Plant Foods for Human Nutrition 43: 71-76, 1993. 1993 Kluwer Academic' Publishers. Printed in the Netherlands.

Control of enzymatic browning in apple slices by using ascorbic acid under different conditions
N A H E D M. EL-SHIMI
Food Science and Technology Department, Faculty of Agriculture, University of Alexandria, Egypt
Received 10 June, 1991; accepted with minor revisions 16 September, 1991

Key words: polyphenol oxidase (PPO), ascorbic acid (AA) Abstract. Control of phenol oxidase activity in apple slices by the use of ascorbic acid at different pH values, temperature and time of incubation was investigated. The enzyme was almost inactivated at 1% and 1.5% ascorbic acid. Ascorbic acid solution (1%) caused a remarkable inhibition with the increasing acidity up to pH = 1. Heating treatments for apple slices dipped in 1% ascorbic acid caused a reduction of enzymatic browning, optimum temperature for inactivation of the enzyme was between 60-70 C for 15 minutes. Increasing the time of dipping apple slices in 1% ascorbic acid solutions and at different pH values reduce phenolase activity.

Introduction

Browning reactions in foods are of wide spread occurence and become evident when, for instance, food material is subjected to processing or to mechanical injury. They are important in terms of the alteration of appearance, flavour and nutritive value. Browning is desirable when it enhances the appearance and flavour of a food products such as coffee, maple syrup and toasting of bread. In case of fruits, vegetables, frozen and dehydrated foods, browning is undesirable which results in off-flavour and poor appearance. Browning of raw fruits and vegetables due to mechanical injury during postharvest handling and processing is an important cause of quality and value loss in affected commodities. Enzymic browning is one of four mechanisms of browning reactions. It occurs in many fruit and vegetable tissues whenever they are injured. The injury can be the result of cutting, freezing or disease. The part of the injured fruit which is exposed to air undergoes a rapid darkening. This darkening reaction results from the polyphenol oxidase (PPO) catalyzed oxidation or phenolic compounds to O-quinones which subsequently polymerize to form

72 dark-colored pigments (Joslyn and Ponting, 1951; Mayer and Hanel, 1979; Vamos Vigiyazo, 1981). The rapid darkening of many fruits and vegetables such as apples, bananas, avocado and potatoes, is a serious problem in their processing, freezing (in case of unblanched materials) and during dehydration where any injury to plant tissue sustained through the use of heat or through poor handling. The enzyme responsible for the initiation of this browning is known as phenolase polyphenol oxidase (O-diphenol: oxygen oxidareductase, Ec 1.10.3.1) (Eskin et al., 1971). Appropriate steps must be taken during food processing against the development of this type of browning. Many methods of potyphenol oxidase inhibition are known such as heating, sulfur dioxide and sulfites, exclusion of oxygen, sodium chloride, methylation of phenolase substrates, acids, boric acid and borates (Mapson and Swain, 1961). Probably the simplest and the most straight forward method of inactivating polyphenol oxidase, along with all other enzymes present, is to apply heat, since enzymes are protein and are therefore easily denatured by heat, but several problems may arise. The fruits or vegetables become cooked which in turn leads to unfavourable texture changes and development of off-flavour (Schultz, 1960). A partial blanch was used by Guandagni and Nimmo, (1957) for enzyme inhibition in sliced peaches before freezing. The widely used method for controlling the enzymic browning is the application of acids especially the ones which naturally occure in tissues such as citric, malic, phosphoric and ascorbic acids. In general, their action is to lower the tissue pH and thus to decrease the rate of enzymic browning. The optimum pH of phenolase lies within the range 6-7; below pH 3 there is no enzymic activity (Eskin, 1971). Taeufel and Voigt (1964) stated that ascorbic acid is the most significant inhibitor of phenolase because it has no detectable flavour at the concentration used which would interfere with the acceptability of the final processed product. Also it has no corrosive action upon metals, in addition to its vitamin value. Ascorbic acid reduces O-quinone to limit browning through process known as 'reaction deactivation' (Powers et al., 1958, Luh et al., 1975) who described this mode of action as forming a barrier to oxygen diffusion into the product. The mode of ascorbic acid action upon phenolase can be summarized as follows: O -- diphenol + 1 02 ~ O - quinone +

H20

O - Quinone + A.A. ~ O diphenol + dehydro A.A A A + 1 02 -+ dehydro A A +

H20

73 Borenstein (1965), Sapers and Dougles (1987) and Sapers and Ziolkowski (1987) stated that ascorbic acid is a more effective inhibitor of enzymic browning than are sulfites or erythorbic acid. The objective of this work is to study the effectiveness of ascorbic acid as enzymic browning inhibitor in apple slices under different temperature, time and concentrations.

Materials and methods

Five kilograms of apples were purchased from retail markets on the morning of the same day of the experiments and immediately kept at 4 C until used. The apples were sliced by using a slicer machine. The apple slices were used in carrying out three treatments as follows: 1. The submerging in three ascorbic acid concentrations (0.5, 1 and 1.5 percent) for 6 periods (0.5, 1, 1.5, 2, 2.5 and 3 hours). 2. The dipping in 1% ascorbic solution differing in their pH values (from 1-7). The pH value was adjusted by using few drops of HC1 (1%) and sodium hydroxide (1%). 3. The soaking in six ascorbic acid solutions (1%) differing in their temperature (30, 40, 50, 60, 70 and 80C) for different times (5, 10, 15, 20 minutes). 4. Determination of phenolase activity: 5 gm of the examined sample were extracted with 50ml acidified methanol (methanol: HC1 97: 13v/v) (Ranganna, 1979). Enzymatic activity of PPO was determined at the rate of increase absorbance at 410 nm. An Apye Unicam Spectrophotometer was used. A reaction mixture contained 1 ml 0.05 M catechol, varying amounts of enzyme extract and 0.20 M phosphate buffer (pH 6.8) in a final volume of 5 ml. The catechol solution and enzyme extract were also in 0.2M phosphate buffer. The temperature of the assay was 30 C. The rate of the reaction was calculated from the initial linear slops of activity curve (Augustin et al., 1985).

Results and discussion

Effect of ascorbic acid concentration

Table 1 data show that phenolase activity was decreased with the increase of ascorbic acid concentration. The higher the ascorbic acid concentration, the lower was the phenolase activity. Compared with water as a control,

74
Table 1. Effect of ascorbic acid concentration at different times (hours) on the activity of polyphenol oxidase in apple slices Concentration % 0.5 h 1h 1.5 h 2h 2,5 h 3h

Polyphenol oxidase activity % Water 0 . 5 % a s c o r b i c acid 1% ascorbic acid 1.5%ascorbic acid 70 60 60 45 90 55 53 40 95 48 44 32 95 45 35 12 95 27 18 10 98 25 15 10

0.5% ascorbic acid lowered phenolax activity to one fourth after 3 hours of treatment, whereas 15% and 10% from the phenolase activity was found after using 1% and 1.5% ascorbic acid, respectively, for 3 hours. This means that 1 to 1.5% ascorbic acid is very effective in considerably reducing enzyme browning in apple slices. Ascorbic acid itself is not an inhibitor for polyphenol oxidase; it must be oxidized indirectly by the enzyme before it can inhibit the enzyme activity. So if sufficient ascorbic acid is present, polyphenoloxidase will oxidize its natural substrate and the oxidation product will be immediately reduced by ascorbic acid (Ingraham, 1956; Schultz, 1960). Eskin et al. (1971) reported that food material must be treated with an adequate amount of ascorbic acid, otherwise browning is only slightly delayed, up to the point at which all the ascorbic acid is oxidized.

Effect of pH
Data presented in Table 2 revealed the effect o f p H on the phenolase activity in apple slices. It was found that below pH 3 the phenolase activity is very weak, while it has a considerable activity within the range of pH 5-7. This is in agreement with Schulize (1960) who found that levels of pH below 2.5 to 2.7 were very suitable for inactivation of enzymic browning in apple. The
Table 2. Effect of different pH values and time (hours) on the polyphenol oxidase activity in apple slices pH 0.5h 1h 1.5h 2h 2.5h 3h

Polyphenol oxidase activity % 7 6 5 4 3 2 1 70 65 65 48 30 28 25 65 59 53 42 25 25 20 58 50 43 30 18 t0 10 50 45 35 25 15 10 10 40 40 21 18 I0 8 5 35 30 18 15 5 5 5

75

Table 3. Effect of temperature and time (minutes) on the activity of polyphenol oxidase in apple slices
Temperature (C) 5min 10rain 15rain 20min

Polyphenol oxidase activity 30 40 50 60 70 80 80 80 65 42 30 25 84 85 50 35 28 20 90 90 45 30 20 10 95 95 32 30 10 10

results showed that dipping the apple slices in ascorbic acid solution at pH 2 and 3 (Table 2) decreased phenolase activity to 5% after 3 hours. Eskin et al. (1971) reported that immersing apple slices in 1.5% ascorbic acid solution up to pH 1-2 to 1.5 hour caused about 90% inhibition of phenolase which in turn lowered the rate of enzymic browning.

Effect o f temperature
The data given in Table 3 show that the inhibitory effect of ascorbic acid was evident at temperatures of 70-80 C. Temperatures from 30-40 C had no effect on phenolase activity even after 20 minutes. Heating the apple slices at 60-70 C for 15 minutes showed a remarkable decrease in the phenolase activity. Ten percent of phenolase activity was found after 20 minutes of heating apple slices at 70-80 C. According to Walker et al. (1955) enzymic browning was prevented when the internal temperature of the apple slices was over 50 C. The trouble with heating the fruit to high temperature for a long time is that little flavor will be left and the texture of the fruit will get softer. So blanching processors usually struggle to achieve suitable enzyme inactivation with a miniumnm number of undesirable changes in flavor and texture. In conclusion, lowering the pH of the apple during processing can reduce the temperature and time of heat treatment. This can be achieved by using an ascorbic acid solution which in addition has no detectable flavour and is an important vitamin.

References

Augustin MA, Ghazali HM, Hasinal Hashim (1985) Polyphenol oxidase from Guava (Psidium guajave L.). J Sci Food Agric 36:1259 Borenstein B (1965) The comparativeproperties of ascorbic acid and erythorbicacid. Food

76
Technol 19:1719 Dauerfeind JC, Pinkert DM (1970) Food processing with added ascorbic acid. Adv Food Res 18:219 Eskin NAM, Henderson HM, Toznsend RJ (1971) Biochemistry of foods, Academic Press. New York, San-Fransisco, London Guandagni DO, Nimmo CC (1957) The time-temperature tolerance of frozen foods. III-Effectiveness of vacuum oxygen removal, and milk heat in controlling browning in frozen peaches. Food Technol 11:43 Joslyn MA, Ponting JD (1951) Enzyme catalyzed oxidative browning of fruit products. Advan Food Res 3:1 Ingraham LL (1956) Effect of ascorbic acid on polyphenol oxidase. J Am Chem Soc 18:50-95 Luh BS, Feinberg B and Chung JI (1975) Freezing of fruits. In 'Commercial fruit processing' Avipublishing Co, Inc, West Port, CT, p. 266 Mapson LW, Swain I (1961) Oxidative of ascorbic acid and phenolic constituents in 'Production and application of enzyme preparations in food manufacture', Sci (Soc Chem Ind London). Monogr 11, p. 121 Mayer AM, Hanel E (1979) Polyphenol oxidase in plant's phytochemistry, 18:193 Powers M J, Talburt WF, Jackson R, Lazar ME (1958) Dehydra canned apples. Food Technol 8:417 Rangannas M (1977) Manual of analysis of fruits and vegetable products. Tata McGraw Hill Publishing Company Limited, New Delhi Sapers MG, Dougles FW (1987) Measurement of enzymatic browning at cut surface and in juice of raw apple and pear fruits. J Food Sci 52:1258 Sapers MG, Ziolokowski AM (1987) Comparison of erythorbic and ascorbic acids as inhibitors of enzymatic browning in apples. J Food Sci 52:1732 Schultz HW (1960) Food enzymes. The Avi publishing Company Inc, West Port, CT Taeufel K and Voight J (1964) Sodium chloride as inhibitor in enzymic browning of apples. Nahrung 8:80 Vamos-Vigyaza L (1981) Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci Nutr 15:49 Walker LH, Power M J, Taylor DH (1955) Factors in processing methods which affect the quality of dehydro frozen apple slices. Food Technol 9:576