Cucurbit Genetics Cooperative Report 31-32:5-7 (2008-2009) / 5

A Rapid Spectrophotometric Method to Determine!-Carotene Content in Cucumis melo germplasmAngela R. Davis*, Wayne W. Fish, and Penelope Perkins-VeazieUSDA, ARS, South Central Agricultural Research Lab, Lane, Oklahoma 74555

Abstract: !-carotene is a carotenoid that has anti-oxidant properties, is a precursor of Vitamin A, and im-parts the orange color in some fruits and vegetables. Thiscompound is the major carotenoid in cantaloupe. Be-cause of its health benefits, the !-carotene content infruits is of interest to the food industry and to melonbreeders. Current methods to assay !-carotene contentin fruit are time consuming, expensive, and use hazard-ous organic solvents. In this report, preliminary data isshown for a method to quantify !-carotene content ofcantaloupe puree using light absorbance measured witha xenon flash colorimeter/spectrophotometer. Absor-bance of twenty seven cantaloupe purees from one vari-ety demonstrated a linear correlation coefficient (R2=0.7)with !-carotene content determined by hexane extrac-tion/spectrophotometry. This linear correlation showsthat this method may be suitable for quantifying !-caro-tene content in purees of fresh cantaloupe. Since puree-ing the sample is the only processing required and nochemicals are needed, the method is rapid, inexpensiveand produces no hazardous waste.

Materials and Methods: Sample Preparation. Allsteps were performed under subdued lighting at roomtemperature. Cantaloupe flesh tissue was cut into ap-proximately 2 to 4 cm cubes. Samples (25 to 500 g) werehomogenized in a Waring blender until particle sizeswere less than 4 x 4 mm. All samples were then pureedusing a Brinkmann Polytron Homogenizer (BrinkmannInstruments, Inc., Westbury, New York) with a 20 mmO.D. blade to produce a uniform slurry with particlessmaller than 2 x 2 mm. The samples were not allowed toheat or froth. A water soluble form of !-carotene wasdiluted in water to use as a control (BASF The ChemicalCompany, Ludwigshafen, Germany).

Low Volume Hexane Extraction Method (LVH): Thelow volume hexane extraction method was performedas in Fish et al. (2002). Approximately 0.6 g (determinedto the nearest 0.001 g) duplicate samples were weighedfrom each puree into 2 forty ml amber screw-top vials(Fisher, #03-391-8F) that contained 5 ml of 0.05% (w/v)BHT in acetone, 5 ml of 95% ethanol, and 10 ml of hex-ane. Purees were stirred on a magnetic stirring plateduring sampling. Samples were extracted on an orbitalshaker at 180 rpm for 15 min on ice. After shaking, 3 ml

of deionized water were added to each vial and thesamples were shaken for an additional 5 min on ice.The vials were left at room temperature for 5 min to al-low for phase separation. The absorbance of the upper,hexane layer was measured in a 1 cm path length quartzcuvette at 479 nm blanked with hexane. The !-carotenecontent was then estimated using absorbance at 479 nmand factoring in the sample weight (Zechmeister andPolgar 1943; Beerh and Siddappa 1959; Fish et al. 2002).

Puree Absorbance Method: The puree absorbancemethod was modified from a lycopene detection methodin watermelon and tomato (Davis et al. 2003a, b). Briefly,the Hunter UltraScan XE was standardized as per com-pany specifications each day the instrument was used.Purees were mixed well by gently shaking in a sealedplastic bottle and approximately 20 ml of the samplewere immediately poured into a 1 cm, 20 ml SR101Acuvette (Spectrocell, Oreland, PA). Samples werescanned in the transmittance (TTRAN) mode under thefollowing settings: the large reflectance port (1.00"),Illuminant at D65, MI Illuminant Fcw, and observer 10o.The instrument was blanked on the empty cuvette. Trip-licate readings were taken. Absorbance at 750 nm wassubtracted from absorbance at 520 nm for analysis.

Results and Discussion: Absorbance of !-carotenestandard in water. A serial dilution in water of a BASF !-carotene standard was performed. An aliquot was readusing the LVH method to check for accurate preparationfor each dilution. Additionally, each dilution was readon the UltraScan XE and the absorbance was comparedto the percent of the standard starting solution and themeasured !-carotene concentration using the LVHmethod. The UltraScan XE readings to the LVH esti-mated !-carotene concentrations were compared (Fig-ure 1). This figure demonstrates that the BASF standardfollows the Beer-Lambert law when diluted in water andwhen read on the UltraScan XE up to an absorbance ofthree, which is the ceiling for this instrument. This dataalso demonstrates that the UltraScan XE provides moreconsistent readings than the LVH method. This findingindicates that an aqueous fruit puree should also obeythe Beer-Lambert law.

Absorbance behavior of puree as related to !-carotenecontent: Based on spectral results, we investigated the

6 / Cucurbit Genetics Cooperative Report 31-32:5-7 (2008-2009)

possibility of employing absorbance measurements at430 nm, 490 nm, and 520 nm for cantaloupe puree as ameans to estimate the !-carotene content. Samples in-cluded tissue from 27 cantaloupe fruit (1 variety). Theabsorbance reading of the puree at 520 nm gave a highercorrelation with the hexane extraction method.

The absorbance at 520 nm measured for each pu-ree as adjusted for scatter by subtraction of the absor-bance at 750 nm was plotted against its !-carotene con-tent as measured by hexane extraction (Figure 2). Thescatter adjusted absorbances at 520 nm of the pureesappear to obey Beer’s law with respect to !-carotene con-tent of the puree. The absorbance reading is linearly cor-related with !-carotene content, and the linear leastsquares fit to the data yields the equation: y = 23.694x +5.7785.

Freeze-thawed samples can not be compared withfresh samples. The freeze-thawed samples exhibit a dif-ferent conversion equation than fresh samples (Data notshown.). For each level of !-carotene, fresh samples readwith a higher absorbance than frozen samples. This islikely due to protein and cell wall breakdown in the fro-zen tissue

Conclusions: In the search for a rapid and reliableway to quantitate !-carotene levels in cantaloupe tissuefor screening large numbers of germplasm samples, weare developing a method that utilizes an instrument thatcan measure actual light absorbance of compounds in aslurried, aqueous medium. The method is simple, usesno hazardous chemicals, and is faster and less expen-sive than currently used methods. More cantaloupe va-rieties are being evaluated to determine the accuracy ofthis method.

Acknowledgements: We would like to thank An-thony Dillard and Amy Helms for providing valuabletechnical support.

Disclaimer: Mention of trade names or commer-cial products in this article is solely for the purpose of

providing specific information and does not imply rec-ommendation or endorsement by the U.S. Department ofAgriculture. All programs and services of the U.S. De-partment of Agriculture are offered on a nondiscrimina-tory basis without regard to race, color, national origin,religion, sex, age, marital status, or handicap. The ar-ticle cited was prepared by a USDA employee as part ofhis/her official duties. Copyright protection under U.S.copyright law is not available for such works. Accord-ingly, there is no copyright to transfer. The fact that theprivate publication in which the article appears is itselfcopyrighted does not affect the material of the U.S. Gov-ernment, which can be freely reproduced by the public.

Correspondence to: Angela R. Davis, United StatesDepartment of Agriculture, Agricultural Research Ser-vice, South Central Agricultural Research Laboratory,PO Box 159, Lane, OK 74555, USA. Phone: 580-889-7395; Fax: 580-889-5783; email: angela.davis@lane-ag.org

ReferencesBeerh, O.P., Siddappa, G.S. 1959. A rapid spectrophotometric

method for the detection and estimation of adulterants intomato ketchup. Food Technol. 13:414-418.

Davis, A. R., Fish, W. W. and Perkins-Veazie, P. 2003a. A rapidspectrophotometric method for analyzing lycopene contentin tomato and tomato products. Postharvest Biol. Technol.28:425-430.

Davis, A.R., Fish, W.W., Perkins-Veazie, P. 2003b. A rapid hex-ane-free method for analyzing lycopene content in water-melon. J. Food Sci. 68:328-332.

Fish, W.W., Perkins-Veazie, P., Collins, J.K. 2002. A quantitativeassay for lycopene that utilizes reduced volumes of organicsolvents. J. Food Composition and Anal. 15:309-317.

Zechmeister, L., Polgar, A. 1943. cis-trans Isomerization andspectral characteristics of carotenoids and some relatedcompounds. J. Am. Chem. Soc. 65:1522-1528.

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Figure 1: Demonstration of a !!!!!-carotene standard obeying Beer-Lambert law when diluted in waterand analyzed using the UltraScan XE.Wavelengths 460 and 490 nm werechosen since they provided thehighest readings of the scannedstandard and the cantaloupe purees.Wavelength 520 nm was chosenbecause cantaloupe shows a peak atthis wavelength. R2 value for eachlinear least squared best fit line wereall 0.99.

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Figure 2: Results of absorbance of 27cantaloupe purees for !!!!!-caroteneusing the Hunter Lab UltraScan XE.Absorbance is plotted versus the !!!!!-carotene content of the cantaloupedetermined by the low volumehexane method. Absorbance at 520nm was adjusted for scatter bysubtracting the absorbance at 750 nm.The absorbance reading was linearlycorrelated with !!!!!-carotene content,and the linear least squares fit to thedata yielded the equation: y =23.694x + 5.7785. The R2 value forthis linear correlation was 0.7.