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Changes in post-harvest phytochemical qualities of broccoli florets during ambient and refrigerated storage
Food Chemistry 127 (2011) 1510–1514
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Changes in post-harvest phytochemical qualities of broccoli florets during ambient and refrigerated storage A. Nath ⇑, B. Bagchi, L.K. Misra, Bidyut C. Deka Division of Horticulture, ICAR Research Complex, Umiam, Meghalaya 793103, India
a r t i c l e
i n f o
Article history: Received 28 September 2010 Received in revised form 23 December 2010 Accepted 1 February 2011 Available online 2 March 2011 Keywords: Broccoli Phytochemicals Chlorophyll b-Carotene Antioxidant activity Post-harvest storage
a b s t r a c t The effect of ambient and refrigerated storage temperature on post-harvest phytochemical qualities of broccoli florets was investigated during storage. Fresh broccoli florets were packed in polypropylene (PP) micro-perforated film bags and stored, under open ambient storage conditions (15 ± 1 °C, 55 ± 2% RH), and laboratory refrigerated storage (4 ± 0.5 °C, 50 ± 2% RH) for a total period of 144 h. Quality of broccoli florets was evaluated in terms of physiological weight loss (PLW), ascorbic acid content, chlorophyll content, b-carotene and total antioxidant activity. Samples packed in PP micro-perforated film showed significantly (P < 0.05) lower losses of PLW, ascorbic acid, chlorophyll, b-carotene and total antioxidant activity (5.51%, 4.53%, 18.9%, 4.04% and 16.4%, respectively), during storage for up to 144 h under refrigerated conditions. For better phytochemical retention, the broccoli florets should be packed in PP micro-perforated film bags and stored under refrigerated conditions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Broccoli (Brassica oleracea L., Italica group) is a high-priced green vegetable. It is highly valued due to its richness in vitamins, antioxidants, anti-carcinogenic compounds (Nestle, 1998) and health-promoting phytochemicals (Yuan, Sun, Yuan, & Wang, 2010). Epidemiological studies have shown an inverse association between the consumption of Brassica vegetables and the risk of cancer (Day et al., 1994). Of the case-controlled studies, 56% demonstrate a strong association between increased broccoli consumption and protection against cancer (Verhoeven, Goldbohm, Van Poppel, Verhagen, & Van den Brandt, 1996). This protective effect has largely been attributed to the complement of phytochemicals in broccoli, which include vitamins C and E, the flavonols quercetin, kaempferol, the carotenoids b-carotene, lutein, and the glucosinolates (Podsedek, 2007). Green-coloured florets of broccoli (looking fresh) are preferred by consumers. Broccoli reaches the retail markets at least one or two days after harvest and, by the time it reaches market, the quality begins to deteriorate. This crop is generally sold in the Asian retail market without any packaging or is sometimes packed in polyethylene bags of 250–500 g. Most of the time, consumers (after purchase if not consumed immediately) keep it under ambient or
⇑ Corresponding author. Tel.: +91 9612106156. E-mail address: [email protected] (A. Nath). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.007
common refrigerated conditions (at 4 °C) with or without packaging. The deterioration in quality of this vegetable is very rapid in both situations. Different reports are available on degradation of chlorophyll content during controlled atmosphere (CA) storage and also at specified storage temperatures under modified atmosphere packaging (MAP). When broccoli was stored at 0 °C and 95% relative humidity, its green colour was maintained for much longer than when unrefrigerated (Wang & Hruschka, 1977). Lebermann, Nelson, and Steinberg (1968) found that total chlorophyll content of broccoli heads decreased after 16 days of storage and the loss was much greater at 7 °C than at 1 °C. Deschene, Paliyath, Lougheed, Dumbroff, and Thompson (1991) reported that florets from freshly cut heads of broccoli rapidly senesced when stored in air at 23 or 10 °C. Chlorophyll levels declined by 80–90% within 4 days at 23 °C and within 10 days at 10 °C. Takeda, Yoza, Nogata, and Ohta (1993) found that chlorophyll levels in broccoli decreased during storage at 20 or 23 °C whereas, at 2 °C, chlorophyll levels remained nearly constant. Storage, transport and processing significantly influence the levels of ascorbic acid, glucosinolates and flavonoids in broccoli (Leja, Mareczek, Starzynska, & Rozek, 2001; Vallejo, Tomas-Barberan, & Garcia-Viguera, 2003). Vallejo et al. (2003) demonstrated major losses of total glucosinolates (71–80%) and total flavonoids (62–59%), but not vitamin C, in broccoli inflorescences stored for 7 days at 1 °C in mimic cold storage, followed by a 3-day period at 15 °C in a mimic retail period. Conversely, Winkler, Faragher, Franz, Imsic, and Jones (2007) showed that extended storage (1
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or 4 °C for up to 28 days), followed by a 3 day period, did not influence glucosinolates or flavonoids in broccoli inflorescences. To date, there is no systematic study representing the changes that take place in physiological loss in weight (PLW), ascorbic acid, chlorophyll content, b-carotene and total antioxidant activity during ambient and refrigerated storage of broccoli. Therefore, a study was conducted to monitor the changes in phytochemical qualities of broccoli florets during ambient and refrigerated storage.
was repeated until the residue was colourless. The volume was then made up to 100 ml with 80% acetone. The optical densities at 645 and 663 nm were recorded for each sample and the total chlorophyll was estimated by the following formula. Total Chlorophyll ðmg=gÞ ¼
ð20:2 OD at 645 nm þ 8:02 OD at 663 nm VÞ 100 W
where, OD is optical density, V is volume of the extract (ml) and W is weight of the sample (g).
2. Materials and methods 2.5. b-Carotene
2.1. Broccoli (B. oleracea L.) Broccoli (cv. ‘Pushpa’ very common and suitable for hill regions of India) florets, at commercial stage, were harvested early in the morning from the Horticulture Firm, Division of Horticulture, ICAR Research Complex, Umiam, Meghalaya. For this experiment, broccoli heads, of 100–250 g size of inflorescence, were harvested. The harvested heads were immediately brought to the Post-harvest Technology Laboratory, ICAR Research Complex, Umiam, Meghalaya for further studies. In this experiment, one set of samples, having a total weight of 200–250 g (8–10 florets) was kept in plastic perforated trays, both under open ambient storage (BTAT) conditions (15 ± 1 °C and 55 ± 2% RH) and laboratory refrigerated storage (BTRS) conditions (4 ± 0.5 °C and 50 ± 2% RH). In another set, 8–10 florets/bag were packaged, using a commercial polypropylene (PP) film with 10 pin holes, and stored under ambient (BPPAT) conditions (15 ± 1 °C and 55 ± 2% RH) and laboratory refrigerated storage (BPPRT) conditions (4 ± 0.5 °C and 50 ± 2% RH). Each treatment was replicated five times. Broccoli florets were sampled at 0, 48, 96 and 144 h intervals for further analysis. 2.2. Physiological loss in weight (PLW) PLW was calculated as cumulative% loss in weight, based on the initial weight (before storage) and loss in weight was recorded at the time of periodical sampling during storage. 2.3. Ascorbic acid Ascorbic acid content was determined by using the 2,6-dichlorophenol-indophenol dye method of Freed (1966). The 2.5 g of broccoli floret samples were ground with about 25 ml of 4% oxalic acid and filtered through Whatman No. 4 filter paper. The filtrate was collected in a 50 ml volumetric flask and the volume was made up with 4% oxalic acid and titrated against the standard dye to a pink point. The amount of ascorbic acid was calculated, using the formula given below, and expressed as mg/100 g on a fresh weight (FW) basis.
Ascorbic acid ðmg=100 gÞ ¼
b-Carotene content of broccoli samples was determined by using the colourimetric method of Srivastava and Kumar (2002). Five grams of fresh sample were crushed in 10–15 ml acetone by adding a few crystals of anhydrous Na2SO4. The supernatant was decanted into a beaker and the process was repeated twice. The combined supernatant was transferred to a separating funnel and 10–15 ml of petroleum ether were added and mixed thoroughly. After discarding the lower layer, the upper layer was collected in a 100 ml volumetric flask and the volume was made up with petroleum ether. The OD was recorded at 452 nm and b-carotene was expressed as mg/100 g on a fresh weight (FW) basis by the following formula
b Carotene ðmg=100gÞ ¼
OD 13:9 104 100 weight of the sample 560 1000
2.6. Antioxidant activity Antioxidant activity of the sample was determined using the ferric reducing antioxidant power (FRAP) assay of Benzic and Strain (1996) as a measure of ‘‘Antioxidant Power.’’ The working FRAP reagent was prepared by mixing 10 ml of 300 mM acetate buffer, 1 ml of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM hydrochloric acid and 1 ml of 20 mM ferric chloride. The 100 ll (0.1 ml) of extract were added to 3.9 ml of freshly prepared FRAP reagent and mixed thoroughly, and a reading was taken at 593 nm in a UV–Vis spectrophotometer. A standard curve was prepared using different concentrations of trolox. The results were expressed as micromoles of trolox equivalents per gramme fresh weight. 2.7. Statistical analysis Results were analysed by analysis of variance (ANOVA) and standard error (SE) of mean was also calculated for presentation in figures.
Titre value Dye factor Volume made up 100 Aliquot of extract taken for estimation Wt: or vol: of the sample taken for estimation
2.4. Total chlorophyll content
3. Results and discussion
Total chlorophyll content of broccoli sample on a fresh weight (FW) basis was determined by using the colourimetric method of Singh (1977). About 2 g of finely cut and well mixed representative broccoli floret sample were placed in a clean mortar. This tissue was ground to a fine pulp by adding 20 ml of 80% acetone. Then the material was centrifuged (5000 rpm for 5 min) and supernatant was transferred to a 100 ml volumetric flask. This procedure
3.1. Changes in cumulative PLW Results on PLW of broccoli florets, irrespective of treatments during storage, indicated that weight loss was gradually increased (Fig. 1). This could be mainly due to continuous loss of moisture due to transpiration from the fruit and respiration. The maximum weight loss (41.7%) was recorded in the samples stored in open
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Fig. 1. Effect of storage atmosphere on PLW (%) of broccoli florets during short term storage (means ± SE, n = 5).
ambient in plastic trays, followed by open refrigerated conditions in plastic trays (26.96%) and PP micro-perforated-packed ambient-stored florets (13.6%) on 144 h of storage. However, among the different treatments, the minimum weight loss (5.51%) was observed in micro-perforated packed florets in refrigerated storage. Minimum weight loss in micro-perforated polythene packed florets could be due to less availability of oxygen for respiration, which ultimately retarded the rate of respiration, thereby lowering the moisture loss due to transpiration. Barth, Perry, Schmidt, and Klein (1992) also reported similar findings in broccoli stored under non-packaged conditions. Moisture loss of broccoli heads (Barth, Kerbel, Perry, & Schmidt, 1993) and florets (Barth, Weston, & Langlois, 1994) during storage was reduced when broccoli was packaged in vented polymeric, perforated films. 3.2. Changes in ascorbic acid The initial ascorbic acid content of fresh broccoli florets was found to be 130 mg/100 g and it decreased linearly during storage under different treatments (Fig. 2). However, broccoli florets packed in micro-perforated PP, and stored under refrigerated conditions (4 °C), showed no significant change in ascorbic acid content by the end of storage (144 h). The ascorbic acid content decreased rapidly in the florets kept in open ambient plastic tray compared to the florets kept in ambient PP micro-perforated packets and open plastic refrigerated trays (r2 = 0.989). By 144 h of storage, ascorbic acid content decreased from 130 mg/100 g to 35.3 mg/100 g FW, a 72.9% reduction, in the open ambient plastic tray samples, while it decreased from 130 to 92 mg/100 g FW, a 29.2% decline, in the open refrigerated samples of broccoli florets in plastic trays. During storage, the samples in open ambient plas-
Fig. 2. Effect of storage atmosphere on ascorbic acid content (mg/100 g FW) of broccoli florets during short term storage (means ± SE, n = 5).
tic trays had significantly lower ascorbic acid contents than did the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated (in plastic trays) or ambient PP micro-perforatedstored samples (P < 0.05) at 96 h (Fig. 2). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had a significantly lower ascorbic acid content than had the PP microperforated-packed refrigerated stored samples (P < 0.01). These results were in agreement with the earlier report of MAP of broccoli in similar packaging material (Rai, Tyagi, Jha, & Mohan, 2008). Wang and Win (2002) and Leja et al. (2001) also reported rapid decrease in ascorbic acid content of broccoli and other vegetables during storage at 15 °C. Higher ascorbic acid (71.9–95.1%) retention in PP micro-perforated-packed broccoli might be due to limited atmospheric oxygen available for oxidation. During storage, oxidising enzymes, e.g. ascorbic acid oxidase, peroxodase, catalase and polyphenol oxidase, might help to reduce the ascorbic acid of the fruits and vegetables (Mapson, 1970). 3.3. Changes in total chlorophyll retention During storage at 4 °C, no significant change was observed in total chlorophyll content of broccoli florets in the PP micro-perforated-packed stored samples (Fig. 3). However, total chlorophyll contents linearly declined, in both the open ambient in plastic tray and ambient micro-perforated-packed florets, and also in open plastic tray refrigerated stored samples (r2 = 0.989). By 144 h of storage, total chlorophyll content decreased from 0.402 to 0.135 mg/g FW, a 66.4% reduction in the open ambient plastic tray samples. Total chlorophyll level decreased from 0.402 to 0.178 mg/ g FW, a 55.7% decline in the open refrigerated samples of broccoli florets in plastic trays. During storage, the samples in open ambient plastic trays had significantly lower levels of total chlorophyll than had the PP micro perforated packed refrigerated and open refrigerated (in plastic tray)-stored samples at 96 h, (P < 0.05). However, no significant difference in total chlorophyll content was found in ambient PP micro-perforated-packed florets (Fig. 3). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had significantly lower levels of total chlorophyll than had the PP micro-perforated-packed refrigerated stored samples (P < 0.01). During the post-harvest period, broccoli deterioration was described as flower head yellowing, turgor loss, and flaccid branchlets and florets (King & Morris, 1994). It has been demonstrated that losses in total chlorophyll (Aiamla-or, Kaewsuksaeng, Shigyo, & Yamauchi, 2010) and weight loss (Barth et al., 1992; Barth et al., 1993) positively correlate with broccoli deterioration. In the present study, it was observed that broccoli florets, in either open ambient and refrigerated storage or in PP micro-perforatedstorage, underwent chlorophyll degradation. It was also noticed
Fig. 3. Effect of storage atmosphere on chlorophyll content (mg/g FW) of broccoli florets during short term storage (means ± SE, n = 5).
A. Nath et al. / Food Chemistry 127 (2011) 1510–1514
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that a higher temperature caused more degradation of total chlorophyll in the broccoli florets (Fig. 3) after 144 h of storage and a lower temperature resulted in less deterioration during post-harvest storage. 3.4. Changes in b-carotene content The b-carotene content of broccoli florets, under different storage conditions, is presented in Fig. 4. Initial b-carotene content of broccoli was found to be 26.5 mg/100 g and it decreased with the advancement of storage time, irrespective of treatments (Fig. 4). However, broccoli florets packed in PP micro-perforated and stored under refrigerated conditions (4 °C) showed no significant change in b-carotene content by the end of storage (144 h). b-Carotene content decreased more rapidly in the florets of open ambient plastic tray samples than in ambient PP micro-perforated-packed florets or open plastic tray refrigerated samples (r2 = 0.995). After144 h of storage, b-carotene content decreased from 26.5 to 7.62 mg/100 g FW, a 71.3% reduction in the open ambient plastic tray samples while, b-carotene content decreased from 26.5 to 16.2 mg/100 g FW, a 38.9% decline, in the open refrigerated samples in plastic trays. During storage, the samples in open ambient plastic trays had significantly lower b-carotene than had the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated in plastic tray or ambient PP micro-perforated-stored samples (P < 0.05) at 96 h, (Fig. 4). The open ambient in plastic tray-stored broccoli florets registered significantly lower b-carotene contents than did the PP micro-perforated-packed refrigerated stored samples (P < 0.01) at the end of storage (144 h). These results were in agreement with an earlier report on MAP of broccoli (Kidmose & Hansen, 1999). Hussein et al. (2000) also reported decrease in b-carotene content of broccoli during storage at higher temperature. Higher b-carotene retention in PP micro-perforated-packed broccoli might be due to the limited atmospheric oxygen available for oxidation and, therefore, oxidising enzymes are less active. During storage, oxidising enzymes, e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX), might have helped to reduce the b-carotene of the broccoli (Li, Maezawa, & Nakano, 2004). 3.5. Changes in antioxidant activity The antioxidant activity of fresh harvested broccoli florets, before storage, was found to be 3.96 lmol trolox/g. Changes in antioxidant activities of broccoli florets during storage, under different treatments, are presented in Fig. 5. A linear decrease in antioxidant activities was recorded in broccoli samples, irrespective of treatments, with the advancement of storage time (Fig. 5). However,
Fig. 5. Effect of storage atmosphere on total antioxidant activity (lmol trolox/g) of broccoli florets during short term storage (means ± SE, n = 5).
broccoli florets packed in PP micro-perforated film and stored under refrigerated conditions (4 °C) showed no significant change in antioxidant activities by the end of storage (144 h). Antioxidant activities decreased more rapidly in the florets of open ambient plastic tray samples than in ambient PP micro-perforated-packed florets or open plastic tray refrigerated samples (r2 = 0.964). After 144 h of storage, antioxidant activities decreased from 3.96 to 1.83 lmol trolox/g FW, a 53.8% reduction in the open ambient plastic tray samples, while antioxidant activities decreased from 3.96 to 2.00 lmol trolox/g FW, a 49.5% decline, in the open refrigerated samples of broccoli florets in plastic trays. During storage, the open ambient (in plastic tray treated)-samples had significantly lower antioxidant activities than had the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated in plastic trays or ambient PP micro-perforated-stored samples (P < 0.05) at 96 h (Fig. 5). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had significantly lower antioxidant activities than had the PP micro-perforated-packed refrigerated stored samples (P < 0.01). Costa, Civello, Chaves, and Martinez (2005) reported similar results in hot air-treated broccoli florets during storage at 20 °C. However, by contrast, Leja et al. (2001) reported increase in antioxidant activity of broccoli (Cv. Lord) flower buds during short-term storage at 20 and at 5 °C for 3 and 7 days, respectively, either non-packaged or packaged in polymeric films. Higher antioxidant activities in PP micro-perforated-packed broccoli might be due to the limited available atmospheric oxygen. 4. Conclusions Chlorophyll, ascorbic acid, b-carotene and total antioxidant content of broccoli florets underwent rapid changes during post-harvest storage. In preventing deterioration of broccoli during storage, temperature and packaging materials played significant roles in retention of phytochemicals. Broccoli florets stored under open ambient conditions (15 °C) showed greater losses in chlorophyll, ascorbic acid, b-carotene and total antioxidant contents than did those in other treatments. Among the treatments, PP microperforated film bag samples stored under refrigerated conditions (4 °C) were able to retain maximum phytochemicals during storage for up to 144 h. References
Fig. 4. Effect of storage atmosphere on b-carotene content (mg/100 g FW) of broccoli florets during short term storage (means ± SE, n = 5).
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Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Changes in post-harvest phytochemical qualities of broccoli florets during ambient and refrigerated storage A. Nath ⇑, B. Bagchi, L.K. Misra, Bidyut C. Deka Division of Horticulture, ICAR Research Complex, Umiam, Meghalaya 793103, India
a r t i c l e
i n f o
Article history: Received 28 September 2010 Received in revised form 23 December 2010 Accepted 1 February 2011 Available online 2 March 2011 Keywords: Broccoli Phytochemicals Chlorophyll b-Carotene Antioxidant activity Post-harvest storage
a b s t r a c t The effect of ambient and refrigerated storage temperature on post-harvest phytochemical qualities of broccoli florets was investigated during storage. Fresh broccoli florets were packed in polypropylene (PP) micro-perforated film bags and stored, under open ambient storage conditions (15 ± 1 °C, 55 ± 2% RH), and laboratory refrigerated storage (4 ± 0.5 °C, 50 ± 2% RH) for a total period of 144 h. Quality of broccoli florets was evaluated in terms of physiological weight loss (PLW), ascorbic acid content, chlorophyll content, b-carotene and total antioxidant activity. Samples packed in PP micro-perforated film showed significantly (P < 0.05) lower losses of PLW, ascorbic acid, chlorophyll, b-carotene and total antioxidant activity (5.51%, 4.53%, 18.9%, 4.04% and 16.4%, respectively), during storage for up to 144 h under refrigerated conditions. For better phytochemical retention, the broccoli florets should be packed in PP micro-perforated film bags and stored under refrigerated conditions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Broccoli (Brassica oleracea L., Italica group) is a high-priced green vegetable. It is highly valued due to its richness in vitamins, antioxidants, anti-carcinogenic compounds (Nestle, 1998) and health-promoting phytochemicals (Yuan, Sun, Yuan, & Wang, 2010). Epidemiological studies have shown an inverse association between the consumption of Brassica vegetables and the risk of cancer (Day et al., 1994). Of the case-controlled studies, 56% demonstrate a strong association between increased broccoli consumption and protection against cancer (Verhoeven, Goldbohm, Van Poppel, Verhagen, & Van den Brandt, 1996). This protective effect has largely been attributed to the complement of phytochemicals in broccoli, which include vitamins C and E, the flavonols quercetin, kaempferol, the carotenoids b-carotene, lutein, and the glucosinolates (Podsedek, 2007). Green-coloured florets of broccoli (looking fresh) are preferred by consumers. Broccoli reaches the retail markets at least one or two days after harvest and, by the time it reaches market, the quality begins to deteriorate. This crop is generally sold in the Asian retail market without any packaging or is sometimes packed in polyethylene bags of 250–500 g. Most of the time, consumers (after purchase if not consumed immediately) keep it under ambient or
⇑ Corresponding author. Tel.: +91 9612106156. E-mail address: [email protected] (A. Nath). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.007
common refrigerated conditions (at 4 °C) with or without packaging. The deterioration in quality of this vegetable is very rapid in both situations. Different reports are available on degradation of chlorophyll content during controlled atmosphere (CA) storage and also at specified storage temperatures under modified atmosphere packaging (MAP). When broccoli was stored at 0 °C and 95% relative humidity, its green colour was maintained for much longer than when unrefrigerated (Wang & Hruschka, 1977). Lebermann, Nelson, and Steinberg (1968) found that total chlorophyll content of broccoli heads decreased after 16 days of storage and the loss was much greater at 7 °C than at 1 °C. Deschene, Paliyath, Lougheed, Dumbroff, and Thompson (1991) reported that florets from freshly cut heads of broccoli rapidly senesced when stored in air at 23 or 10 °C. Chlorophyll levels declined by 80–90% within 4 days at 23 °C and within 10 days at 10 °C. Takeda, Yoza, Nogata, and Ohta (1993) found that chlorophyll levels in broccoli decreased during storage at 20 or 23 °C whereas, at 2 °C, chlorophyll levels remained nearly constant. Storage, transport and processing significantly influence the levels of ascorbic acid, glucosinolates and flavonoids in broccoli (Leja, Mareczek, Starzynska, & Rozek, 2001; Vallejo, Tomas-Barberan, & Garcia-Viguera, 2003). Vallejo et al. (2003) demonstrated major losses of total glucosinolates (71–80%) and total flavonoids (62–59%), but not vitamin C, in broccoli inflorescences stored for 7 days at 1 °C in mimic cold storage, followed by a 3-day period at 15 °C in a mimic retail period. Conversely, Winkler, Faragher, Franz, Imsic, and Jones (2007) showed that extended storage (1
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or 4 °C for up to 28 days), followed by a 3 day period, did not influence glucosinolates or flavonoids in broccoli inflorescences. To date, there is no systematic study representing the changes that take place in physiological loss in weight (PLW), ascorbic acid, chlorophyll content, b-carotene and total antioxidant activity during ambient and refrigerated storage of broccoli. Therefore, a study was conducted to monitor the changes in phytochemical qualities of broccoli florets during ambient and refrigerated storage.
was repeated until the residue was colourless. The volume was then made up to 100 ml with 80% acetone. The optical densities at 645 and 663 nm were recorded for each sample and the total chlorophyll was estimated by the following formula. Total Chlorophyll ðmg=gÞ ¼
ð20:2 OD at 645 nm þ 8:02 OD at 663 nm VÞ 100 W
where, OD is optical density, V is volume of the extract (ml) and W is weight of the sample (g).
2. Materials and methods 2.5. b-Carotene
2.1. Broccoli (B. oleracea L.) Broccoli (cv. ‘Pushpa’ very common and suitable for hill regions of India) florets, at commercial stage, were harvested early in the morning from the Horticulture Firm, Division of Horticulture, ICAR Research Complex, Umiam, Meghalaya. For this experiment, broccoli heads, of 100–250 g size of inflorescence, were harvested. The harvested heads were immediately brought to the Post-harvest Technology Laboratory, ICAR Research Complex, Umiam, Meghalaya for further studies. In this experiment, one set of samples, having a total weight of 200–250 g (8–10 florets) was kept in plastic perforated trays, both under open ambient storage (BTAT) conditions (15 ± 1 °C and 55 ± 2% RH) and laboratory refrigerated storage (BTRS) conditions (4 ± 0.5 °C and 50 ± 2% RH). In another set, 8–10 florets/bag were packaged, using a commercial polypropylene (PP) film with 10 pin holes, and stored under ambient (BPPAT) conditions (15 ± 1 °C and 55 ± 2% RH) and laboratory refrigerated storage (BPPRT) conditions (4 ± 0.5 °C and 50 ± 2% RH). Each treatment was replicated five times. Broccoli florets were sampled at 0, 48, 96 and 144 h intervals for further analysis. 2.2. Physiological loss in weight (PLW) PLW was calculated as cumulative% loss in weight, based on the initial weight (before storage) and loss in weight was recorded at the time of periodical sampling during storage. 2.3. Ascorbic acid Ascorbic acid content was determined by using the 2,6-dichlorophenol-indophenol dye method of Freed (1966). The 2.5 g of broccoli floret samples were ground with about 25 ml of 4% oxalic acid and filtered through Whatman No. 4 filter paper. The filtrate was collected in a 50 ml volumetric flask and the volume was made up with 4% oxalic acid and titrated against the standard dye to a pink point. The amount of ascorbic acid was calculated, using the formula given below, and expressed as mg/100 g on a fresh weight (FW) basis.
Ascorbic acid ðmg=100 gÞ ¼
b-Carotene content of broccoli samples was determined by using the colourimetric method of Srivastava and Kumar (2002). Five grams of fresh sample were crushed in 10–15 ml acetone by adding a few crystals of anhydrous Na2SO4. The supernatant was decanted into a beaker and the process was repeated twice. The combined supernatant was transferred to a separating funnel and 10–15 ml of petroleum ether were added and mixed thoroughly. After discarding the lower layer, the upper layer was collected in a 100 ml volumetric flask and the volume was made up with petroleum ether. The OD was recorded at 452 nm and b-carotene was expressed as mg/100 g on a fresh weight (FW) basis by the following formula
b Carotene ðmg=100gÞ ¼
OD 13:9 104 100 weight of the sample 560 1000
2.6. Antioxidant activity Antioxidant activity of the sample was determined using the ferric reducing antioxidant power (FRAP) assay of Benzic and Strain (1996) as a measure of ‘‘Antioxidant Power.’’ The working FRAP reagent was prepared by mixing 10 ml of 300 mM acetate buffer, 1 ml of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM hydrochloric acid and 1 ml of 20 mM ferric chloride. The 100 ll (0.1 ml) of extract were added to 3.9 ml of freshly prepared FRAP reagent and mixed thoroughly, and a reading was taken at 593 nm in a UV–Vis spectrophotometer. A standard curve was prepared using different concentrations of trolox. The results were expressed as micromoles of trolox equivalents per gramme fresh weight. 2.7. Statistical analysis Results were analysed by analysis of variance (ANOVA) and standard error (SE) of mean was also calculated for presentation in figures.
Titre value Dye factor Volume made up 100 Aliquot of extract taken for estimation Wt: or vol: of the sample taken for estimation
2.4. Total chlorophyll content
3. Results and discussion
Total chlorophyll content of broccoli sample on a fresh weight (FW) basis was determined by using the colourimetric method of Singh (1977). About 2 g of finely cut and well mixed representative broccoli floret sample were placed in a clean mortar. This tissue was ground to a fine pulp by adding 20 ml of 80% acetone. Then the material was centrifuged (5000 rpm for 5 min) and supernatant was transferred to a 100 ml volumetric flask. This procedure
3.1. Changes in cumulative PLW Results on PLW of broccoli florets, irrespective of treatments during storage, indicated that weight loss was gradually increased (Fig. 1). This could be mainly due to continuous loss of moisture due to transpiration from the fruit and respiration. The maximum weight loss (41.7%) was recorded in the samples stored in open
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Fig. 1. Effect of storage atmosphere on PLW (%) of broccoli florets during short term storage (means ± SE, n = 5).
ambient in plastic trays, followed by open refrigerated conditions in plastic trays (26.96%) and PP micro-perforated-packed ambient-stored florets (13.6%) on 144 h of storage. However, among the different treatments, the minimum weight loss (5.51%) was observed in micro-perforated packed florets in refrigerated storage. Minimum weight loss in micro-perforated polythene packed florets could be due to less availability of oxygen for respiration, which ultimately retarded the rate of respiration, thereby lowering the moisture loss due to transpiration. Barth, Perry, Schmidt, and Klein (1992) also reported similar findings in broccoli stored under non-packaged conditions. Moisture loss of broccoli heads (Barth, Kerbel, Perry, & Schmidt, 1993) and florets (Barth, Weston, & Langlois, 1994) during storage was reduced when broccoli was packaged in vented polymeric, perforated films. 3.2. Changes in ascorbic acid The initial ascorbic acid content of fresh broccoli florets was found to be 130 mg/100 g and it decreased linearly during storage under different treatments (Fig. 2). However, broccoli florets packed in micro-perforated PP, and stored under refrigerated conditions (4 °C), showed no significant change in ascorbic acid content by the end of storage (144 h). The ascorbic acid content decreased rapidly in the florets kept in open ambient plastic tray compared to the florets kept in ambient PP micro-perforated packets and open plastic refrigerated trays (r2 = 0.989). By 144 h of storage, ascorbic acid content decreased from 130 mg/100 g to 35.3 mg/100 g FW, a 72.9% reduction, in the open ambient plastic tray samples, while it decreased from 130 to 92 mg/100 g FW, a 29.2% decline, in the open refrigerated samples of broccoli florets in plastic trays. During storage, the samples in open ambient plas-
Fig. 2. Effect of storage atmosphere on ascorbic acid content (mg/100 g FW) of broccoli florets during short term storage (means ± SE, n = 5).
tic trays had significantly lower ascorbic acid contents than did the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated (in plastic trays) or ambient PP micro-perforatedstored samples (P < 0.05) at 96 h (Fig. 2). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had a significantly lower ascorbic acid content than had the PP microperforated-packed refrigerated stored samples (P < 0.01). These results were in agreement with the earlier report of MAP of broccoli in similar packaging material (Rai, Tyagi, Jha, & Mohan, 2008). Wang and Win (2002) and Leja et al. (2001) also reported rapid decrease in ascorbic acid content of broccoli and other vegetables during storage at 15 °C. Higher ascorbic acid (71.9–95.1%) retention in PP micro-perforated-packed broccoli might be due to limited atmospheric oxygen available for oxidation. During storage, oxidising enzymes, e.g. ascorbic acid oxidase, peroxodase, catalase and polyphenol oxidase, might help to reduce the ascorbic acid of the fruits and vegetables (Mapson, 1970). 3.3. Changes in total chlorophyll retention During storage at 4 °C, no significant change was observed in total chlorophyll content of broccoli florets in the PP micro-perforated-packed stored samples (Fig. 3). However, total chlorophyll contents linearly declined, in both the open ambient in plastic tray and ambient micro-perforated-packed florets, and also in open plastic tray refrigerated stored samples (r2 = 0.989). By 144 h of storage, total chlorophyll content decreased from 0.402 to 0.135 mg/g FW, a 66.4% reduction in the open ambient plastic tray samples. Total chlorophyll level decreased from 0.402 to 0.178 mg/ g FW, a 55.7% decline in the open refrigerated samples of broccoli florets in plastic trays. During storage, the samples in open ambient plastic trays had significantly lower levels of total chlorophyll than had the PP micro perforated packed refrigerated and open refrigerated (in plastic tray)-stored samples at 96 h, (P < 0.05). However, no significant difference in total chlorophyll content was found in ambient PP micro-perforated-packed florets (Fig. 3). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had significantly lower levels of total chlorophyll than had the PP micro-perforated-packed refrigerated stored samples (P < 0.01). During the post-harvest period, broccoli deterioration was described as flower head yellowing, turgor loss, and flaccid branchlets and florets (King & Morris, 1994). It has been demonstrated that losses in total chlorophyll (Aiamla-or, Kaewsuksaeng, Shigyo, & Yamauchi, 2010) and weight loss (Barth et al., 1992; Barth et al., 1993) positively correlate with broccoli deterioration. In the present study, it was observed that broccoli florets, in either open ambient and refrigerated storage or in PP micro-perforatedstorage, underwent chlorophyll degradation. It was also noticed
Fig. 3. Effect of storage atmosphere on chlorophyll content (mg/g FW) of broccoli florets during short term storage (means ± SE, n = 5).
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that a higher temperature caused more degradation of total chlorophyll in the broccoli florets (Fig. 3) after 144 h of storage and a lower temperature resulted in less deterioration during post-harvest storage. 3.4. Changes in b-carotene content The b-carotene content of broccoli florets, under different storage conditions, is presented in Fig. 4. Initial b-carotene content of broccoli was found to be 26.5 mg/100 g and it decreased with the advancement of storage time, irrespective of treatments (Fig. 4). However, broccoli florets packed in PP micro-perforated and stored under refrigerated conditions (4 °C) showed no significant change in b-carotene content by the end of storage (144 h). b-Carotene content decreased more rapidly in the florets of open ambient plastic tray samples than in ambient PP micro-perforated-packed florets or open plastic tray refrigerated samples (r2 = 0.995). After144 h of storage, b-carotene content decreased from 26.5 to 7.62 mg/100 g FW, a 71.3% reduction in the open ambient plastic tray samples while, b-carotene content decreased from 26.5 to 16.2 mg/100 g FW, a 38.9% decline, in the open refrigerated samples in plastic trays. During storage, the samples in open ambient plastic trays had significantly lower b-carotene than had the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated in plastic tray or ambient PP micro-perforated-stored samples (P < 0.05) at 96 h, (Fig. 4). The open ambient in plastic tray-stored broccoli florets registered significantly lower b-carotene contents than did the PP micro-perforated-packed refrigerated stored samples (P < 0.01) at the end of storage (144 h). These results were in agreement with an earlier report on MAP of broccoli (Kidmose & Hansen, 1999). Hussein et al. (2000) also reported decrease in b-carotene content of broccoli during storage at higher temperature. Higher b-carotene retention in PP micro-perforated-packed broccoli might be due to the limited atmospheric oxygen available for oxidation and, therefore, oxidising enzymes are less active. During storage, oxidising enzymes, e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX), might have helped to reduce the b-carotene of the broccoli (Li, Maezawa, & Nakano, 2004). 3.5. Changes in antioxidant activity The antioxidant activity of fresh harvested broccoli florets, before storage, was found to be 3.96 lmol trolox/g. Changes in antioxidant activities of broccoli florets during storage, under different treatments, are presented in Fig. 5. A linear decrease in antioxidant activities was recorded in broccoli samples, irrespective of treatments, with the advancement of storage time (Fig. 5). However,
Fig. 5. Effect of storage atmosphere on total antioxidant activity (lmol trolox/g) of broccoli florets during short term storage (means ± SE, n = 5).
broccoli florets packed in PP micro-perforated film and stored under refrigerated conditions (4 °C) showed no significant change in antioxidant activities by the end of storage (144 h). Antioxidant activities decreased more rapidly in the florets of open ambient plastic tray samples than in ambient PP micro-perforated-packed florets or open plastic tray refrigerated samples (r2 = 0.964). After 144 h of storage, antioxidant activities decreased from 3.96 to 1.83 lmol trolox/g FW, a 53.8% reduction in the open ambient plastic tray samples, while antioxidant activities decreased from 3.96 to 2.00 lmol trolox/g FW, a 49.5% decline, in the open refrigerated samples of broccoli florets in plastic trays. During storage, the open ambient (in plastic tray treated)-samples had significantly lower antioxidant activities than had the PP micro-perforated-packed refrigerated samples (P < 0.01), open refrigerated in plastic trays or ambient PP micro-perforated-stored samples (P < 0.05) at 96 h (Fig. 5). At the end of storage (144 h), the open ambient (in plastic tray)-stored broccoli had significantly lower antioxidant activities than had the PP micro-perforated-packed refrigerated stored samples (P < 0.01). Costa, Civello, Chaves, and Martinez (2005) reported similar results in hot air-treated broccoli florets during storage at 20 °C. However, by contrast, Leja et al. (2001) reported increase in antioxidant activity of broccoli (Cv. Lord) flower buds during short-term storage at 20 and at 5 °C for 3 and 7 days, respectively, either non-packaged or packaged in polymeric films. Higher antioxidant activities in PP micro-perforated-packed broccoli might be due to the limited available atmospheric oxygen. 4. Conclusions Chlorophyll, ascorbic acid, b-carotene and total antioxidant content of broccoli florets underwent rapid changes during post-harvest storage. In preventing deterioration of broccoli during storage, temperature and packaging materials played significant roles in retention of phytochemicals. Broccoli florets stored under open ambient conditions (15 °C) showed greater losses in chlorophyll, ascorbic acid, b-carotene and total antioxidant contents than did those in other treatments. Among the treatments, PP microperforated film bag samples stored under refrigerated conditions (4 °C) were able to retain maximum phytochemicals during storage for up to 144 h. References
Fig. 4. Effect of storage atmosphere on b-carotene content (mg/100 g FW) of broccoli florets during short term storage (means ± SE, n = 5).
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