J . Sci. Food Agric.

1985,36,877-880

The Peroxidase Enzyme Activity of some Vegetables and its Resistance to Heat
Nezih Muftugil
TUBITAK, Marmara Research Institute, Nutrition and Food Technology Department, Gebze, Kocaeli, Turkey (Manuscript received 12 April 1985)

The peroxidase enzyme activities of some fresh vegetables (cabbage, leeks, carrot, spinach, celery, squash, potatoes, onions and green beans) were determined. The peroxidase activities of cabbage and green beans were high. Onions showed very little peroxidase activity. The vegetables were blanched in hot water at 75",85" and 95C. Peroxidase inactivation was faster at the higher temperature blanch. Blanching of green beans, potatoes and squash at 75C for 30 min was not sufficient for complete inactivation of peroxidase enzyme. Peroxidase enzyme inactivation was also affected by the type of vegetable and the size of vegetable pieces. Keywords: Peroxidase; vegetables; blanching; heat treatment; enzyme activity.

1. Introduction
Blanching is an important process in the preparation of vegetables for canning, freezing and dehydration. It consists of a mild heat treatment accomplished by exposing the vegetables to hot water or steam at 60-100C for several minutes. This treatment is quite acceptable since most vegetables are cooked prior to consumption. Raw and underblanched vegetables discolour, lose flavour and develop off-odours during frozen and dried storage. It is now widely recognised that the loss of quality that unblanched vegetables experience during frozen and dried storage is caused by enzymic action.'-3 Despite the efforts of many investigators over the past 30 years the enzymes responsible for quality deterioration have not been positively identified.4 There is a general agreement that where flavour is concerned several enzymic systems may be involved, working in sequence or simultaneously. Peroxidase, a member of a large group of enzymes called the oxidoreductases, is considered to have an empirical relationship to off-flavours and off-colours in raw and unblanched vegetables, although the reactions involved have not been conclusively identified.5 It has been found that enzyme peroxidase present in all vegetables is the most heat stable enzyme in plank5 For this reason peroxidase activity is widely used as an index of blanching. In this study the peroxidase enzyme activity of some vegetables was determined and the effect of blanching temperature on the inactivation of peroxidase enzyme was investigated.

2. Experimental
2.1 Materials In this study nine different kinds of vegetables were used as the raw materials (Table 1). Vegetables were size graded, trimmed, washed and some of them were peeled. Cabbage (cut 5mm), leek (cut lOmm), carrot (cut lOmm), spinach (leaves), celery (cut lOmm), squash (cut lOmm), potato (10mm cubes), onion (cut 5mm) and green beans (cut 20mm) were used, one commercial variety of each blanched in water at three different temperatures (75,85 and 95C).
58
877

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N. Miiftugil
Table 1. The peroxidase activity of some fresh vegetables

Veeetable Cabbage Leeks Carrots Spinach Celery Squash Potatoes Onions Green beans

Botanical name
Brassica oleracea Allium porrum Daucus carota Spinacia oleracea Apium graveolens Cucurbito pep0 Solanum tuberosum Allium cepa Phaseolus vulgaris

Peroxidase enzyme activity (unit 100n") 56.20 3.73 1.24 19.46 4.05 36.10 4.02 0.80 51.46

Blanching was performed in a blanching kettle with 8 parts of water to 1 part vegetable. Temperature of the blanching water was controlled by an immersed thermocouple connected to a digital thermometer. After blanching the vegetables were cooled in cold water.
2.2 Method

The quantitative peroxidase activities of raw and blanched vegetables were assayed according to the method described by Harvey et aL6 Vegetable purees were made by blending 50g of the sample with 50ml of 0 . 2 ~ acetate buffer (pH 5.6). A known weight of puree (approximately 50mg) was weighed into each of two spectrophotometer cuvettes; 2ml of acetate buffer was added to the sample cuvette whilst 2.7ml of acetate buffer was added to the sample cuvette whilst 2.7ml of the same buffer was added to the reference cuvette. The contents of the cuvette were transferred to, and homogenised in, a small tissue grinder. After homogenisation the suspended vegetable tissue was returned to the cuvettes; 0.7ml of an aqueous mixture (1:l) of 0.5% guaicol and 0.1% hydrogen peroxidase was added to the sample cuvette. Both cuvette were then shaken prior to determining the rate of formation of brown colour in the sample by measuring the absorbance of light at a wavelength of 420nm in a Varian Techtron Model 635 spectrophotometer. This rate was generally linear with time in the early stages of the reaction and the gradient of the line was taken to be proportional to the concentration of peroxidase in the sample.

3. Results and discussion

Peroxidase was found in all samples of fresh vegetables investigated (Table 1). Cabbage and green beans had high enzyme activities whereas in ordon and carrots the peroxidase enzyme activity was low.

Table 2. The inactivation of peroxidase during the blanching of some vegetables in water at 75C

Peroxidase activity 01 fresh vegetable (W) Vegetable Cabbage Leeks Carrots Spinach Celery Squash Potatoes Onion Green beans
0

15

30

60

90

120

Blanching: time (s) 180 240 300 26.56 14.45 1.15

510 2.10

600

900

1200

1500 18M

100 100 100 100 100 100 100 100 100

66.40 45.07 64.50 59.60 31.50 22.61 62.46 42.72 73.87 59.70 57.96 68.75 -

53.13 47.43 40.80 37.95 30.02 12.08 51.61 27.41 11.62 2.43 6.41 0.32 55.40

1.63

45.70 61.44

39.88 64.17 41.10

27.14 20.0 32.8

89.10

86.37

79.90

68.20 58.02 37.31

Peroxidase enzyme activity of vegetables

879

Table 3. The inactivation of peroxidase during the blanching of some vegetables in water at 85C
~~ ~

Peroxidase activity of fresh vegetable (%)

-

Vegetable Cabbage Leeks Carrots Spinach Celery Squash Potatoes Onion Green beans

0
100

15

30

60

90
2.88

Blanching time (s) 120 180 240 360 1.79 26.17 16.80 6.13 2.57 7.91 0.57 2.15

420

600

900

1080 1200

18.02 11.62 100 39.90 35.00 32.17 100 69.35 40.30 14.19 100 11.76 100 43.90 38.02 2.46 100 94.50 84.76 100 72.13 59.70 100 37.75 100

40.60

26.59 13.18 10.72

14.42

17.72 9.27

11.80 3.40 7.77 2.8'

Table 4. The inactivation of peroxidase during the blanching of some vegetables in water at 95C Peroxidase activity of fresh vegetable (%) Vegetable Cabbage Leeks Carrots Spinach Celery Squash Potatoes Onion Green beans
0

15 16.25 32.97 53.20 0.20 41.48 47.92 72.88 26.25 73.17

30
4.10 24.93 27.45
-

45

Blanching time (s) 60 90 120 2.82 21.85 0.85 9.18 1.05

180 0.92

210

240

100 100 100 100 100 100 100 100 100

4.83 1.97 33.14

26.17

23.62 6.46 23.82 8.12 5.94

6.26 3.76

1.46 1.77 0.50

56.95

The effect of blancing temperature on the inactivation of peroxidase enzyme is given in Tables

2, 3 and 4.The intensity of heat treatment applied during blanching caused differences in enzyme
activity of the vegetables. Peroxidase enzyme was most easily inactivated at the high temperature blanch. Blanching of green beans, potatoes and cabbage at 75C for 30min was not sufficient for complete inactivation of peroxidase. A full account of the biochemistry of this enzyme has been given by Saunders et aL7 It has been stated that various types of peroxidase exist and the properties depend to some extent on its source.8 Jansen reported that when peroxidase is considered responsible for changes in frozen vegetables, the locale and occurrence of isoenzymes should also be considered.' If one or more isoenzyme is responsible for adverse changes in frozen vegetables, then the measurement of total peroxidase and of the loss therein could have little meaning. During blanching, peroxidase inactivation was also affected by the size of vegetable pieces. The enzyme inactivation in spinach and cabbage samples which have large surface to volume ratios and comparably high enzyme activities was faster. Fawpenetration of heat to the centres could explain fast enzyme inactivation during blanching. Gelatinisation of starch in potatoes by heat caused formation of a thin film on the surface of the potatoe cubes and thereby decreased the inactivation rate of peroxidase enzyme.
4. Conclusions

Peroxidase enzyme inactivation time depended on the temperature of the blanching water, size of vegetables and the nature of the enzyme present. In industrial blanching processes the peroxidase enzyme test is commonly used to determine whether a sufficient blanch has been given. Attempts to inhibit all peroxidase activity is not strictly necessary; a significant proportion

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N. Miiftiigil

of active peroxidase can be left in many vegetables and a long shelf life in frozen storage can still be achieved." There is some concern that the heat treatment required to give complete inactivation may represent an excessive blanch in many cases. However, for some vegetables complete inactivation of peroxidase enzyme is necessary in order to obtain good quality frozen stored products." Some observations support the view that the enzyme lipoxygenase in green beans, spinach, peas and cauliflowers is responsible for the development of off-flavour during frozen storage12 but its low measurable activity in the fresh products makes this enzyme less suitable as a blanching parameter. Acknowledgement This work forms part of a research project sponsored by the Scientific Affairs of NATO, Science for Stability Programme to whom thanks are due. References
1. Hartzler, E.R.; Guerrant, N.B. Effect of blanching and of frozen storage of vegetables on ascorbic acid retention and the concomitant activity of certain enzymes. Food Res. 1952 17, 15-29. 2. Rhee, K.S.; Watts, B.M. Lipid oxidation in frozen vegetables in relation to flavour change. 1. Food Sci. 1966, 31, 657-679. 3. Chow, L.; Watts, B.M. Origin of off-odors in frozen green beans. Food Technol. 1969, 23, 11S114. 4. Adams, J.B. Thermal requirements for blanching of fruits and vegetables to be frozen. Proceedings of Internationa[ Meeting Day of 'Blanching of Fruits and Vegetables to be Fro Zen-Present Knowledge and Future Perspectives'. C.O.S.T. 91, Paris 1981, 12-32. 5 . Burnette, F.S. Peroxidase and its relationship to food flavour and quality. A review. J . Food Sci. 1977, 42, 1-5. 6 . Harvey, P.; Butler, R.C.; Powel, S.J.; Ongley, M.H.; Adams. M.J. Subjective and Objective Methods Used in Blanching Research in 1976177. Technical Memorandum. No. 178. The Campden Food Preservation Research Association, Gloucestershire, 1977, pp. 1&11. 7. Saunders, B.C.; Holmes-Siedle, A.G.; Stark, B.P. Peroxidase. Butlenvorths, Kent, 1964, pp. 2-32. 8. Aylward, F.; Haisman, D.R. Oxidation systems in fruits and vegetables. Adv. Food Res. 1969, 17, 1-76. 9. Jansen, E.F. Quality related chemical and physical changes in frozen foods. In: Quality and Stabiliry of Frozen Foods. (Van Arsdel, W.B.; Copley, M.S.; Olson, R.L., Eds), Wiley, New York, 1964, pp. 19-42. 10. Bottcher, H. The enzyme content and the quality of frozen vegetables. I. Residual peroxidase activity. Nahrung. 1975, 19, 17S179. 11. Lindquist, F.E.; Dietrich, W.C.; Masure, M.P.; Boggs, M.M. Effect of enzyme inactivation on quality retention in frozen Brussel Sprouts. Food Technol. 1951, 5, 198-199 12. Baardseth, P. Quality changes of frozen vegetables. Food Chem. 1978,3,271-282.