot, which can be stored for months, will start to deteriorate when peeled, cut and frozen, but swede, onion and leek do not. 169 Fd. Chem. 0308-8146/80/0005-0169/$02-25 © Applied Science Publishers Ltd, London, 1980 Printed in Great Britain

170

PERNILLE BAARDSETH, ERIK SLINDE

The enzymes lipoxygenase and phenolase are involved in undesirable quality changes in food. Peroxidase and catalase, on the other hand, are often reported to cause off-flavour, but the reactions involved have not been conclusively identified (Svensson, 1977). The aim of this investigation was to study the presence, pH optima and heat stabilities of these four enzymes in carrot, swede and Brussels sprouts. These vegetables differ in storage stability, and it was of interest to correlate enzyme activities with shelf life.

MATERIALS AND METHODS

Materials Carrot (Daucus carota, var. Nantes Duke), swede (Brassica napus var. napobrassica, var. Bangholm) a n d Brussels sprouts (Brassica oleracea var. gemmifera, var. Jade Cross E) used in the experiments were obtained locally. Methods The vegetable samples were prepared by washing, peeling and cutting (Weisser cutting machine). The cut vegetables (20 g) were homogenised (Ultra-Turrax TP 18/10, Janke & Kunkel KG) with 20 ml 0' 1 Mphosphate buffer, pH 7.0, at 4 °C. The supernatant obtained after centrifugation (600 000 gay.min at 4 °C with a Sorvall centrifuge and rotor SS 34) was used in the enzyme assays. Enzyme reaction velocities were calculated from the initial slopes of the absorbance/time curves. The enzyme activities were determined at 25 °C in a thermostatically controlled chamber of a recording spectrophotometer (Shimadzu UV 300), and the pH of the reaction mixtures was measured. To cover the actual pH range and elucidate the presence of ionic effects, different buffers were selected as shown in Figs. 1 and 2. Heat inactivation was performed in triplicate in glass tubes (0.5 mm walls) covered with a marble. The tubes were placed in a circulating water bath at 70 °C for different lengths of time and cooled in ice water before enzyme assays were performed. The peroxidase activity was determined at 420 nm using guaiacol and hydrogen peroxide as substrates, as described by Lu & Whitaker (1974). Catalase activity was measured at 230 nm using hydrogen peroxide (absorbance at 230 nm = 1.0)as substrate (Bergmeyer et al., 1974). Lipoxygenase was determined essentially as described by Sekiya et al. (1977), but Brij 58 (a nonionic detergent, Polyoxyethylene 20 cetyl ether, Sigma, St. Louis, USA) was used to emulsify the linolenic acid. Brij 58 has the advantage that it does not absorb significantly at 230 nm, and the solutions did not show any sign of clouding under the conditions employed. The assay was standardised using lipoxygenase from peas. Phenolase activity was assayed essentially according to Sat6 (1976) at 500 nm, but 20 mM catechol and sulphanilic acid were used. Potato phenolase was used to standardise the assay. Protein was determined by the method of Lowry et al. (1951).

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172

PERNILLE BAARDSETH, ERIK SLINDE RESULTS AND DISCUSSION

Peroxidase and catalase are widely distributed in higher plants (Whitaker, 1972), and the presence of these enzymes in carrot, swede and Brussels sprouts was confirmed. On the other hand, phenolase and lipoxygenase activities were not detected with the present techniques. Pinsky et al. (1971) reported no activity of lipoxygenase in carrot while swede and Brussels sprouts were not investigated. The amounts of protein in extracts of the three vegetables were determined as carrot, 3-4mg/ml; swede, 5.5mg/ml and Brussels sprouts 63.7mg/ml. The pH values of water extracts were 5.6 for carrot, and 5.8 for both swede and Brussels sprouts. A number of peroxidase activities exist, and these belong to various classes (for review, see Whitaker, 1972). Since the optimal pH for the peroxidase activities are different in the three vegetables (Fig. I), indications are that the enzymes responsible for the activities are somewhat different. However, the peroxidase activities of the three vegetables are nearly maximal at the pH values (5.6-5-8) found within the vegetables. The pH optima of the catalase activities (Fig. 2) were not found to coincide with the pH of the vegetables. Furthermore, a pronounced variation was observed at the same pH with the different buffers especially in the case of carrot (Fig. 2(B)). Thus, the three enzymes seem to be very heterogeneous. A variety of acids react with catalase to give inactive compounds (Schonbaum & Chance, 1976). This may explain the activity differences. Heat inactivation of the enzymes at 70 °C for 1.5 min gave residual activities of approx. 45 9/0 for peroxidase and 20 ~o for catalase (Fig. 3). In the response to heat 1

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Heat inactivation (70 °C with 0.1 Mphosphate buffer pH 7.0) of peroxidase (A) and catalase (B) from (I-2) Brussels sprouts, (A) carrot, and ((3) swede as a function of time.

HEAT INACTIVATION AND

pH

OPTIMA OF PEROX1DASE AND CATALASE

}73

inactivation peroxidase shows greater variation among the vegetables than catalase (Fig. 3). Swede peroxidase seems to be more heat stable than peroxidase from Brussels sprouts and carrot. The living vegetable contains numerous enzymes that are active in different metabolic processes, and in some cases the presence of certain deteriorative enzymes in fresh and harvested vegetables can change the quality within days or weeks (Svensson, 1977). The absence of the deteriorative enzymes lipoxygenase and phenolase in the vegetables investigated favours extended shelf life and may partly explain why they can be stored for the lengths of time stated. On the other hand, the different storage stabilities of the Brussels sprouts, carrot and swede could be due to the differences in their contents of peroxidase and catalase. For the three vegetables studied, a correlation exists between the storage stability and the activity of catalase and peroxidase, since swede has a high activity of peroxidase and a medium catalase activity, carrot has a medium peroxidase and a high catalase activity, while Brussels sprouts have a relatively low activity of both enzymes. However, it must be kept in mind that the amount of protein is highly different in the three vegetable extracts and that the activity per volume of Brussels sprouts is relatively high for at least peroxidase. In the cut state, swede has a long storage stability in contrast to carrot. When vegetables are injured, the deteriorative enzymes.are often released or activated and enzyme reactions utilising oxygen proceed at a much higher rate. The swede contains the highest amount of ascorbic acid per mg protein (Paul & Southgate, 1978). Ascorbic acid is a reducing agent and an endogenous substrate to the respiratory chains which give ATP and remove oxygen. This, in conjunction with the absence of deteriorative enzymes, may partly explain the long storage stability of swede when cut.

REFERENCES

BAARDSETH, P. (1978). Quality changes of frozen vegetables. Food Chem., 3, 271-83. BERG, VANDER,L. & LE•TZ, C. P. (1977). Effect of relative humidity of storage of vegetables. Acta Hortic. (The Hague), 62, 19%208. BERGMEYER,H. U,, GAWEHN, K. & GRASSL, M. (1974). Cataiase. In: Methods oJ en:ymic analysis. (2nd ed.) (H. U. Bergmeyer, Ed.) Verlag Chemie, Weinheim, 438-9. LowRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RAr~DALL,R. J. (1951). Protein measurement wit h t he Folin phenol reagent. J. Biol. Chem., 193, 265-73. Lu, A. T. & WmxArd~g, J. R. (1974). Some factors affecting rates of heat inactivation and reactivation of horseradish peroxidase. J. Food Sci., 39, 1173-8. PAUL, A. A. & SOUTnGATE,D. A. T. (1978). The composition ofJoods. (4th ed.), Elsevier/North-Holland Biomedical Press, Amsterdam, 172-84. PINSg£, A., GROSSM^N, S. & TROP, M. (1971). Lipoxygenase content and antioxidant activity of some fruits and vegetables. J. Food Sci., 36, 571-2. SAT6, M. (1976). Phenolase of spinach roots, Phytoehem., 15, 1845-7. S¢8ONSAUM, G. R. & C,ANCE, B. (1976). Catalase. In The en-ymes, (3rd Ed.) (P. D. Boyer, (Ed.)) Vol. XIII, Academic Press, New York, 363-408.

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SEKIYA, J., AOSmMA, M., K~olWmtA, T., Toc,o, T. & HATANAKA,A. (1977). Purification and some properties of potato tuber lipoxygenas¢ and dection of linoleic acid radical in the enzyme reaction. Agric. BioL Chem., 41,827-32. SVENSSON,S. G. (1977). Inactivation of enzymes during thermal processing. In: Physical, chemical and biological changes in food caused by thermal processing, (T. Heyem & O. KvAle (Eds.)) Applied Science Publishers, Ltd, London, 202-17. WmTAr,ER, J. 'R. (1972). Catalase and peroxidase. In: Principles of enzymology for the food sciences. Marcel Dekker, New York. (Food Science 2), 591-605.