and browning), and weight loss were also evaluated. The influence of different .... vapour transmission rate of 0.090 g/m2 24 h (all values at 83% RH and 5 °C).
Lebensm.-Wiss. u.-Technol., 29, 708–713 (1996)
Minimally Processed Pomegranate Seeds Maria I. Gil*, Juan A. Mart´ınez and Francisco Artes ´ Postharvest and Refrigeration Laboratory, Food Science and Technology Department, CEBAS (CSIC), PO Box 4195, Murcia 30080 (Spain) (Received March 3, 1995; accepted January 4, 1996)
Ready-to-eat pomegranate seeds were prepared and their quality attributes and appearance was followed during storage for 7 d under different conditions. Quality attributes, titratable acidity, total soluble solids, L* values, absorbance at 510 nm (anthocyanin content) and absorbance at 446 nm (brown metabolites) were analysed. Respiratory activity, appearance of the seeds (shriveling and browning), and weight loss were also evaluated. The influence of different washing treatments, storage temperatures (8, 4 and 1 °C) and actively or passively modified atmosphere packaging on the quality of the minimally processed pomegranate seeds was studied. Washing the seeds with chlorine and chlorine followed by ascorbic and citric acid were selected, and storage at 1 °C led to the best quality preservation. The best overall results were obtained for seeds washed with chlorine (100 mg/kg) plus antioxidants (5 g/L ascorbic acid and 5 g/L citric acid), and sealed in oriented polypropylene film using an initial atmosphere actively modified to 20 mL/L O2 and 0 mL/L CO2. Under these conditions, minimally processed pomegranate seeds maintained good quality and appearance for 7 d of storage at 1 °C without fungal attacks or off-flavour development. ©1996 Academic Press Limited
Introduction Pomegranate is the fruit of Punica granatum L. (Punicaceae) which is widely grown in Mediterranean countries, and has been introduced to most parts of the tropics and subtropics (1). The seeds are the edible part of the fruit which are normally consumed fresh. One of the main features of its quality is the red colour of both its seeds and juice (2–4). However, pomegranate consumption is limited due to difficulties in peeling to obtain the seeds. Therefore, production of pomegranate seeds in ‘ready-to-eat’ form would be a convenient and desirable alternative to the consumption of fresh fruits and may increase pomegranate demand by consumers. Minimally processed pomegranate seeds have a greatly reduced post-harvest life compared to whole fruit. Modified atmosphere packaging (MAP) may be an excellent method of extending shelf-life of pomegranate seeds (3). To prevent microbial development, washing lightly processed products with chlorine solutions has proven essential (5). In pigmented products, an additional problem is the discolouration caused by oxidation of phenolic pigments and other phenolic compounds catalysed by phenolases or peroxidases (6). Since the colour of pomegranate seeds is the most important quality attribute for consumers, its stability must be preserved (4). Washing with antioxidant solutions might therefore prove useful. Selected conditions for pomegranate seed preparation have been described (7)
*To whom correspondence should be addressed.
0023-6438/96/080708 + 06$25.00/0
but little work on minimally processed pomegranate seeds has been published so far. The purpose of this work was the preparation of pomegranate seeds and the study of the effect of storage temperature, pretreatments and MAP technique on the keeping quality of this product.
Materials and Methods Plant material Pomegranates (cv. Mollar) were harvested (October 3, 1994) in the typical production area of Elche (Alicante, Spain), located at the Mediterranean coast, and transported 60 km the same day by car to the laboratory and kept at 5 °C until used the next day. Seeds were obtained by manual peeling under refrigerated conditions (13 °C).
Washing treatments Six different washing treatments were tested including distilled water, chlorinated water and solutions of ascorbic and/or citric acids with and without chlorine. Concentrations and rinsing times are shown in Table 1. Seeds were divided into uniform groups (250 g) and each was dipped in 5 L of appropriate solution. Washing treatments were carried out at 13 °C. After the rinsing time indicated for each experiment (Table 1), seeds were dried in a manual spin-dryer for 1 min and placed at 1 °C for 1 h to remove residual water before analysis. ©1996 Academic Press Limited
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Table 1 Washing treatments Washing Water Chlorine (100 mg/kg) Ascorbic acid (5 g/L) Citric acid (10 g/L) Ascorbic acid (5 g/L)+citric acid (5 g/L) Chlorine (100 mg/kg)+ ascorbic acid (5 g/L)+citric acid (5 g/L)
Rinsing time 5 min 5 min 30 s 30 s 30 s 5 min 30 s
Storage conditions Packaged and unpackaged seeds were stored for 7 d at different temperatures (8, 4 and 1 °C) to select the best storage conditions. Relative humidity was 90 ± 5% controlled by a humidostat (cen-frio, Spain) and measured by a thermo-hygrometer (Solomat, mod. MPM 500e, U.K.) for all treatments. The storage period of 7 d was considered adequate as a retail sale period. After peeling, seeds were divided into two batches. One batch was dipped in chlorine (100 mg/kg) for 5 min and the other batch was first dipped in chlorine (100 mg/kg) for 5 min followed by dipping for 30 s in a solution of ascorbic acid (5 g/L) and citric acid (5 g/L). Seeds were dried as described above. For experiments with unpackaged seeds, 50 g were placed in open plastic trays (9 cm diameter and 1 cm thick). In addition, 50 g of seeds were placed in heat-sealed pouches (9 3 12 cm) made of oriented polypropylene (OPP) film of 40 µm thickness (Derfilm 40 DF 300, Derprosa, Spain), with an O2 permeability of 290 mL/m2 24 h bar, a CO2 permeability of 1112 mL/m2 24 h bar, and a water vapour transmission rate of 0.090 g/m2 24 h (all values at 83% RH and 5 °C). Handling was carried out at 13 °C.
MAP treatments To study the effect of MAP treatments, seeds were washed and handled as in the storage study and then packaged in sealed OPP pouches with the characteristics described above, and perforated oriented polypropylene film (POPP) (33 holes of 2 mm per dm2) which allowed a free diffusion of gases, as a control. A modified atmosphere was passively established within each heat-sealed pouch (9 3 12 cm) as a consequence of both the respiration of the seeds and the permeability of the film. Storage conditions were 1 °C and 90 ± 5% RH for 7 d. Initial gas concentrations (140 mL/L O2 and 80 mL/L CO2 for the CO2 treatment and 20 mL/L O2 and 0 mL/ L CO2 for the N2 treatment) were established by active modification, by flushing a specific gas in a gas exchange device with a vacuum packaging machine Egarvac and protective mixing gas KM 100-3M. Changes in concentrations of O2 and CO2 within the packages were monitored daily using a Perkin Elmer Autosystem gas chromatograph equipped with a thermal conductivity detector (TCD).
Analytical evaluation Quality attribute measurements were carried out on the juice obtained by squeezing 50 g of seeds with a commercial turmix blender (Moulinex). Titratable acidity (TA) was determiend by titrating juice samples with 0.1 mol/L NaOH (8) and was expressed as g of citric acid/100 mL. Total soluble solids (TSS) was measured with an Atago N1 refractometer (refractometric reading at 20 °C) and expressed in °Brix. Colour components L*, a* and b* were measured with a tristimulus colorimeter (Chroma Meter CR-300, Minolta) using a liquid sample holder CR-A70 (Minolta) where 35 mL of juice were analysed. Two millilitres of juice were centrifuged in an Eppendorf centrifuge at 1200 3 g for 2 min and the anthocyanin content and browning compounds were measured at 510 nm and 446 nm, respectively, using a Pye-Unicam SP-8-100 spectrophotometer. Respiratory activity The respiration rate (RR) was determined by the Gas Stream Method (9), placing 50 g of seeds into a 100 mL gas-tight glass jar at 20 °C and using 0.2 L/min continuous flow of humidified air (above 95% RH) free of CO2. The increase in CO2 content in the head space over a fixed period of time (60 min) was measured. CO2 emissions were determined using a gas chromatograph Hewlett Packard 5370 A model, equipped with a Porapack-Q column and a thermal conductivity detector (TCD). Results were expressed as mL CO2/kg/h. Helium was used as carried gas. Sensory evaluation The subjective quality of minimally processed pomegranate seeds after the different treatments was assessed by a panel of five trained judges (10). Two women and three men were instructed to judge appearance (browning, shriveling, skin defects) and visual fungal attacks. Statistical analysis Five replicates of each treatment were examined. Data were analysed by analysis of variance (P < 0.05). Results and Discussion Effect of washing with different solutions TA, TSS, L* value and absorbance at 510 nm were compared to assess if the different washing treatments affected the colour and composition of pomegranate seeds and if they lead to loss of quality prior to storage (Table 2). No significant differences (P < 0.05) were found in TA and TSS among treatments. L* parameter was a good indicator of changes in the seed juice brightness. Minimal values were obtained with citric and ascorbic acid treatment and no differences were found among the other washing solutions. In general, all the assayed treatments led to a slight
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Table 2 Changes in some selected quality attributes of pomegranate seeds after different washing treatments Washing treatments
Titrable acidity (g/100 mL)
Not washed Water Chlorine Ascorbic acid Citric acid Ascorbic+citric acid Chlorine+ascorbic+citric acid
0.26 ± 0.01 0.26 ± 0.01 0.25 ± 0.01 0.27 ± 0.01 0.26 ± 0.01 0.26 ± 0.01 0.26 ± 0.01
Total soluble solids (°B)
L*
Absorbance at 510 nm
17.32 ± 0.11 17.00 ± 0.14 17.08 ± 0.23 17.28 ± 0.23 17.12 ± 0.11 17.28 ± 0.11 17.00 ± 0.20
36.32 ± 0.82 37.16 ± 1.06 36.84 ± 1.39 37.16 ± 0.92 36.05 ± 0.72 33.23 ± 0.85 34.70 ± 0.33
1.69 ± 0.09 1.45 ± 0.06 1.46 ± 0.03 1.39 ± 0.07 1.44 ± 0.08 1.45 ± 0.02 1.45 ± 0.06
Means (n=5) ± standard deviation. For washing treatments see Table 1.
decrease in the juice anthocyanin content and to a small pigmentation of the washing liquids, indicating that some leakage of juice from arils, which might have been slightly damaged by the peeling procedure, had occurred. There is a slight decrease in absorbance at 510 nm with washing, suggesting a leakage of juice from the seeds to the washing solutions. Since no significant differences were observed among the different washing treatments, and chlorine was recommended to prevent microbial development (4), washing with chlorine and that with addition of both antioxidants were selected for the rest of experiments.
Effect of storage temperature The effect of different storage temperatures (8, 4 and 1 °C) with the selected washing solutions (chlorine and chlorine followed by ascorbic and citric acid) on the quality parameters is shown in Table 3a and b. After unpacked storage, changes were observed in seed weight due to water loss (Table 4). Therefore, quality parameters changed remarkably, and increases in TA, TSS and pigmentation of the juice (Abs 510 nm) were observed for all temperatures and for both washing treatments (Table 3a). On the other hand, the L* value of seed juice decreased, showing a decrease in bright-
Table 3 Effect of washing treatment, packaging and storage temperatures on some quality attributes of pomegranate seeds stored for 7 d Initial
8 °C
4 °C
1 °C
0.30 ± 0.01 18.04 ± 0.17 40.55 ± 2.40 0.95 ± 0.06 1.56 ± 0.05 0.61±0.03
0.43 ± 0.03 23.56 ± 1.69 39.24 ± 1.08 1.61 ± 0.10 1.85 ± 0.16 0.87 ± 0.03
0.37 ± 0.01 25.04 ± 1.21 37.53 ± 2.11 1.52 ± 0.18 2.05 ± 0.13 0.74 ± 0.04
0.34 ± 0.03 21.33 ± 0.51 37.05 ± 1.67 1.22 ± 0.07 1.93 ± 0.06 0.63 ± 0.05
0.30 ± 0.01 18.04 ± 0.17 40.55 ± 2.40 0.95 ± 0.06 1.56 ± 0.05 0.61 ± 0.03
0.26 ± 0.01 16.60 ± 0.14 42.21 ± 2.26 1.10 ± 0.04 1.62 ± 0.03 0.68 ± 0.01
0.27 ± 0.01 17.28 ± 0.44 43.07 ± 0.58 1.06 ± 1.06 1.61 ± 0.05 0.66 ± 0.03
0.28 ± 0.01 17.44 ± 0.05 43.45 ± 0.67 0.99 ± 0.06 1.57 ± 0.03 0.63 ± 0.04
(a) Washed with chlorine (100 mg/kg) Unpacked TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510 Packaged TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510
(b) Washed with chlorine (100 mg/kg) followed by ascorbic acid (5 g/L) and citric acid (5 g/L) Unpacked TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510 Packaged TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510
0.31 ± 0.01 18.20 ± 0.50 41.35 ± 1.27 0.85 ± 0.03 1.50 ± 0.03 0.57±0.03
0.41 ± 0.04 23.44 ± 1.00 37.77 ± 2.39 1.36 ± 0.09 2.01 ± 0.10 0.68 ± 0.02
0.39 ± 0.08 23.12 ± 1.20 37.57 ± 1.11 1.33 ± 0.10 1.95 ± 0.15 0.68 ± 0.01
0.35 ± 0.02 23.04 ± 0.38 35.08 ± 0.66 1.30 ± 0.15 1.81 ± 0.07 0.72 ± 0.09
0.31 ± 0.01 18.20 ± 0.5 41.35 ± 1.27 0.85 ± 0.03 1.50 ± 0.03 0.57±0.03
0.30 ± 0.02 16.40 ± 0.14 46.30 ± 1.84 0.76 ± 0.03 1.16 ± 0.03 0.66 ± 0.02
0.38 ± 0.04 16.28 ± 0.33 46.41 ± 1.38 0.76 ± 0.02 1.19 ± 0.03 0.64 ± 0.02
0.29 ± 0.01 17.34 ± 0.12 45.28 ± 1.42 0.76 ± 0.06 1.43 ± 0.03 0.53 ± 0.04
Mean (n=5) ± standard deviation. TA=titratable acidity; TSS=total soluble solids; Abs=absorbance.
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Table 4 Influence of storage temperature, washing treatment and packaging on weight loss, shriveling and browning of pomegranate seeds 8 °C
4 °C
Chlorine* Chlorine+antiox.**
Chlorine
1 °C
Chlorine+antiox.
Chlorine
Chlorine+antiox.
Unpacked Weight loss % Shriveling % Browning %
32.33 ± 4.58 20.04 ± 1.45 4.08 ± 1.27
33.84 ± 4.02 24.44 ± 2.96 3.52 ± 1.81
35.40 ± 4.70 21.72 ± 1.59 3.80 ± 1.26
36.04 ± 4.15 25.40 ± 3.13 2.24 ± 1.14
26.20 ± 2.14 19.72 ± 1.22 4.00 ± 0.99
26.44 ± 1.21 22.22 ± 1.67 2.62 ± 0.83
OPP packed Weight loss % Browning %
0.70 ± 0.27 9.40 ± 1.99
0.72 ± 0.32 8.12 ± 3.27
0.56 ± 0.37 8.60 ± 3.25
0.84 ± 0.23 7.56 ± 0.52
0.52 ± 0.41 7.72 ± 1.79
0.68 ± 0.18 7.14 ± 1.03
Means (n=5) ± standard deviation. *Washing with chlorine (100 mg/kg); **Washing with chlorine (100 mg/kg) followed by ascorbic acid (5 g/L) and citric acid (5 g/L).
ness, this being more marked in seeds washed with chlorine plus antioxidants. The absorbance at 446 nm and the Abs 446 nm/Abs 510 nm ratio increased as well and indicated an increase in the concentration of browning products. Dehydration and shriveling were observed when seeds were stored unpacked, leading to an appearance and quality which were considered unacceptable (Table 4). For pomegranate fruits cv. ‘Mollar’ there is some indication about the limits of acceptance which are 0.4 g/kg maximum TA and 14°Brix of minimum TSS(3). Packaged seeds washed with chlorine showed a decrease in TA and TSS and a slight increase in absorbance at 446 nm and the Abs 446/Abs 510 ratio, but nonsignificant differences (P < 0.05) were observed for L* value and absorbance at 510 nm (Table 3a). Packaged seeds washed with chlorine, ascorbic acid and citric acid showed differences when stored at all temperatures with the exception of storage at 1 °C, that showed similar values to those of the initial sample. No significant differences were found between storage at 8 and 4 °C except a decrease in pigmentation at 8 °C and an increase in browning (Table 3b). When seeds were stored in OPP bags, at different temperatures, the quality was preserved much better than in those stored unpacked. Hence, only slight differences were observed for TA in all cases. TSS slightly decreased when stored at 8 and 4 °C and this value was similar to the initial values when seeds were stored at 1 °C. The L* parameter increased in all cases, showing an opposite effect to unpacked seeds. The juice pigmentation, measured at 510 nm, showed nonsignificant changes in those seeds washed with chlorine and stored at all the assayed temperatures and it can be concluded that the pigmentation is quite well preserved under the different storage conditions. On the contrary, a decrease in the anthocyanin content was observed in seeds washed with chlorine plus antioxidants, being lower in those seeds stored at 1 °C. Browning slightly increased with increasing storage temperatures (increase in the absorbance at 446 nm and of the Abs 446/Abs 510 nm ratio), but these values were lower when seeds were washed with chlorine plus anti-
oxidants, and no significant differences (P < 0.05) were found compared to the initial seeds, for those seeds stored at 1 °C. The respiration rates were 1.94, 1.30 and 0.53 mL/(kg.h) CO2 at 8, 4 and 1 °C, respectively. Since the respiration rate of seeds is important in storage life of minimally processed products, the respiration rate is an important criterion for temperature selection and should be minimal (11). Thus, storage temperature at 1 °C was selected as the best for keeping quality.
Effect of MAP treatments Pomegranate seeds washed with chlorine and chlorine plus antioxidants were stored at 1 °C, and packaged in sealed polymeric films under different initial atmosphere compositions in order to generate different modified atmospheres and test their influence on the preservation of minimally processed pomegranate seed quality attributes. The steady state of CO2 and N2 treatments were reached on the 1st day and for the OPP treatment on the 3rd day. The composition of the different atmospheres at steady state were: 188 mL/L O2 and 22 mL/L CO2 for the OPP treatment; 206 mL/L O2 and 3 mL/L CO2 for the CO2 treatment; and 203 mL/L O2 and 4 mL/L CO2 for the N2 treatment. In spite of the very different initial concentrations of O2 and CO2 that were established by active modification of atmospheres, due to the high O2 and CO2 permeability of the OPP film used in this work, very similar atmospheres resulted at steady state CO2. This could explain the small differences in quality attributes observed for the three different MAP treatments assayed. The effects of the different modified atmospheres on the quality attributes of the seeds and their visual appearance are shown in Table 5 and Table 6, respectively. The results showed that both TA and TSS increased when seeds were stored in perforated films (POPP). This increase was due to water loss observed only in this treatment for both washings (Table 6). For the other three treatments, TA and TSS remained unchanged. Again, the L* value of the juice showed significant changes with storage under all conditions.
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Table 5 Effect of modified atmosphere packaging and washing treatments on some quality attributes of pomegranate seeds stored at 1 °C for 7 d Initial
POPP
OPP
OPP-CO2
OPP-N2
0.28 ± 0.02 17.82 ± 0.15 40.68 ± 1.36 0.86 ± 0.03 1.48 ± 0.06 0.58 ± 0.00
0.26 ± 0.03 17.08 ± 0.11 43.45 ± 0.67 0.95 ± 0.02 1.54 ± 0.04 0.62 ± 0.01
0.27 ± 0.01 17.16 ± 0.22 44.24 ± 0.51 0.89 ± 0.04 1.50 ± 0.07 0.59 ± 0.00
0.27 ± 0.03 16.92 ± 0.18 44.18 ± 0.71 0.98 ± 0.07 1.61 ± 0.06 0.61 ± 0.05
(a) Washed with chlorine (100 mg/kg) TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510
0.25 ± 0.01 17.08 ± 0.18 36.84 ± 1.39 0.79 ± 0.01 1.46 ± 0.03 0.54 ± 0.00
(b) Washed with chlorine (100 mg/kg) followed by ascorbic acid (5 g/L) and citric acid (5 g/L) TA (g/100 mL) TSS (°B) L* Abs 446 nm Abs 510 nm 446/510
0.26 ± 0.01 17.0 ± 0.20 36.65 ± 0.68 0.81 ± 0.03 1.45 ± 0.06 0.55 ± 0.01
0.29 ± 0.01 17.48 ± 0.11 44.63 ± 0.53 0.99 ± 0.07 1.61 ± 0.08 0.62 ± 0.03
0.28 ± 0.01 17.04 ± 0.26 45.08 ± 1.17 0.92 ± 0.03 1.58 ± 0.05 0.58 ± 0.00
0.28 ± 0.01 16.72 ± 0.18 44.48 ± 1.55 0.91 ± 0.02 1.55 ± 0.02 0.59 ± 0.01
0.27 ± 0.01 16.52 ± 0.23 47.39 ± 1.11 0.83 ± 0.03 1.44 ± 0.06 0.58 ± 0.01
Means (n=5) ± standard deviation. POPP=perforated oriented polypropylene film; OPP=oriented polypropylene film (40 µm); OPP-CO 2=oriented polypropylene film with initial 0.14 O2 and 0.08 CO2 concentration; OPP-N2=oriented polypropylene film with initial 0.02 O2 and 0.00 CO2.
There was an increase in this value indicating an increase in lightness of the juice. The pigmentation of the juice (Abs 510 nm) was well maintained, and in some cases a slight increase was observed. In general a small increase in absorbance at 446 nm and the Abs446/Abs510 ratio were also observed and none of the modified atmospheres led to the good results. However, washing with chlorine plus antioxidants led to reduced values of these browning parameters and should be recommended. No visual fungal attacks or off-flavours were detected in any treatment, probably due to high pomegranate quality at harvest and proper measures during processing. In conclusion, the experiment showed that the best keep ability was obtained when pomegranate seeds were washed with chlorine, followed by antioxidant solution packaged in polypropylene films, and stored at 1 °C. An initial atmosphere containing reduced levels of oxygen (20 mL/L) and carbon dioxide (0 mL/L) reduced total losses and improved the visual appearance of the pomegranate seeds (Table 6) compared with air atmosphere. More extensive and detailed studies using films with reduced O2 and CO2 permeability should be used in future studies in order to assess the effect of reduced
levels of O2 and increased levels of CO2 in the keeping quality of minimally processed pomegranate seeds.
Acknowledgements We acknowledge financial support for this work by the Spanish CICYT (Projects ALI92-0534 and ALI940721) and the technical assistance of C. Martinez Ataz. M.I.G. is indebted to the Spanish CSIC (Contrato temporal de Personal Investigador) and J.A.M. to the Instituto de Fomento de la Region ´ de Murcia for fellowships. Thanks are due to Agrudeco S.A. for providing pomegranates.
References 1 GIL, M. I., CHERIF, J., AYED, N., ARTES, F. AND TOMASBARBERAN, F. A. Influence of cultivar, maturity stage and geographical location on the juice pigmentation of Tunisian pomegranates. Zeitschrift fur ¨ Lebensmittel- Untersuchung und - Forschung, 201, 361–364 (1995) ´ y tipificacion ´ varietal de granado 2 MELGAREJO, P. Seleccion (Punica granatum L.). Doctoral Thesis, Universidad Politecnica, ´ Valencia, Spain. (1994)
Table 6 Influence of modified atmosphere packaging at 1 °C on weight loss and browning after 7 d of storage of pomegranate seeds POPP Chlorine*
Chlorine+ antiox**
Weight loss % 2.00 ± 0.80 2.40 ± 0.24 Browning % 7.27 ± 3.46 7.12 ± 1.38
OPP Chlorine
OPP-CO2
Chlorine+ antiox
0.49 ± 0.14 0.69 ± 0.10 9.60 ± 3.10 7.84 ± 2.40
Chlorine
Chlorine+ antiox
0.58 ± 0.22 0.64 ± 0.15 10.20 ± 2.92 8.87 ± 3.01
OPP-N2 Chlorine
Chlorine+ antiox
0.57 ± 0.21 0.56 ± 0.27 9.60 ± 2.84 7.25 ± 2.63
Mean (n=5) ± standard deviation. *Washing with chlorine (100 mg/kg); **Washing with chlorine (100 mg/kg) followed by ascorbic acid (5 g/L) and citric acid (5 g/L).
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