JFS C: Food Chemistry and Toxicology

Effects of Extraction Conditions on Improving the Yield and Quality of an Anthocyanin-Rich Purple Corn (Zea mays L.) Color Extract
P. JING AND M.M. GIUSTI
ABSTRACT: Purple corn (Zea mays L.) is a rich and economic source of anthocyanin colorants and functional ingredients. However, high levels of anthocyanin-rich waste are generated during processing, reducing the yields and increasing the costs of the final product. This waste has been associated with anthocyanin complexation with tannins and proteins. Our objective was to evaluate anthocyanin extraction methods to reduce purple corn waste. Different solvents (water, 0.01%-HCl-acidified water, and 0.01%-HCl-acidified ethanol), temperatures (room temperature, 50, 75, and 100 C), and times of exposure to the solvents were investigated. Acetone (70% acetone in water) extraction was used as control. Anthocyanins, total phenolics, tannins, and proteins in extracts were measured by the pH differential, Folin-Ciocalteu, protein precipitation, and BCA assay methods. Qualitative analyses were done by HPLC coupled to a PDA detector and SDS-PAGE analysis. Water at 50 C achieved the highest yield of anthocyanins (0.94 0.03 g per 100 g dry corncob) with relatively low tannins and proteins, comparable to the anthocyanin yield obtained by 70% acetone (0.98 0.08 g per 100 g dry corncob). Extending the extraction time from 20 to 60 min and using consecutive reextraction procedures reduced anthocyanin purity, increasing the yields of other phenolics. A neutral protease was applied to the extracts and effectively decomposed the major protein that was believed to contribute to the development of anthocyanin complexation and waste generation. Extraction time, consecutive reextraction procedures, and enzyme hydrolysis should be considered for high yield of anthocyanins and waste reduction. Keywords: anthocyanins, extraction, protein, purple corncob, tannin

Introduction
nthocyanins are the largest and most important group of water-soluble pigments in nature, contributing to the attractive orange, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins, which have been consumed for many years without any apparent adverse effects, have bright pHdependent colors (Mazza and Miniati 1993). Interest in anthocyanins has increased due to their color properties and potential health benefits as an alternative to the use of synthetic dyes (Giusti and Wrolstad 2003). Close to 25% of the population perceives foods without artificial ingredients as desirable, this being very important in their food and beverage purchase decisions in the United States (Sloan 2005). Similar consumer attitudes are found in many countries around the world. Recently, anthocyanins have been reported to have various potential health benefits such as antioxidant capacity (Ghiselli and others 1998; Noda and others 2000; Wang and Lin 2000; Prior 2003), antimutagenic activity (Gasiorowski and others 1997; Peterson and Dwyer 1998), and chemopreventive activity (Koide and others 1997; Zhao and others 2004), contributing to a reduced incidence of chronic diseases. Researchers have shown that an anthocyanin-based food colorant from purple corn was able to inhibit cell mutation (Aoki and others 2004), reduce chemically induced colorectal carcinogenesis (Hagiwara and others 2001), and may aid in the prevention of obesity and diabetes (Tsuda and others 2003).
MS 20070062 Submitted 1/26/2007, Accepted 5/13/2007. Authors are with Dept. of Food Science and Technology, The Ohio State Univ., Columbus, Ohio 43210-1096, U.S.A. Direct inquiries to author Giusti (E-mail: giusti.6@osu.edu).

Purple corn (Zea mays L.), rich in anthocyanins, has been cultivated in South America, mainly in Peru and Bolivia, and used for centuries to prepare drinks and desserts. A colorant from purple corn is widely used in Asia, South America, and Europe. Cyanidin 3-glucoside is the major anthocyanin in purple corncob (Styles and Ceska 1972; Nakatani and others 1979). Glucosylated derivatives of cyanindin, pelargonidin, and peonidin have been found in maize plants as well as their respective malonyl derivatives (Aoki and others 2002; Pascual-Teresa and others 2002; Jing and Giusti 2005). Large quantities of anthocyanin-rich waste (ARW) are generated during the industrial preparation of an anthocyanin colorant. The food colorant industry produces anthocyanin extracts from purple corncobs by using hot acidified water, followed by sedimentation. Both supernatant and precipitated portions were separated and spray-dried. The precipitated portion has limited application in foods due to its low solubility in acidified aqueous systems (Jing and Giusti 2005), and it is considered as waste material. Protein and other complex molecules such as tannins exist in purple corncobs, which may form complexes with anthocyanins, resulting in a loss of solubility under typical conditions of anthocyanin food applications. In this study, we evaluated the extraction conditions to maximize the yield of anthocyanins while decreasing the yield of tannins and protein, consequently reducing the generation of purple corn waste during the processing of purple corn colorant.

Materials and Methods

urple corncobs (Zea mays L.) were donated by Globenatural Intl. S.A. (Chorrillos-Lima, Peru). The dry purple corncob was crushed into coarse particles and milled into powder by PERTEN laboratory mill 3600 (Perten Instruments Inc., Ill., U.S.A.).
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Effects of extraction conditions . . .

Multifect neutral enzyme and Enzeco fungal acid protease were obtained from Genencor Intl. (Rochester, N.Y., U.S.A.) and Enzyme Development Corp. (New York, N.Y., U.S.A.). Folin and Ciocalteau phenol reagent and the gallic acid standard (crystalline gallic acid, 98% purity) were purchased from Sigma (St. Louis, Mo., U.S.A.). Bovine serum albumin (BSA) and PIERCE BCA (bicinchoninic acids) protein assay kit were purchased from Fisher Scientific (Fair Lawn, N.J., U.S.A.). Multicolored protein marker (6.5 to 205 kD) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, Mass., U.S.A.). The 10% to 20% gradient gel was obtained from BioWhittaker (Walkersville, Md., U.S.A.). All high-performance liquid chromatography (HPLC) grade solvents and other chemicals (analytic grade) were obtained from Fisher Scientific.

Protein analysis
Total protein concentration was measured by PIERCE BCA (bicinchoninic acids) protein assay kit purchased from Fisher Scientific. The absorbance of the samples, standards of bovine serum albumin (BSA), and controls (samples in deionized water instead of BCA reagent B) was measured at 562 nm on a Shimadzu spectrophotometer described above. Anthocyanin color background was deducted from the final sample color. Total protein was quantified as BSA equivalents, based on a standard curve of 0, 25, 125, 250, 500, 750, and 1000 g/mL of BSA.

Total phenolics
Total phenolics were measured using a modified FolinCiocalteu method (Singleton and Rossi 1965). Briefly, a series of tubes were prepared with 15-mL water and 1-mL Folin-Ciocalteau reagent. Then 1 mL of the samples, gallic acid dilutions and water blank was added in tubes, mixed and let stand at room temperature for 10 min. The 20% Na 2 CO 3 solution (3 mL) was added to each test tube and mixed well before they were put in a Fisher isotemp dry-bath incubator (Fisher Scientific) at 40 C for 20 min. After incubation, the tubes were cooled in an ice bath to room temperature. The absorbance of samples and standards was measured at 755 nm using a Shimadzu UV-visible spectrophotometer after zeroing the spectrophotometer with a water blank. Total phenolics were calculated as gallic acid equivalents based on a gallic acid standard curve. Disposable cuvettes of 1-cm path length were used. Each sample was evaluated using 4 replications.

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Anthocyanin extraction: effects of times and temperatures of extraction

Effects of solvent, temperature, and acidity: 1 g of purple corncob powder was added to 25 mL of deionized water, 0.01%-HClacidified water, 0.01%-HCl-acidified ethanol, or 70% aqueous acetone. The samples were shaken in a NBS C76 Water Bath Shaker (New Brunswick Scientific) at 80 rpm at room temperature for 1 h. Additionally, extraction with water/0.01%-HCl-acidified water was also performed at 50, 75, or 100 C to evaluate if extraction efficiency with water could be increased at higher temperatures. The resulting extracts were filtered through a Whatman No. 1 filter pa per (Whatman Inc., N.J., U.S.A.) under vacuum using a Buchner funnel. Acetone and ethanol were removed using a rotary evaporator at 40 C under vacuum, and the residue was reconstituted to 25 mL with 0.01%-HCl-acidified water. Every treatment was done in duplicates. In addition, we evaluated the efficiency of extraction times, using 70% acetone as the test solvent. One gram of purple corncob powder was added to 25 mL of 70% (v/v) aqueous acetone in a flask. The mixture was stirred at room temperature for 20 or 60 min and filtered by the Whatman No. 1 filter paper using a Buchner funnel under vacuum condition. The cake was reextracted using 15 mL of 70% aqueous acetone solvent for 10 min until no more color could be obtained (for a total of 5 consecutive extractions). The acetone was removed using a rotary evaporator at 40 C under vacuum. Every treatment and control had 4 replications.

Enzyme treatment and SDS analysis

Two different enzymes, Multifect neutral enzyme (Genencor Intl. Inc.), a food grade enzyme derived from bacteria, with optimal pH of 7 and temperature of 40 to 60 C, and an Enzeco fungal acid protease (Enzyme Development Corp.), with optimal conditions at pH 3 and 50 to 60 C, were tested to hydrolyze the purple corncob proteins. Purple corn powder (100 mg) and 1% (v/w) Enzyme Multifect neutral enzyme or 0.1% (w/w) Enzeco fungal acid protease (as recommended by the manufacturer) were placed in a microcentrifuge tube and taken to 2 mL with pH 7 deionized water or pH 3 water acidified with HCl, and incubated in a shaking water bath at 50 C for 2 h. The mixtures were then centrifuged at 1.3 104 rpm for 5 min at 4 C. The supernatant was transferred from the tubes to volumetric flask, added up to 10 mL by deionized water for SDS-PAGE analysis. SDS-PAGE analysis was applied to determine changes in protein composition obtained by the use of the enzyme treatment. Extracts or multicolored protein markers (6.5 to 205 kD) (PerkinElmer Life and Analytical Sciences Inc. Boston, Mass., U.S.A.) were prepared by mixing samples with the same volume of SDS-PAGE sample buffer (3% SDS, 5% 2-mercaptoethanol, 10% glycerol, 62.5 mmol/L Tris/HCl) to dilute 1-fold, heated at 95 C for 3 min, and cooled down to room temperature. Denatured extracts (50 L) or multicolored protein markers (5 L) were loaded in 10% to 20% gradient gel (BioWhittaker Inc.) and run at 150 V for 75 min. Proteins that were not decomposed by proteases were visualized by double-staining with Coomassie blue.

Monomeric anthocyanins
The total monomeric anthocyanin content was measured by the pH-differential method (Giusti and Wrolstad 2001). A Shimadzu UV-visible spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was used to measure absorbance at the visible lambda max (510 nm) and at 700 nm. The total monomeric anthocyanins were calculated as cyanidin-3-glucoside, using the extinction coefficient of 26900 L/(cm)(mg) a molecular weight of 449.2 g/L. Disposable cuvettes of 1-cm path length were used.

Tannins analysis
Tannins were determined by the protein precipitation method (Hagerman and Bulter 1978). Briefly, 1 mL of BSA was mixed with 0.5 mL sample at pH 4.9 and sat for 16 h at room temperature, then centrifuged for 8 min at 1.3 104 rpm. The pellet was reconstituted into 2-mL sodium dodecyl sulfate (SDS)/triethanolamine (TEA) (1% w/v SDS, 5% v/v TEA). And then 0.5 ml of 0.01 mol/L FeCl 3 solution was added to form the colored ionphenolate complex. After 15-min standing, the absorbance of the samples was measured at 510 nm.
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Analytical chromatography
An HPLC system (Waters Delta 600 systems) equipped with a photodiode array detector (Water 996), autosampler (Waters 717 plus), and Millennium32 software (Waters Corp.) was used. Separation was conducted using a reversed phase 5 m Symmetry C18 column (4.6 150 mm, Waters Corp.) fitted with a 4.6 22 mm Symmetry 2 microguard column (Waters Corp., Mass.,

Effects of extraction conditions . . .

U.S.A.). The solvents used were A, 1% phosphoric acid/10% acetic acid/5% acetonitrile in water, and B, 100% acetonitrile. Anthocyanins were separated by using a linear gradient from 0% to 30% A in 30 min. An injection volume of 50 L with a 1 mL/min of flow rate was used. Spectral information over the wavelength range of 260 to 600 nm was collected. Solvents and samples were filtered through 0.45-m poly(tetrafluorothylene) membrane filters (Pall Life Sciences, Mich., U.S.A.) and 0.45-m polypropylene filters (Whatman Inc.), respectively. control, without significant difference between them (P > 0.10) in Table 1. High temperature can increase compound solubility and diffusion and decrease the viscosity of solvents, thereby resulting in improvement of the efficiency of the extraction (Escribano-Bailon and Santos-Buelga 2003). Nevertheless, anthocyanins are sensitive to heat and can easily convert to the colorless chalcone form during heating (Wrolstad and others 2002). In addition, high temperature might affect monomeric anthocyanin concentration indirectly by favoring more tannin extraction and proteinanthocyanin complexations. On the other hand, the consistent decrease in protein absorbance at 100 C compared to 75 C could be indicative of protein denaturation and precipitation at the higher temperature, which could result again in more anthocyanin complexation and precipitation. These effects of temperature on extractability and stability of anthocyanins, proteins, and tannins may account for the increased yield obtained at a moderate temperature (50 C). Anthocyanins are sensitive to pH and undergo reversible transformations from acid to base environment due the gain or loss of a proton: flavylium cation, carbinol pseudobase, colorless chalcone, and quinonoidal base. The flavylium cation, most abundant at the low pH condition, is the most stable form of those. Accordingly, acids are usually used in anthocyanin extraction because of the anthocyanin stability and increased polarity by the positive charge as well (Wrolstad and others 2002). Interestingly, this study found that water without acidification achieved higher yields of anthocyanins, proteins, and tannins than acidified water at the same extraction temperature. This was particularly noticeable for the extraction of tannins (P < 0.05), where the yields obtained by deionized water were over twice as much as those obtained with acidified water at the same temperature (Table 1). The possible reason is that tannins provided hydroxyl groups to form hydrogen bonds with carboxyl groups from proteins, developing complexes that can precipitate on acid environment (Haslam 1996). In addition, anthocyanins are more electrophilic at acid environment due to the charged C-ring and could easily copigment in a interaction

Statistical analysis
The least significance difference test (LSD) was performed in general linear univariate model to identify differences among means of monomeric anthocyanins, tannins, and proteins obtained by different conditions (solvents and temperature), as well as mean differences of monomeric anthocyanins and total phenolics in consecutive reextraction procedure. Students t -test was performed to evaluate the mean differences between the control and treatment group in heat treatment for anthocyanin stability. All analyses were performed by SPSS (version 14.0, SPSS Inc., Ill., U.S.A.) software. For all statistics, P < 0.05 was considered to be statistically significant.

Results and Discussion

Effect of temperature and extraction solvent on yields of anthocyanins, tannins, and proteins
Temperature and solvents significantly affected (P < 0.001) the final yields of anthocyanins, protein, and tannins (Table 1). The highest yields of monomeric anthocyanins were extracted at 50 C by deionized water and acidified water (0.94 and 0.84 mg per 100-mg dry mass), respectively. These values were slightly (but not significantly, P > 0.10) higher than the yields at 75 C, and significantly higher (P < 0.05) than those obtained using the same exaction solvents at room temperature and 100 C. The yields obtained at 50 C were also comparable to those obtained with the

Table 1 --- Concentrations of monomeric anthocyanins, tannins, and proteins of extracts by different extraction methods Yields Solvents Water Temperature ( C) RT 50 75 100 Acidied water RT 50 75 100 Acidied ethanol Control (70% acetone) RT RT Anthocyanins (g/100 g DW) 0.68 0.05 (0.001) 0.94 0.03 (0.502) 0.83 0.03 (0.043) 0.52 0.07 (0.000) 0.60 0.02 (0.000) 0.84 0.03 (0.055) 0.77 0.04 (0.008) 0.63 0.06 (0.000) 0.14 0.01 (0.000) 0.98 0.08 (1.000) Proteins (absorbance) 1.27 0.02 (0.000) 1.54 0.04 (0.000) 1.85 0.02 (0.000) 1.50 0.03 (0.000) 1.08 0.03 (0.001) 1.38 0.03 (0.000) 1.69 0.03 (0.000) 1.33 0.02 (0.000) 0.13 0.04 (0.000) 0.74 0.04 (1.000) Tannins (absorbance) 0.23 0.02 (0.433) 0.48 0.02 (0.000) 0.62 0.01 (0.000) 0.76 0.02 (0.000) 0.11 0.02 (0.000) 0.18 0.01 (0.000) 0.26 0.02 (0.842) 0.36 0.02 (0.020) 0.10 0.02 (0.000) 0.25 0.02 (1.000)

Extracting solvents are deionized water, 0.01%-HCl-acidied water, 0.01%-HCl-acidied ethanol, and 70% aqueous acetone at room temperature (RT), 50, 75, and 100 C for 1 h. Extracts were analyzed for monomeric anthocyanins, tannins, and protein after 1-h extracting. Values are represented as mean standard error (P value) (n = 2). The P value was the signicance of paired mean comparison with the control.

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with nucleophiles such as tannins (Waterhouse 2002). It is possible that anthocyanintannin copigments could precipitate by binding with proteins under acidic conditions. As a result, the acid may contribute to the formation of complexes of anthocyanins, tannins, and proteins that precipitate, consequently decreasing levels of anthocyanin, tannins, and proteins present in the final extracts after filtration and centrifugation. Acetone (70%), a typical solvent used in the laboratory for anthocyanin extraction (Rodriguez-Saona and Wrolstad 2001), achieved the highest yield of anthocyanins (0.98 0.08 g per 100 g corncob, P < 0.01) with relatively low tannin and protein content. Deionized water was a very good and economic solvent with a high yield of monomeric anthocyanins (about 0.94 0.03 g per 100 g corncob) at 50 C, and low tannin and protein yields. Acidified water was a good overall solvent that achieved lower yields of proteins and tannins with a little sacrifice on yield of anthocyanins. In addition, anthocyanins in acidified media would be expected to exhibit more stability over time. The yield of monomeric anthocyanins, proteins, and tannins was significantly lower with acidified ethanol than with other solvents (P < 0.05), showing that 100% ethanol was not a good solvent for anthocyanin extraction from purple corn. The effect of extraction conditions on the anthocyanin profile of purple corncobs was investigated. For the extraction at room temperature, 50, and 75 C, anthocyanin profiles were very similar to the control (data not shown). However, at extraction condition of 100 C, a difference was found in the anthocyanin profiles with lower proportions of the peonidin derivatives (peaks 3 and 8 in Figure 1A). Eight anthocyanins were identified as the 3-glucoside and 3-malonyl glucoside derivatives of cyanidin, pelargonidin, and peonidin (Figure 1B) by LC-MS (Jing and others 2007).

Optimal time and consecutive reextraction procedures

Consecutive reextraction resulted in an increased recovery of total phenolics with little increase on anthocyanin yield, resulting in a decreased purity. In Figure 2, extraction with 70% aqueous acetone for 60 min followed by reextraction (4 times 10 min) yielded around 18% more total phenolics, including tannins (P <0.05)

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Figure 1 --- HPLC proles of anthocyanins extracted from purple corncob by 70% acetone and acidied boiling water. 1. Cyanidin-3-glucoside; 2. pelargonidin-3glucoside; 3. Peonidin-3-glucoside; 4. cyanidin-3maloylglucoside; 5. cyanidin-3maloylglucoside; 6. unknown; 7. pelargonidin-3maloylglucoside; 8. peonidin-3malonylglucoside; 9. cyanidin-3dimaloylglucoside. , denoted to peak 3 and peak 8.

Figure 2 --- Yield of monomeric anthocyanins and total phenolics during consecutive reextraction procedures. Extraction with 70% aqueous acetone for 20 or 60 min (fraction 1) was followed by 4 reextraction procedures of 10 min each (fractions 2 to 4). Different superscript letters (a,b,c ) were assigned to statistical signicant at the 0.05 level (n = 4). Values are represented as mean standard error (n = 4).

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than the 20-min extraction with the same reextraction procedure, with both procedures yielding similar amounts of monomeric anthocyanins (P > 0.05). Therefore, more pure monomeric anthocyanins were obtained with a shorter extraction time. Stirring for 20 min and 60 min plus 1 reextraction generated 88.36% and 87.64% total monomeric anthocyanins. More reextractions did not contribute much to the total monomeric anthocyanins and total phenolics. Anthocyanins normally occur in flower petals, fruits, stems, roots, and leaves, accumulating in vacuoles of epidermal and subepidermal cells (Strack and Wray 1994). A short extraction time was not sufficient to allow solvents to penetrate deeply into particles and released efficiently other phenolics with more nonpolarity rather than anthocyanins. Therefore a short time (about 20 min) and 1 reextraction would be recommended to efficiently yield a high amount of monomeric anthocyanins and a low level of total phenolics using 70% acetone as extracting solvent. For other extracting solvents, a similar study should be carried out to determine the optimum conditions to efficiently achieve the desired purity of anthocyanins. matic hydrolysis of this protein could be an approach to reduce proteintannin complexes with anthocyanin and decrease purple corn waste.

Conclusions
xtraction conditions, including the type of solvent, temperature, and time of exposure, can be modulated in order to produce pigment of higher quality and reduce losses during production. Deionized water at a mild temperature (50 C) was a good and economic solvent that produced a high yield of monomeric anthocyanins (about 0.94 0.03 g per 100 g corncob) as compared to other solvents and temperatures with low levels of tannins and proteins. Reduced levels of tannins and proteins may reduce pigment complexation and precipitation and reduce the waste. Length of extraction and number of reextraction procedures will affect anthocyanin recovery and purity and should also be considered for colorant production.

Acknowledgments
We thank Globenatural Intl. S.A. (Chorrillos-Lima, Peru) for providing samples and funding to support this research. Part of the experiments was carried out at the Univ. of Maryland, College Park.

Enzyme hydrolysis and SDS-PAGE

In our previous study (Jing and Giusti 2005), it was determined that high protein concentrations correlated with the formation of anthocyanin complexes with limited solubility in acidic environment. One major protein band was found at a molecular weight of 29 KD in purple corncob by SDS-PAGE analysis. The major storage proteins in corn (Zea mays L.) endosperm are zeins that are alcoholsoluble proteins (Wilson 1991). Six classes of zein proteins have been identified: zein-A (21 to 26 KD), zein-B (18 to 24 KD), zeinC (17 KD), zein-D (14 KD), zein-E (27 to 31 KD), and zein-F (18 KD) by SDS-PAGE and HPLC (Wilson 1991). According to our results, the proteins in purple corncob may be zein-E or zein-A because of their molecular weight. Zeins are a class of proline-rich proteins, which bind most strongly to tannins due to an open, random coil type of conformation with a high affinity for tannins (Haslam 1996). An enzyme treatment with Multifect neutral protease resulted in the disappearance of the 29 KD band, while samples treated with Enzeco fungal acid protease did show the band (Figure 3). This suggested that the protein could be hydrolyzed by the neutral protease but not by the acid protease. Preliminary studies (data not published) showed that treatment of anthocyanin complexes with neutral proteases was not effective for releasing the anthocyains from the complexes. Therefore, we suggest the use of the protease to prevent complexation during the process of extraction. The enzy-

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Figure 3 --- SDS-PAGE graph of protein after protein enzymatic hydrolysis. Enzyme 1: Enzeco fungal acid protease; Enzyme 2: Multifect neutral enzyme.

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