Extraction modeling and activities of antioxidants from pomegranate marc

Journal of Food Engineering 99 (2010) 16–23

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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng

Extraction modeling and activities of antioxidants from pomegranate marc Wenjuan Qu a,c, Zhongli Pan b,c,*, Haile Ma a a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China Processed Foods Research Unit, USDA–ARS West Regional Research Center, 800 Buchanan Street, Albany, Berkeley, CA 94710, USA c Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA b

a r t i c l e

i n f o

Article history: Received 26 August 2009 Received in revised form 13 November 2009 Accepted 23 January 2010 Available online 28 January 2010 Keywords: Pomegranate Marc Phenolic compound Antioxidant extraction Kinetics Activity Particle size Temperature Time Solvent/solid ratio

a b s t r a c t To develop value-added antioxidants from the peel and seeds of pomegranate marc, a by-product after pomegranate juice processing, the effects of drying before extraction and processing parameters on the extraction kinetics and product properties were systematically studied using water as an environmental friendly solvent for the extraction. The results showed that the drying process did not significantly affect the yield, content, and activity of antioxidants from either the peel or seeds. The antioxidants extracted from the peel had higher yield and content than those from the seeds. The yield and content of antioxidants increased with reduced particle size and increased water/sample ratio and temperature, but antioxidant activity was low when extraction temperature was high. By considering the antioxidant activity and operation cost, the recommended extraction conditions were peel particle size of 0.2 mm, water/peel ratio of 50/1 (w/w), temperature of 25 °C, and extraction time of 2 min, which gave the high antioxidant yield (11.5%) and content (22.9%), and DPPH scavenging activity of 6.2 g/g. Kinetic models were successfully developed for describing the extraction processes with different processing parameters. Published by Elsevier Ltd.

1. Introduction It has been reported that consumption of pomegranate fruits has nutritional and medical benefits, including reduced oxidative stress, atherogenic modifications to LDL, and platelet aggregation (Aviram et al., 2000), as well as anticancer, antibacterial, and antiviral activities (Negi and Jayaprakasha, 2003; Zhang et al., 1995). Because of the benefits, pomegranate production has increased rapidly in the United States. California alone now produces about 20.5 thousand tons of pomegranate fruits each year with 75% consumed as fresh and 25% processed for juice production. The juice yield measured in our laboratory is about 332 L per ton of pomegranate fruit (Wonderful variety), which corresponds to about 3.3 thousand tons of processing by-products created. The by-product is called pomegranate marc and contains about 78% peel and 22% seeds based on wet weight. At present, most of the studies focus on the extraction and product properties of antioxidants, oil, and juice from pomegranate fruits (Guo et al., 2003; Schubert et al., 1999; Yasoubi et al., 2007). Little information that has been found is about value-added utilization of the by-product. Pomegranate

* Corresponding author. Address: Processed Foods Research Unit, USDA–ARS West Regional Research Center, 800 Buchanan Street, Albany, Berkeley, CA 94710, USA. Tel.: +1 510 559 5861; fax: +1 510 559 5851. E-mail addresses: [email protected], [email protected] (Z. Pan). 0260-8774/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2010.01.020

marc is normally used as cattle feeds with low value or directly disposed in the field which could cause environmental problem. Our previous research has shown that pomegranate marc is a good raw material for producing natural antioxidants because of its high content of antioxidants (Qu et al., 2009). In general, extraction solvent, temperature, solid–liquid ratio, and particle size are influential parameters for extraction process (Bucic-Kojic et al., 2007; Lapornik et al., 2005; Petersson et al., 2006). It has been reported that the solvents may not affect the characteristics of antioxidants, but the composition of the pomegranate extracts (Kulkarni et al., 2004; Negi and Jayaprakasha, 2003; Singh et al., 2002). Water as an environmental friendly solvent has been found to be very effective for antioxidant extraction from pomegranate marc in our previous study and reported research (Qu et al., 2009; Singh et al., 2002). Therefore, water was used as the extraction solvent in this research. Because the antioxidant extraction from pomegranate marc is a solid–liquid extraction process, the determination of kinetic parameters is very important for designing an efficient extraction process for antioxidant production from pomegranate marc. The typical kinetic models of solid–liquid extractions include unsteady diffusion (Stankovic et al., 1994), film theory (Pekic et al., 1988), empirical equation of Ponomaryov (Ponomaryov, 1976), Peleg model (Peleg, 1988), and Fick’s law of diffusion (Cacace and Mazza, 2003). Arrhenius model is commonly used to describe rate–temperature

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W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

Nomenclature t Ce, Ct

k h k0 E R T, Ta L

extraction time (min) equilibrium concentration of total phenolics and total phenolic concentration in the liquid extract at a given extraction time t (g/L) second-order extraction rate constant (L/g min) initial extraction rate (g/L min) temperature-independent factor (L/g min) activation energy of extraction (kJ/mol) gas constant (8.314 J/mol K) extraction temperature (°C) or (K) particle size (mm)

relations in thermally-activated processes (Cohen and Saguy, 1985; Mossel et al., 2000). However, no information is available for kinetic modeling of water extraction of antioxidants from pomegranate marc. It is known that antioxidants are mainly constituted by phenolic compounds and have strong potential in scavenging free radicals (Gil et al., 2000; Noda et al., 2002). Because the potential of scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical has been widely used to evaluate the activity of antioxidants (Awika et al., 2003; Bondet et al., 1997), it was also used in this study. The objective of this research was to develop environmental friendly and efficient extraction methods for producing antioxidants from pomegranate marc by studying the extraction kinetics and the yield, content, and activity of antioxidants using water as the solvent. Because wet pomegranate marc from juice processing may not be able to be utilized right away, it needs to be preserved by drying for a later extraction. Therefore, the effects of drying process on the yield, content, and activity of antioxidants were investigated. The influences of processing parameters, including particle size (L), water/sample ratio (Z), and extraction temperature (T), on the yield, content, and activity of antioxidants were also determined. The kinetic models of extraction processes under different parameters were established for predicting the extraction processes and revealing the extraction mechanism. 2. Materials and methods 2.1. Materials Pomegranate marc was obtained from Stiebs Pomegranate Products (Madera, CA, USA), a commercial pomegranate juice processor, after juice processing of pomegranate fruit (Wonderful variety). Samples were stored at 18 °C at the Western Regional Research Center of the United States Department of Agriculture (USDA) – Agricultural Research Service in Albany, CA until use. Prior to the antioxidant extraction, 30 kg of pomegranate marc were thawed at 4 °C and then a part of sample was dried at 40 °C using hot air in a cabinet drier (CPM Wolverine Proctor LLC, Horsham, PA, USA). Then the peel and seeds in both wet and dry samples were manually separated and ground using a mill (WBB-6, Gruendler Pulverizing Co., St. Louis, MO, USA), equipped with a screen of 5.0 mm opening. The ground samples were used to study the effects of drying process on the yield, content, and activity of antioxidants. The dry peel powders were screened by using a Tyler Sieve Shaker (RO-TAP Testing Sieve Shaker, W.S. Tyler Co. Cleveland, OH, USA) with sieves of 2.0, 0.8, 0.6, and 0.4 mm openings to produce samples with different particle sizes. The obtained samples had particle sizes of 2.0–5.0, 0.8–2.0, 0.6–0.8, 0.4–0.6, and 0– 0.4 mm which were reported as averages of 3.5, 1.4, 0.7, 0.5, and 0.2 mm in this research.

Z water/sample ratio, w/w Cc, Cs, Cb DPPH concentration equivalents in the control solution, sample solution, and blank solution (g/L) n dilution factor of liquid extract total volume of liquid extract at a given extraction time Vt t (L) dry weight of sample (g) W1 dry weight of liquid extract (g) W2

2.2. Effect of drying process The antioxidant extraction was performed using distilled (DI) water in a beaker with stirring speed of 1200 rpm. During the extraction process, the beaker was held in a thermostat-controlled water bath at temperature of 25 °C and covered with an aluminum–foil paper to prevent oxidative changes from light. The experimental conditions were determined based on our preliminary tests. They were extraction water/sample ratio of 50/1 (w/w), and extraction time of 4 h for the wet and dry peels and 8 h for the wet and dry seeds. The liquid extract was separated from the residue by centrifugation (Marathon 21000R, Fisher Scientific Inc., Pittsburgh, PA, USA) with 3500 rpm for 20 min at 4 °C. The antioxidants in the liquid extract was analyzed to quantify the yields and contents (in terms of tannic acid equivalents), and DPPH scavenging activities. 2.3. Effects of extraction parameters Three processing parameters, including particle size, water/ sample ratio and temperature, were studied in this research. To study the effects of particle sizes, each sample (2 g) of different particle sizes (3.5, 1.4, 0.7, 0.5, and 0.2 mm) was extracted with DI water (100 g) at temperature of 25 °C and stirring speed of 1200 rpm for 2, 30, 60, and 90 min, respectively. Because the preliminary experiment showed that antioxidants can be effectively extracted from the sample with small particle size, to determine the effects of water/sample ratios, dry peel powder with particle size of 0.2 mm was selected and five samples with weights of 2, 2.5, 3.3, 5, and 10 g were separately mixed with 100 g of DI water to produce the water/sample ratios of 50/1, 40/1, 30/1, 20/1, and 10/1. The extraction was performed at 25 °C for 0.17, 0.33, 0.5, 1, 1.5, 2, 3, and 4 min for each sample. The preliminary study also showed that high water/sample ratio benefited the extraction process. To measure the effects of extraction temperatures, therefore, water/sample ratio of 50/1 was used. The extraction temperatures were 25, 40, 60, 80, and 95 °C for each extraction time of 0.17, 0.33, 0.5, 1, 1.5, 2, 3, and 4 min, respectively. The liquid extracts from all samples were also separated and then the antioxidants were analyzed by using the same methods described in the Section 2.2. 2.4. Kinetic model for evaluating extraction process of antioxidants To quantify the extraction rate of antioxidants, the second-order rate law was proposed based on the reported studies (Rakotondramasy-Rabesiaka et al., 2007, 2009). The general second-order kinetic model can be written as:

dC t ¼ kðC e  C t Þ2 dt

ð1Þ

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W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

where k is the second-order extraction rate constant (L/g min), Ce is the equilibrium concentration of total phenolics in the liquid extract (g/L), and Ct is the total phenolic concentration in the liquid extract at a given extraction time t (g/L). The integrated rate law for a second-order extraction under the boundary conditions t = 0 to t and Ct = 0 to Ct, can be written as an Eq. (2) or a linearized Eq. (3):

Ct ¼

C 2e kt

1 þ C e kt t 1 t ¼ þ C t kC 2e C e

ð2Þ ð3Þ

Then when t approaches 0, initial extraction rate, h (g/L min), can be written as: 2

h ¼ kC e

ð4Þ

After rearranging the Eqs. (3) and (4), Ct can be expressed as:

Ct ¼

t ð1=hÞ þ ðt=C e Þ

ð5Þ

The h, Ce, and k were determined experimentally from the slope and intercept by plotting t/Ct against t. It was assumed that the second-order kinetic model could be applied to measure the influences of variables (L, Z, and T). Therefore, the h, Ce, and k had relations with those variables and were fitted by functional models, using Origin Pro 7.5SR1 (V 7.5776, Origin Lab Corporation, Northampton, MA, USA). Arrhenius equation was used to describe the relationship between extraction rate constant (k) and temperature (Ta), which is written as:

  1000E k ¼ k0 exp  RT a

ð6Þ

where k0 is the temperature-independent factor (L/g min), E is the activation energy of extraction (kJ/mol), R is the gas constant (8.314 J/mol K), and Ta is extraction temperature (K). The plot of ln(k) against 1000/Ta was used for the calculation of k0 and E. 2.5. Analysis assay 2.5.1. Determinations of yield and content of antioxidants The antioxidants in the extracts were determined using the total phenolics in terms of tannic acid equivalents, according to a modified Folin–Ciocalteu method (Li et al., 2006). Folin–Ciocalteu reagent, tannic acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich Company (St. Louis, MO, USA). Methanol and sodium carbonate (Na2CO3) were obtained from Fisher Scientific Inc. (Pittsburgh, PA, USA). An extract sample of 60 lL was mixed with 2 mL of Na2CO3 (7.5%) and 2.5 mL of 10-fold diluted Folin–Ciocalteu reagent thoroughly using a vortex mixer (K-550-G Vortex-Genie, Scientific Industries Inc., Bohemia, NY, USA). The mixed solution was held in a water bath for 30 min at 25 °C and then its absorbance was measured at 760 nm using a spectrophotometer (Genesys 10Bio UV–vis spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA). Three measurements were conducted for each liquid sample and the test was replicated three times. The blank was prepared using above procedure, but the extract was replaced by the same volume of DI water. The total phenolic yield and content, %, were calculated using Eqs. (7) and (8):

Ct V t  100% 100W 1 Ct V t Total phenolic content ¼  100% 100W 2 Total phenolic yield ¼

ð7Þ ð8Þ

where Vt is the total volume of liquid extract at a given extraction time t (L), W1 is the dry weight of sample (g), and W2 is the dry weight of extract (g). The moisture contents of all samples were determined with an oven method by drying each sample to a constant weight at 105 °C (APHA et al., 1998). 2.5.2. Determination of antioxidant activity The antioxidant activity was determined using the DPPH equivalent, according to an adapted colorimetric procedure (Singh et al., 2002). An extract sample of 60 lL was reacted with 3 mL of DPPH solution in methanol (0.05 g/L). The sample solution was mixed thoroughly using a vortex mixer and held in a water bath for 20 min at 25 °C. The sample absorbance was measured at 517 nm using a spectrophotometer. Three measurements were conducted for each liquid sample and the test was replicated three times. The control solution included 60 lL of DI water and 3 mL of DPPH solution in methanol. The blank solution contained 60 lL of extract and 3 mL of methanol. The DPPH scavenging activity, g/g, was calculated using Eq. (9):

DPPH scavenging activity ¼

nV t ½C c  ðC s  C b Þ Ct V t

ð9Þ

where Cc is the DPPH concentration equivalent in the control solution (g/L), Cs is the DPPH concentration equivalent in the sample solution (g/L), Cb is the DPPH concentration equivalent in the blank solution (g/L), and n is the dilution factor of liquid extract. All reported weights and percentages were dry basis (d.b.) unless specified otherwise. All extraction trials were carried out in triplicate. The reported results are means of replicated experiments. 2.6. Statistical analysis Tukey’s studentized range (HSD) test, using a SAS software (Ver. 9.2., SAS Institute Inc., Cary, NC, USA) was performed to determine if there were significant differences in the yields, contents, and DPPH scavenging activities of antioxidants from the peel and seeds in both wet and dry forms, as well as the DPPH scavenging activities of antioxidants obtained with different extraction parameters, particle size, water/sample ratio, and extraction temperature. The significance was determined using least significant difference (LSD) (a = 0.05). 3. Results and discussion 3.1. Effect of drying Fig. 1 shows the yields, contents, and activities of antioxidants (total phenolics) from the peel and seeds in both wet and dry forms. The measured moisture contents of wet peel, wet seeds, dried peel, and dried seeds were 74.3%, 64.3%, 11.7%, and 6.9%, respectively. Based on the results of Tukey’s tests, there were no significant differences (P < 0.05) in the total phenolic yields, total phenolic contents, and DPPH scavenging activities between the wet and dry forms of raw materials. This indicated that the drying process did not significantly affect the measured yield, content, and activity of antioxidants from either the peel or seeds. Therefore, it is reasonable to believe that pomegranate marc could be dried for antioxidant production in a later date when it is necessary. However, it is also important to note that the effect of drying on individual components in the extract was not measured and needs to be studied in the future. It is clear that the yield and content of antioxidants from the peel were higher than those from the seeds. The results further revealed that the peel had higher antioxidant extraction rate (total phenolic yield gain per unit of extrac-

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W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

25

a

20

15 a

a

10 a

a

a

a

b

b

100

20 Particle size (mm) =

3.5 1.4 Total phenolic yield Total phenolic content

0.7

0.5

0.2

80

15

60

10

40

5

20

Total phenolic content (%)

a

Total phenolic yield (%)

25

Total phenolic yield Total phenolic content DPPH scavenging activity

5 b 0

b

0 0

Dry peel

Wet peel

Dry seeds

10

20

Fig. 1. Total phenolic yields, total phenolic contents, and DPPH scavenging activities from peel and seeds in both wet and dry forms. Different letters mean significant differences among the samples at P < 0.05. Extraction parameters are water/sample ratio of 50/1 (w/w), temperature of 25 °C, and extraction time of 4 h for wet and dry peels and 8 h for wet and dry seeds.

tion time) than the seeds because a shorter extraction time was used for the peel (4 h) compared to for the seeds (8 h). We speculated that was mainly due to the inherent physical and chemical differences between the peel and seeds. The peel contains much less cellulosic compounds and has a looser physical structure than the seeds, which could allow more efficient extraction. The DPPH scavenging activities of antioxidants extracted from the peel and seeds were not statistically different (P < 0.05). Therefore, the peel of pomegranate marc could be a better source for producing antioxidants because of its higher antioxidant yield than the seeds. For example, one ton of dry peel of pomegranate marc could produce 102 kg of antioxidants with content of 20.1% in the extract and DPPH scavenging activity of 6.5 g/g. The total phenolic yields and contents from the peel and seeds obtained in this research were higher than those reported by Singh et al. (2002). They also reported that the DPPH scavenging activity of antioxidants from pomegranate peel was 9.2 and 6.0 g/g from pomegranate seeds. The differences might be due to different varieties of pomegranates used and extraction procedures. Because the drying process did not significantly affect the yield, content, and activity of antioxidants and the peel had high yield and content of antioxidants, only dry peel powder was used for the rest of the tests. 3.2. Influence of extraction parameters 3.2.1. Effect of particle size The yields and contents of antioxidants from dry peel powders with different particle sizes at different extraction times are shown in Fig. 2(a). It can be seen that the total phenolic yield and content increased rapidly and then reached the final stabilization with the increase in extraction time. This indicated that the yield and content of antioxidants were closely related to the extraction time. For different particle sizes of 0.2, 0.5, 0.7, 1.4, and 3.5 mm, the antioxidant yields were 11.5%, 11.4%, 11.6%, 11.2%, and 9.4% after reaching equilibrium at corresponding extraction times of 2, 30, 30, 60, and 90 min. The corresponding contents of antioxidants were 22.9%, 22.7%, 22.2%, 21.1%, and 17.6%. As expected, the results revealed that smaller particle size could prominently shorten the extraction time to achieve the highest yield and content of antioxidants. That was because smaller particle size means a shorter mass transfer distance and larger resolve surface area, which ulti-

40

30

Wet seeds

50

60

70

80

0 90 100

Extraction time (min) (a) 6.50

DPPH scavenging activity (g/g)

Total phenolic yield (%) Total phenolic content (%) DPPH scavenging activity (g/g)

30

6.25 6.00 5.75 5.50 5.25 Particle size (mm) =

3.5 0.5

5.00

1.4 0.2

0.7

4.75 4.50

0

10

20

30

40

50

60

70

80

90 100

Extraction time (min) (b) Fig. 2. Total phenolic yields, total phenolic contents, and DPPH scavenging activities of antioxidants from dry peel powders at different extraction times and particle sizes with the extraction temperature of 25 °C and water/sample ratio of 50/1 (w/w) (dash lines are the non-linear fitting curves).

mately reduces the extraction time and increases the extraction efficiency. Similarly, Landbo and Meyer (2001) also reported that the total phenolic yield significantly increased with a reduction in particle size during the extraction of antioxidants from blank currant juice press residues. The antioxidants extracted from dry peel powders with different particle sizes had fluctuating DPPH scavenging activities with increased extraction times from 2 min to 90 min (Fig. 2(b)). The statistical analysis results also showed that the antioxidant activities were not significantly affected by the particle size at P < 0.05 level. In general, smaller particle size was preferred for processors to shorten the extraction time even though the energy used for grinding needs to be considered. Because smaller particle size dramatically increased the antioxidant yield and content without much effect on the antioxidant activity, the sample with the smallest particle size of 0.2 mm was used for the rest of the tests. 3.2.2. Effect of water/sample ratio Fig. 3(a) shows the yields and contents of antioxidants from dry peel powder under different extraction times and water/sample ra-

W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

Water/sample ratio, w/w =

10/1 40/1

20/1 50/1

30/1

80

Total phenolic yield Total phenolic content

15

60

10

40

5

20

Total phenolic yield (%)

20

100

Total phenolic content (%)

Total phenolic yield (%)

25

25

100

20

80

15

60

10

40

20

5 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Temperature (°C) = 25 40 Total phenolic yield Total phenolic content

0 4.5

Extraction time (min) (a)

0.0

95

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 4.5

4.0

4.5

Extraction time (min) (a)

7.5 Water/sample ratio, w/w =

10/1 40/1

20/1 50/1

30/1

10

7.0

9

6.5

8

6.0 5.5 5.0 4.5

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Extraction time (min) (b) Fig. 3. Total phenolic yields, total phenolic contents, and DPPH scavenging activities of antioxidants from dry peel powders at different extraction times and water/sample ratios with the extraction temperature of 25 °C and particle size of 0.2 mm (dash lines are the non-linear fitting curves).

tios. As it can be seen, the total phenolic yield and content rapidly increased before 0.5 min and then showed a slow increase from 0.5 min to 2 min before reaching equilibrium. Also, the total phenolic yield and content generally increased with the increase in water/sample ratio at 25 °C. Because higher solvent/solid ratio resulted in a larger concentration gradient during the diffusion from internal material into the solution, the extraction efficiency increased. Similarly, Li et al. (2005) also reported that the extraction efficiency of chlorogenic acid from Eucommia ulmodies increased with the increase of the solvent/sample ratio. The antioxidant yields and contents under different water/sample ratios of 10/1, 20/1, 30/1, 40/1, and 50/1, w/w reached equilibriums at the same extraction time of 2 min. The corresponding equilibrium yields were 7.6%, 9.4%, 9.8%, 11.0%, and 11.5% and equilibrium contents were 20.2%, 21.3%, 21.6%, 22.3%, and 22.9%. DPPH scavenging activities for the antioxidants produced with different extraction times and water/sample ratios are shown in Fig. 3(b). The DPPH scavenging activities of antioxidants did not significantly change with different water/sample ratios (P < 0.05). This indicated that the antioxidant activity was independent of water/sample ratio. The antioxidants extracted with different water/sample ratios had DPPH scavenging activities in the range

DPPH scavenging activity (g/g)

DPPH scavenging activity (g/g)

80

0

8.0

4.0 0.0

60

Total phenolic content (%)

20

Temperature (°C) =

25 80

40 95

60

7 6 5 4 3 2 1 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Extraction time (min) (b) Fig. 4. Total phenolic yields, total phenolic contents, and DPPH scavenging activities of antioxidants from dry peel powders at different extraction times and temperatures with the extraction water/sample ratio of 50/1 (w/w) and particle size of 0.2 mm (dash lines are the non-linear fitting curves).

of 5.1–6.4 g/g during the short extraction time duration from 0.17 min to 4 min. The obtained results clearly demonstrated that higher water/ sample ratio was better for increasing the antioxidant yield and content without much effect on the antioxidant activity. However, higher water/sample ratio may mean more water usage in extraction and energy consumption for concentration in a later processing stage. Therefore, the recommended water/sample ratio is not higher than 50/1 (w/w).

3.2.3. Effect of extraction temperature The extraction temperatures had remarkable effects on the total phenolic yields and contents (Fig. 4(a)). The total phenolic yields and contents rapidly increased almost linearly within the first 0.5 min, and then displayed a slow extraction until reaching equilibrium. The total phenolic yield and content were significantly increased with the increased extraction temperature. This might be due to increased solubility and diffusion coefficient of antioxidants

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W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23 Table 1 Parameters of second-order kinetic model for antioxidant extraction from dry peel powders with different particle sizes, water/sample ratios, and extraction temperatures. Equilibrium concentration of total phenolics, Ce (g/L)

R2

0.375 1.575 2.678 9.307 44.229

0.115 0.340 0.516 1.803 7.685

1.806 2.152 2.278 2.272 2.399

0.997 0.999 0.999 0.999 1.0

10 20 30 40 50

50.905 33.899 20.495 13.239 12.967

0.603 1.210 1.774 2.242 2.575

9.188 5.293 3.399 2.430 2.244

0.999 0.999 0.999 0.999 0.999

25 40 60 80 95

10.512 20.217 35.532 59.192 100.721

1.948 2.687 3.212 4.621 6.314

2.323 2.743 3.326 3.579 3.994

0.999 0.995 0.999 0.996 0.999

Observation

Particle size, L (mm)

3.5 1.4 0.7 0.5 0.2

Water/sample ratio Z (g/g)

Extraction temperature, T (°C)

Initial extraction rate, h (g/L min)

Temperatureindependent factor, k0 (g/L min)

Activation energy, E (kJ/mol)

R2

677.9

14.541

0.976

Initial extraction rate (g/L.min)

120

10

100

8 h

80 k 60 4 40

Ce 2

20

0 0

Initial extraction rate (g/L.min)

60

10 h

50

8 Ce

40

6

30 4

20 2

10 k

0

0

10

20 30 40 Water/sample ratio, w/w

Extraction rate constant (L/g.min) Equilibrium concentration of total phenolics (g/L)

Fig. 5. Equilibrium concentration of total phenolics (Ce), extraction rate constant (k), and initial extraction rate (h) with different particle sizes.

50

Fig. 6. Equilibrium concentration of total phenolics (Ce), extraction rate constant (k), and initial extraction rate (h) with different water/sample ratios.

6

10

20

30

40

50

60

70

80

90

0 100

Extraction rate constant (L/g.min) Equilibrium concentration of total phenolics (g/L)

Extraction rate constant, k (L/g min)

Variable type

Extraction temperature (°C) Fig. 7. Equilibrium concentration of total phenolics (Ce), extraction rate constant (k), and initial extraction rate (h) with different extraction temperatures.

at a high temperature (Spigno and De Faveri, 2007). The antioxidant yields and contents at different extraction temperatures of 25, 40, 60, 80, and 95 °C respectively reached equilibrium at extraction times of 2, 2, 1.5, 1.5, and 1 min. The corresponding equilibrium yields were of 11.5%, 14%, 16.3%, 16.8%, and 19.5% and equilibrium contents were 22.9%, 29.8%, 31.5%, 31.3%, and 36.1%. Fig. 4(b) shows that the DPPH scavenging activities of antioxidants significantly decreased with the increase in extraction temperatures from 25 to 95 °C (P < 0.05), which was opposed to the trends of antioxidant yields and contents. This revealed that the extraction temperature had a negative effect on the antioxidant activity. That could be due to the fact that phenolic compounds are thermo-sensitive substances and therefore high temperature might reduce their activities, which needs to be further studied. Similarly, Miranda et al. (2009) reported that the antioxidant capacity of Aloe Vera gel decreased as the temperature increased. The DPPH scavenging activities were 5.5–6.2, 5.0–5.4, 4.0–4.8, 3.5–3.8, and 3.1–3.4 g/g at 25, 40, 60, 80, and 95 °C, respectively.

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W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

Based on the present results, the highest total phenolic yield (19.5%) and content (36.1%) were achieved at peel particle size of 0.2 mm, water/peel ratio of 50/1, w/w, temperature of 95 °C, and extraction time of 1 min, but the DPPH scavenging activity was low as 3.1 g/g. The highest DPPH scavenging activity (6.2 g/g) of antioxidants was obtained at peel particle size of 0.2 mm, water/ peel ratio of 50/1, temperature of 25 °C, and time of 2 min, despite relatively low yield (11.5%) and content (22.9%). By considering the antioxidant activity and operation cost, the recommended temperature is 25 °C.

3.3. Kinetic model of antioxidant extraction The h, Ce, and k values for different L, Z, and T were respectively obtained from the slopes and intercepts by plotting t/Ct against t (Eq. (3)) and listed in Table 1. These kinetic parameters decreased with the increase of particle sizes as expected based on the experimental results. Because the h, k, and Ce were dependent on L, the h, k, and Ce, values for different L values were fitted by linear and power functions with high coefficients of determination (R2 = 0.950–0.986). The functions are plotted in Fig. 5 and expressed as: 2

C eðLÞ ¼ 0:17 L þ 2:396 R ¼ 0:986

ð10Þ

2

1:462

kL ¼ 0:567 L

R ¼ 0:971

ð11Þ

hL ¼ 2:565 L1:652

R2 ¼ 0:950

ð12Þ

The Ct as a function of L can be obtained by substituting Eqs. (10) and (12) into Eq. (5). The relationship is obtained as:

t  C t;L ¼  1=ð2:565 L1:652 Þ þ ðt=ð0:17 L þ 2:396ÞÞ

ð13Þ

This equation can be used to predict the antioxidant extraction under different particle sizes at a given time with the extraction temperature of 25 °C and water/sample ratio of 50/1, w/w. The extraction at water/sample ratio of 10/1 displayed the highest h and Ce values compared to those at ratios of 20/1, 30/1, 40/1, and 50/1. That was due to the highest amount of raw material in the solvent. However, the highest k value was achieved at ratio of 50/1, followed by that at ratios of 40/1, 30/1, 20/1, and 10/1. An increase on the extraction rate was caused by the high water/ sample ratio, which was in agreement with the experimental results. According to the model assumption, the parameters (h, k, and Ce) were expressed by the variable of Z. Therefore, the relationships between kinetic parameters and Z were nonlinearly fitted by second-order polynomial functions (R2 = 0.995–0.999). The functions are plotted in Fig. 6 and written as:

C eðZÞ ¼ 0:006Z 2  0:525Z þ 13:708 R2 ¼ 0:995 2

ð14Þ

2

kZ ¼ 0:005Z þ 0:077Z  0:134 R ¼ 0:999

ð15Þ

hZ ¼ 0:028Z 2  2:663Z þ 75:070 R2 ¼ 0:999

ð16Þ

Substituting the hz and Ce(z) into Eq. (5), the relationship is described as: t    C t;Z ¼  1=ð0:028Z 2  2:663Z þ 75:070Þ þ t=ð0:006Z 2  0:525Z þ 13:708Þ ð17Þ

This equation can be used to predict the antioxidant extraction under different water/sample ratios at a given time with the particle size of 0.2 mm and extraction temperature of 25 °C. The highest h, k, and Ce values were obtained at temperature of 95 °C, followed by those at 80, 60, 40, and 25 °C. Temperature had an accelerative influence on these kinetic parameters. The relationships between kinetic parameters and L were fitted by linear, second-order polynomial, and exponential functions (R2 = 0.985– 0.988). The functions are plotted in Fig. 7 and the equations are:

C eðTÞ ¼ 0:023T þ 1:805 R2 ¼ 0:985

ð18Þ

2

kT ¼ 1:250 expð0:017TÞ R ¼ 0:986

ð19Þ

hT ¼ 0:017T 2  0:875T þ 23:833 R2 ¼ 0:988

ð20Þ

Substituting the hT and Ce(T) into Eq. (5), the relationship is obtained as:

t  C t;T ¼  1=ð0:017T 2  0:875T þ 23:833Þ þ ðt=ð0:023T þ 1:805ÞÞ ð21Þ This equation can be used to predict the antioxidant extraction under different temperatures at a given time with the particle size of 0.2 mm and water/sample ratio of 50/1, w/w. When the Arrhenius equation (Eq. (6)) was used to determine the relationship between k and Ta, the k0 and E were determined from the plot of ln(k) against 1000/Ta. The k0 and E were 677.9 L/ g min and 14.541 kJ/mol (Table 1). The high coefficient of determination (R2) of 0.976 confirmed that Arrhenius equation can be used to describe the relationship between second-order extraction rate constant with temperature. Therefore, the relationship of k and T (°C) is written as:

k ¼ 677:9 exp 

!

14:541

ð22Þ

8:314  103 ðT þ 273:15Þ

Empirical Eqs. (13), (17), and (21) are the kinetic models for predicting antioxidant extraction from dry peel of pomegranate marc. Even though the statistical models might not completely account for the phenomena governing extraction processes, they still could be used to determine the influences of particle size, temperature,

Table 2 Experimental verification with model prediction in total phenolic concentration (Ct) at different extraction times (t), particle sizes (L), water/sample ratios (Z), and temperatures (T). Extraction temperature of 25 °C, water/sample ratio of 50/1 w/w, and particle size of 0.2 mm. Ct,

L

C0.4

(g/L) min,

0.2 mm

Pred.a Exp.b Errorc a b c

2.034 2.067 1.622

Ct, C0.6

min,

0.2 mm

2.133 2.134 0.047

C0.9

min,

0.2 mm

2.204 2.192 0.544

C1.1

min,

0.2 mm

2.231 2.255 1.076

C1.8

min,

0.2 mm

2.280 2.270 0.439

Z

C0.7

(g/L) min,

50/1 w/w

1.899 1.896 0.158

Ct, C0.8

min,

50/1 w/w

1.954 1.955 0.051

C1.2

min,

50/1 w/w

2.098 2.083 0.720

C1.3

min,

50/1 w/w

2.122 2.130 0.376

C1.6

min,

50/1 w/w

2.177 2.172 0.230

T

(g/L)

C0.7 min,

C0.8

C1.2

C1.3

C1.6

95 °C

min, 95

min, 95

min, 95

min, 95

°C

°C

°C

°C

3.789 3.790 0.026

3.854 3.824 0.778

3.864 3.871 0.181

3.887 3.897 0.257

3.762 3.749 0.346

Pred. is the predictive value of total phenolic concentration by using respective mathematic model of each variable. Exp. is the experimental value of total phenolic concentration. Error is the difference between experimental and predicted values and is expressed as percentage (%) of experimental value.

W. Qu et al. / Journal of Food Engineering 99 (2010) 16–23

and water/sample ratio on the antioxidant extraction capacity versus extraction times. The results obtained from these models should provide the guidance for the improvement of extraction process, and reductions in extraction operating costs and times. 3.4. Experimental fitting Table 2 shows the predictive and experimental values of total phenolic concentrations under the extraction conditions different from the ones used for the model development. The satisfactory fits with low errors ranging from 0.778% to 1.622% were observed. This indicated that the developed models can be used for predicting the extraction performances. 4. Conclusions When water was used as the solvent for extraction of antioxidants from pomegranate marc, the results showed that the drying process of pomegranate marc had no significant effect on the yield, content, and activity of antioxidants from either the peel or seeds. The antioxidants extracted from the peel had higher yield (10.2%) and content (20.1%) compared to those from the seeds, whereas all antioxidants gave similar DPPH scavenging activities. Therefore, the peel of pomegranate marc is a good source for producing antioxidants. The research results also showed that the yield and content of antioxidants increased with reduced particle size, increased water/peel ratio and extraction temperature, but the antioxidant activity decreased with the increased temperature. By considering the antioxidant activity and operation cost, the recommended conditions are peel particle size of 0.2 mm, water/peel ratio of 50/1 (w/ w), temperature of 25 °C, and extraction time of 2 min, which gave the antioxidant yield of 11.5%, antioxidant content of 22.9%, and DPPH scavenging activity of 6.2 g/g. The kinetic models were successfully developed for describing the extraction processes under different extraction parameters, including particle size, water/peel ratio, and extraction temperature. The activation energy of antioxidant extraction was determined as 14.541 kJ/mol based on the Arrhenius model. Acknowledgements This research was conducted at the Western Regional Research Center of USDA–ARS and Department of Biological and Agricultural Engineering, University of California, Davis, USA. The authors thank Mr. Donald Olson and Dr. Griffiths G. Atungulu for their support in this research and Stiebs Pomegranate Inc. for providing the pomegranate marc materials. References APHA et al., 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed., Washington, DC. Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R., Hayek, T., Presser, D., Fuhrman, B., 2000. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. American Journal of Clinical Nutrition 71 (5), 1062–1076. Awika, J.M., Rooney, L.W., Wu, X.L., Prior, R.L., Cisneros-Zevallos, L., 2003. Screening methods to measure antioxidant activity of sorghum (sorghum bicolor) and sorghum products. Journal of Agricultural and Food Chemistry 51 (23), 6657– 6662. Bondet, V., Brand-Williams, W., Berset, C., 1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. LWT – Food Science and Technology 30 (6), 609–615.

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