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Effects of Extraction Conditions on Improvingthe Yield andQuality of an Anthocyanin-RichPurple Corn (ZeamaysL.) Color ExtractP. JING AND M.M. GIUSTI

ABSTRACT: Purple corn (ZeamaysL.)is a rich and economic source of anthocyanin colorants and functional ingre-dients. However, high levels of anthocyanin-rich waste are generated during processing, reducing the yields and in-creasing the costs of the final product. This waste has been associated with anthocyanin complexation with tanninsand proteins. Our objective was to evaluate anthocyanin extraction methods to reduce purple corn waste. Differentsolvents(water, 0.01%-HCl-acidified water, and 0.01%-HCl-acidifiedethanol),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 dif-ferential, Folin-Ciocalteu, protein precipitation, and BCA assay methods. Qualitative analyses were done by HPLCcoupled to a PDA detector and SDS-PAGE analysis. Water at 50 C achieved the highest yield of anthocyanins (0.94

0.03 g per 100g dry corncob)with relatively low tannins andproteins, comparable to theanthocyaninyieldobtainedby 70% acetone (0.98 0.08 g per 100 g dry corncob). Extending the extraction time from 20 to 60 min and usingconsecutive reextraction procedures reduced anthocyanin purity, increasing the yields of other phenolics. A neutralprotease was applied to the extracts and effectively decomposed the major protein that was believed to contributeto the development of anthocyanin complexation and waste generation. Extraction time, consecutive reextractionprocedures, and enzyme hydrolysis should be considered for high yield of anthocyanins and waste reduction.

Keywords: anthocyanins, extraction, protein, purple corncob, tannin

Introduction

Anthocyanins are the largest and most important group ofwater-soluble pigments in nature, contributing to the attrac-tive orange, red, purple, violet, and blue colors of fruits, vegeta-bles, and flowers. Anthocyanins, which have been consumed for

many years without any apparent adverse effects, have bright pH-

dependent colors (Mazza and Miniati 1993). Interest in antho-

cyanins 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 coun-

tries around the world. Recently, anthocyanins have been reported

to have various potential health benefits such as antioxidant ca-

pacity (Ghiselli and others 1998; Noda and others 2000; Wang and

Lin 2000; Prior 2003), antimutagenic activity (Gasiorowski and oth-ers 1997; Peterson and Dwyer 1998), and chemopreventive activity

(Koide andothers 1997; Zhao andothers 2004),contributing to a re-

duced 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 in-

duced colorectal carcinogenesis (Hagiwara and others 2001), and

may aid in the prevention of obesity and diabetes (Tsuda and oth-

ers 2003).

MS 20070062 Submitted 1/26/2007, Accepted 5/13/2007. Authors are withDept. of Food Science and Technology, The Ohio State Univ., Colum-bus, Ohio 43210-1096, U.S.A. Direct inquiries to author Giusti (E-mail:giusti.6@osu.edu).

Purple corn (Zea maysL.), rich in anthocyanins, has been culti-

vated 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. Cyanidin3-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 oth-

ers 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 pur-

ple corncobs by using hot acidified water, followed by sedimenta-

tion. 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), andit is considered as waste material. Protein andother 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 applica-

tions. In this study, we evaluated the extraction conditions to maxi-

mize theyieldof anthocyaninswhile decreasing theyieldof tannins

and protein, consequently reducing the generation of purple corn

waste during the processing of purple corn colorant.

Materials and Methods

Purple corncobs (Zea mays L.) were donated by Globenatu-ral Intl. S.A. (Chorrillos-Lima, Peru). The dry purple corn-cob was crushed into coarse particles and milled into powder by

PERTEN laboratory mill 3600 (Perten Instruments Inc., Ill., U.S.A.).

C 2007 Institute of Food Technologists Vol. 72, Nr. 7, 2007JOURNAL OF FOOD S CIENCE C363doi: 10.1111/j.1750-3841.2007.00441.xFurther reproduction without permission is prohibited

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Multifect neutral enzymeand Enzeco fungal acid protease were ob-

tained from Genencor Intl. (Rochester, N.Y., U.S.A.) and Enzyme

Development Corp. (New York, N.Y., U.S.A.).

Folinand 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 Analyt-

ical Sciences (Waltham, Mass., U.S.A.). The 10% to 20% gradientgel 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.

Anthocyanin extraction: effects of timesand temperatures of extraction

Effects of solvent, temperature, and acidity: 1 g of purple corn-

cob powder was added to 25 mL of deionized water, 0.01%-HCl-

acidified water, 0.01%-HCl-acidified ethanol, or 70% aqueous ace-

tone. 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 wasalso performed at 50, 75, or 100 C to evaluate if extraction effi-

ciency 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 evapo-

rator 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 pow-

der 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 funnelunder 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. Ev-

ery treatment and control had 4 replications.

Monomeric anthocyaninsThe total monomeric anthocyanin content was measured by

the pH-differential method (Giusti and Wrolstad 2001). A Shi-

madzu 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 antho-

cyanins were calculated as cyanidin-3-glucoside, using the extinc-tion coefficient of 26900 L/(cm)(mg) a molecular weight of 449.2

g/L. Disposable cuvettes of 1-cm path length were used.

Tannins analysisTannins 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 recon-

stituted 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

FeCl3solution was added to form the colored ionphenolate com-

plex. After 15-min standing, the absorbance of the samples was

measured at 510 nm.

Protein analysisTotal protein concentration was measured by PIERCE BCA

(bicinchoninic acids) protein assay kit purchased from Fisher Sci-

entific. 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 spec-

trophotometer described above. Anthocyanin color background

was deducted from the final sample color. Total protein was quan-

tified as BSA equivalents, based on a standard curve of 0, 25, 125,

250, 500, 750, and 1000 g/mL of BSA.

Total phenolicsTotal 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 2CO3solution (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 ab-

sorbance of samples and standards was measured at 755 nm using

a Shimadzu UV-visible spectrophotometer after zeroing the spec-trophotometer with a water blank. Total phenolics were calculated

as gallicacid equivalents based on a gallic acid standard curve. Dis-

posable cuvettes of 1-cm path length were used. Each sample was

evaluated using 4 replications.

Enzyme treatment and SDS analysisTwo differentenzymes,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 pro-

tease (Enzyme Development Corp.), with optimal conditions at pH

3 and 50 to 60 C, were tested to hydrolyze the purple corncob pro-

teins. Purple corn powder (100 mg) and 1% (v/w) Enzyme Multifect

neutral enzyme or 0.1% (w/w) Enzeco fungal acid protease (as rec-

ommended 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 vol-

umetric 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 cooleddown to room temperature. Denatured extracts (50L) or multicol-

ored protein markers (5L) wereloaded 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.

Analytical chromatographyAn 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 Sym-

metry C18 column (4.6 150 mm, Waters Corp.) fitted with a

4.6 22 mm Symmetry 2 microguard column( Waters Corp., Mass.,

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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. Antho-

cyanins wereseparated byusing a linear gradient from 0%to 30% A

in 30 min. An injectionvolume of 50L witha 1 mL/minof flowrate

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 Sci-

ences, Mich., U.S.A.) and 0.45-m polypropylene filters (Whatman

Inc.), respectively.

Statistical analysisThe least significance difference test (LSD) was performed in

general linear univariate model to identify differences among

means of monomeric anthocyanins, tannins, and proteins ob-

tained by different conditions (solvents and temperature), as well

as mean differences of monomeric anthocyanins and total phe-

nolics 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 wasconsidered to be sta-

tistically significant.

Results and Discussion

Effect of temperature and extraction solventon yields of anthocyanins, tannins, and proteins

Temperature and solvents significantly affected (P 0.10) higher than the yields at 75 C, and sig-

nificantly higher (P< 0.05) than those obtained using the same

exaction solvents at room temperature and 100 C. The yields ob-

tained 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 meth-ods

Yields

Anthocyanins Proteins TanninsSolvents Temperature (C) (g/100 g DW) (absorbance) (absorbance)

Water RT 0.68 0.05 1.27 0.02 0.23 0.02(0.001) (0.000) (0.433)

50 0.94 0.03 1.54 0.04 0.48 0.02(0.502) (0.000) (0.000)

75 0.83 0.03 1.85 0.02 0.62 0.01(0.043) (0.000) (0.000)

100 0.52 0.07 1.50 0.03 0.76 0.02(0.000) (0.000) (0.000)

Acidified water RT 0.60 0.02 1.08 0.03 0.11 0.02(0.000) (0.001) (0.000)

50 0.84 0.03 1.38 0.03 0.18 0.01(0.055) (0.000) (0.000)

75 0.77 0.04 1.69 0.03 0.26 0.02(0.008) (0.000) (0.842)

100 0.63 0.06 1.33 0.02 0.36 0.02(0.000) (0.000) (0.020)

Acidified ethanol RT 0.14 0.01 0.13 0.04 0.10 0.02(0.000) (0.000) (0.000)

Control RT 0.98 0.08 0.74 0.04 0.25 0.02(70% acetone) (1.000) (1.000) (1.000)

Extracting solvents are deionized water, 0.01%-HCl-acidified water, 0.01%-HCl-acidified ethanol, and 70% aqueous acetone at room temperature (RT), 50, 75, and100 C for 1 h. Extracts were analyzed for monomeric anthocyanins, tannins, and protein after 1-h extracting. Values are represented as mean standard error (Pvalue) (n= 2). The Pvalue was the significance of paired mean comparison with the control.

control, without significant difference between them (P>0.10) in

Table 1.

High temperature can increase compound solubility and diffu-

sion and decrease the viscosity of solvents, thereby resulting in im-

provement 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 dur-

ing heating (Wrolstad and others 2002). In addition, high tem-

perature might affect monomeric anthocyanin concentration indi-

rectly by favoring more tannin extraction and proteinanthocyanincomplexations. On the other hand, the consistent decrease in

protein absorbance at 100 C compared to 75 C could be in-

dicative of protein denaturation and precipitation at the higher

temperature, which could result again in more anthocyanin com-

plexation and precipitation. These effects of temperature on ex-

tractability and stability of anthocyanins, proteins, and tannins

may account for the increased yield obtained at a moderate tem-

perature (50 C).

Anthocyanins are sensitive to pH and undergo reversible trans-

formations 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 an-

thocyanin 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 antho-

cyanins, proteins, and tannins than acidified water at the same

extraction temperature. This was particularly noticeable for the ex-

traction of tannins (P< 0.05), where the yields obtained by deion-

ized water were over twice as much as those obtained with acidi-

fied 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, an-

thocyanins are more electrophilic at acid environment due to the

charged C-ring and could easily copigment in a interaction

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with nucleophiles such as tannins ( Waterhouse 2002). It is possi-

ble that anthocyanintannin copigments could precipitate by bind-

ing 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 an-

thocyanin, tannins, and proteins present in the final extracts after

filtration and centrifugation.

Acetone (70%), a typical solvent used in the laboratory for antho-

cyanin 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 tan-

nins with a little sacrifice on yield of anthocyanins. In addition, an-

thocyanins 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

Figure 1 --- HPLC

profiles ofanthocyaninsextracted from purplecorncob by 70%acetone and acidifiedboiling water. 1.Cyanidin-3-glucoside;2. pelargonidin-3-glucoside; 3.Peonidin-3-glucoside;4. cyanidin-3-maloylglucoside; 5.cyanidin-3-maloylglucoside; 6.unknown; 7.pelargonidin-3-maloylglucoside; 8.

peonidin-3-malonylglucoside; 9.cyanidin-3-dimaloylglucoside. ,denoted to peak 3 andpeak 8.

Figure 2 --- Yield of monomericanthocyanins and total phenolicsduring consecutive reextractionprocedures. Extraction with 70%

aqueous acetone for 20 or 60 min(fraction 1) was followed by 4reextraction procedures of 10min each (fractions 2 to 4).Different superscript letters (a,b,c)were assigned to statisticalsignificant at the 0.05 level (n=4). Values are represented asmean standard error (n= 4).

other solvents (P< 0.05),showingthat 100% ethanol wasnot 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 simi-

lar to the control (data not shown). However, at extraction condi-

tion 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, andpeonidin (Figure 1B) by LC-MS (Jing and others 2007).

Optimal time and consecutivereextraction procedures

Consecutive reextraction resulted in an increased recovery of to-

tal phenolics with little increase on anthocyanin yield, resulting in

a decreased purity. In Figure 2, extraction with 70% aqueous ace-

tone for 60 min followed by reextraction (4 times 10 min) yielded

around 18% more total phenolics, including tannins (P

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than the 20-min extraction with the same reextraction procedure,

with both procedures yielding similar amounts of monomeric an-

thocyanins (P > 0.05). Therefore, more pure monomeric antho-

cyanins 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 andsubepidermal cells (Strack and Wray 1994). A short extraction time

was not sufficient to allow solvents to penetrate deeply into par-

ticles and released efficiently other phenolics with more nonpo-

larity 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 to-

tal phenolics using 70% acetone as extracting solvent. For other ex-

tracting solvents, a similar study shouldbe carried outto determine

the optimum conditions to efficiently achieve the desired purity of

anthocyanins.

Enzyme hydrolysis and SDS-PAGE

In our previous study (Jing and Giusti 2005), it was deter-mined that high protein concentrations correlated with the for-

mation 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 incorn (ZeamaysL.)endosperm arezeins that arealcohol-

soluble proteins (Wilson 1991). Six classes of zein proteins have

been identified: zein-A (21 to 26 KD), zein-B (18 to 24 KD), zein-

C (17 KD), zein-D (14 KD), zein-E (27 to 31 KD), and zein-F (18 KD)

by SDS-PAGE andHPLC (Wilson1991). According to ourresults, the

proteins in purplecorncob maybe zein-Eor zein-Abecause 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

Enzecofungalacid protease didshow theband (Figure 3). This sug-

gested that the protein could be hydrolyzed by the neutral protease

but not by the acid protease. Preliminary studies (data not pub-

lished) showed that treatment of anthocyanin complexes with neu-

tral 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-

Figure 3 --- SDS-PAGE graph of protein after protein enzy-matic hydrolysis. Enzyme 1: Enzeco fungal acid protease;

Enzyme 2: Multifect neutral enzyme.

matic hydrolysis of this protein could be an approach to reduce

proteintannin complexes with anthocyanin and decrease purple

corn waste.

Conclusions

Extraction conditions, including the type of solvent, tempera-ture, and time of exposure, can be modulated in order to pro-duce pigment of higher quality and reduce losses during produc-

tion. Deionized water at a mild temperature (50 C) was a good and

economic solvent that produced a high yield of monomeric antho-

cyanins (about 0.94 0.03 g per 100 g corncob) as compared to

other solvents and temperatures with low levelsof tannins andpro-

teins. Reduced levels of tannins and proteins may reduce pigment

complexation and precipitationand reduce the waste. Length of ex-

traction and number of reextraction procedures will affect antho-

cyanin recovery and purity and should also be considered for col-

orant production.

AcknowledgmentsWe thank Globenatural Intl. S.A. (Chorrillos-Lima, Peru) for provid-

ing samples and funding to support this research. Part of the exper-

iments was carried out at the Univ. of Maryland, College Park.

ReferencesAoki H, Kuze N, Kato Y. 2002. Anthocyanins isolated from purple corn (Zea maysL.).

Foods & Food Ingredients J Japan 199:415.Aoki H, Wada K, Kuzo N, Ogawa Y, Koda T. 2004. Inhibitory effect of anthocyanin

colors on mutagenicity induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Foods & Food Ingredients J Japan 209: 2406.

Escribano-Bailon MT, Santos-BuelgaC. 2003. Ch1.Polyphenol extraction fromfoods.In: Santos-Buelga C, Williamson G, editors. Methods in polyphenol analysis.Cambridge: The Royal Society of Chemistry. p 2.

Gasiorowski K, Szyba K, Brokos B, Koczyska B, Jankowiak-Wodarczyk M, OszmiaskiJ. 1997. Antimutagenic activity of anthocyanins isolated fromAronia melanocarpafruits. Cancer Lett 119:3746.

Ghiselli A, Nardini M, Baldi A, Scaccini C. 1998. Antioxidant activity of different phe-nolic fractions separated from an Italian red wine. J Agric Food Chem 46:3617.

Giusti MM, Wrolstad RE. 2001. Characterization and measurement of anthocyaninsby UV-visible spectroscopy. In: Wrolstad RE, Acree TE, An H, Decker EA, PennerMH, Reid DS, Schwartz SJ, Shoemaker CF, Sporns P, editors. Current protocols in

food analytical chemistry. 1st ed. New York: John Wiley & Sons, Inc. p F1.2.1F.2.13.Giusti MM, Wrolstad RE. 2003. Acylated anthocyanins from edible sources and their

applications in food systems. Biochem Eng J 14:21725.Hagerman AE, Bulter LG. 1978. Protein precipitation method for the quantitative de-

termination of tannins. J Agric Food Chem 26:80912.Hagiwara A, Miyashita K, Nakanishi T, Sano M, Tamano S, Kadota T, Koda T, Naka-

mura M, Imaida K, Ito N, Shirai T. 2001. Pronounced inhibition by a natural antho-cyanin, purple corn color, of 2- amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-dimethylhydrazine. Cancer Lett 171:1725.

Haslam E. 1996. Natural polyphenols (vegetable tannins) as drugs: possible modes ofaction. J Nat Prod 59:20515.

Jing P, Giusti MM. 2005. Characterization of purple corncobs (Zea mays L)anthocyanin-rich waste and its application in dairy products. J Agric Food Chem53:877581.

JingP, Noriega V, Schwartz SJ, Giusti MM. 2007. Effectsof growing conditions on Pur-ple corncob (Zea maysL.) anthocyanins. J Agric Food Chem, In press.

Koide T, Hashimoto Y, Kamei H, Kojima T, Hasegawa M, Terabe K. 1997. Antitumoreffect of anthocyanin fractions extracted from red soybeans and red beansin vitroandin vivo. Cancer Biother Radiopharm 12:27780.

Mazza G, Miniati E. 1993. Anthocyanins in fruits, vegetables and grains. Boca Raton,Fla.: CRC Press Inc.

Nakatani N, FukudaH, FuwaH. 1979. Major anthocyanin of Bolivianpurple corn(ZeamaysL.). Agric Bio Chem Tokyo 43:38991.

NodaY, Kneyuki T, IgarashiK, Mori A, Packer L. 2000. Antioxidant activityof nasunin,an anthocyanin in eggplant peels. Toxicology 148:11923.

Pascual-Teresa S, Santos-Buelga C, Rivas-Gonzalo JC. 2002. LC-MS analysis of antho-cyanins from purple corn cob. J Sci Food Agric 82:10036.

Peterson J, Dwyer J. 1998. Flavonoids: dietary occurrence and biochemical activity.Nutr Res 18:19952018.

Prior RL. 2003. Fruits and vegetables in the prevention of cellular oxidative damage.Am J Clin Nutr 78:570S-8S.

Rodriguez-Saona LE, Wrolstad RE 2001. Extraction, isolation, and purification of an-thocyanins. In: Wrolstad RE, Acree TE, An H, Decker EA, Penner MH, Reid DS,Schwartz SJ, Shoemaker CF, Sporns P, editors. Current protocols in food analyti-cal chemistry. 1st ed. New York: John Wiley & Sons, Inc. p F1.1.1F1.1.11.

Singleton V, Rossi JL. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:14458.

Sloan AE. 2005. Healthy vending and other emerging trends. Food Technol-Chicago

59:2635.

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