Culinary Method Affects the Antioxidant Activity of Collard Greens ( Brassica oleracea )

The antioxidant activity of collard greens was determined after exposed to eight different thermal treatments: 1) untreated raw group, 2) short simmer 3short simmer water 4) short simmer + saute’, 5) saute’ 6) long simmer 7) long simmer water 8) long simmer + saute’. After treatment, total phenolic content (TPC) expressed in gallic acid equivalents/sample concentration (GAE/conc.), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferrous ion chelating (FIC) antioxidant assays were determined. The sauté treated group showed the highest TPC (8.2858 GAE/conc.) followed by the raw group (8.0361) and the short simmer + sauté group (7.6227). The raw group showed the highest DPPH activity (7.7952% inhibition/conc.) followed by the sauté group (7.5877) and the short simmer + sauté group (7.4753). In both of these assays the addition of a sauté treatment to either short or long simmered treatment increased the antioxidant activity of samples compared to just the short or long simmer treatment alone. Additionally both TPC and DPPH assays showed greater antioxidant activity in the cooking water reserved from a long simmer treatment compared to the reserved cooking water of a short simmer treatment suggesting significant (p≤0.05) leeching of antioxidants from collard greens into the water related to the duration of aquathermal treatment. Similar trends were not found in the results of the FIC chelating assay where both long and short simmer treatment groups showed the highest chelating abilities and the reserved cooking water from both treatments showed the lowest chelating abilities. This suggests that chelators contained in collard greens were not relatively water soluble and therefore not negatively affected byaquathermal treatments.


Antioxidants
Antioxidants continue to be a focus in food research, marketing and nutrition.While synthetic antioxidants are available to supplement processed foods in order to mediate the effects of lipid peroxidation, there are many natural sources available containing antioxidants (Moure et al., 2001).The benefits of antioxidants extend beyond just enhancing shelf life of products.Antioxidants are also highly prized by consumers for health benefits (Bazzano et al., 2002).From potentially reducing cancer risks, cardiovascular disease and Type II diabetes, and antioxidants are drawing attention in food and diet.There are different antioxidant assays available to determine the antioxidant content or power of foods.Tests can be direct in assessing a samples concentration of antioxidant compounds or alternatively can determine a samples ability to reduce a stable free radical or chelate a metal both of which can initiate or propagate the lipid peroxidation process (Prior, Wu, & Schaich, 2005).

Cooking and Antioxidants
Cooking food emerged possibly as early as the Middle Pleistocene era (400,000-125,000 years ago) when some archeologists believe evidence exists of charred bones in hearths (Jurmain, Nelson, Kilgore, & Trevathan, 1999).While the cooking of produce is most often done for alteration of flavor and texture, cooking can lead to beneficial changes in edibility with the removal or inactivation of harmful or anti-nutritive components (Shahidi, Janitha, & Wanasundara, 1992).Antioxidant assays of unprocessed food items may not accurately represent the health impact of foods that were cooked or thermally processed prior to consumption.Different cooking methods can affect various indicators of antioxidant activity in produce.Total reducing capacity of vegetables can be significantly reduced by processing and thermal treatment.For a large part, this reduction is due to an overall loss via leeching of hydrophilic antioxidants while there is a relative increase in the lipophilic reducing capacity via concentration of non-hydrophilic compounds as a result of almost all thermal treatments.This increase in lipophilic reducing capacity may also be due to an increase in the bioavailability of lipophilic antioxidants or the development of Maillard reaction products that can exhibit antioxidant properties themselves (Greco, Riccio, Bergero, Del Re, & Trevisan, 2007).

Hypothesis
Methods of thermal treatments in these studies are often times simplified cooking applications that are seldom used by the home cook, making the results of these studies potentially not applicable to food service as well as the home setting.Temperatures obtained in home cooking are capable of altering enzymatic content of vegetables (McGee, 1984) and some methods such as simmering and boiling can diminish nutritional content (The Culinary Institute of America, 2000).Thus, the objective of this study was to determine how commonly used cooking treatments affect the antioxidant content of collard greens (Brassica oleracea).

Treatments
The experiment was conducted in a Randomized Complete Block Design.Each block consisted of one head of collard greens.To prepare each head of collard greens, the leafy portions were removed from the thick stalks and triple soaked in a vegetable preparation sink with cold water.Once collard greens were air dried in a large colander for 30 minutes, they were sliced into approximately one-eighth to one-quarter inch wide ribbons using the culinary chiffonade technique where the leaves are rolled crosswise and then sliced in the crosswise with an 8" Henkel Chef Knife.Twenty-five gram samples of cut collard greens were chosen through simple random sampling from the sliced head of collard greens and assigned to one of eight treatments whose order had been randomly created for each different block:(1 Raw; (2 Short Simmer (heated 5 min @ 82 C in 250 ml, drained); (3 Short simmer water (water drained from short simmer); (4 Short simmer+saute' (short simmered then sauted for 4 min @ 82C); (5 Saute' (heated 4 min @ 82C in saucepan); (6 Long simmer (heated 20 min @ 82C in 250 ml, drained); (7 Long simmer water (water drained from long simmer); (8 Long simmer+saute' (long simmered then sauted for 4 min a @ 82C) Water for simmering was prepared ahead of time in a large 4qt stock pot and kept at a steady 82˚C.
If assigned to the control group, the sample was placed in a plastic bag and immediately placed under refrigeration at 3˚ ± 2˚C.The remaining treatments were as follows: Short simmer; samples were added to 250 ml of deionized water in a 1-qt sauce pan heated to 82˚C and cooked for 5 minutes then drained in a colander for 10 minutes, stored in a plastic bag and immediately placed under refrigeration at 3˚ ± 2˚C.Short simmer reserved water; collard greens assigned to this group were cooked identically to the short simmer method however the drained water was reserved and stored while the collard greens were then disposed and the water held for analysis at 3˚ ± 2˚C.Short simmer + sauté; collard greens samples were cooked according to the short simmer method and after draining for 10 minutes in a colander, were further cooked in an 8 sauté pan with 50ml of 82˚C deionized water over medium heat for 4 minutes before being bagged and stored identically to all solid treatment samples.Sauté; collard green samples were cooked in an 8" sauté pan over medium heat for 4 minutes with 50ml of 82˚C deionized water before baggage and storage.Long simmer; collard green samples were cooked in 250ml of 82˚C deionized water for 20 minutes in a covered 1-qt sauce pan, then drained for 10 minutes in a colander, bagged and stored.Long simmer reserved water; samples assigned to this group were cooked identically to the long simmer treatment group and as with the short simmer reserved water group, the drained liquid was reserved for analysis, while the solids were then disposed.For the final treatment (long simmer + sauté), collard green samples were cooked according to the long simmer method before being drained in a colander for 10 minutes and then undergoing the sauté treatment method.

Sample Extraction
Extraction procedures were based on Lim, Lim, and Tee (2007).For all but the reserved simmer water treatments, 6-10g of each sample were added to a blender with 100ml of 50% ethanol (v/v) and homogenized for 60 seconds.The homogenized mixture was further agitated for 10 minutes at 100 rpm with an Orbital Shaker model 3520 (Lab-Line/Thermo Fischer Scientific.Waltham, Massachusetts).Each mixture was then filtered through #40 filter paper (Whatman.Florham Park, New Jersey).Once yield was assessed volumetrically, 50ml of each sample was reserved and stored for no longer than one week at -20˚C.

Solids Determination
The oven drying method was used to determine solids content.Approximately 2-3 g of post-treatment collard greens were added to marked, dried and pre-weighed aluminum trays.Filled and weighed trays were placed into a model 130DM Thelco Laboratory Convection Oven (Precision Scientific/Thermo Fischer Scientific.Waltham, Massachusetts) and heated at 150˚C.After 24 hours trays were equilibrated to room temperature in a desiccator, weighed again and sample moisture percentage was determined according to the following formula: where T D1 is the mass of the oven dried tray with sample, T 1 is the premeasured mass of the empty tray and T F1 is the mass of the filled pre-oven dried tray with sample.Solids percentage was then used to express each sample concentration in mg/ml format.

DPPH Assay
DPPH assay procedures were based on (Ardestani & Yazdanparast, 2007).For each reading, 150 µl of sample were added to 850 µl of 50% (v/v) ethanol.To this 1 ml of 0.2 mM DPPH in 50% (v/v) ethanol solution was added.Mixtures were then vortexed and held for 30 minutes at room temperature after which, absorbance readings were recorded at 517 nm.Absorbance readings were compared to a negative control of 50% (v/v) ethanol and expressed as a percentage change per sample concentration (mg/ml) according to the following formula: Free radical scavenging activity (%/mg/ml) = [(A 0 -A 1 )/A 0 ] x 100%/Conc 1 where A 0 is the absorbance of the control, A 1 is the absorbance of sample and Conc 1 is the mg/ml concentration of the sample.

Total Phenolics
Total phenolic content was measured according to the Folin-Ciocalteu reaction (Singleton, Orthofer, & Lamuela-Ravnetos, 1999).Briefly, 200 µl of sample were added to 1.00 ml of Folin-Ciocalteu reagent and resulting mixture was vortexed for five seconds then held for 1 minute.To this mixture, 800 microliters of 7.5% NaHCO 3 was added then vortexed for 5 seconds and allowed to react for 15 minutes before absorbance was read at 765 nm.A standard curve was obtained using a freshly made gallic acid solution with varying degrees of concentration ranging from 0-300 mg/ml in 50% EtOH and reacting in place of samples.Using the resulting standard curve (R 2 =0.998) with a slope of 0.006 and a y-intercept of 0.016 (y=0.006x+0.016),sample absorbance readings were converted into Gallic Acid Equivalents (GAE) and expressed in terms of GAE/mg/ml of sample according to the formula: where A 1 is the absorbance of the sample and Conc 1 is the concentration of sample.

Ferrous Ion Chelating
Chelating ability of the samples was determined by the ferrous ion chelating assay (Gulcin et al., 2008).From each of the sample extracts, 0.4 ml was taken and mixed with 0.2ml of 2mM FeCL 2 .To this 3ml of 50% ethanol was added and then reaction was initiated by addition of 0.4ml of 5mM ferrozine.The mixture was allowed to sit at room temperature (~24˚C) for 10 minutes before absorbance was measured at 562nm.A negative control of 50% (v/v) ethanol without sample was used for calculations.The samples ability to prevent formation of ferrozine-Fe 2+ complex was reported in relation to samples dry mass by using the formula: Ferrous ion chelating activity (%/mg/ml) = [(A 0 -A 1 )/A 0 ] x 100%/Conc 1 where A 0 is the absorbance of the control, A 1 is the absorbance of sample and Conc 1 is the mg/ml concentration of the sample.

Statistical Analysis
A Randomized Complete Block Design was used for assigning samples to treatment groups.Statistical analysis was carried out in SPSS release 15.0.0 using the General Linear Model univariate analysis with assigned blocks and treatments with a Fisher's Least Significant Difference post hoc test to determine significant differences (p≤0.05) between treatments.

Total Phenolics
Cooking method resulted in differences in TPC for collard greens with the sautéed collard greens containing the highest amount of gallic acid equivalents (Table 1).The raw (R) followed by the short simmer + sauté (SS+S) treatments contained the next highest TPC.The long simmer water (LSW) was not different from the short simmer group (SS) (p=0.246).Gallic acid equivalents descended through the remaining treatments of long simmer + sauté (LS+S), short simmer water (SSW) and long simmer (LS).All but the long simmer water and short simmeredgreens were found to be different (p≤0.05) from other groups showing a total phenolics strength in the order listed above.Table 1.Total phenolic content (TPC), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and ferrous chelating capacity (FIC) of collard greens prepared using different cooking methods TPC DPPH FIC Raw 8.03b 7.79a 4.12e Short Simmer (heated 5 min @ 82 C in 250 ml, drained) Short simmer water (water drained from short simmer) Short simmer+saute' (short simmered then sauted for 4 min @ 82C) Saute' (heated 4 min @ 82C in saucepan) Long simmer (heated 20 min @ 82C in 250 ml, drained) Long simmer water (water drained from long simmer) Long simmer+saute' (long simmered then sauted for 4 min a@82C) Standard error of the mean 6.73d TPC = total phenolic content (gallic acid equivalentsmg/ml).DPPH = 1.1-diphenyl-2-picrylhydrazyl (% change in absorbance (517 nm)/mg/ml).FIC = ferrous iron chelating capacity (% change in absorbance (562 nm) /mg/ml).

DPPH
The DPPH assay showed similar results in ranking of treatments as the TPC assay.The order of the groups from strongest radical scavenging effect to lowest was R, S, SS+S, SS, LSW and LS+S, SSW and lastly LS (Table 1).
Treatments not differing in DPPH activity were the LSW and LS+S groups (p=0.590).All other groups were significantly different at (p≤0.05).

Ferrous Iron Chelating
Results for the Ferrous Ion Chelating assay showed different trends compared to the other two assays.The treatment that was able to chelate the greatest amount of Fe 2+ was the SS (Table 1).The next different treatment was the LS.The LS+S and the SS+S did not differ (p=0.083) in chelating capacity.The fourth and fifth best chelating treatments were the S and R groups, respectively.The remaining two treatments of LSW and SSW had the lowest chelating capacity and did not significantly differ from each other (p=0.409).

Discussion
The order of antioxidant strength of cooking treatments for both the total phenolics and DPPH assays followed a similar trend (Figure 1).In both cases the weakest group was LS, followed by SSW and then LS+S.In both assays the two strongest groups were S and R and the third strongest treatment in both assays was Short simmer + sauté.Other researchers have found similar relationships between TPC and DPPH assays (Chew, Lim, Omar, & Khoo, 2008;Sikora, Cieslik, Leszczynska, Filipiak-Florkiewicz, & Pisulewski, 2008;Xu & Chang, 2008;Roy et al., 2007).Other positively correlated values between antioxidant tests have been shown in vegetables with TPC, Trolox Equivalent Antioxidant Capacity (TEAC), Ferric Reducing Ability of Plasma (FRAP) and Oxygen Radical Absorbance Capacity (ORAC) assays (Proteggente et al., 2002) and in fruits with DPPH, FRAP, ORAC and 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assays (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006).In testing the effects of thermal treatments on the content of various polyphenols in artichoke, Ferracane et al. (2008) reported that most thermal treatments increased the bioavailability of polyphenols most likely as a result of a weakening of the plant biomass.A similar mechanism could also account for the increase in total phenolic content found in sauté collard greens when compared to raw greens in the current study.When an aquathermal treatment was applied, measures for both TPC and DPPH decreased with longer simmering times.Aquathermal treatment of kale, another Brassica variant, also caused reductions in total phenolics and radical scavenging activity (Sikora et al., 2008).
The order trend of the FIC assay was noticeably different from both the TPC and DPPH assays (Figure 2).Both of the simmer water treatments (SSW and LSW) showed the lowest ferrous ion chelating ability of any of the treatments.Sauté and Raw groups, which were the two strongest groups in the other two assays, were 4 th and 3 rd weakest respectively in the FIC assay.The Folin-Ciocalteu reagent used in the TPC assay has been found to react with chelating molecules et al., 2005) yet in various experiments there was no strong correlation between TPC and FIC assay results (Chew et al., 2008;Gulcin & Kirecci, 2008;Sun, Chou, & Yu, 2009).As opposed to the DPPH and TPC assays where antioxidant activity and phenolics were lost to the simmer water during aquathermal treatment, similar leeching was not observed with metal chelating compounds found in collard greens.Metal chelating substances found in plant material include chlorophyll, phytic acid, phospholipids, oxalates and possibly carotenoids.Since length of aquathermal treatment did not affect the chelating ability of the simmer water the chelating compounds in collard greens may be predominately lipid soluble.Based on the current study, simmer treatments were the most optimal at increasing the activity of metal chelators.This increase is possibly due to degradation of biomass since the sauté treatment also increased the metal chelating activity compared to other treatments.
Figure 2. Relative rank of each antioxidant assay for each cooking method.Each antioxidant assay was ranked from lowest (1) to highest (8) for each cooking method Primary antioxidants as measured in the electron transfer based DPPH and Folin-Ciocalteu assays were negatively impacted by aquathermal treatment.Total antioxidant measures decreased from an increase in treatment duration and more antioxidant activity was found in the wastewater of the long simmer treatments compared to the short simmer treatments.Both antioxidant capacity and total phenolics were found to increase when aquathermal treatments were subjected to additional sauté treatment with deionized water.Further increases could possibly be measured by using the wastewater of a simmer method as the liquid for the sauté or even with using a regular vegetable oil as in a traditional sauté method.Kalogeropoulos, Chiou, Mylona, Ioannou, and Andikopoulos (2007) found that they were able to transfer a significant amount of polyphenols from olive oil into fish fried in the olive oil.However because this supplementation would provide lipophilic polyphenols, it would not be a direct replacement for the most likely hydrophilic polyphenols that are lost during aquathermal treatment.
While DPPH and TPC assays followed similar ranking order, they varied on which group was the strongest.For the DPPH assay, the raw group was significantly higher than the sauté group while in the TPC assay the sauté group actually showed greater values than the untreated raw group.Such an increase in total phenolics could be explained by the thermal treatment's ability to break down the biomass of the collard greens allowing for greater availability of polyphenols contained inside the cell walls (Ferracane et al., 2008).
Following a different ranking trend than seen in DPPH and TPC assays, the FIC assay found greater chelating ability in the samples that had undergone aquathermal treatment compared to raw and sauté groups.Additionally very low values of chelating ability were found inthe reserved wastewater samples for both the long and short simmer treatments that were not significantly different from each other.The low chelating content of the reserved water reinforces the need for more than just water as a solvent when testing for chelating compounds in collard greens.
Further experimentation would be useful not only in determining if the above assumption of antioxidant retention through using simmer wastewater as a liquid in sautéing is correct but also as to how such a use would affect overall taste acceptance of the resulting product.While the combined values of the simmer and reserved water groups exceeded that of the other groups antioxidant assays were not performed specifically on combined samples such as this.Because of the lack of actual testing on combined simmer + reserved water groups it is not possible to reliably compare results to the measured treatments.Traditional comfort food cooking of collard greens often times uses the wastewater as a flavoring liquid in the final dish, referring to it as the "pot liquor".As the results of this experiment show, this pot liquor has significant amounts of antioxidants making it quite beneficial to consume.Additionally polyphenolic content is not the only thing lost during aquathermal treatment.In a similar experiment with kale, it was found that aquathermal treatment caused a significant decrease in the amino acid content of the samples (Lisiewska et al., 2008) and so follow-up experiments would be useful in determining the effect of cooking techniques used in this study on more than just primary and secondary antioxidant measures of collard greens.
Through the use of three different assays it was determined that aquathermal treatments had a significant effect on the various antioxidants modes of activity.In the cases where hydrophilic compounds appeared to constitute a considerable portion of total antioxidant power, aquathermal treatments such as a short term simmer and a long term simmer were shown to decrease the antioxidant activity.Through the use of an additional cooking treatment of sauté both long term and short term simmer treatments increased antioxidant activity.As such this study supports the use of a two-step cooking method for collard greens to maximize antioxidant activity when water cooking is used.The importance of this two-step method is restricted to hydrophilic antioxidants.
means with different letter within columns are significantly different (p≤0.05).

Figure 1 .
Figure 1.Total phenolic content (TPC) and DPPH activity for collard greens prepared using different cooking methods.Standard error of the mean for TPC = 0.11, standard error of the mean for DPPH = 0.13