SUMMARY
Enzymatic browning, mainly catalyzed by Polyphenol Oxidase (PPO) Enzyme, is one of the main problems that affect quality and limit the shelf life of minimally processed fruits and vegetables. The compounds traditionally used to inhibit PPO are sulfites. However, its use in the food industry has been discouraged because cases of allergic reactions have been reported, especially in asthmatic individuals. As a consequence, the use of other compounds as potential enzyme inhibitors is currently being evaluated to guarantee fresh and natural products. In the present work, the evolution of the PPO enzyme activity and the chromatic characteristics of the pulp of apple slices cv. Granny Smith, treated by immersion in a solution of additives. The treatments used were: I. 2% Ascorbic Acid + 1% Citric Acid + 0.5% EDTA, II. 1% Ascorbic Acid + 0.5% Citric Acid + 0.25% EDTA and III. water, used as control. During storage, the color coordinates of the CIE L*a*b* space of the slices were evaluated and it was shown that treatments I and II were effective in preventing fruit browning. The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors The treatments used were: I. 2% Ascorbic Acid + 1% Citric Acid + 0.5% EDTA, II. 1% Ascorbic Acid + 0.5% Citric Acid + 0.25% EDTA and III. water, used as control. During storage, the color coordinates of the CIE L*a*b* space of the slices were evaluated and it was shown that treatments I and II were effective in preventing fruit browning. The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors The treatments used were: I. 2% Ascorbic Acid + 1% Citric Acid + 0.5% EDTA, II. 1% Ascorbic Acid + 0.5% Citric Acid + 0.25% EDTA and III. water, used as control. During storage, the color coordinates of the CIE L*a*b* space of the slices were evaluated and it was shown that treatments I and II were effective in preventing fruit browning. The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors 25% EDTA and III. water, used as control. During storage, the color coordinates of the CIE L*a*b* space of the slices were evaluated and it was shown that treatments I and II were effective in preventing fruit browning. The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors 25% EDTA and III. water, used as control. During storage, the color coordinates of the CIE L*a*b* space of the slices were evaluated and it was shown that treatments I and II were effective in preventing fruit browning. The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors The spectrophotometric evaluation of the enzymatic activity of the PPO, in extracts of apples submitted to the different treatments, showed that the most severe (I) was the one that produced the highest degree of inhibition of the enzyme, in all the times analyzed. It is proposed to evaluate in the future the effectiveness of these inhibitors“in vitro”, in order to compare them with the results obtained in apple slices.
Keywords: Polyphenol oxidase; Ascorbic acid; Citric acid; EDTA; minimal processing; apples.
ABSTRACT
Enzymatic browning, which is mainly catalyzed by the enzyme polyphenol oxidase (PPO), is one of the major problems affecting quality and limiting shelf life of fresh cut fruits and vegetables. Traditionally, sulfites are used to inhibit the enzyme. However, its presence in the food has induced allergenic reactions, particularly in asthmatic persons. Consequently, it has been evaluated the effect of other PPO inhibitors in order to obtain fresh and natural products. In the present work, the evolution of PPO activity and the chromatic characteristics of the pulp were evaluated in cv. Granny Smith apple slices. The slices were submitted to three treatments: I. 2% ascorbic acid + 1% citric acid + 0.5% EDTA; II. 1% ascorbic acid + 0.5% citric acid + 0.25% EDTA; and III. water, used as control. During the storage, parameters of the CIE L*a*b* color space of the slices were evaluated, indicating that both treatments containing additives were effective in preventing browning. The specific activity of PPO was determined spectrophotometrically in apple extracts obtained from each treatment. The results indicated that the stronger treatment (I) had induced the most effective inhibition of the enzyme. On view of the present results, It is proposed to evaluate the “in vitro” effectiveness of the inhibitors in order to compare these results with the ones obtained with apple slices. The specific activity of PPO was determined spectrophotometrically in apple extracts obtained from each treatment. The results indicated that the stronger treatment (I) had induced the most effective inhibition of the enzyme. On view of the present results, It is proposed to evaluate the “in vitro” effectiveness of the inhibitors in order to compare these results with the ones obtained with apple slices. The specific activity of PPO was determined spectrophotometrically in apple extracts obtained from each treatment. The results indicated that the stronger treatment (I) had induced the most effective inhibition of the enzyme. On view of the present results, It is proposed to evaluate the “in vitro” effectiveness of the inhibitors in order to compare these results with the ones obtained with apple slices.
Keywords: Polyphenol oxidase; Fresh cut fruit; Ascorbic acid; Citric acid; EDTA; Apples .
INTRODUCTION
Lately, the production of minimally processed fruits and vegetables (FyHMP) has grown remarkably both in quantity and variety (Watada, 1996), because they provide the consumer with a fresh-like product with a relatively long shelf life and, at the same time , guarantee their safety, nutritional and sensory quality (Wiley, 1997).
Appearance, determined mainly by color, is one of the most used attributes by consumers to assess the quality of FyHMP. The color is due to natural pigments, such as chlorophylls, carotenoids and anthocyanins, or to pigments resulting from enzymatic browning reactions. This process, related to the activity of the PPO enzyme, does not occur in cells of the intact plant because the substrates (phenolic compounds) are contained in cytoplasmic cellular vacuoles, separated from the enzyme. Once the cell tissue is damaged, either by handling itself or by cutting the fruit, compartmentalization is lost, the enzyme and the substrate come into contact, and browning reactions are triggered (Toivonen &
The Enzyme Commission (EC-Enzyme Commission) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), classifies PPO as EC 1.10.3.1., belonging to the oxidoreductases that act on diphenols with oxygen as an acceptor (Nevin, 2009). This enzyme acts on two types of substrates, monohydroxyphenols (example p-cresol), hydroxylating them in the ortho position with respect to the original hydroxyl group (EC 1.14.18.1), and on o-dihydroxyphenols (example catechol) oxidizing them to benzoquinone by removal of hydrogen from the hydroxyl group (EC 1.10.3.1) (Ramírez and Whitaker, 2003; Ayaz et al.., 2007). In turn, the non-enzymatic formation of melanins (polymeric compounds that give brown, red and/or black colorations) occurs.
The most important structural feature of PPO is the presence, in its active center, of two copper atoms bound to histidine. Around them, hydrophobic amino acids with aromatic rings are located, important for their binding to substrates (Calvo, 2007). The first methodology used to control enzymatic browning consisted of the addition of sulfites (Sapers, 1993) that act as reducing agents transforming o-quinones into less reactive diphenols to prevent the development of melanins. Although enzymatic browning is prevented in this way, its use has recently been restricted in vegetables and fruits by the US Food and Drug Administration (Langdon, 1987). In our country,et al ., 1993).
Enzymatic browning reactions can also be controlled by physical methods including reduction of temperature and/or the availability of molecular oxygen, the use of modified atmospheres or edible coatings, and treatments with gamma irradiation or high hydrostatic pressures. On the other hand, chemical methods can be used based on the use of compounds that inhibit the enzyme, reduce the availability of the substrate and/or the products of enzymatic catalysis that prevent the formation of colored products. Its application in food is restricted due to relevant considerations such as toxicity, health and the effect it may cause on taste, texture or eventually because of cost.
Among the reducing agents currently used is ascorbic acid, which reduces o-benzoquinones to o-diphenols (Whitaker and Lee, 1995). Given that during the reaction this compound is consumed by oxidation, the protection it confers is only temporary and organic acids can also be applied to control enzymatic browning that manage to lower the pH and guarantee microbiological safety. In addition, some of them can act as fungicides/fungistatics and as inhibitors of the growth of a large part of the flora of deterioration.
Acidulants are frequently used in combination with other anti-browning agents, because it is very difficult to achieve complete inhibition of browning by pH control alone. The most widely used is citric acid, which reduces browning by capturing or chelating copper from the active site of the PPO, and potentiates the effect of compounds such as ascorbic acid (Wiley, 1997).
Another compound of interest is ethylenediamine tetraacetic acid (EDTA) which inactivates the enzyme and forms complexes with heavy metal ions such as copper from PPO.
In accordance with what has been expressed, the objective of the present work is to evaluate the activity of the PPO, the chromatic characteristics and its evolution during refrigerated storage (1.5 ºC) for 16 days, in slices of apples cv. Granny Smith, treated by immersion with a combination of additives (citric acid, ascorbic acid and EDTA) in different proportions.
MATERIALS AND METHODS
Plant material
Apples cv. Granny Smith, chosen category, purchased in the Central Market of Buenos Aires, Argentina and from the Alto Valle del Río Negro whose average weight per fruit was 180 grams.
Processing
The fruits were pre-washed by immersion for two minutes in a sodium hypochlorite solution (200 ppm), peeled, cored and cut into slices. Subsequently, the batches of fruit were subjected for five minutes to an immersion bath with different additives.
The treatments applied were: I. 2% Ascorbic Acid + 1% Citric Acid + 0.5% EDTA; II. 1% Ascorbic acid + 0.5% Citric acid + 0.25% EDTA and III water, used as control, for which, in each treatment, three replicates were carried out.
Subsequently, the slices were drained, conditioned in polyethylene trays (10 slices with approximately 200 g per tray) and covered by a food grade film (70% polyvinyl chloride (PVC) resin), 9 µm thick, permeability to O 2 of 1536 cm 3 /m 2 .24 h.1 atm and CO2 of 3690 cm 3 /m 2 .24 h.1 atm measured at 0% RH and 23ºC and the vapor permeability of H 2 O 99 g/ m 2.24 h measured at 50% RH and 23ºC). The slices were stacked in five pairs per tray and were later stored for 16 days in a climate chamber at 1.5 ºC, whose soluble solids content in the slices at the beginning of the treatment was 9.9 °Brix, with malic acidity of 7.6 g/l, and the pH of 3.32.
Experimental design
The data obtained in the study of the chromatic characteristics and in the determination of the PPO activity, were analyzed by means of a balanced factorial design of two factors: treatment (I, II and III) and time (1, 7 and 16 days) where each tray contained apple slices as an experimental unit.
The applied analysis was carried out using the statistical package SPSS® version 12 (SPSS Inc., Illinois). In the case of the data corresponding to the determination of PPO activity, the Taylor transformation was applied to homogenize the data.
Analytical determinations
All evaluations were performed at 0, 7, and 16 days of refrigerated storage.
Color characteristics
The color characteristics of the CIE L*a*b* space were evaluated with a Konica Minolta model CR-400 Illuminant D 65 colorimeter . The determinations were made on the exposed face of the five upper apple slices of each tray, taking four cross values (total: 20 determinations per sample and per date).
PPO activity
The extraction of the enzyme to determine its activity was done according to the method of Yemenicioglu et al.(1997). To do this, 200 g of apple slices were weighed, cut into small pieces and 250 ml of cold (-20 °C) acetone were added. The preparation was homogenized in a Waring Blender processor for 2 minutes at maximum speed. The homogenate was filtered through a Buchner funnel, using Whatman No. 1 paper, and the residue obtained was extracted again with 200 ml of cold acetone and filtered under the same conditions. This procedure was repeated three more times, finally obtaining an acetone powder that was dried in a vacuum oven for four hours at room temperature, and subsequently stored at -20 °C until use. For the extraction of the PPO, 2 g of acetone powder were taken and suspended in 120 ml of 0.05 M potassium phosphate buffer and pH 6.8. The suspension was homogenized by shaking for 30 minutes at 4 °C. Then, the extract was clarified by gauze filtration and, subsequently, the solution was centrifuged at 3000 xg for 10 minutes at 4 °C. The supernatant obtained was used as an enzyme source for all the measurements carried out. The PPO activity was evaluated at 420 nm with the use of a Perkin Elmer UV-visible spectrophotometer, Lambda Bio 20 model. The values obtained were recorded at various time intervals, for three minutes and the result of each enzymatic measurement corresponded to the average of three measurements. The test conditions for the determination of the PPO activity were: 0.4 ml of the enzyme extract, 0.1 ml of catechol (17 mM), 0.05 M sodium acetate buffer pH 5, 2 csp a final volume of 2.5 ml. All determinations were carried out at 30 °C, in triplicate. Enzyme activity (EU) was defined as the amount of enzyme that catalyzes an increase of 0.1 absorbance units (AU) per minute at 420 nm and 30 ºC (Flurkey and Jen, 1978). The enzyme activity was calculated from the linear portion of the curve and the protein concentration of the enzyme extract was determined by the Lowry method.et al. (1951) using BSA (0.2 mg/l) as standard. The specific activity (AE) was defined as the enzymatic activity per protein unit (EU/mg protein).
RESULTS AND DISCUSSION
Chromatic characteristics
Table 1 shows the average results obtained for the parameter L* (luminosity) in apple slices cv. Granny Smith subjected to different treatments during refrigerated storage. Statistical analysis indicated that the treatment-time interaction was not significant (p>0.05). Therefore, the comparison between the means of the factors Treatment and Conservation time was made independently.
Table 1. Means obtained for the parameter L* (luminosity) in apple slices cv. Granny Smith subjected to different treatments during refrigerated storage.
Ref. AA (Ascorbic acid), AC (Citric acid). Uppercase and lowercase letters indicate statistically significant differences for the Treatment effect and Time effect, respectively (Tukey, p < 0.05)
Treatments I and II were not significantly different from each other (p>0.05) and both showed significant differences (p<0.05) with respect to the control for all times analyzed. The apple slices subjected to treatments I and II (combination of additives), showed a greater luminosity with respect to the samples used as control (immersion in water). A decrease in the luminosity value (L*) is associated with an increase in enzymatic browning (Rocha and Morais, 2003). In the samples subjected to the treatments that use combinations of additives, the value of L* was significantly higher (p<0.05) than in the control sample, immediately after treatment, which indicated a lighter coloration.
Regarding the evolution of this parameter during refrigerated storage, it was observed that on the first day (time=0), the L* values of all the samples were the highest (p<0.05). From day 7, the L* values decreased significantly (p<0.05), suggesting that the samples were taking on a darker coloration. These values remained unchanged (p> 0.05) until the end of the period analyzed. table 2, shows the average results obtained for the a* parameter (green-red component) where the samples subjected to treatments I and II had significantly lower a* values (p<0.05) than those samples subjected to the control treatment in all the period studied. No significant differences were observed between both treatments with additives, for which it functioned as an indicator of the change in chromaticity from green (negative values) to red (positive values), which is positively correlated with the color changes of fresh apples (Goupy et al . to the., nineteen ninety five). Enzymatic browning is also evidenced by an increase in the a* parameter, indicating a greater tendency towards red color (Rocha and Morais, 2003) than was observed only in control samples (III). During the period studied, the apples treated in water showed changes towards less negative values (from -3.48 to -2.39). While, the samples subjected to treatments I and II, did not show significant changes in a* during cold storage.
Table 2. Means obtained for parameter a* in apple slices cv. Granny Smith subjected to different treatments during refrigerated storage
Ref. AA (Ascorbic acid), AC (Citric acid). Different letters indicate statistically significant differences in the Treatment-Time interaction effect (Tukey, p < 0.05).
Determination of the PPO Activity
Table 3 shows the average results obtained in the determination of the specific activity (AE) of the PPO in each of the treatments applied where the treatment-time interaction was not significant (p>0, 05). However, the comparison between the means of the factors treatment and conservation time was made independently. When comparing the results of the treatments with each other, it was observed that the enzyme activity in treatment I was significantly lower (p<0.05) than that corresponding to treatments II and III at all times evaluated (0, 7 and 16 days) and that suggested a greater degree of inhibition of the enzyme.
Table 3. Means obtained for the specific Activity of the PPO (UE/mg protein) for each combination Treatments and Storage time
Ref. AA (Ascorbic acid), AC (Citric acid). Uppercase and lowercase letters indicate statistically significant differences for the Treatment effect and Time effect, respectively (Tukey, p < 0.05).
After 16 days, the comparison of the three treatments showed that the concentrations of additives in treatment II were not enough to inhibit the enzyme in the whole fruit, consequently, the AE of the enzyme increased, in a similar way. to the AE obtained in the control fruit.
CONCLUSIONS
When comparing the AE of the enzyme with the instrumental analysis of the color in the apple slices, for the different treatments and conservation times, it was noted that there is a certain disparity in the results obtained. In the instrumental analysis, those slices that were immersed in treatments I and II did not show evidence of enzymatic browning. On the contrary, the results obtained in the measurement of the AE of the PPO showed that the only treatment that was effective in achieving the inhibition of the enzyme was treatment I.
The results obtained in the present study lead to future tests to test the effectiveness of the inhibitors directly on extracts obtained from the enzyme, regarding the determination of activity, and that would allow relating these results with those obtained in the apple slices.
Another important aspect is to carry out a sensory analysis of the product in the future by trained panelists to evaluate and determine if there are differences in the sensory properties of the fruits, other than color, due to the treatments applied. It is important to be able to offer a product on the market that is preserved over time by reducing or suppressing enzymatic browning, but it is also important that they retain their original sensory properties and “fresh” characteristics in order to thus achieve better acceptability by consumer part.
THANKS
This research was financed by the INTA – AETA PE 1671 project. The collaboration of the assistants of the Food Technology Institute of the CNIA-INTA Castelar is gratefully acknowledged.
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