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Effect of l -cysteine, kojic acid and 4-hexylresorcinol combination on inhibition of enzymatic browning in Amasya apple juice
Journal of Food Engineering 62 (2004) 299–304 www.elsevier.com/locate/jfoodeng
Effect of L -cysteine, kojic acid and 4-hexylresorcinol combination on inhibition of enzymatic browning in Amasya apple juice N.F. I_ yido gan, A. Bayındırlı
*
Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey Received 8 February 2003; accepted 22 June 2003
Abstract In this study, the effect of different anti-browning agents on the enzymatic browning in the cloudy apple juice from Amasya cultivar was compared by considering colour changes. For this purpose, L -cysteine, kojic acid and 4-hexylresorcinol were used at different concentrations in the apple juice (0–4 mM). They were found as effective inhibitors. The combination of L -cysteine, kojic acid and 4-hexylresorcinol was also studied for the control of enzymatic browning. The results showed that 89.2% inhibition was obtained at the end of 24 h storage period with the combination of 3.96 mM L -cysteine, 2.78 mM kojic acid and 2.34 mM 4-hexylresorcinol in the cloudy apple juice. L -Cysteine was found to be the most significant anti-browning agent. The interaction of kojic acid and 4-hexylresorcinol was found to have a positive effect on the inhibition of enzymatic browning. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Enzymatic browning; Polyphenol oxidase; Anti-browning agents; Apple juice; Response surface methodology
1. Introduction Turkey is one of the leading fruit producing countries. Starking Delicious, Golden Delicious and Amasya are the main apple cultivars grown in Turkey (Eksßi, 1997). The Amasya cultivar has a naturally low acid content (0.9–1.9 g/L) and low mass ratio of glucose to fructose (0.17–0.21). Optimum pH values for partially purified Amasya apple PPO were 7.0, 9.0, 8.6 and 6.6 on substrates catechol, 4-methyl catechol, pyrogallol and L -dopa respectively. Catechol was the most suitable substrate for Amasya apple PPO. The optimum temperature for maximum PPO activity was 18 °C with catechol. Effectiveness of anti-browning agents on partially purified PPO increased in the order of thiourea, glutathione, beta-mercaptoethanol, sodium cyanide, ascorbic acid, sodium metabisulfide, and L -cysteine. Three PPO isoenzymes with high thermostability (as high as 80 °C) were found in Amasya apples (Oktay, K€ ufrevioglu, Kocacßalısßkan, & S glu, 1995; Yemenicioglu, ß akiro € zkan, & Cemero O glu, 1997).
*
Corresponding author. Tel.: +90-312-210-5629; fax: +90-312-2101270. E-mail address: [email protected] (A. Bayındırlı). 0260-8774/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00243-7
Cloudy apple juice, a light, whitish-yellow juice, contains a high proportion of pulp in suspension showing a definite cloudiness. Cloudy apple juice has a growing share in the market due to its sensory and nutritional qualities. The successful process will depend on maintaining the stability of the cloud and the assurance of colour (Genovese, Elustondo, & Lozano, 1997; Tronc, Lamarche, & Maklouf, 1997). Polyphenol oxidase (PPO, o-diphenol: oxygen oxidoreductase) in the presence of oxygen catalyses two different reactions: the hydroxylation of monophenols to o-diphenols (monophenolase/cresolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase/ catecholase activity). Then quinones may condense and react non-enzymatically with other phenolic compounds, amino acids, proteins, and other cellular constituents to produce coloured polymers or pigments. Therefore, enzymatic browning have been extensively studied in apples and apple products (Janovitz-Klapp, Richard, Goupy, & Nicolas, 1990; Martinez & Whitaker, 1995; McEvily, Iyengar, & Otwell, 1992; Nicolas, Richard-Forget, Goupy, Amiot, & Aubert, 1994; Sapers et al., 1989; Walker, 1995). The use of sulfites, a powerful anti-browning chemical, is discussed more and more because of the potential hazards. Thus, an active field of research is currently
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under development to find non-sulfite anti-browning agents for food industry (Lambrecht, 1995; Son, Moon, & Lee, 2001). Ascorbic acid is that the most frequently used chemical product for the chemical reduction of oquinones back into o-diphenolic compounds for browning control of apple products. But, its effect is € zo temporary (Nicolas et al., 1994; O glu & Bayındırlı, 2002). Sulfhydryl compounds have been shown to be effective in the control of enzymic browning in fruit products. Several thiol-containing compounds such as L -cysteine, gluthatione, mercaptoethanol, and thiourea have been investigated as inhibitors of enzymatic browning. Cysteine is the most effective one in this group (Dudley & Hotchkiss, 1989; Molnar-Perl & Friedman, € zo 1990a, 1990b; O glu & Bayındırlı, 2002; RichardForget, Goupy, Nicolas, Lacombe, & Pavia, 1991; Richard-Forget, Goupy, & Nicolas, 1992). The action of cysteine is complex. Sulfhydryl-containing amino acids like L -cysteine prevent brown pigment formation by reacting with the quinone intermediates to form stable colourless compounds. Cysteine-quinone adducts are proved to be competitive inhibitors of polyphenol oxidase. Direct inhibition of polyphenol oxidase by cysteine through the formation of stable complexes with copper has also been proposed (Nicolas et al., 1994). Among the recently discovered agents, the substituted resorcinols are structurally related to phenolic substrates. 4-Hexylresorcinol is an effective agent for the enzymatic browning control of apple slices as well as in apple juice. It has a long history of use in pharmaceutical and exhibits no systemic toxicity and safe as a food additive (Monsalve-Gonzalez, Barbosa-Canovas, Cavalieri, McEvily, & Iyengar, 1993; Nicolas et al., 1994; Vamos-Vigyazo, 1995; Son et al., 2001). Kojic acid is found in many fermented Japanese foods. Son et al. (2001) reported that kojic acid showed the highest inhibitory activity on apple slice browning among phenolic acids tested (caffeic, chlorogenic, cinnamic, coumalic, ferulic, gallic, kojic). Some researchers studied on mushroom, potato, apple, white shrimp, and spiny lobster PPO, proposed that kojic acid acts as a reducing agent as well as an inhibitor to the enzyme (Chen, Wei, & Marshall, 1991; Chen et al., 1991). According to their studies, Kojic acid exhibited a competitive inhibition for the oxidation of chlorogenic acid and catechol by potato and apple PPO. This compound showed a mixed-type inhibition for white shrimp, grass prawn, and lobster PPO when D L -beta-3,4-dihydroxyphenylalanine and catechol were used as substrates. A mixture of ascorbic acid and kojic acid has been patented for use as an anti-browning agent in foods (McEvily et al., 1992). Although the presence of kojic acid in certain foods occurs as a natural fermentation product, the use of kojic acid in the food industry may be restricted due to the difficulty of large-scale production and its high cost.
Due to the properties of Amasya apple juice such as high juice yield but higher rates of browning, it will be beneficial to investigate the effectiveness of different antibrowning agents on enzymatic browning in the Amasya apple juice. The main objective of this work was to study the inter-related effects of L -cysteine, kojic acid and 4hexylresorcinol on enzymatic browning in cloudy apple juice by using a response surface methodology.
2. Materials and methods 2.1. Materials Amasya apples were obtained from the producers in Turkey (Tokat) and kept at 4 °C until use. Kojic acid (Sigma), 4-hexylresorcinol (Sigma), L -cysteine (Sigma) and ascorbic acid (Sigma) were used as anti-browning agents. 2.2. Preparation of samples and control of enzymatic browning Apples were washed, peeled and cored. Apple juice was obtained by using a domestic food processor. Juice was poured in 50 mL beakers containing anti-browning agent solutions and then stirred with a magnetic stirrer for approximately 10 s. The concentrations of antibrowning agents were 0, 1, 2 and 4 mM in apple juice samples (pH ¼ 4 0:1). After approximately 1 min, the first measurements were done. The enzymatic browning was followed for 24 h at room temperature (25 ± 2 °C). All the experiments were replicated twice and the analyses were carried out in duplicate for each replication. DuncanÕs multiple range tests (p 6 0:05) were performed to determine any significant difference. 2.3. Determination of enzymatic browning Hunter L, a and b values were determined by Spectrophotometer (Shimadzu UV-2100, illuminant C, colour system Lab). The instrument was calibrated using a standard white reflector plate. The browning was determined by using the following equation: browning ¼ DL ¼ Linitial Lat a given time Total colour change (DE) was also used to evaluate the browning potential as: h i0:5 2 2 2 DE ¼ ðLt Linitial Þ þ ðat ainitial Þ þ ðbt binitial Þ The degree of inhibition was calculated from L values of the anti-browning agent added samples and the corresponding controls as follows: Degree of inhibition ¼ ðDLcontrol DLtreatment Þ=DLcontrol
N.F. I_ yido gan, A. Bayındırlı / Journal of Food Engineering 62 (2004) 299–304
2.4. Measurement of enzyme activity PPO activity was determined by using catechol as substrate in McIlvane buffer (pH 6.5) according to the € zo procedure given in the study of O glu and Bayındırlı (2002). One unit of enzyme activity for polyphenol oxidase was defined as 0.001 DA420 s1 under the assay conditions.
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The statistical significance of results and also the coefficients of the response functions were determined by using the linear regression enter method of SPSS statistical software program. The values correspond to maximum degree of inhibition with minimum amounts of agents determined by using Mathematica software program. 3. Results and discussion
2.5. Experimental design Amasya apple has one of the highest rates of enzymatic browning among several apple cultivars. The Residual enzyme activity (%)
Box–Behnken design was used to determine the combined effect of anti-browning agents (Box & Behnken, 1960; Thompson, 1982). It was assumed that a mathematical function exists for the response variable (Y , degree of inhibition) in terms of three independent coded variables X1 (cysteine), X2 (kojic acid), and X3 (hexylresorcinol) as follows: Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b12 X1 X2 þ b13 X1 X3 þ b23 X2 X3 þ b11 X1 X1 þ b22 X2 X2 þ b33 X3 X3
100 90 80 70 60 50 40 30 20 10 0
Exp. 1 Exp. 2 Exp. 3 Exp. 4
0
Concentrations of anti-browning agents were 0, 2 and 4 mM at )1, 0 and +1 coded variable levels (X1 , X2 , and X3 ), respectively.
5
10
15
20
25
30
time (h)
Fig. 1. Residual enzyme activity in cloudy apple juice from Amasya cultivar (4 replications at different seasonal times).
Table 1 Enzymatic browning (DL) and total colour change (DE) in the presence of L -cysteine, kojic acid and 4-hexylresorcinol at different concentrations in apple juice Time (h)
Enzymatic browning (DL)
Total colour change (DE)
Control
1 mM
2 mM
4 mM
Control
1 mM
2 mM
4 mM
0.05 0.75 1.75 2.75 3.75 4.75 24.0 25.0
0 3.284 ± 0.95 9.537 ± 0.49 13.606 ± 0.45 17.49 ± 0.79 21.71 ± 0.36 28.56 ± 0.57 28.72 ± 0.27
0 1.846 ± 0.22 6.59 ± 0.76 13.536 ± 0.77 22.11 ± 0.68 30.76 ± 0.46 51.07 ± 0.64 50.83 ± 1.02
0 0.78 ± 0.48 0.28 ± 0.20 3.80 ± 1.07 5.77 ± 0.19 6.47 ± 1.11 17.01 ± 0.82 16.84 ± 1.12
0 0.79 ± 0.15 1.71 ± 0.25 2.41 ± 0.34 3.46 ± 0.45 3.53 ± 0.72 7.08 ± 0.39 7.31 ± 0.48
0 3.79 ± 1.46 11.76 ± 2.58 16.81 2.89 21.74 ± 1.70 26.95 ± 1.53 34.22 ± 2.00 34.34 ± 1.86
0 2.23 ± 1.09 7.78 ± 1.74 17.09 ± 1.46 29.19 ± 2.86 40.21 ± 1.19 59.52 ± 3.93 59.45 ± 3.06
0 1.09 ± 0.50 1.99 ± 1.17 4.8 ± 1.61 7.57 ± 1.79 9.15 ± 2.35 21.84 ± 2.01 22.02 ± 1.39
0 1.26 ± 0.48 2.32 ± 0.57 3.15 ± 0.41 4.71 ± 0.25 4.56 ± 0.58 8.70 ± 1.25 8.99 ± 1.14
Kojic acid 0.05 0.50 1.00 2.00 3.00 4.00 24.0 25.0
0 6.63 ± 0.91 10.38 ± 0.96 13.283 ± 0.60 16.88 ± 0.23 15.48 ± 0.99 24.95 ± 0.86 26.78 ± 1.02
0 0.45 ± 0.27 3.12 ± 0.63 6.58 ± 0.15 9.48 ± 0.41 6.92 ± 0.12 16.14 ± 0.37 17.11 ± 0.97
0 0 1.88 ± 0.84 5.30 ± 0.17 7.94 ± 0.21 5.07 ± 0.35 9.45 ± 0.37 10.34 ± 0.10
0 0 1.3 ± 0.99 3.52 ± 0.84 5.23 ± 0.32 2.86 ± 0.98 7.78 ± 0.34 6.86 ± 0.17
0 4.91 ± 1.84 9.03 ± 2.61 12.32 1.08 16.54 ± 2.94 13.73 ± 2.78 22.53 ± 1.37 24.66 ± 1.67
0 3.56 ± 1.65 5.88 ± 1.16 9.58 ± 1.84 12.94 ± 1.56 8.83 ± 1.91 17.03 ± 0.27 18.15 ± 0.96
0 3.45 ± 1.53 5.41 ± 0.99 8.69 ± 0.48 11.38 ± 1.28 8.08 ± 1.44 10.07 ± 2.03 10.84 ± 2.70
0 2.64 ± 0.95 4.29 ± 0.64 6.67 ± 0.11 8.82 ± 0.59 7.75 ± 0.33 10.47 ± 1.51 10.28 ± 1.33
0 4.29 ± 0.22 5.86 ± 0.41 7.16 ± 0.74 7.71 ± 0.79 8.36 ± 0.17 13.97 ± 0.65 14.93 ± 0.94
0 8.75 ± 0.88 9.66 ± 0.74 10.84 ± 0.37 9.93 ± 0.46 11.35 ± 0.32 13.75 ± 0.86 13.82 ± 0.47
0 0.90 ± 0.66 1.20 ± 0.67 1.01 ± 0.35 1.69 ± 0.82 1.21 ± 0.25 2.13 ± 0.13 2.46 ± 0.42
0 7.78 ± 1.25 14.50 ± 1.85 19.70 ± 2.61 25.22 ± 2.94 29.81 ± 2.78 37.58 1.37 37.99 ± 1.67
0 5.38 ± 1.09 7.01 ± 1.74 8.38 ± 2.67 8.97 ± 1.65 9.61 ± 2.67 15.38 ± 2.59 16.45 ± 1.11
0 10.29 ± 2.17 11.43 ± 2.61 12.76 ± 1.79 11.86 ± 2.35 13.32 ± 2.01 15.77 ± 1.38 15.65 ± 3.03
0 2.58 ± 1.25 3.14 ± 1.42 3.16 ± 1.39 3.59 ± 1.38 2.97 ± 1.42 3.56 ± 1.60 3.88 ± 1.65
L -Cysteine
4-Hexylresorcinol 0.05 0 0.75 4.98 ± 0.54 1.75 11.36 ± 0.25 2.75 16.57 ± 0.64 3.75 21.69 ± 0.69 4.75 26.31 ± 0.43 24.5 35.69 ± 0.28 25.5 35.65 ± 0.73
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Table 2 Box–Behnken experimental design and dataa Experiment no.
Codesb
Degree of inhibitionc
X1
X2
X3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+1 )1 +1 )1 0 +1 )1 +1 )1 0 0 0 0 0 0
+1 +1 )1 )1 0 0 0 0 0 0 +1 )1 +1 )1 0
0 0 0 0 0 +1 +1 )1 )1 0 +1 +1 )1 )1 0
0.80 0.75 0.71 0.62 0.70 0.65 0.41 0.77 0.49 0.72 0.69 0.25 0.16 0.45 0.68
a
Experimental runs were performed in random order. X1 : code of cysteine, X2 : code of kojic acid, X3 : code of 4-hexylresorcinol; )1, 0 and +1 levels correspond to 0, 2 and 4 mM concentrations. c Degree of inhibition ¼ ðDLcontrol DLtreatment Þ=DLcontrol . b
change of PPO activity in the apple juice with respect to storage time is represented in Fig. 1. The decrease in PPO activity with time probably results from the formation of oxidation reaction products derived from the natural substrates which are inhibitors of PPO activity and possibly the protein degradation during storage at 25 ± 2 °C. Table 1 shows the effect of L -cysteine, kojic acid and 4-hexylresorcinol on enzymatic browning in apple juice stored at 25 ± 2 °C. Initial L, a and b values of the cloudy apple juice were 49.96 ± 2.4, 14.2 ± 2.02 and 29.99 ± 0.84 respectively. Twenty-four hour observation is sufficient in order to see the end of enzymatic browning (no more colour change). L -Cysteine prevents brown pigment formation by reacting with the quinone intermediates to form stable colourless compounds. At l mM concentration, cysteine lost its effect, since the o-quinones formed in excess can cooxidize cysteinequinone addition compound, leading to phenol regeneration with a deep colour formation (Vamos-Vigyazo, 1995). The efficiency of browning inhibition by cysteine depended on the thiol:phenol ratio. The relative cysteine concentration must be sufficient to transform all the substrate into colourless adducts. L -Cysteine at 4 mM concentration was significantly effective in preventing the browning of apple juice (p < 0:05). Increasing the concentration of cysteine increased its effectiveness to prevent discolouration. However, high cysteine concentrations may sometimes be inconsistent with good organoleptic properties of the final product due to offodor formation. Kojic acid is a significantly effective anti-browning agent at the selected concentration range (p < 0:05). Kojic acid exhibits inhibition of PPO and also bleaches melanin due to chemical reduction of the browning pigment to colourless compounds. 4Hexylresorcinol was significantly effective against enzy-
Table 3 Estimated coefficients of the second order model Coefficients
Values
Standard error
Significance of t
b0 b1 b2 b3 b11 b22 b33 b12 b13 b23
0.700 0.083 0.045 0.018 0.107 )0.087 )0.227 )0.008 )0.012 0.180
0.060 0.037 0.037 0.037 0.054 0.054 0.054 0.052 0.052 0.052
0.0001 0.0715 0.2730 0.6500 0.1025 0.1653 0.0083 0.8903 0.8292 0.0174
Multiple regression: R2 ¼ 0:90 Standard error ¼ 0.1034 Significance of F ¼ 0:0463
matic browning in the apple juice at high (4 mM) concentration level (p < 0:05), it has long term inhibitory effect. It has several advantages over using other agents, including its specific mode of inhibitory action, its inability to bleach pigments and its chemical stability. The total colour change (DE) during enzymatic browning in the presence of L -cysteine, kojic acid and 4hexylresorcinol, are given in Table 1. In the colour space, L is the intensity, a is the position on the green ()) to red (+) axis and b is the position on the blue ()) to yellow (+) axis. For browning estimation, the L value has been considered as one of the best colour index. Generally, it can be said that DL and DE values were highly correlated due to the negligible change in a and b values compared to the change in L values. However, in the case of a bleaching effect the same correlation can not be observed, since the total colour difference gives the distance in the colour space between two colours,
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enzymatic browning inhibition based on L measurements was considered. The data obtained for the inhibition of enzymatic browning according to Box–Behnken design are given in Table 2. Table 3 shows the regression coefficients obtained by fitting the experimental data to the second order response model: b0 is the value of the fitted response at the center of the design, b1 , b2 and b3 are linear, b11 , b22 , b33 are quadratic and b13 , b23 and b12 are cross product (interaction) regression terms. The positive values of linear regression terms indicate that inhibition increased with increasing the amount of anti-browning agents. Among the linear regression terms, L -cysteine was found to be the most significant anti-browning agent in the combination according to the t-test. When the interactions between the anti-browning agents are considered, the interaction of kojic acid and 4-hexylresorcinol was found to have a positive effect on the inhibition of enzymatic browning. There were no significant interaction between L -cysteine and kojic acid and also between L -cysteine and 4-hexylresorcinol. The predictive model was used to graphically represent the system. Fig. 2 is an example for response surfaces. A formulation of the anti-browning reagents were found: X1 , X2 and X as 0.98 (3.96 mM L -cysteine), 0.39 (2.78 mM kojic acid) and 0.17 (2.34 mM 4-hexylresorcinol), respectively. The calculated inhibition was 89.2% at the end of 24 h storage period. For shorter storage times, this combination will also delay or prevent enzymatic browning that it is especially important in the application of non-thermal processes for the production of fruit juices in terms of the retardation of the enzymatic browning in the process line before enzyme inactivation. Also the use of combination of anti-browning agents lowers the required concentrations that it is important for the organoleptic properties of the juice.
Fig. 2. Three dimensional plot representations for inhibition of enzymatic browning based on measurements of DL at X1 ¼ 0 (a), X2 ¼ 0 (b), X3 ¼ 0 (c) (Y ¼ degree of inhibition).
but not the direction in which they differ. Therefore the evaluation of browning in terms of DL values may be more reliable. The mechanism of inhibition differs widely for each of the categories of anti-browning agents. The combination of different agents may prevent browning better than a specific chemical alone. Since L -cysteine (sulfur-containing amino acid), kojic acid (phenolic acid) and 4-hexylresorcinol (substituted resorcinols, structurally related to phenolic substrates) were found to be effective antibrowning agents on enzymatic browning, in the second part of the study, the combined effect of these agents on the enzymatic browning reaction was determined at the end of the 24 h storage period. For this purpose, the
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Effect of L -cysteine, kojic acid and 4-hexylresorcinol combination on inhibition of enzymatic browning in Amasya apple juice N.F. I_ yido gan, A. Bayındırlı
*
Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey Received 8 February 2003; accepted 22 June 2003
Abstract In this study, the effect of different anti-browning agents on the enzymatic browning in the cloudy apple juice from Amasya cultivar was compared by considering colour changes. For this purpose, L -cysteine, kojic acid and 4-hexylresorcinol were used at different concentrations in the apple juice (0–4 mM). They were found as effective inhibitors. The combination of L -cysteine, kojic acid and 4-hexylresorcinol was also studied for the control of enzymatic browning. The results showed that 89.2% inhibition was obtained at the end of 24 h storage period with the combination of 3.96 mM L -cysteine, 2.78 mM kojic acid and 2.34 mM 4-hexylresorcinol in the cloudy apple juice. L -Cysteine was found to be the most significant anti-browning agent. The interaction of kojic acid and 4-hexylresorcinol was found to have a positive effect on the inhibition of enzymatic browning. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Enzymatic browning; Polyphenol oxidase; Anti-browning agents; Apple juice; Response surface methodology
1. Introduction Turkey is one of the leading fruit producing countries. Starking Delicious, Golden Delicious and Amasya are the main apple cultivars grown in Turkey (Eksßi, 1997). The Amasya cultivar has a naturally low acid content (0.9–1.9 g/L) and low mass ratio of glucose to fructose (0.17–0.21). Optimum pH values for partially purified Amasya apple PPO were 7.0, 9.0, 8.6 and 6.6 on substrates catechol, 4-methyl catechol, pyrogallol and L -dopa respectively. Catechol was the most suitable substrate for Amasya apple PPO. The optimum temperature for maximum PPO activity was 18 °C with catechol. Effectiveness of anti-browning agents on partially purified PPO increased in the order of thiourea, glutathione, beta-mercaptoethanol, sodium cyanide, ascorbic acid, sodium metabisulfide, and L -cysteine. Three PPO isoenzymes with high thermostability (as high as 80 °C) were found in Amasya apples (Oktay, K€ ufrevioglu, Kocacßalısßkan, & S glu, 1995; Yemenicioglu, ß akiro € zkan, & Cemero O glu, 1997).
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Corresponding author. Tel.: +90-312-210-5629; fax: +90-312-2101270. E-mail address: [email protected] (A. Bayındırlı). 0260-8774/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0260-8774(03)00243-7
Cloudy apple juice, a light, whitish-yellow juice, contains a high proportion of pulp in suspension showing a definite cloudiness. Cloudy apple juice has a growing share in the market due to its sensory and nutritional qualities. The successful process will depend on maintaining the stability of the cloud and the assurance of colour (Genovese, Elustondo, & Lozano, 1997; Tronc, Lamarche, & Maklouf, 1997). Polyphenol oxidase (PPO, o-diphenol: oxygen oxidoreductase) in the presence of oxygen catalyses two different reactions: the hydroxylation of monophenols to o-diphenols (monophenolase/cresolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase/ catecholase activity). Then quinones may condense and react non-enzymatically with other phenolic compounds, amino acids, proteins, and other cellular constituents to produce coloured polymers or pigments. Therefore, enzymatic browning have been extensively studied in apples and apple products (Janovitz-Klapp, Richard, Goupy, & Nicolas, 1990; Martinez & Whitaker, 1995; McEvily, Iyengar, & Otwell, 1992; Nicolas, Richard-Forget, Goupy, Amiot, & Aubert, 1994; Sapers et al., 1989; Walker, 1995). The use of sulfites, a powerful anti-browning chemical, is discussed more and more because of the potential hazards. Thus, an active field of research is currently
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under development to find non-sulfite anti-browning agents for food industry (Lambrecht, 1995; Son, Moon, & Lee, 2001). Ascorbic acid is that the most frequently used chemical product for the chemical reduction of oquinones back into o-diphenolic compounds for browning control of apple products. But, its effect is € zo temporary (Nicolas et al., 1994; O glu & Bayındırlı, 2002). Sulfhydryl compounds have been shown to be effective in the control of enzymic browning in fruit products. Several thiol-containing compounds such as L -cysteine, gluthatione, mercaptoethanol, and thiourea have been investigated as inhibitors of enzymatic browning. Cysteine is the most effective one in this group (Dudley & Hotchkiss, 1989; Molnar-Perl & Friedman, € zo 1990a, 1990b; O glu & Bayındırlı, 2002; RichardForget, Goupy, Nicolas, Lacombe, & Pavia, 1991; Richard-Forget, Goupy, & Nicolas, 1992). The action of cysteine is complex. Sulfhydryl-containing amino acids like L -cysteine prevent brown pigment formation by reacting with the quinone intermediates to form stable colourless compounds. Cysteine-quinone adducts are proved to be competitive inhibitors of polyphenol oxidase. Direct inhibition of polyphenol oxidase by cysteine through the formation of stable complexes with copper has also been proposed (Nicolas et al., 1994). Among the recently discovered agents, the substituted resorcinols are structurally related to phenolic substrates. 4-Hexylresorcinol is an effective agent for the enzymatic browning control of apple slices as well as in apple juice. It has a long history of use in pharmaceutical and exhibits no systemic toxicity and safe as a food additive (Monsalve-Gonzalez, Barbosa-Canovas, Cavalieri, McEvily, & Iyengar, 1993; Nicolas et al., 1994; Vamos-Vigyazo, 1995; Son et al., 2001). Kojic acid is found in many fermented Japanese foods. Son et al. (2001) reported that kojic acid showed the highest inhibitory activity on apple slice browning among phenolic acids tested (caffeic, chlorogenic, cinnamic, coumalic, ferulic, gallic, kojic). Some researchers studied on mushroom, potato, apple, white shrimp, and spiny lobster PPO, proposed that kojic acid acts as a reducing agent as well as an inhibitor to the enzyme (Chen, Wei, & Marshall, 1991; Chen et al., 1991). According to their studies, Kojic acid exhibited a competitive inhibition for the oxidation of chlorogenic acid and catechol by potato and apple PPO. This compound showed a mixed-type inhibition for white shrimp, grass prawn, and lobster PPO when D L -beta-3,4-dihydroxyphenylalanine and catechol were used as substrates. A mixture of ascorbic acid and kojic acid has been patented for use as an anti-browning agent in foods (McEvily et al., 1992). Although the presence of kojic acid in certain foods occurs as a natural fermentation product, the use of kojic acid in the food industry may be restricted due to the difficulty of large-scale production and its high cost.
Due to the properties of Amasya apple juice such as high juice yield but higher rates of browning, it will be beneficial to investigate the effectiveness of different antibrowning agents on enzymatic browning in the Amasya apple juice. The main objective of this work was to study the inter-related effects of L -cysteine, kojic acid and 4hexylresorcinol on enzymatic browning in cloudy apple juice by using a response surface methodology.
2. Materials and methods 2.1. Materials Amasya apples were obtained from the producers in Turkey (Tokat) and kept at 4 °C until use. Kojic acid (Sigma), 4-hexylresorcinol (Sigma), L -cysteine (Sigma) and ascorbic acid (Sigma) were used as anti-browning agents. 2.2. Preparation of samples and control of enzymatic browning Apples were washed, peeled and cored. Apple juice was obtained by using a domestic food processor. Juice was poured in 50 mL beakers containing anti-browning agent solutions and then stirred with a magnetic stirrer for approximately 10 s. The concentrations of antibrowning agents were 0, 1, 2 and 4 mM in apple juice samples (pH ¼ 4 0:1). After approximately 1 min, the first measurements were done. The enzymatic browning was followed for 24 h at room temperature (25 ± 2 °C). All the experiments were replicated twice and the analyses were carried out in duplicate for each replication. DuncanÕs multiple range tests (p 6 0:05) were performed to determine any significant difference. 2.3. Determination of enzymatic browning Hunter L, a and b values were determined by Spectrophotometer (Shimadzu UV-2100, illuminant C, colour system Lab). The instrument was calibrated using a standard white reflector plate. The browning was determined by using the following equation: browning ¼ DL ¼ Linitial Lat a given time Total colour change (DE) was also used to evaluate the browning potential as: h i0:5 2 2 2 DE ¼ ðLt Linitial Þ þ ðat ainitial Þ þ ðbt binitial Þ The degree of inhibition was calculated from L values of the anti-browning agent added samples and the corresponding controls as follows: Degree of inhibition ¼ ðDLcontrol DLtreatment Þ=DLcontrol
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2.4. Measurement of enzyme activity PPO activity was determined by using catechol as substrate in McIlvane buffer (pH 6.5) according to the € zo procedure given in the study of O glu and Bayındırlı (2002). One unit of enzyme activity for polyphenol oxidase was defined as 0.001 DA420 s1 under the assay conditions.
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The statistical significance of results and also the coefficients of the response functions were determined by using the linear regression enter method of SPSS statistical software program. The values correspond to maximum degree of inhibition with minimum amounts of agents determined by using Mathematica software program. 3. Results and discussion
2.5. Experimental design Amasya apple has one of the highest rates of enzymatic browning among several apple cultivars. The Residual enzyme activity (%)
Box–Behnken design was used to determine the combined effect of anti-browning agents (Box & Behnken, 1960; Thompson, 1982). It was assumed that a mathematical function exists for the response variable (Y , degree of inhibition) in terms of three independent coded variables X1 (cysteine), X2 (kojic acid), and X3 (hexylresorcinol) as follows: Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b12 X1 X2 þ b13 X1 X3 þ b23 X2 X3 þ b11 X1 X1 þ b22 X2 X2 þ b33 X3 X3
100 90 80 70 60 50 40 30 20 10 0
Exp. 1 Exp. 2 Exp. 3 Exp. 4
0
Concentrations of anti-browning agents were 0, 2 and 4 mM at )1, 0 and +1 coded variable levels (X1 , X2 , and X3 ), respectively.
5
10
15
20
25
30
time (h)
Fig. 1. Residual enzyme activity in cloudy apple juice from Amasya cultivar (4 replications at different seasonal times).
Table 1 Enzymatic browning (DL) and total colour change (DE) in the presence of L -cysteine, kojic acid and 4-hexylresorcinol at different concentrations in apple juice Time (h)
Enzymatic browning (DL)
Total colour change (DE)
Control
1 mM
2 mM
4 mM
Control
1 mM
2 mM
4 mM
0.05 0.75 1.75 2.75 3.75 4.75 24.0 25.0
0 3.284 ± 0.95 9.537 ± 0.49 13.606 ± 0.45 17.49 ± 0.79 21.71 ± 0.36 28.56 ± 0.57 28.72 ± 0.27
0 1.846 ± 0.22 6.59 ± 0.76 13.536 ± 0.77 22.11 ± 0.68 30.76 ± 0.46 51.07 ± 0.64 50.83 ± 1.02
0 0.78 ± 0.48 0.28 ± 0.20 3.80 ± 1.07 5.77 ± 0.19 6.47 ± 1.11 17.01 ± 0.82 16.84 ± 1.12
0 0.79 ± 0.15 1.71 ± 0.25 2.41 ± 0.34 3.46 ± 0.45 3.53 ± 0.72 7.08 ± 0.39 7.31 ± 0.48
0 3.79 ± 1.46 11.76 ± 2.58 16.81 2.89 21.74 ± 1.70 26.95 ± 1.53 34.22 ± 2.00 34.34 ± 1.86
0 2.23 ± 1.09 7.78 ± 1.74 17.09 ± 1.46 29.19 ± 2.86 40.21 ± 1.19 59.52 ± 3.93 59.45 ± 3.06
0 1.09 ± 0.50 1.99 ± 1.17 4.8 ± 1.61 7.57 ± 1.79 9.15 ± 2.35 21.84 ± 2.01 22.02 ± 1.39
0 1.26 ± 0.48 2.32 ± 0.57 3.15 ± 0.41 4.71 ± 0.25 4.56 ± 0.58 8.70 ± 1.25 8.99 ± 1.14
Kojic acid 0.05 0.50 1.00 2.00 3.00 4.00 24.0 25.0
0 6.63 ± 0.91 10.38 ± 0.96 13.283 ± 0.60 16.88 ± 0.23 15.48 ± 0.99 24.95 ± 0.86 26.78 ± 1.02
0 0.45 ± 0.27 3.12 ± 0.63 6.58 ± 0.15 9.48 ± 0.41 6.92 ± 0.12 16.14 ± 0.37 17.11 ± 0.97
0 0 1.88 ± 0.84 5.30 ± 0.17 7.94 ± 0.21 5.07 ± 0.35 9.45 ± 0.37 10.34 ± 0.10
0 0 1.3 ± 0.99 3.52 ± 0.84 5.23 ± 0.32 2.86 ± 0.98 7.78 ± 0.34 6.86 ± 0.17
0 4.91 ± 1.84 9.03 ± 2.61 12.32 1.08 16.54 ± 2.94 13.73 ± 2.78 22.53 ± 1.37 24.66 ± 1.67
0 3.56 ± 1.65 5.88 ± 1.16 9.58 ± 1.84 12.94 ± 1.56 8.83 ± 1.91 17.03 ± 0.27 18.15 ± 0.96
0 3.45 ± 1.53 5.41 ± 0.99 8.69 ± 0.48 11.38 ± 1.28 8.08 ± 1.44 10.07 ± 2.03 10.84 ± 2.70
0 2.64 ± 0.95 4.29 ± 0.64 6.67 ± 0.11 8.82 ± 0.59 7.75 ± 0.33 10.47 ± 1.51 10.28 ± 1.33
0 4.29 ± 0.22 5.86 ± 0.41 7.16 ± 0.74 7.71 ± 0.79 8.36 ± 0.17 13.97 ± 0.65 14.93 ± 0.94
0 8.75 ± 0.88 9.66 ± 0.74 10.84 ± 0.37 9.93 ± 0.46 11.35 ± 0.32 13.75 ± 0.86 13.82 ± 0.47
0 0.90 ± 0.66 1.20 ± 0.67 1.01 ± 0.35 1.69 ± 0.82 1.21 ± 0.25 2.13 ± 0.13 2.46 ± 0.42
0 7.78 ± 1.25 14.50 ± 1.85 19.70 ± 2.61 25.22 ± 2.94 29.81 ± 2.78 37.58 1.37 37.99 ± 1.67
0 5.38 ± 1.09 7.01 ± 1.74 8.38 ± 2.67 8.97 ± 1.65 9.61 ± 2.67 15.38 ± 2.59 16.45 ± 1.11
0 10.29 ± 2.17 11.43 ± 2.61 12.76 ± 1.79 11.86 ± 2.35 13.32 ± 2.01 15.77 ± 1.38 15.65 ± 3.03
0 2.58 ± 1.25 3.14 ± 1.42 3.16 ± 1.39 3.59 ± 1.38 2.97 ± 1.42 3.56 ± 1.60 3.88 ± 1.65
L -Cysteine
4-Hexylresorcinol 0.05 0 0.75 4.98 ± 0.54 1.75 11.36 ± 0.25 2.75 16.57 ± 0.64 3.75 21.69 ± 0.69 4.75 26.31 ± 0.43 24.5 35.69 ± 0.28 25.5 35.65 ± 0.73
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Table 2 Box–Behnken experimental design and dataa Experiment no.
Codesb
Degree of inhibitionc
X1
X2
X3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+1 )1 +1 )1 0 +1 )1 +1 )1 0 0 0 0 0 0
+1 +1 )1 )1 0 0 0 0 0 0 +1 )1 +1 )1 0
0 0 0 0 0 +1 +1 )1 )1 0 +1 +1 )1 )1 0
0.80 0.75 0.71 0.62 0.70 0.65 0.41 0.77 0.49 0.72 0.69 0.25 0.16 0.45 0.68
a
Experimental runs were performed in random order. X1 : code of cysteine, X2 : code of kojic acid, X3 : code of 4-hexylresorcinol; )1, 0 and +1 levels correspond to 0, 2 and 4 mM concentrations. c Degree of inhibition ¼ ðDLcontrol DLtreatment Þ=DLcontrol . b
change of PPO activity in the apple juice with respect to storage time is represented in Fig. 1. The decrease in PPO activity with time probably results from the formation of oxidation reaction products derived from the natural substrates which are inhibitors of PPO activity and possibly the protein degradation during storage at 25 ± 2 °C. Table 1 shows the effect of L -cysteine, kojic acid and 4-hexylresorcinol on enzymatic browning in apple juice stored at 25 ± 2 °C. Initial L, a and b values of the cloudy apple juice were 49.96 ± 2.4, 14.2 ± 2.02 and 29.99 ± 0.84 respectively. Twenty-four hour observation is sufficient in order to see the end of enzymatic browning (no more colour change). L -Cysteine prevents brown pigment formation by reacting with the quinone intermediates to form stable colourless compounds. At l mM concentration, cysteine lost its effect, since the o-quinones formed in excess can cooxidize cysteinequinone addition compound, leading to phenol regeneration with a deep colour formation (Vamos-Vigyazo, 1995). The efficiency of browning inhibition by cysteine depended on the thiol:phenol ratio. The relative cysteine concentration must be sufficient to transform all the substrate into colourless adducts. L -Cysteine at 4 mM concentration was significantly effective in preventing the browning of apple juice (p < 0:05). Increasing the concentration of cysteine increased its effectiveness to prevent discolouration. However, high cysteine concentrations may sometimes be inconsistent with good organoleptic properties of the final product due to offodor formation. Kojic acid is a significantly effective anti-browning agent at the selected concentration range (p < 0:05). Kojic acid exhibits inhibition of PPO and also bleaches melanin due to chemical reduction of the browning pigment to colourless compounds. 4Hexylresorcinol was significantly effective against enzy-
Table 3 Estimated coefficients of the second order model Coefficients
Values
Standard error
Significance of t
b0 b1 b2 b3 b11 b22 b33 b12 b13 b23
0.700 0.083 0.045 0.018 0.107 )0.087 )0.227 )0.008 )0.012 0.180
0.060 0.037 0.037 0.037 0.054 0.054 0.054 0.052 0.052 0.052
0.0001 0.0715 0.2730 0.6500 0.1025 0.1653 0.0083 0.8903 0.8292 0.0174
Multiple regression: R2 ¼ 0:90 Standard error ¼ 0.1034 Significance of F ¼ 0:0463
matic browning in the apple juice at high (4 mM) concentration level (p < 0:05), it has long term inhibitory effect. It has several advantages over using other agents, including its specific mode of inhibitory action, its inability to bleach pigments and its chemical stability. The total colour change (DE) during enzymatic browning in the presence of L -cysteine, kojic acid and 4hexylresorcinol, are given in Table 1. In the colour space, L is the intensity, a is the position on the green ()) to red (+) axis and b is the position on the blue ()) to yellow (+) axis. For browning estimation, the L value has been considered as one of the best colour index. Generally, it can be said that DL and DE values were highly correlated due to the negligible change in a and b values compared to the change in L values. However, in the case of a bleaching effect the same correlation can not be observed, since the total colour difference gives the distance in the colour space between two colours,
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enzymatic browning inhibition based on L measurements was considered. The data obtained for the inhibition of enzymatic browning according to Box–Behnken design are given in Table 2. Table 3 shows the regression coefficients obtained by fitting the experimental data to the second order response model: b0 is the value of the fitted response at the center of the design, b1 , b2 and b3 are linear, b11 , b22 , b33 are quadratic and b13 , b23 and b12 are cross product (interaction) regression terms. The positive values of linear regression terms indicate that inhibition increased with increasing the amount of anti-browning agents. Among the linear regression terms, L -cysteine was found to be the most significant anti-browning agent in the combination according to the t-test. When the interactions between the anti-browning agents are considered, the interaction of kojic acid and 4-hexylresorcinol was found to have a positive effect on the inhibition of enzymatic browning. There were no significant interaction between L -cysteine and kojic acid and also between L -cysteine and 4-hexylresorcinol. The predictive model was used to graphically represent the system. Fig. 2 is an example for response surfaces. A formulation of the anti-browning reagents were found: X1 , X2 and X as 0.98 (3.96 mM L -cysteine), 0.39 (2.78 mM kojic acid) and 0.17 (2.34 mM 4-hexylresorcinol), respectively. The calculated inhibition was 89.2% at the end of 24 h storage period. For shorter storage times, this combination will also delay or prevent enzymatic browning that it is especially important in the application of non-thermal processes for the production of fruit juices in terms of the retardation of the enzymatic browning in the process line before enzyme inactivation. Also the use of combination of anti-browning agents lowers the required concentrations that it is important for the organoleptic properties of the juice.
Fig. 2. Three dimensional plot representations for inhibition of enzymatic browning based on measurements of DL at X1 ¼ 0 (a), X2 ¼ 0 (b), X3 ¼ 0 (c) (Y ¼ degree of inhibition).
but not the direction in which they differ. Therefore the evaluation of browning in terms of DL values may be more reliable. The mechanism of inhibition differs widely for each of the categories of anti-browning agents. The combination of different agents may prevent browning better than a specific chemical alone. Since L -cysteine (sulfur-containing amino acid), kojic acid (phenolic acid) and 4-hexylresorcinol (substituted resorcinols, structurally related to phenolic substrates) were found to be effective antibrowning agents on enzymatic browning, in the second part of the study, the combined effect of these agents on the enzymatic browning reaction was determined at the end of the 24 h storage period. For this purpose, the
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