- Email: [email protected]
Study on the Mechanism of Browning of Pomegranate (Punica granatum L. cv. Ganesh) Peel in Different Storage Conditions
Available online at www.sciencedirect.com " a,?
Agricultural Sciences in China
*'
Q#
2008, 7(1): 65-73
ScienceDirect
January 2008
Study on the Mechanism of Browning of Pomegranate (Punica granafum L. cv. Ganesh) Peel in Different Storage Conditions ZHANG You-lin and ZHANG Run-pang College of Food Engineering and Nurrirional Science, Shaanni Normal Universiry, Xi'an 710062, P.R.China
Abstract Peel browning of pomegranate fruit is the key problem during storage. The mechanism of it during storage in relation to the technology of the browning control was studied in this paper. The effects of storage temperature, gas component and pH value on peel browning of sweet pomegranate fruit in Lintong, Shaanxi Province, China, were investigated. Change in tannin content was measured and the relationship between the activities of ascorbic acid oxidase (AAO), polypenoloxidase (PPO), peroxidase (POD), catalase (CAT) and peel browning was analysed. The results showed tannin was the basic substance of pomegranate peel browning. The activities of the browning index of peel were correlated positively with AAO, PPO and POD, correlated negatively with CAT activity. Application of intermittent warming for 24 h at (15 *0.5)"C every 5 days for pomegranate fruit storage under the conditions of pH 4.0, (5.0i0.5)"C and 5.0% C0,+8.0% 0,+87.0% N, gas component could delay browning, with the browning index being 0.15 after 120 days of storage. Enzymatic tannin denaturation was the main cause that led to peel browning of pomegranate fruit during storage. The browning control of pomegranate fruit peel was effective using intermittent warming storage with suitable pH, temperature and gas component.
Key words: pomegranate (Punica granatum L.), stcxage, peel, browning
INTRODUCTION Pomegranate (Punica granatum L.) is a non-ciimacteric respiratory fruit (Elyatem and Kader 1984). The key to extend the storage life and maintain the quality of pomegranate fruit lies in preventing peel from browning. Browning of pomegranate peel has been mainly attributed to many factors, including chilling injury (Elyatem and Kader 1984; Kader et al. 1984), water loss, and the development of physiological disorder during storage. Kahn (1983) found that the injured plant surface or the tissue browning was mainly because the phenolic compounds were oxidized into quinone compounds under aerobic conditions by
polyphenoloxidase, and the quinone compounds underwent polymerization forming brown polymeric pigments, leading to browning. Sapers et al. (1989) discerned that vitamin C could d e o x i d i z e q u i n o n e c o m p o u n d s into phenolic compounds, which prevent from browning. But the reaction above was inhibited while vitamin C was destroyed at low temperature or low humidity conditions, which resulted in the accumulation of quinone and then browning. Studies performed by Alkahtani (1992) showed that there were lots of pores in pomegranate peel tissue, which made peel lose free water easily and become brown. This finding was further supported by the case that Lintong Tianhongdan pomegranates were stored by different packages (Liu et al. 1997). It was
This paper is translated from its Chinese version in Scientia Agricultura Sinica ZHANG You-lin. Ph 0,Professor, Tel: +86-29-85300865. E-mail: [email protected]; Correspondence ZHANG Run-guang, Tel: +86-29-8.53 10520, E-mail: [email protected]
62008,CAAS. All nghts reserved. Publishedby Elsevier Ltd.
ZHANG You-lin et al.
66
found that the peel lost free water and became brown seriously when pomegranate fruit was packed in carton. However, if they were packed in 0.03 mm polythene (PE) bags, peel showed little loss of water and light browning. Ben-Arie and Or (1986) reported that although pomegranate was a non-climacteric respiratory fruit, an appropriate ratio of 0, and CO, could control peel browning. When the ratio was combined with the fitting storage temperature, it had a better effect on antibrowning. According to Gao et al. (1987), there was a correlation between catechol content and peel browning in pomegranate fruit harvested in the late season, and there was a stable catechol level in the little brown peel during storage. In enzymatic browning researches, Ben-Arie and Or ( 1986) believed that polypenoloxidase (PPO) activity of pomegranate peel had no obvious correlation with peel browning. However, Jiang et al. (1991) reported that the occurrence of banana peel browning depended on the ratio of dissociative PPO and associative PPO, and the higher the ratio was, the more serious was peel browning. Low temperature could increase the ratio. Liu et al. (1995) found that Tianhongdan pomegranate fruit treated with Na,S,O, could reduce peel browning. The reason was maybe that Na,S,O, released SO, and inhibited the activity of PPO, which suggested that pomegranate peel browning was enzymatic browning. Many factors affect pomegranate peel browning. Many studies have examined the effect of browning with single factor, but few have focused on the browning mechanism, especially lacking the further research. In this article, we studied the mechanism of pomegranate peel browning, analyzed the relationship between the activities of several enzymes and the browning index, and explored the effect of combination of different storage conditions for delaying peel browning, in order to provide the theoretical basis of the pomegranate peel browning and the technical parameters of controlling browning during storage.
MATERIALS AND METHODS Materials Sweet pomegranate fruits in Litong, Shaanxi Province,
China, were selected in the light of full maturity, uniform color and size, while the diseased or injured fruits were discarded. The soluble solid content of the fruit juice was above 15% and the weight of each fruit was about 300 g. Pomegranate fruit were harvested with stalks and precooled at (6+0.5)"C for 3 days prior to experiments. There were four replicates per treatment (n=4). Each replicate weighed 6 kg. The samples were selected randomly and were analyzed at 30-day intervals during 120 days storage.
Methods Storage temperature test The pomegranate fruit was packed individually in 0.03 mm thick PE bags with a 5 mm diameter hole at the bottom and stored at (-1 f O S ) , ( 2 f 0 . 3 , (5f0.3,and (8fOS)"C, respectively, with 90-95% relative humidity (RH). Storage atmosphere test The pomegranate fruit were placed in five controlled atmosphere test-boxes (volume of 57 c m x 5 7 c m x 5 8 cm), in which the atmosphere mixture was designed as follows: treatment 1,3.0% CO, +3.0% 0,+94.0% N,; treatment 2, 5.0% CO,+ 8.0% 0,+87.0% N,; treatment 3,8.0% C0,+5.0% 0,+87.0% N,; treatment 4, 12.0% CO, + 12.0% 0, + 76.0% N,; control group was the natural air. The desired gas mixture was changed once every 10 days. The fruits were stored at (5.0 f 0 3 ° C . pH coating test A 0.5% carboxymethyl cellulose (CMC) solution was prepared by mixing 10.0 g sodium carboxymethyl cellulose (CMC-Na) powder and a little 95% alcohol into 2000 mL of distilled water maintained at 40°C with sufficient agitation to form a smooth suspension. After cooling, the pH was adjusted to 1.0, 3.5, 5.0, 7.0, and 9.0, respectively, with 20% citric acid solution and 10%NaOH solution. The fruits (peel pH 3.8-4.0) were dipped into the CMC solution of different pH for 15 s and air-dried at ambient temperature for 24 h. The fruit without dipping was taken as control. After treatment, the pomegranate fruit were packed individually in PE bags with a hole and stored at (5.0+ 0.5)"C with 90-95% relative humidity. Intermittent warming test The pomegranate fruit packed individually in PE bags with a hole were kept in opening cartons and stored at (5.0 2 0.5)"C. Cartons
82008.CAAS. All rights resewed.Published by Elsevier LM.
Study on the Mechanism of Browning of Pomegranate (Punica granarurn L. cv. Ganesh) Peel in Different
were placed at (15.0+0.5)"C for 24 h every 3 or 6 days, respectively, and then placed in the original condition, (5.OkOS)"C. The fruit kept at the stable temperature of (5.0 + 0.5)"C was taken as the control. Optimization of methods controlling browning test The optimum method of controlling peel browning was investigated by using L,(34) orthogonal test involving the storage temperature, atmosphere, pH coating and intermittent warming. The concrete method was as follows: The pomegranate fruits were coated with CMC film of different pH, dried naturally, and placed in the controlled atmosphere test-boxes. The boxes were filled with differently designed gas mixture, which was changed once every 10 days. The fruits were stored at the designed temperature and treated as intermittent warming experiment. Repeat the procedure above during the 120 days of storage.
Mensuration The browning index of peel was calculated by the following formula: C (Browning scale x Percentage of corresponding fruit within each class)/100. The appearance scale was as follows: 0 (0 score), no browning and glabrous surface; 1 (25 score), a slight browning and smooth surface; 2 (50 score), obvious browning and rough surface; 3 (75 score), serious browning and hollow surface depression; and 4 (100 score), black and encrusting surface. The tannin content of the peel was measured by the Folin-Denis colorimetry (Hou 2004); ascorbic acid oxidase (AAO) activity by iodine titration (Tang 1999); PPO activity by colorimetry (Pizzocaro et al. 1993); peroxidase (POD) activity by guaiacol colorimetry (Xu and Ye 1989); and catalase (CAT) activity by potassium permanganate titration (Li et al. 2000).
Statistical analysis
61
were significant and at the 1% level were extremely significant.
RESULTS Effect of storage temperature on pomegranate peel browning As shown in Fig. 1 , the browning index of pomegranate peel increased with storage time. The browning index of peel stored at (- 1 k 0 3 ° C rose markedly and reached the maximum of 1.0 by day 60 (P 0.05, compared between them), whereas it was only 0.32 at (5kOS)"C (P
Effect of storage atmosphere on pomegranate peel browning As shown in Fig.2, the browning index of peel increased in all treatments during storage. The browning indices of treatments 1 and 2 were only 0.21 and 0.23 after 120 days storage ( P > 0.05, compared between them; P < 0.01, compared with other treatments) and the pomegranate peel was still kept fresh and bright.
1.o
0.8 0.6 0.4
0.2
The data were analyzed using ANOVA. Mean differences were established by Duncan's multiple range test and r-test. All the data were expressed as means & standard error (X+ SE) and were calculated after 3 days of precooling. Differences between means at the 5% level
0
0
30
60
90
I20
Duration of storage (d)
Fig. 1 Changes of peel browning index of pomegranate fruit during storage at different temperatures (n =4).
02008,CAAS. All rights reserved. Publishedby ElsevlerLtd.
ZHANG You-lin et al.
68
However, there was serious peel browning in fruit by treatments 3 and 4, especially for treatment 4 with the index up to 1.00 by day 120.
Effectof pH coating solution on pomegranatepeel browning As shown in Fig.3, the browning index of peel increased in all treatments with storage time. The browning index of the pH 9.0 CMC coating fruit went up sharply and reached the top to 1.0 by day 60, (Pe 0.0 1, compared with other treatments), which was
x c
0
/
0.8 l'O[ 0.6
X
8
.2 .$
g
0.4
0.2 0
0
30
60
90
120
Duration of storage (d)
+Treatment 1 3.0% C0,+3.0% 0,+94.0%
N,
-A- Treatment 2 5.0% C0,+8.0% 0,+87.0% N,
+Treatment 3 8.0% C0,+5.0%
probably caused by the anthocyanin and tannin in the pomegranate peel that changed into blue or black pigment in alkaline condition, resulting in serious peel browning, wherein the browning indices of pH 1.0 and 3.5 CMC coating fruit were relatively lower ( P < 0.05, compared with control; P c 0.01, compared with pH 9.0; P>0.05, compared with pH 5.0 and 7.0) than other treatments after 120 days of storage, which indicated that the acidic condition conduced to maintaining better peel appearance.
Effect of intermittent warming on pomegranate peel browning As shown in Fig.4, intermittent warming treatments could inhibit peel browning during storage. The browning index of the fruit stored by intermittent warming every 6 days was just 0.21 but that of the control was 0.36 after 120 days of storage. It was probable that intermittent warming could prevent the chilling injury, protect the peel cells against harm, and keep the fruit from browning. There is a significance ( P < 0.05) among the three treatments after 120 days of storage and the intermittent warming every 6 days could control the peel browning effectively.
0,+87.0% N, Treatment 4 12.0% CO,+ 12.0% 0,+76.0% N, Control air
-0-
I .o
--C Control
Fig. 2 Changes of peel browning index of the pomegranate fruits during storage under different gas components (n = 4).
-6d
0
30
60
90
120
Duration of storage (d)
Fig. 4 Changes of peel browning index of the pomegranate fruits during storage using different intermittent warming (n =4). 0
30
90
60
120
Optimum storage conditions
Duration of storage (d) 1.0
+3.5
--t 7.0
*9.0
5.0 --O- Control
Fig. 3 Changes of peel browning index of the pomegranate fruits during storage under different pH values (n=4).
On the basis of single factor tests above, the L,(34) orthogonal experiment of the pomegranate fruits was carried out to study compositive effects of storage temperature, atmosphere, pH coating solution and in-
02008,CAAS. All rights RSBN&.Publishedby Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica grunaturn L. cv. Ganesh) Peel in Different
69
Table Results of L,(34) orthogonal experiment of the pomegranate fruits during storage and analysis of extremal deviations (n =4) Test 1 2 3
A Storage temperature ("C) A, (4.0k0.5) A, A,
4
A, (5.0i0.5)
5 6
A2
B Gas component C 0 2and O,(%) B, (4.0, 7.0) B, (5.0, 8.0) B, (6.0.9.0) Bl B2
A, A, (6.0i0.5)
B3
7
8
A3
9
A3 0.26 0.22
B* B3 0.26 0.26 0.27 0.01
Kl K2 K3
Range Sequence Optimal combination
0.30 0.08
Bl
C Different pH
D Intermittent warming (d)
Browning index Of peel
*
0.35 0.029 ab
0.24*0.020 cde 0.20*0.020 ef 0.22iO.039 def 0.15*0.015f 0.29*0.035 bed 0.21 k0.019 def 0.38*0.029 a 0.32 kO.025 abc
CADB A1B2C3D2
The different small letters indicate significant difference at P = 0.05.
termittent warming on the peel browning, and the optimum method of controlling the browning was determined. Samples were selected at random and their peel browning indices were measured after 120 days of storage. The results of orthogonal experiment and the analysis of extremal deviation were shown in Table. The optimum storage combination that could control p o m e g r a n a t e p e e l b r o w n i n g effectively w a s A,B,C,D,, which was (5.0 +0.5)"C, 5.0% CO,+ 8.0% O,+ 87.0% N,, pH 4.0 coating and intermittent warming for every 6 days. This result was inconsistent with the better combination of A,B,C,D,, which is (5.0*0.5)°C, 5.0% C0,+8.0% 0,+87.0% N,, pH 4.0 coating and intermittent warming for every 5 days. Therefore, in the following year, pair comparison test was set up between A,B,C,D, and A,B,C,D,. The test was replicated three times and there was no significance between two treatments ( P >0.05). Considering the intermittent warming and ventilation treatment, A,B,C,D, was determined as the final optimum combination during storage.
Relationship between tannin content and pomegranate peel browning As shown in Fig.5, tannin content of pomegranate peel decreased slowly for the first 90 days, then decreased sharply during remaining period. After 120 days of storage, tannin content was 0.083,0.096, and 0.15 1%, at (8.0 k 0.5), (2.0 k 0.5), and (5.0 k 0.5)"C, respectively. The content at (5.0k 0.5)"C storage was significantly higher than the other two groups. Fig.5
showed that peel browning index increased gradually with the decrease in tannin content in pomegranate peel. There was a negative interrelationship between them. This is probably the reason that the tannin was a substrate of pomegranate peel browning.
Relationship between AAO activity and pomegranate peel browning The AAO in plants exposed to the air could catalyze molecular oxygen, which oxidized ascorbic acid into dehydroascorbate acid, thereby made the antioxidation of ascorbic acid lost, leading to peel browning. As shown in Fig.6, AAO activity of pomegranate peel increased during storage in all the treatments. The increase was more rapid after 60 days of storage. AAO activity reached the highest value at (8.0 f 0.5)"C and the lowest value at (5.0kOS)"C after 120 days of storage. Generally, the lower the storage temperature, the lower was the AAO activity. However, the AAO activity at (2.0 k 0.5)"C was higher than that at (5.0 f 0.5)"C by day 90. In the later period of storage, chilling injury led to the physiological metabolic disorder in pomegranate fruits at (2.0 k0.5)"C. As a result, AAO activity increased. The results showed that there was a positive correlation between AAO activity of pomegranate peel and peel browning index.
Relationship between PPO activity and pomegranate peel browning Plant surface or tissue browning is mainly due to the
02008, CAAS. Ail rights resewed. Publishedby Elsevier Ltd.
ZHANG You-lin et al.
70
0.5
rT
1
2
0.4
0.8
E
0.3
0.6
I
c X
4
.-
P
8
0.2
0.4
.?
g
0.1
0.2
g
.-
C
B
0
0
30
0
60
90
120
Duration of storage (d)
--o-O-
-0-
8°C. tannin content 2°C. tannin content 8°C. browning index
5°C tannin content 2"C, browning index 5°C browning index
-&--t
-A-
Fig. 5 Relationship between tannin content and peel browning index of the pomegranate fruits (n=3).
0.8 L
oxidation of phenolic compounds into quinone compounds under aerobic conditions by polyphenol oxidase, then the quinone compounds undergoes polymerization forming brown polymeric pigments, leading to browning. As shown in Fig.7, PPO activity of pomegranate peel has risen quickly within 90 days of storage, reached the peak value by day 90, and then decreased slowly. PPO activity went down with the temperature decreasing before 90 days of storage, but it didn't obey this rule during the last 30 days of storage. PPO activity of the pomegranate peel stored at (2.0+0.5)"C was higher than that stored at (5.0+0.5)"C after 120 days of storage. It probably resulted from abnormal physiological metabolism caused by chilling injury. There was a significant correlation between PPO activity and peel browning index.
Relationship between POD activity and pomegranatepeel browning
0.6
d
m 0 0
30
60
90
120
-
Duration of storage (d)
-
-c+
-0-
-A-
8"C, AAO activity 2°C. AAO activity 8"C, browning index
5°C. AAO activity 2°C. browning index 5°C. browning index
-&-
Fig. 6 Relationship between AAO activity and peel browning index of the pomegranate fruits (n=3).
250 r
1
POD is another major enzyme in enzymatic browning reaction. It can oxidize polyphenol substances quickly in the presence of H,O, and cause fruits or vegetables to brown with PPO synergizing. As shown in Fig.8, POD activity of pomegranate peel increased gradually from beginning to end with great fluctuation during storage. Different temperatures had little effect on POD activity. By day 120, POD activity was 4 856 (8.O&0S0C), 5306 (5.0&0.5"C), and 5064 U g-' (2.0 + 0.5"C), respectively. The results showed that there was a positive correlation between POD activity of
1.0
200
0.8
150
0.6
;:
100
0.4
g
50
0.2
x
2
0
'il
8000
3.-
6 000
0.6
6s
4 000
0.4
2 000
0.2
-
-0-
-C-
,?
0 0
30
60
90
0.8
8
0
0
120
0
Duration of storage (d)
8°C. PPO activity 2"C, PPO activity 8°C. browning index
1
10000
M
2
1.o
g
30
60
90
120
Duration of storage (d)
-h-
5°C.PPO activity
-A-
5 ° C browning index
+ 2°C. browning index
Fig. 7 Relationship between PPO activity and peel browning index of the pomegranate fruits (n=3).
8"C, POD activity
-A-
5'C. POD activity
+2°C. POD activity
-m-
2°C. browning index browning index
-0-
--t
8°C. browning index
-5'C.
Fig. 8 Relationship between POD activity and peel browning index of the pomegranate fruits (n = 3).
02008,CAAS. All rights taserved. Publishedby Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica Rranafum L. cv. Ganesh) Peel in Different
pomegranate peel and peel browning index.
Relationship between CAT activity and pomegranatepeel browning CAT exists in the ferritin of fruit and vegetable tissues widely. Its activity has a certain relationship with metabolic intensity, anticoldness and antidisease of fruits and vegetables. In the course of respiratory metabolism, CAT could decompose H,O, into H,O and 0,, which prevented H,O, from accumulating in fruit and vegetable tissues. As shown in Fig.9, CAT activity of pomegranate peel had a downtrend during storage, while it declined rapidly at (8.0+0.5)"C but went down slowly at two other temperatures. By day 120, CAT activity was 4.22 (8.0k0S°C), 5.49 (5.0+0.5"C), and 5.06 (2.OkOS"C) mg H,O, g-' min", respectively. The results showed that there was a negative correlation between CAT activity of pomegranate peel and peel browning index.
10
1
0
30
60
90
120
Duration of storage (d) 8°C. CAT activity 2"C, CAT activity +8°C. browning index -c-
-A-
-c-
-w-A-
5°C. CAT activity 2 ° C browning index 5°C. browning index
Fig. 9 Relationship between CAT activity and peel browning index of the pomegranate fruits (n = 3).
DISCUSSION Many phenolic compounds, such as tyrosine, 3,4dihydroxyphenyl alanine, dopamine, hydroquinone, catechol, 4-methyl catechol, protocatechuic acid, caffeic acid, chlorogenic acid, anthocyanidin, flavonoid compounds, tannin, and sumac hydroxyketone, can cause the fruits browning. The main browning sub-
71
strates of coconut fruit were dopamine and chlorogenic acid (Jiang et al. 1992), the latter was also the browning substrate of pear, apple and peach (Wu et al. 1992). In the litchi peel, the substrates were catechol compounds, which formed browning substances as treated with PPO (Jiang 1991). Free polyphenols were the main browning substrates of longan peel, whereas the effect of anthocyanin was insignificant (Wu et al. 1999). Catechin tannin in the fruit and vegetable tissues was due to the ramification of catechol, and they were easily oxidized in the air and formed brown or black pigments. Tannin was involved in the banana peel browning (Bao et al. 2005). The higher was the tannin content, the easier was peel browning, especially in alkaline conditions. This result was confirmed by the fact that the tannin content of peach fruit decreased slowly during storage and the reduction of tannin content coincided with the Snow peach browning (Wang and Liu 1996). In this article, the browning index of sweet pomegranate peel in Lintong, Shaanxi, was gradually increased as the tannin content decreased during storage (Fig.5), which indicated that tannin was a substrate for browning. This is in accord with the theory that tannin resulted in peel browning proposed by predecessors. In addition, the pomegranate peel browned seriously in alkaline conditions (Fig.3) under the high oxygen concentration (Fig.2). The result fitted the tannin's character of oxidized browning or alkaline browning, suggesting that the pomegranate peel browning might be the tannin browning. Generally, phenolic compounds in plant organs or tissues are oxidized into quinone and water under enzymatic catalysis, and then the quinone is polymerized into brown polymeric pigments without enzymes, namely, the enzymatic browning. Substrates (phenolic compounds), enzymes and oxygen are three conditions for this reaction (Lin 2002). PPO is the general designation of the catechol oxidase and laccase (He and Bin 2001). They almost exist in all fruits as studied but different types of fruit have different nature of PPO. PPO of litchi peel could oxidize pyrocatechol and pdihydroxybenzene, and had a stronger effect on the former. However, it could not oxidize monoprotic phenol and resorcinol (Jiang 1991); PPO of longan peel could oxidize pyrogallol, pyrocatechol and 4-methyl-
Q2008. CAAS.All rightsreserved.Published by Elsevier Ltd.
ZHANG You-lin et a/.
12
catechol but not monoprotic phenol and resorcinol (Jiang 1999); PPO of loquat fruit could oxidize pyrocatechol but not monoprotic phenol, p-dihydroxydroxybenzene and resorcinol (Ding et al. 1998). POD could oxidize and polymerize phenols and flavonoids in the presence of H,O,, resulting in the tissue browning (Mayer and Hare1 1979; Lin et al. 1988; Jiang 1991). POD content of litchi peel was high. The increase of peel browning index was in accordance with the rapid rise of POD activity at room temperature during storage (Lin et al. 1988; Chen and Wang 1989). The higher was the PPO activity of peel, the easier was peel browning (Jiang 1991). Therefore, it was believed that POD took an important role in fruit browning (Chen and Wang 1989; Underhill and Critchley 1995). In this experiment, it was found that activities of PPO and POD in pomegranate peel correlated with the peel browning positively (Figs.7 and 8). Ascorbic acid in fruit and vegetable tissues is a strong antioxidant, which can prevent the fruits and vegetables from browning effectively. AAO exposed to the air can oxidize ascorbic acid into dehydroascorbate, which lead to the antioxidation of ascorbic acid losing and contributing to the tannin oxidation into browning substances. In this article, it was found that AAO activity in pomegranate peel correlated with the browning index positively (Fig.6). H,O, in the fruit and vegetable tissues has a strong oxidizability. It is the main reason for fruits and vegetable browning. CAT can decompose H,O, into H,O and 0,, prevent the accumulation of H,O, and control the peel browning. In the experiment, it was found that CAT activity in pomegranate peel correlated with the browning index negatively (Fig.9). It may be the reason that the accumulation of H,O, with the decline of CAT activity made the tannin oxidize to browning substances. The gas component is another factor for fruit browning. The browning was controlled effectively under the suitable gas component (low 0, and high CO,) (Fig.2). However, if 0, concentration was too low or CO, concentration was too high, reactive oxygen (O,, H,O,) would be produced by disorders of physiological metabolism and increase enzymatic browning (TomasBarberan and Espin 2001). Unsuitable gas component led to pomegranate peel browning during storage.
In addition, tannin of the pomegranate peel needs to be studied further, including its physical structure, chemical properties and biochemical mechanism on peel browning.
CONCLUSION According to the results, enzymatic browning was the main reason for peel browning of pomegranate fruit during storage and tannin was the main browning substrate. After precooling, the pomegranate fruits were stored by the following treatment: coated with 0.5% CMC solution (pH 4.0), placed under the gas component of 5.0% CO,+ 8.0% 0, + 87.0% N, as designed, stored at (5.0+0.5)”C, ventilated every 10 days and then treated with the intermittent warming for 24 h at (15.0+0.5)”C every 5 days. Repeat the operation in cycles during storage. Under above conditions, the pomegranate fruit had a good quality with 0.15 browning index and 97.8% good fruit rate after 120 days storage.
Acknowledgements This work was financed by the Natural Science Foundation of Shaanxi Province, China (2004C109), Key Technology R&D Program of Shaanxi Province, China (2003K02-G6), and Agricultural Science and Technology Development of Xi’an City, China (NG200313).
References Alkahtani H A. 1992. Intercultivar differences in quality and postharvest life of pomegranate influenced by partial drying. Journal of the American Society for Horticultural Science, 117, 100-104. Bao J Y,Liang S R, Zhao G J, Yang G M . 2005. Study on extraction technology and relations with browning of tannins from banana peel. Food Research and Development, 26,3-6. (in Chinese) Ben-Arie R, Or E. 1986. The development and control of husk scald on “Wonderful” pomegranate fruit during storage. Journal of the American Society for Horticultural Science, ll!, 395-399. Chen Y Z, Wang Y R. 1989. A study on peroxidase in litchee pericarp. Acta Botanica Austro Sinica, 5,47-52.(in Chinese) Ding C K, Chachin K, Ueda Y, Imahori Y. 1998. Purification and properties of polyphenol oxidase form loquat fruit. Journal
02008,CAAS.All rightsreserved. Published by Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica granatum L. cv. Ganesh) Peel in Different
of Agricultural and Food Chemistiy, 46,4144-4149. Elyatem S M, Kader A A. 1984. Post-harvest physiology and storage behaviour of pomegranate fruits. Scientia Horticulturae, 24, 287-298. Gao M Y, Shi F H, Chang N J. 1987. Experiment of pomegranate storage. Deciduous Fruit Tree, (3), 28-30. (in Chinese) He L H, Bin J H. 2001. Polyphenol oxidase in higher plants. Plant Physiology Communications, 37,340-345. (in Chinese) Hou M L. 2004. Food Analysis. Chemical Industry Press, Beijing. pp. 134-141. (in Chinese) Jiang Y M, Chen M D, Lin Z F, Chen F. 1991. Enzymatic browning of banana during low temperature storage. Acta Phytophysiologica Sinica, 17, 157-163. (in Chinese) Jiang Y M. 1991. Browning of fruit and its control. Journal of Plant, 18,22-23. (in Chinese) Jiang Y M. 1999. Purification and some properties of polyphenol oxidase of longan fruit. Food Chemistry,.66,75-79. Jiang Y M, Zhang D L, Liu S X, Chen F, Li Y B. 1992. A primary study on browning substrates of coconut. Plant Physiology Communications, 28,347-349. (in Chinese) Kader A A, Chordas A, Elyatem S M. 1984. Responses of pomegranates to ethylene treatment and storage temperature. California Agriculture, 38, 14-15. Kahn V. 1983. Multiple effects of hydrogen peroxide on the activity of avocado polyphenol oxidase. Phytochemistry, 22, 2155-2159. Li H S, Sun Q, Zhao S J, Zhang W H. 2000. The Theory and Technology of Plant Physiology and Biochemistry. Higher Education Press, Beijing. pp, 165-167. (in Chinese) Lin H T, Xi Y F, Chen S J. 2002. A review of enzymaticbrowning in fruit during storage. Journal of Fuzhou University, 30 (Suppl.), 696-703. (in Chinese) Lin Z F, Li S S, Zhang D L, Liu S X, Li Y B, Lin G Z, Chen M D. 1988. The changes of oxidation and peroxidation in postharvest litchi fruit. Acta Botanica Sinica, 30, 382-387. (in Chinese) Liu X H, Cai J, Huo S, Xu H D. 1995. Study on postharvest physiological characters of Tianhongdan pomegranate. Tianjin Agricultural Sciences, 1,30-31. (in Chinese) Liu X H, Hu Q X, Kou L P, Xu H D. 1997. The study on
13
pomegranate husk scald during storage. Journal of Northwest Forestry College, 12,93-96. (in Chinese) Mayer A M, Hare1 E. 1979. Polyphenol oxidases in plants. Phytochemistry, 18, 193-215. Pizzocaro F, Torreggiani D, Gilardi G. 1993. Inhibition of apple polyphenoloxidase (PPO) by ascorbic acid, citric acid and sodium chloride. Journal of Food Processing and Preservation, 17, 21-30. Sapers G M, Hicks K B, Phillips J G, Garzarella L, Pondish D L, Matulaitis R M, McCormack T J, Sondey S M, Seib PA, ElAtawy Y S. 1989. Control of enzymatic browning in apple with ascorbic acid derivatives,polyphenol oxidase inhibitors, and complexing agents. Journal of Food Science, 54, 9971002,1012. Tang Z C. 1999. Guide to the Experiment of Modern Plant Physiology. Science Press, Beijing. pp. 316-3 17. (in Chinese) Tomas-Barberan F A, Espin J C. 2001. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. Journal of the Science of Food and Agriculture, 81, 853-876. Underhill S J R, Critchley C. 1995. Cellular localisation of polyphenol oxidase and peroxidase activity in Litchi chinensis Sonn. pericarp. Australian Journal of Plant Physiology, 22, 627-632. Wang J, Liu C L. 1996. Primaryexplorationon changes of chemical composition of Xue peach under different storage conditions and its browning reason. China Fruit Research, (3), 12-14. (in Chinese) Wu G X, Zhou H W, Wang J M. 1992. Biochemical mechanism and substances determination of enzymic browning of yali pear (Pynrs bretschneideri Rehd.). Acta Horticulturae Sinica, 19, 198-202. (in Chinese) Wu Z X, Han D M, Ji Z L, Chen W X. 1999. Effect of sulphur dioxide treatment on enzymatic browning of longan pericarp during storage. Acta Horticulturae Sinica, 26, 91-95. (in Chinese) Xu L L, Ye M B. 1989. A method of continuous record to test the activity of peroxidase. Journal of Nanjing Agricultural University, 12, 80-83. (in Chinese) (Edited by ZHANG Juan)
~32008, CAAS. All rightsreserved. Publishedby Elsevier Ltd.
Agricultural Sciences in China
*'
Q#
2008, 7(1): 65-73
ScienceDirect
January 2008
Study on the Mechanism of Browning of Pomegranate (Punica granafum L. cv. Ganesh) Peel in Different Storage Conditions ZHANG You-lin and ZHANG Run-pang College of Food Engineering and Nurrirional Science, Shaanni Normal Universiry, Xi'an 710062, P.R.China
Abstract Peel browning of pomegranate fruit is the key problem during storage. The mechanism of it during storage in relation to the technology of the browning control was studied in this paper. The effects of storage temperature, gas component and pH value on peel browning of sweet pomegranate fruit in Lintong, Shaanxi Province, China, were investigated. Change in tannin content was measured and the relationship between the activities of ascorbic acid oxidase (AAO), polypenoloxidase (PPO), peroxidase (POD), catalase (CAT) and peel browning was analysed. The results showed tannin was the basic substance of pomegranate peel browning. The activities of the browning index of peel were correlated positively with AAO, PPO and POD, correlated negatively with CAT activity. Application of intermittent warming for 24 h at (15 *0.5)"C every 5 days for pomegranate fruit storage under the conditions of pH 4.0, (5.0i0.5)"C and 5.0% C0,+8.0% 0,+87.0% N, gas component could delay browning, with the browning index being 0.15 after 120 days of storage. Enzymatic tannin denaturation was the main cause that led to peel browning of pomegranate fruit during storage. The browning control of pomegranate fruit peel was effective using intermittent warming storage with suitable pH, temperature and gas component.
Key words: pomegranate (Punica granatum L.), stcxage, peel, browning
INTRODUCTION Pomegranate (Punica granatum L.) is a non-ciimacteric respiratory fruit (Elyatem and Kader 1984). The key to extend the storage life and maintain the quality of pomegranate fruit lies in preventing peel from browning. Browning of pomegranate peel has been mainly attributed to many factors, including chilling injury (Elyatem and Kader 1984; Kader et al. 1984), water loss, and the development of physiological disorder during storage. Kahn (1983) found that the injured plant surface or the tissue browning was mainly because the phenolic compounds were oxidized into quinone compounds under aerobic conditions by
polyphenoloxidase, and the quinone compounds underwent polymerization forming brown polymeric pigments, leading to browning. Sapers et al. (1989) discerned that vitamin C could d e o x i d i z e q u i n o n e c o m p o u n d s into phenolic compounds, which prevent from browning. But the reaction above was inhibited while vitamin C was destroyed at low temperature or low humidity conditions, which resulted in the accumulation of quinone and then browning. Studies performed by Alkahtani (1992) showed that there were lots of pores in pomegranate peel tissue, which made peel lose free water easily and become brown. This finding was further supported by the case that Lintong Tianhongdan pomegranates were stored by different packages (Liu et al. 1997). It was
This paper is translated from its Chinese version in Scientia Agricultura Sinica ZHANG You-lin. Ph 0,Professor, Tel: +86-29-85300865. E-mail: [email protected]; Correspondence ZHANG Run-guang, Tel: +86-29-8.53 10520, E-mail: [email protected]
62008,CAAS. All nghts reserved. Publishedby Elsevier Ltd.
ZHANG You-lin et al.
66
found that the peel lost free water and became brown seriously when pomegranate fruit was packed in carton. However, if they were packed in 0.03 mm polythene (PE) bags, peel showed little loss of water and light browning. Ben-Arie and Or (1986) reported that although pomegranate was a non-climacteric respiratory fruit, an appropriate ratio of 0, and CO, could control peel browning. When the ratio was combined with the fitting storage temperature, it had a better effect on antibrowning. According to Gao et al. (1987), there was a correlation between catechol content and peel browning in pomegranate fruit harvested in the late season, and there was a stable catechol level in the little brown peel during storage. In enzymatic browning researches, Ben-Arie and Or ( 1986) believed that polypenoloxidase (PPO) activity of pomegranate peel had no obvious correlation with peel browning. However, Jiang et al. (1991) reported that the occurrence of banana peel browning depended on the ratio of dissociative PPO and associative PPO, and the higher the ratio was, the more serious was peel browning. Low temperature could increase the ratio. Liu et al. (1995) found that Tianhongdan pomegranate fruit treated with Na,S,O, could reduce peel browning. The reason was maybe that Na,S,O, released SO, and inhibited the activity of PPO, which suggested that pomegranate peel browning was enzymatic browning. Many factors affect pomegranate peel browning. Many studies have examined the effect of browning with single factor, but few have focused on the browning mechanism, especially lacking the further research. In this article, we studied the mechanism of pomegranate peel browning, analyzed the relationship between the activities of several enzymes and the browning index, and explored the effect of combination of different storage conditions for delaying peel browning, in order to provide the theoretical basis of the pomegranate peel browning and the technical parameters of controlling browning during storage.
MATERIALS AND METHODS Materials Sweet pomegranate fruits in Litong, Shaanxi Province,
China, were selected in the light of full maturity, uniform color and size, while the diseased or injured fruits were discarded. The soluble solid content of the fruit juice was above 15% and the weight of each fruit was about 300 g. Pomegranate fruit were harvested with stalks and precooled at (6+0.5)"C for 3 days prior to experiments. There were four replicates per treatment (n=4). Each replicate weighed 6 kg. The samples were selected randomly and were analyzed at 30-day intervals during 120 days storage.
Methods Storage temperature test The pomegranate fruit was packed individually in 0.03 mm thick PE bags with a 5 mm diameter hole at the bottom and stored at (-1 f O S ) , ( 2 f 0 . 3 , (5f0.3,and (8fOS)"C, respectively, with 90-95% relative humidity (RH). Storage atmosphere test The pomegranate fruit were placed in five controlled atmosphere test-boxes (volume of 57 c m x 5 7 c m x 5 8 cm), in which the atmosphere mixture was designed as follows: treatment 1,3.0% CO, +3.0% 0,+94.0% N,; treatment 2, 5.0% CO,+ 8.0% 0,+87.0% N,; treatment 3,8.0% C0,+5.0% 0,+87.0% N,; treatment 4, 12.0% CO, + 12.0% 0, + 76.0% N,; control group was the natural air. The desired gas mixture was changed once every 10 days. The fruits were stored at (5.0 f 0 3 ° C . pH coating test A 0.5% carboxymethyl cellulose (CMC) solution was prepared by mixing 10.0 g sodium carboxymethyl cellulose (CMC-Na) powder and a little 95% alcohol into 2000 mL of distilled water maintained at 40°C with sufficient agitation to form a smooth suspension. After cooling, the pH was adjusted to 1.0, 3.5, 5.0, 7.0, and 9.0, respectively, with 20% citric acid solution and 10%NaOH solution. The fruits (peel pH 3.8-4.0) were dipped into the CMC solution of different pH for 15 s and air-dried at ambient temperature for 24 h. The fruit without dipping was taken as control. After treatment, the pomegranate fruit were packed individually in PE bags with a hole and stored at (5.0+ 0.5)"C with 90-95% relative humidity. Intermittent warming test The pomegranate fruit packed individually in PE bags with a hole were kept in opening cartons and stored at (5.0 2 0.5)"C. Cartons
82008.CAAS. All rights resewed.Published by Elsevier LM.
Study on the Mechanism of Browning of Pomegranate (Punica granarurn L. cv. Ganesh) Peel in Different
were placed at (15.0+0.5)"C for 24 h every 3 or 6 days, respectively, and then placed in the original condition, (5.OkOS)"C. The fruit kept at the stable temperature of (5.0 + 0.5)"C was taken as the control. Optimization of methods controlling browning test The optimum method of controlling peel browning was investigated by using L,(34) orthogonal test involving the storage temperature, atmosphere, pH coating and intermittent warming. The concrete method was as follows: The pomegranate fruits were coated with CMC film of different pH, dried naturally, and placed in the controlled atmosphere test-boxes. The boxes were filled with differently designed gas mixture, which was changed once every 10 days. The fruits were stored at the designed temperature and treated as intermittent warming experiment. Repeat the procedure above during the 120 days of storage.
Mensuration The browning index of peel was calculated by the following formula: C (Browning scale x Percentage of corresponding fruit within each class)/100. The appearance scale was as follows: 0 (0 score), no browning and glabrous surface; 1 (25 score), a slight browning and smooth surface; 2 (50 score), obvious browning and rough surface; 3 (75 score), serious browning and hollow surface depression; and 4 (100 score), black and encrusting surface. The tannin content of the peel was measured by the Folin-Denis colorimetry (Hou 2004); ascorbic acid oxidase (AAO) activity by iodine titration (Tang 1999); PPO activity by colorimetry (Pizzocaro et al. 1993); peroxidase (POD) activity by guaiacol colorimetry (Xu and Ye 1989); and catalase (CAT) activity by potassium permanganate titration (Li et al. 2000).
Statistical analysis
61
were significant and at the 1% level were extremely significant.
RESULTS Effect of storage temperature on pomegranate peel browning As shown in Fig. 1 , the browning index of pomegranate peel increased with storage time. The browning index of peel stored at (- 1 k 0 3 ° C rose markedly and reached the maximum of 1.0 by day 60 (P
Effect of storage atmosphere on pomegranate peel browning As shown in Fig.2, the browning index of peel increased in all treatments during storage. The browning indices of treatments 1 and 2 were only 0.21 and 0.23 after 120 days storage ( P > 0.05, compared between them; P < 0.01, compared with other treatments) and the pomegranate peel was still kept fresh and bright.
1.o
0.8 0.6 0.4
0.2
The data were analyzed using ANOVA. Mean differences were established by Duncan's multiple range test and r-test. All the data were expressed as means & standard error (X+ SE) and were calculated after 3 days of precooling. Differences between means at the 5% level
0
0
30
60
90
I20
Duration of storage (d)
Fig. 1 Changes of peel browning index of pomegranate fruit during storage at different temperatures (n =4).
02008,CAAS. All rights reserved. Publishedby ElsevlerLtd.
ZHANG You-lin et al.
68
However, there was serious peel browning in fruit by treatments 3 and 4, especially for treatment 4 with the index up to 1.00 by day 120.
Effectof pH coating solution on pomegranatepeel browning As shown in Fig.3, the browning index of peel increased in all treatments with storage time. The browning index of the pH 9.0 CMC coating fruit went up sharply and reached the top to 1.0 by day 60, (Pe 0.0 1, compared with other treatments), which was
x c
0
/
0.8 l'O[ 0.6
X
8
.2 .$
g
0.4
0.2 0
0
30
60
90
120
Duration of storage (d)
+Treatment 1 3.0% C0,+3.0% 0,+94.0%
N,
-A- Treatment 2 5.0% C0,+8.0% 0,+87.0% N,
+Treatment 3 8.0% C0,+5.0%
probably caused by the anthocyanin and tannin in the pomegranate peel that changed into blue or black pigment in alkaline condition, resulting in serious peel browning, wherein the browning indices of pH 1.0 and 3.5 CMC coating fruit were relatively lower ( P < 0.05, compared with control; P c 0.01, compared with pH 9.0; P>0.05, compared with pH 5.0 and 7.0) than other treatments after 120 days of storage, which indicated that the acidic condition conduced to maintaining better peel appearance.
Effect of intermittent warming on pomegranate peel browning As shown in Fig.4, intermittent warming treatments could inhibit peel browning during storage. The browning index of the fruit stored by intermittent warming every 6 days was just 0.21 but that of the control was 0.36 after 120 days of storage. It was probable that intermittent warming could prevent the chilling injury, protect the peel cells against harm, and keep the fruit from browning. There is a significance ( P < 0.05) among the three treatments after 120 days of storage and the intermittent warming every 6 days could control the peel browning effectively.
0,+87.0% N, Treatment 4 12.0% CO,+ 12.0% 0,+76.0% N, Control air
-0-
I .o
--C Control
Fig. 2 Changes of peel browning index of the pomegranate fruits during storage under different gas components (n = 4).
-6d
0
30
60
90
120
Duration of storage (d)
Fig. 4 Changes of peel browning index of the pomegranate fruits during storage using different intermittent warming (n =4). 0
30
90
60
120
Optimum storage conditions
Duration of storage (d) 1.0
+3.5
--t 7.0
*9.0
5.0 --O- Control
Fig. 3 Changes of peel browning index of the pomegranate fruits during storage under different pH values (n=4).
On the basis of single factor tests above, the L,(34) orthogonal experiment of the pomegranate fruits was carried out to study compositive effects of storage temperature, atmosphere, pH coating solution and in-
02008,CAAS. All rights RSBN&.Publishedby Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica grunaturn L. cv. Ganesh) Peel in Different
69
Table Results of L,(34) orthogonal experiment of the pomegranate fruits during storage and analysis of extremal deviations (n =4) Test 1 2 3
A Storage temperature ("C) A, (4.0k0.5) A, A,
4
A, (5.0i0.5)
5 6
A2
B Gas component C 0 2and O,(%) B, (4.0, 7.0) B, (5.0, 8.0) B, (6.0.9.0) Bl B2
A, A, (6.0i0.5)
B3
7
8
A3
9
A3 0.26 0.22
B* B3 0.26 0.26 0.27 0.01
Kl K2 K3
Range Sequence Optimal combination
0.30 0.08
Bl
C Different pH
D Intermittent warming (d)
Browning index Of peel
*
0.35 0.029 ab
0.24*0.020 cde 0.20*0.020 ef 0.22iO.039 def 0.15*0.015f 0.29*0.035 bed 0.21 k0.019 def 0.38*0.029 a 0.32 kO.025 abc
CADB A1B2C3D2
The different small letters indicate significant difference at P = 0.05.
termittent warming on the peel browning, and the optimum method of controlling the browning was determined. Samples were selected at random and their peel browning indices were measured after 120 days of storage. The results of orthogonal experiment and the analysis of extremal deviation were shown in Table. The optimum storage combination that could control p o m e g r a n a t e p e e l b r o w n i n g effectively w a s A,B,C,D,, which was (5.0 +0.5)"C, 5.0% CO,+ 8.0% O,+ 87.0% N,, pH 4.0 coating and intermittent warming for every 6 days. This result was inconsistent with the better combination of A,B,C,D,, which is (5.0*0.5)°C, 5.0% C0,+8.0% 0,+87.0% N,, pH 4.0 coating and intermittent warming for every 5 days. Therefore, in the following year, pair comparison test was set up between A,B,C,D, and A,B,C,D,. The test was replicated three times and there was no significance between two treatments ( P >0.05). Considering the intermittent warming and ventilation treatment, A,B,C,D, was determined as the final optimum combination during storage.
Relationship between tannin content and pomegranate peel browning As shown in Fig.5, tannin content of pomegranate peel decreased slowly for the first 90 days, then decreased sharply during remaining period. After 120 days of storage, tannin content was 0.083,0.096, and 0.15 1%, at (8.0 k 0.5), (2.0 k 0.5), and (5.0 k 0.5)"C, respectively. The content at (5.0k 0.5)"C storage was significantly higher than the other two groups. Fig.5
showed that peel browning index increased gradually with the decrease in tannin content in pomegranate peel. There was a negative interrelationship between them. This is probably the reason that the tannin was a substrate of pomegranate peel browning.
Relationship between AAO activity and pomegranate peel browning The AAO in plants exposed to the air could catalyze molecular oxygen, which oxidized ascorbic acid into dehydroascorbate acid, thereby made the antioxidation of ascorbic acid lost, leading to peel browning. As shown in Fig.6, AAO activity of pomegranate peel increased during storage in all the treatments. The increase was more rapid after 60 days of storage. AAO activity reached the highest value at (8.0 f 0.5)"C and the lowest value at (5.0kOS)"C after 120 days of storage. Generally, the lower the storage temperature, the lower was the AAO activity. However, the AAO activity at (2.0 k 0.5)"C was higher than that at (5.0 f 0.5)"C by day 90. In the later period of storage, chilling injury led to the physiological metabolic disorder in pomegranate fruits at (2.0 k0.5)"C. As a result, AAO activity increased. The results showed that there was a positive correlation between AAO activity of pomegranate peel and peel browning index.
Relationship between PPO activity and pomegranate peel browning Plant surface or tissue browning is mainly due to the
02008, CAAS. Ail rights resewed. Publishedby Elsevier Ltd.
ZHANG You-lin et al.
70
0.5
rT
1
2
0.4
0.8
E
0.3
0.6
I
c X
4
.-
P
8
0.2
0.4
.?
g
0.1
0.2
g
.-
C
B
0
0
30
0
60
90
120
Duration of storage (d)
--o-O-
-0-
8°C. tannin content 2°C. tannin content 8°C. browning index
5°C tannin content 2"C, browning index 5°C browning index
-&--t
-A-
Fig. 5 Relationship between tannin content and peel browning index of the pomegranate fruits (n=3).
0.8 L
oxidation of phenolic compounds into quinone compounds under aerobic conditions by polyphenol oxidase, then the quinone compounds undergoes polymerization forming brown polymeric pigments, leading to browning. As shown in Fig.7, PPO activity of pomegranate peel has risen quickly within 90 days of storage, reached the peak value by day 90, and then decreased slowly. PPO activity went down with the temperature decreasing before 90 days of storage, but it didn't obey this rule during the last 30 days of storage. PPO activity of the pomegranate peel stored at (2.0+0.5)"C was higher than that stored at (5.0+0.5)"C after 120 days of storage. It probably resulted from abnormal physiological metabolism caused by chilling injury. There was a significant correlation between PPO activity and peel browning index.
Relationship between POD activity and pomegranatepeel browning
0.6
d
m 0 0
30
60
90
120
-
Duration of storage (d)
-
-c+
-0-
-A-
8"C, AAO activity 2°C. AAO activity 8"C, browning index
5°C. AAO activity 2°C. browning index 5°C. browning index
-&-
Fig. 6 Relationship between AAO activity and peel browning index of the pomegranate fruits (n=3).
250 r
1
POD is another major enzyme in enzymatic browning reaction. It can oxidize polyphenol substances quickly in the presence of H,O, and cause fruits or vegetables to brown with PPO synergizing. As shown in Fig.8, POD activity of pomegranate peel increased gradually from beginning to end with great fluctuation during storage. Different temperatures had little effect on POD activity. By day 120, POD activity was 4 856 (8.O&0S0C), 5306 (5.0&0.5"C), and 5064 U g-' (2.0 + 0.5"C), respectively. The results showed that there was a positive correlation between POD activity of
1.0
200
0.8
150
0.6
;:
100
0.4
g
50
0.2
x
2
0
'il
8000
3.-
6 000
0.6
6s
4 000
0.4
2 000
0.2
-
-0-
-C-
,?
0 0
30
60
90
0.8
8
0
0
120
0
Duration of storage (d)
8°C. PPO activity 2"C, PPO activity 8°C. browning index
1
10000
M
2
1.o
g
30
60
90
120
Duration of storage (d)
-h-
5°C.PPO activity
-A-
5 ° C browning index
+ 2°C. browning index
Fig. 7 Relationship between PPO activity and peel browning index of the pomegranate fruits (n=3).
8"C, POD activity
-A-
5'C. POD activity
+2°C. POD activity
-m-
2°C. browning index browning index
-0-
--t
8°C. browning index
-5'C.
Fig. 8 Relationship between POD activity and peel browning index of the pomegranate fruits (n = 3).
02008,CAAS. All rights taserved. Publishedby Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica Rranafum L. cv. Ganesh) Peel in Different
pomegranate peel and peel browning index.
Relationship between CAT activity and pomegranatepeel browning CAT exists in the ferritin of fruit and vegetable tissues widely. Its activity has a certain relationship with metabolic intensity, anticoldness and antidisease of fruits and vegetables. In the course of respiratory metabolism, CAT could decompose H,O, into H,O and 0,, which prevented H,O, from accumulating in fruit and vegetable tissues. As shown in Fig.9, CAT activity of pomegranate peel had a downtrend during storage, while it declined rapidly at (8.0+0.5)"C but went down slowly at two other temperatures. By day 120, CAT activity was 4.22 (8.0k0S°C), 5.49 (5.0+0.5"C), and 5.06 (2.OkOS"C) mg H,O, g-' min", respectively. The results showed that there was a negative correlation between CAT activity of pomegranate peel and peel browning index.
10
1
0
30
60
90
120
Duration of storage (d) 8°C. CAT activity 2"C, CAT activity +8°C. browning index -c-
-A-
-c-
-w-A-
5°C. CAT activity 2 ° C browning index 5°C. browning index
Fig. 9 Relationship between CAT activity and peel browning index of the pomegranate fruits (n = 3).
DISCUSSION Many phenolic compounds, such as tyrosine, 3,4dihydroxyphenyl alanine, dopamine, hydroquinone, catechol, 4-methyl catechol, protocatechuic acid, caffeic acid, chlorogenic acid, anthocyanidin, flavonoid compounds, tannin, and sumac hydroxyketone, can cause the fruits browning. The main browning sub-
71
strates of coconut fruit were dopamine and chlorogenic acid (Jiang et al. 1992), the latter was also the browning substrate of pear, apple and peach (Wu et al. 1992). In the litchi peel, the substrates were catechol compounds, which formed browning substances as treated with PPO (Jiang 1991). Free polyphenols were the main browning substrates of longan peel, whereas the effect of anthocyanin was insignificant (Wu et al. 1999). Catechin tannin in the fruit and vegetable tissues was due to the ramification of catechol, and they were easily oxidized in the air and formed brown or black pigments. Tannin was involved in the banana peel browning (Bao et al. 2005). The higher was the tannin content, the easier was peel browning, especially in alkaline conditions. This result was confirmed by the fact that the tannin content of peach fruit decreased slowly during storage and the reduction of tannin content coincided with the Snow peach browning (Wang and Liu 1996). In this article, the browning index of sweet pomegranate peel in Lintong, Shaanxi, was gradually increased as the tannin content decreased during storage (Fig.5), which indicated that tannin was a substrate for browning. This is in accord with the theory that tannin resulted in peel browning proposed by predecessors. In addition, the pomegranate peel browned seriously in alkaline conditions (Fig.3) under the high oxygen concentration (Fig.2). The result fitted the tannin's character of oxidized browning or alkaline browning, suggesting that the pomegranate peel browning might be the tannin browning. Generally, phenolic compounds in plant organs or tissues are oxidized into quinone and water under enzymatic catalysis, and then the quinone is polymerized into brown polymeric pigments without enzymes, namely, the enzymatic browning. Substrates (phenolic compounds), enzymes and oxygen are three conditions for this reaction (Lin 2002). PPO is the general designation of the catechol oxidase and laccase (He and Bin 2001). They almost exist in all fruits as studied but different types of fruit have different nature of PPO. PPO of litchi peel could oxidize pyrocatechol and pdihydroxybenzene, and had a stronger effect on the former. However, it could not oxidize monoprotic phenol and resorcinol (Jiang 1991); PPO of longan peel could oxidize pyrogallol, pyrocatechol and 4-methyl-
Q2008. CAAS.All rightsreserved.Published by Elsevier Ltd.
ZHANG You-lin et a/.
12
catechol but not monoprotic phenol and resorcinol (Jiang 1999); PPO of loquat fruit could oxidize pyrocatechol but not monoprotic phenol, p-dihydroxydroxybenzene and resorcinol (Ding et al. 1998). POD could oxidize and polymerize phenols and flavonoids in the presence of H,O,, resulting in the tissue browning (Mayer and Hare1 1979; Lin et al. 1988; Jiang 1991). POD content of litchi peel was high. The increase of peel browning index was in accordance with the rapid rise of POD activity at room temperature during storage (Lin et al. 1988; Chen and Wang 1989). The higher was the PPO activity of peel, the easier was peel browning (Jiang 1991). Therefore, it was believed that POD took an important role in fruit browning (Chen and Wang 1989; Underhill and Critchley 1995). In this experiment, it was found that activities of PPO and POD in pomegranate peel correlated with the peel browning positively (Figs.7 and 8). Ascorbic acid in fruit and vegetable tissues is a strong antioxidant, which can prevent the fruits and vegetables from browning effectively. AAO exposed to the air can oxidize ascorbic acid into dehydroascorbate, which lead to the antioxidation of ascorbic acid losing and contributing to the tannin oxidation into browning substances. In this article, it was found that AAO activity in pomegranate peel correlated with the browning index positively (Fig.6). H,O, in the fruit and vegetable tissues has a strong oxidizability. It is the main reason for fruits and vegetable browning. CAT can decompose H,O, into H,O and 0,, prevent the accumulation of H,O, and control the peel browning. In the experiment, it was found that CAT activity in pomegranate peel correlated with the browning index negatively (Fig.9). It may be the reason that the accumulation of H,O, with the decline of CAT activity made the tannin oxidize to browning substances. The gas component is another factor for fruit browning. The browning was controlled effectively under the suitable gas component (low 0, and high CO,) (Fig.2). However, if 0, concentration was too low or CO, concentration was too high, reactive oxygen (O,, H,O,) would be produced by disorders of physiological metabolism and increase enzymatic browning (TomasBarberan and Espin 2001). Unsuitable gas component led to pomegranate peel browning during storage.
In addition, tannin of the pomegranate peel needs to be studied further, including its physical structure, chemical properties and biochemical mechanism on peel browning.
CONCLUSION According to the results, enzymatic browning was the main reason for peel browning of pomegranate fruit during storage and tannin was the main browning substrate. After precooling, the pomegranate fruits were stored by the following treatment: coated with 0.5% CMC solution (pH 4.0), placed under the gas component of 5.0% CO,+ 8.0% 0, + 87.0% N, as designed, stored at (5.0+0.5)”C, ventilated every 10 days and then treated with the intermittent warming for 24 h at (15.0+0.5)”C every 5 days. Repeat the operation in cycles during storage. Under above conditions, the pomegranate fruit had a good quality with 0.15 browning index and 97.8% good fruit rate after 120 days storage.
Acknowledgements This work was financed by the Natural Science Foundation of Shaanxi Province, China (2004C109), Key Technology R&D Program of Shaanxi Province, China (2003K02-G6), and Agricultural Science and Technology Development of Xi’an City, China (NG200313).
References Alkahtani H A. 1992. Intercultivar differences in quality and postharvest life of pomegranate influenced by partial drying. Journal of the American Society for Horticultural Science, 117, 100-104. Bao J Y,Liang S R, Zhao G J, Yang G M . 2005. Study on extraction technology and relations with browning of tannins from banana peel. Food Research and Development, 26,3-6. (in Chinese) Ben-Arie R, Or E. 1986. The development and control of husk scald on “Wonderful” pomegranate fruit during storage. Journal of the American Society for Horticultural Science, ll!, 395-399. Chen Y Z, Wang Y R. 1989. A study on peroxidase in litchee pericarp. Acta Botanica Austro Sinica, 5,47-52.(in Chinese) Ding C K, Chachin K, Ueda Y, Imahori Y. 1998. Purification and properties of polyphenol oxidase form loquat fruit. Journal
02008,CAAS.All rightsreserved. Published by Elsevier Ltd.
Study on the Mechanism of Browning of Pomegranate (Punica granatum L. cv. Ganesh) Peel in Different
of Agricultural and Food Chemistiy, 46,4144-4149. Elyatem S M, Kader A A. 1984. Post-harvest physiology and storage behaviour of pomegranate fruits. Scientia Horticulturae, 24, 287-298. Gao M Y, Shi F H, Chang N J. 1987. Experiment of pomegranate storage. Deciduous Fruit Tree, (3), 28-30. (in Chinese) He L H, Bin J H. 2001. Polyphenol oxidase in higher plants. Plant Physiology Communications, 37,340-345. (in Chinese) Hou M L. 2004. Food Analysis. Chemical Industry Press, Beijing. pp. 134-141. (in Chinese) Jiang Y M, Chen M D, Lin Z F, Chen F. 1991. Enzymatic browning of banana during low temperature storage. Acta Phytophysiologica Sinica, 17, 157-163. (in Chinese) Jiang Y M. 1991. Browning of fruit and its control. Journal of Plant, 18,22-23. (in Chinese) Jiang Y M. 1999. Purification and some properties of polyphenol oxidase of longan fruit. Food Chemistry,.66,75-79. Jiang Y M, Zhang D L, Liu S X, Chen F, Li Y B. 1992. A primary study on browning substrates of coconut. Plant Physiology Communications, 28,347-349. (in Chinese) Kader A A, Chordas A, Elyatem S M. 1984. Responses of pomegranates to ethylene treatment and storage temperature. California Agriculture, 38, 14-15. Kahn V. 1983. Multiple effects of hydrogen peroxide on the activity of avocado polyphenol oxidase. Phytochemistry, 22, 2155-2159. Li H S, Sun Q, Zhao S J, Zhang W H. 2000. The Theory and Technology of Plant Physiology and Biochemistry. Higher Education Press, Beijing. pp, 165-167. (in Chinese) Lin H T, Xi Y F, Chen S J. 2002. A review of enzymaticbrowning in fruit during storage. Journal of Fuzhou University, 30 (Suppl.), 696-703. (in Chinese) Lin Z F, Li S S, Zhang D L, Liu S X, Li Y B, Lin G Z, Chen M D. 1988. The changes of oxidation and peroxidation in postharvest litchi fruit. Acta Botanica Sinica, 30, 382-387. (in Chinese) Liu X H, Cai J, Huo S, Xu H D. 1995. Study on postharvest physiological characters of Tianhongdan pomegranate. Tianjin Agricultural Sciences, 1,30-31. (in Chinese) Liu X H, Hu Q X, Kou L P, Xu H D. 1997. The study on
13
pomegranate husk scald during storage. Journal of Northwest Forestry College, 12,93-96. (in Chinese) Mayer A M, Hare1 E. 1979. Polyphenol oxidases in plants. Phytochemistry, 18, 193-215. Pizzocaro F, Torreggiani D, Gilardi G. 1993. Inhibition of apple polyphenoloxidase (PPO) by ascorbic acid, citric acid and sodium chloride. Journal of Food Processing and Preservation, 17, 21-30. Sapers G M, Hicks K B, Phillips J G, Garzarella L, Pondish D L, Matulaitis R M, McCormack T J, Sondey S M, Seib PA, ElAtawy Y S. 1989. Control of enzymatic browning in apple with ascorbic acid derivatives,polyphenol oxidase inhibitors, and complexing agents. Journal of Food Science, 54, 9971002,1012. Tang Z C. 1999. Guide to the Experiment of Modern Plant Physiology. Science Press, Beijing. pp. 316-3 17. (in Chinese) Tomas-Barberan F A, Espin J C. 2001. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. Journal of the Science of Food and Agriculture, 81, 853-876. Underhill S J R, Critchley C. 1995. Cellular localisation of polyphenol oxidase and peroxidase activity in Litchi chinensis Sonn. pericarp. Australian Journal of Plant Physiology, 22, 627-632. Wang J, Liu C L. 1996. Primaryexplorationon changes of chemical composition of Xue peach under different storage conditions and its browning reason. China Fruit Research, (3), 12-14. (in Chinese) Wu G X, Zhou H W, Wang J M. 1992. Biochemical mechanism and substances determination of enzymic browning of yali pear (Pynrs bretschneideri Rehd.). Acta Horticulturae Sinica, 19, 198-202. (in Chinese) Wu Z X, Han D M, Ji Z L, Chen W X. 1999. Effect of sulphur dioxide treatment on enzymatic browning of longan pericarp during storage. Acta Horticulturae Sinica, 26, 91-95. (in Chinese) Xu L L, Ye M B. 1989. A method of continuous record to test the activity of peroxidase. Journal of Nanjing Agricultural University, 12, 80-83. (in Chinese) (Edited by ZHANG Juan)
~32008, CAAS. All rightsreserved. Publishedby Elsevier Ltd.