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DOI : http://dx.doi.org/10.9787/KJBS.2012.44.4.433
Introduction to Colorimetric Analysis for Assessment of Carotenoid Pigments in Squash Germplasm
Shim,
Organic Agriculture Division, National Academy of Agricultural Science, Rural Development Administration
Received on June 25, 2012. Revised on November 19, 2012. Accepted on November 27, 2012
Abstract
Carotenoids of squash play an important role in human health by acting as sources of provitamin A or as protective antioxidants. Among the 60 accessions of squash germplasm, fluorescent yellow and yellow types of flesh color got the highest count, followed by the orange, whitish yellow and greenish yellow. The redness and yellowness values of the flesh powder ranged from -2.45 to 86.09 and from 13.77 to 39.80, respectively. While the lightness and the total color difference values of flesh color varied from 67.64 to 86.09 and from 19.77 to 51.79, respectively. Colorimetric values of redness and yellowness showed positive correlation, and the correlation coefficient (r) was as high as 0.7386. The five accessions represented each flesh color types, IT195043 (orange), IT136696 (fluorescent yellow), IT186365 (yellow), IT137963 (whitish yellow), and IT180449 (greenish yellow). The total amount of carotenoid contents was in the order of orange color (104.64 mg/100 g), greenish yellow color (70.82 mg/100 g), fluorescent yellow color (32.41 mg/100 g), yellow color (8.73 mg/100 g), and whitish yellow color (4.73 mg/ 100 g). Both lutein and β-carotene were the predominant pigments of carotenoids, and lycopene was only separated and identified in the orange color flesh. According to the results, colorimetric analysis can aid breeders interested in increasing carotenoid content in squash, which could be accurately measured using a simple, reliable, and cost- and labor-efficient method for the evaluation of carotenoid pigments.
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INTRODUCTION

 Squash is the principal ingredient of several culinary vegetable utilized at the immature and mature fruit stages. Squash provides a valuable source of carotenoidsamd ascorbic acid that have a major role in nutrition in the form of proviyamin A and vitamin C as antioxidants, when used at repening stage or after storage (Peirce, 1987; Andres, 1990).

Pumpkins and squash (Cucurbita spp.) are excellent dietary sources of carotenoids (Wills et al., 1987; Gross, 1991) and, in 2001, ranked 11th among other vegetables produced around the world (FAOSTAT, 2008). Especially, winter squash cultivars are very good and promising sources of β-carotene (Whang et al., 1999; Chavasit et al., 2002; Gajc-Wolska et al., 2005; Murkovic et al., 2002) 

Carotenoids are a class of more than 600 naturally occurring pigments synthesized de novo by plants, algae, and photosynthetic bacteria. Carotenoids are yellow, orange, and red pigments synthesized by plants. Moreover, numerous aspects of carotenoid metabolism are still unclear (Takeda, 1982; Gross, 1991; Fraser et al., 1994; Kato et al., 2004). 

The most common carotenoids are α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene (Lee et al., 1984; Kim et al., 2003). Carotenoid pigments play a beneficial role in a variety of physiological mechanisms, including free radical scavenging, immune system enhancers, and precursors for vitamin A, included β-carotene, α-carotene, and β-cryptoanthin in pumpkin (Borenslein and Bunnel, 1996; Nishino, 1998; ODS/NIH, 2006). 

The wide range of carotenoids in pumpkins and squash provides fertile ground for genetic improvement. When breeders have reliable information about carotenoid types and concentrations, they traditionally use a method as HPLC. HPLC is highly sensitive and reproducible, but can be expensive and time-consuming. To determine if carotenoid content of pumpkin and squash could be accurately measured using a less-expensive and simpler method, 

Colorimetric analysis is mostly calculated as the L*, a*, b*-value system developed by Judd and Hunter, standardized in 1976. In CIE-LAB color space system, the L*-value indicates the brightness as the position of the bright/dark axis, and also the a*-and b*-value indicates the redness and yellowness as the position of the red/green axis and the position of the blue/yellow axis, respectively. 

Chemical measurement of carotenoid pigments can be accomplished by spectrophotometric analysis of extracts and high pressure liquid chromatography (HPLC) using a Silica gel column. The advantage of HPLC measurement is that individual pigments can be quantified and also isomers can be separated (Gross, 1991; Emenhiser et al., 1995; Whang et al., 1999; Kim et al., 2003; Ha et al., 2009). 

This study aimed to determine the correlation with colorimetric analysis and HPLC method in analyzing the carotenoid content in squash germplasm. The colorimetric analysis method could be accurately measured using a low cost, rapid and simple method. The results of this study can be used by breeders interested in increasing carotenoid content in squash germplasm. 

MATERIALS AND METHODS

Plant materials

The profile of squash germplasms tested was analyzed based on the origin, the country that indtroduced it, and species. As previously reported, there are already information on the agronomic traits of 78 accessions of squash germplasm. From which 60 accessions were regenerated and selected in the National Agrobiodiversity Center of Korea with Genebank management program in 2009 (Kim et al., 2010). About 50 g of squash fruit flesh was harvested from 60 accessions of squash germplasm at 30 days of ripening stage and was frozen at -70℃ in the deep freezer. Each frozen samples was freeze-dried and ground to a fine powder using a grinding mill for colorimetric and HPLC analysis to determine the content of carotenoid pigments.. 

Observation of flesh color and colorimetric analysis

The squash fruit flesh color of 60 accessions of squash germplasm after ripening stage was determined with the naked eye. The color of squash fruit flesh was categorized into the following; five types as orange, fluorescent yellow, yellow, whitish yellow and greenish yellow. 

The L, a, b, and △E-system developed by Judd and Hunter, standardized in 1976 and based on sensitivity is commonly used (CIE, 1976). Colorimetric value of squash flesh powder was obtained using a Minolta CR300 colorimeter (Minolta Co., Japan) and the CIE-LAB color space (CIE, 1976). In this system, the L-value indicates the position of the bright/dark axis where in the minimum L value is zero, which represents black. The a-value indicates the position of the red/green axis wherein positive a is red and negative a is green. The b-value indicates the position of the blue/yellow axis wherein positive b is yellow and negative b is blue. The total color difference, △E, has to be calculated too. The △E is a single value which takes into account the differences between the L, a, and b of the sample and the standard. The L, a, b coordinates are directly related to the standard color values X, Y and Z. 

Extraction procedure for HPLC

The efficient extraction method of carotenoids developed by Kim et al., (2003) and Ha et al. (2009) was modified. For extraction of catotenoids, 0.2 g of the freeze-dried squash flesh powder was homogenized three times with a vortex with 0.1 g MgCO3  and 5 mL of 0.2% ascorbic acid in methanol containing 0.1% BHT (butylated hydroxyl toluene) as antioxidant, and was saponificated on water bath for 10 minutes at 80℃. After saponification, the sample was cooled down on ice. 1.2 μg of β-apo-8'-carotenal (Sigma, USA) was added as internal standard (ISTD) at about the same concentration that was expected to be in the sample. The solvent was removed by filtration and the filter cake was re-extracted twice. From this extraction solvent, the carotenoids were extracted with petroleum benzene (40~60℃ boiling range) after addition of 5 mL aqueous NaCl solution (10%). The water phase was reextracted twice with 5 mL petroleum benzene. After evaporation of solvent the residue was redissolved in 0.5 mL mobile phase. The extracts were evaporated and loaded into a column.

Analysis conditions on HPLC

The separation and quantification of carotenoids were accomplished by HPLC (High-Performance Liquid Chromatography). A total of five samples were selected for HPLC analysis directly following colorimetric analysis of 60 accessions of squash germplasm. For HPLC analysis of extracts 20 μL were injected into a reversed phase column (YMC carotenoid, 4.6 × 250 mm, 5 μm) with a pre-column (ODS1, metal free, 10 × 4.6 mm, 5 μm). The HPLC equipment used was WATERS Liquid Chromatography (Japane) with a photo diode array (PDA) detector. The columns were eluted with two types of mobile phase (mobile phase A : MeOH : TBME : water : triethylamine = 6 : 90 : 4 : 0.1(v/v/v/v) and mobile phase B : MeOH : TBME : water : triethylamine = 81 : 15 : 4 : 0.1(v/v/v/v)) and operated binary linear gradient program (0% A/100% B at 10 min., 50% A/50% B at 40 min., 100% A/0% B at 50 min., 100% A/0% B at 55 min., 0% A/100% B at 60 min., 0% A/100% B at 65 min). The flow rate was 1.0 mL/min and the absorption of the effluent was monitored at a wave length of 450 nm for the determination of the carotenoids. For identification of the carotenoids (capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, β-carotene) the retention times with that of standard substances were compared. The quantification was based on an internal standard method with β–apo-8’-carotenal as the internal standard (Murkovic et al., 2000). 

Statistic analysis

All data were performed by ANOVA procedures appropriated for a randomized complete block design by the GLM procedures of SAS (SAS Institute, 2002). Differences among the colorimetric values of flesh color were determined by Duncan’s new multiple range test (Duncan, 1955), and significance was defined at P < 0.05. 

RESULTS

Observation of flesh color and colorimetric analysis

In Table 1, the flesh color variation of squash germplasm was determined with the observation of naked eye which was categorized into five types of flesh color, orange, fluorescent yellow, yellow, whitish yellow and greenish yellow at 30 days of ripening stage. 

9b877528b8bb0e087e7eee6310148c8e.JPG.resized.JPGTable 1. Estimation of the variation of flesh color of 60 accessions of squash germplasm by color and difference meter.

534505de2bf828a57ac18b15ead1b563.JPG.resized.JPGTable 1. Continued.

The colorimetric values, the Lightness (L), the redness (a) and yellowness (b) of a standard white reference tile were 92.75, -0.76 and -0.07, respectively. The colorimetric values, lightness (L), redness (a), and yellowness (b) were obtained from the reflectance’s value of the fine flesh powder from the 60 accessions of squash germlasm using a Minolta CR300 colorimeter and the CIE-LAB color space. Redness (a) and yellowness (b) values of the flesh powder color ranged from -2.45 to 86.09 and from 13.77 to 39.80, respectively. The redness (a) value of greenish yellow types of flesh color was mostly calculated as negative values. On the other hand, the redness value (a) of orange types of flesh color was calculated more than 9.76. Lightness (L) and the total color difference (△E) values of flesh powder color varied from 67.64 to 86.09 and from 19.77 to 51.79, respectively (Table 1). 

Among the 60 accessions of squash germplasm, fluorescent yellow and yellow type of flesh color got the highest count with 28 (46.7%) and 16 (26.7%) accessions of squash germplasm, respectively, and followed by whitish yellow, greenish yellow and the orange types with seven, five and five accessions, respectively (Table 2). 

d215b690f4a87a8854ad16810981c589.JPG.resized.JPGTable 2. Hunt color values of flesh color of 60 accessions of squash germplasm at different rate

As the result of ANOVA analysis of colorimetric values of lightness (L), statistical significance with P value (P<0.05) of 0.214 was not recognized. There is no difference between the averages of the lightness values of five types of flesh color (Table 2). 

However, the ANOVA analysis of colorimetric values of redness (a), yellowness (b) and total color difference values (△E) were significantly recognized with P values (P<0.05) of 0.822, 0.634 and 0.460, respectively (Table 2). There were significant difference between the average means of colorimetric values of redness (a), yellowness (b) and total color difference (△E) for five types of flesh color (Table 2). 

Colorimetric values of redness (a) and yellowness (b) showed positive correlation, and the correlation coefficient (r) was as high as 0.7386 (Fig. 1, A). On the other hand, the flesh color types demonstrated a negative correlation with the two colorimetric values, redness (a) and yellowness (b), respectively. The correlation coefficient (r) of the redness values and the yellowness values with the flesh color types were slightly low as 0.6947 and 0.5123, respectively (Fig. 1, C, D). Also, the correlation of lightness and the total color difference value was negative, with low correlation coefficient (r) of 0.6544 (Fig. 1, B). 

3b6d6afc3e6545e045d7b9e52063eb8d.JPG.resized.JPGFig. 1. Quantitative relationship between redness (a) and yellowness value (b) (A), lightness (L) and the total color difference (△E) value (B), redness (a) and flesh color types (C), and yellowness (b) and flesh color types (D) obtained from the colorimetric value of carotenoid pigments of squash germplasm with colorimeter. Flesh color types: 1, Orange; 2, Fluorescent Yellow; 3, Yellow; 4, Whitish Yellow; 5, Greenish Yellow.

Identification and separation of carotenoid pigments with HPLC

Out of 60 accessions of squash germplasm, five accession of each flesh color types with the colorimetric values, namely; IT195043 (orange), IT136696 (fluorescent yellow), IT186365 (yellow), IT137963 (whitish yellow), and IT180449 (greenish yellow) were selected and prepared for the separation and identification of carotenoid pigments (Fig. 2). 

3ff7d0d75d51b8be183711c27ee1c46a.JPG.resized.JPGFig. 2. Distribution of five types of flesh color (A: IT195045, B: IT136696, C: IT186365, D: IT137963, E: 180449) and flesh powder color (A: Orange, B: Fluorescent Yellow, C: Yellow, D: Whitish Yellow, and E: Greenish Yellow) for matured squash germplasm after 30 days of ripening stage in 2009.

Carotenoid pigments were extracted from flesh powder and subjected to HPLC analysis. In order to determine the carotenoid pigment patterns, the carotenoid extracts of the five color types of flesh powder were compared using seven standard carotenoid substances, capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, and β -carotene (Fig. 3). 

34be243c1188ad2c42c30325e6d5f060.JPG.resized.JPGFig. 3. HPLC standard chromatograms of carotenoids (S: capsanthin, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene) and comparison of carotenoids HPLC chromatograms in pumpkin lipid extracts (A: orange, B: greenish yellow, C: fluorescent yellow, D: yellow, E: whitish yellow). ISTD is abbreviated as internal standard.

The total amount of carotenoid pigment contents were in the order, orange color flesh (104.64 mg/100 g), greenish yellow color flesh (70.82 mg/100 g), fluorescent yellow color flesh (32.41 mg/100 g), yellow color flesh (8.73 mg/ 100 g), and whitish yellow color flesh (4.73 mg/100 g) in Table 3. 

251657d3d38b3e9281132d03b4927a73.JPG.resized.JPGTable 3. Variations of carotenoid pigments on the five types of squash flesh color identified and separated with HPLC.

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 Both lutein and β-carotene were the predominant pigments of carotenoids identified in all of the five types of representative flesh color (Fig. 3 and Table 3). The next predominant pigments identified were capsanthin, zeaxanthin, and α-carotene of the six carotenoid pigments. Out of the six carotenoid pigments, lycopene was only separated and identified in the orange color flesh powder (Fig. 3, A). Fluorescent yellow flesh color was absent α-carotene and lycopene of the six carotenoid pigments (Table 3).

Moreover, lutein, α-carotene, and β-carotene was detected from whitish yellow color of flesh powder (Fig, 3, E), which had the lowest amount of carotenoid pigments compared with the other flesh color types (Table 3). 

DISCUSSION

In Korea, there are a number of studies on the ecological and morphological characteristics of the Korean landrace Squash (Chung et al., 1998), the quality and quantity of carotenoid pigments of pumpkin cultivated in Korea (Whang et al., 1999) and the characterization of 63 accessions of landrace squash, C. mosachata, at central districts in Korea (An et al., 1994). 

We previously reported the agronomic traits of 78 accessions of squash germplasm including 60 accessions of squash germplasm tested in this study (Kim et al., 2010). However, only two species of squash germplasm, C. mosachata and C. maxima, were used in this study. 

In this study, the flesh color types of squash germplasm were grouped into orange, fluorescent yellow, yellow, whitish yellow and greenish yellow based on the observation of eye at 30 days of ripening stage. The predominant flesh color types squash germplasm were fluorescent yellow and yellow followed by orange, whitish yellow and greenish yellow in decreasing order. 

Generally, the flesh color of pumpkins and squash includes a wide range of white, yellow, and orange (Gross, 1971). These colors are based on the specific carotenoid type and concentrations that are influenced by genetic and environmental factors (Paris and Nelson, 2005; Tadmor et al., 2005). In some studies reported that pumpkins consist of β-carotene and lycopene (Azizah et al., 2009; Amotz and Fishler, 1998). 

Based on the results of this study, the redness (a) and yellowness (b) values of the flesh powder ranged from -2.45 to 20.66 and from 13.77 to 39.80, respectively. The Lightness (L) and the total color difference (△E) values of flesh color varied from 67.64 to 86.09 and from 19.77 to 51.79, respectively. The redness (a) value of greenish yellow type of flesh color was mostly calculated as negative values towards green direction. On the other hand, the redness (a) value of orange type of flesh color was calculated as positive values towards red direction.

However, carotenoids are responsible for the pleasing appearance of yellow, orange or red color foods. The family of yellow to red pigments responded to the striking orange hues of pumpkins and the familiar red color of vine-ripping tomatoes (Olson and Krinsky, 1995; Murkovic et al., 2002). 

Recently, because of high antioxidant activity and nutritional properties of squash, the ready-to-eat dried winter squash snack was developed with high carotenoid content and good sensory prosperties (Konopacka et al, 2010). 

In this study, colorimetric values of redness (a) and yellowness (b) gave positive correlation, and the correlation coefficient (r) were as high as 0.7386. On the other hand, the flesh color types were negatively correlated with two colorimetric values, redness (r=0.6947) and yellowness (r=0.5123), respectively. This result indicated the effectiveness of selection for color would be moderate to high. Subjective observation of flesh color were consistent with the color space value corresponded to more orange-red flesh and yellow flesh. 

Strong correlation was observed between redness value (a*) and total carotenoids and yellowness value (b*) and chroma with lutein (Rachel and Eileen, 2009). In color analysis, yellowness value of hot air dried pumpkin powder decreased under the bright and dark condition from 10 days storage at 20℃ (Park et al., 1997). Heat treatments increased the red and yellow color of the flesh of buttercup squash (Bruce et al., 1999) and flesh color deepens during storage, because of the accumulation of carotenoids in winter squash (Arvayo-Ortiz et al., 1994). 

Our result showed that the total amount of carotenoid contents analyzed with HPLC was in the order, orange color flesh type, greenish yellow color flesh type, fluorescent yellow color flesh type, yellow color flesh type, and whitish yellow color flesh type. 

In this study, both lutein and β-carotene were the predominant pigments of carotenoids identified in all of the five types of representative flesh color. Capsanthin, zeaxanthin, and α-carotene, were second predominant pigments of the seven carotenoid pigments. Out of the seven carotenoid pigments, lycopene was only separated and identified in the orange color flesh powder. 

The yellow to orange color of the pumpkin flesh indicates presence of the carotenoids, and the visual characterization of the color correlates well with the carotenoid content (Murkovic et al., 2002). 

In three different species, C. pepo, C. moschata, and C. maxima, β-carotenoid was the most abundant carotenoid (Murkovic et al., 2002). Also, β-carotene of zucchini, green pumpkin, and sweet pumpkin was higher than the standard contents (Kim et al., 2004). Also, the principal carotenoids in C. moschata were β-carotene and α-carotene, whlereas lutein and β-carotene dominate in C. maxima and C. pepo as the major carotenoids (Azevedo-Meleio and Rodriguez- Amaya, 2007). 

The total amounts of carotenoids in the dehydrated pumpkin powder was reduced by 65~70% without effect of light after one month. Leutin increased from 15 days of storage under blight condition, but under dark condition, no change it observed for one month. On the other hand, β-carotene decreased from 15 days of storage under blight condition, and also decreased from 25 days under dark condition. The contents of lycopene and α-carotene did not show any significant change throughout the storage period. (Park et al., 1997) 

Also in this study, the smallest amount of lutein, α- carotene, and β-carotene were detected in whitish yellow color of flesh powder. However, these results indicate that lutein, α-carotene, and β-carotene pigments related with yellow and orange types of flesh color. 

Nakkanong et al (2012) reported that C. moschata had the lowest total carotenoid content and mainly accumulated α-carotene, and β-carotene, as expected in a fruit with paleorange flesh. The predominant carotenoids in C. maxima were violaxanthin and lutein, which produced a corresponding yellow flesh color in mature fruit. 

Higher significant difference would be expected when comparison is made from the immature stage. This was indeed the case of Cucurbita moschata cultivar Menina Verde. α-Carotene and β-carotene increased dramatically during maturation (Pedrosa et al., 1983; Lee et al., 1984; Chavasit et al., 2002; Kato et al., 2004). 

Among the factors related to the amount of carotenoids, climatic effects are also seen in fruits of the same cultivars produced in regions of different climates. Elevated temperature and greater exposure to sunlight increased carotenogenesis (Ramos and Rodriguez-Amaya, 1987; Arima and Rodriguez-Amaya, 1988). 

Of the visual method, color difference meter, spectrophotometer and HPLC were used for evaluation of colored flesh fruit (Park et al., 1995; Kim et al., 2003). The breeding programs focused on the selection of winter squash varieties with a high β-carotene content have been successful as demonstrated by the production of seeds of new promising cultivars and hybrids now made available (Gajc-Wolska et al., 2005; Bognar, 2006). 

Furthermore, previous studies have correlated color measurement system with carotenoid content in vegetable crops such as tomato (D’Souza et al., 1992; Arias et al., 2000), sweet potato (Simonne et al., 1993; Ameny and Wilson, 1997), pepper (Reeves, 1987; Lee and Lee, 1992), carrot (Park et al., 1995) and winter squash (Francis, 1962; Whang et al., 1999; Seroczynska et al., 2006; Rachel and Eileen, 2009; Konopacka et al., 2010) 

This study has provided that a colorimetric or spectrophotometer can be used to measure any test on a colored substance or any substance which reacts to produce color. In fact, the simple definition of colorimetric value is the measurement of color, and a colorimetric method is any technique used to evaluate an unknown color in reference to known colors. This will assure that indirect selection for high carotenoid content within pumpkin and squash breeding material will be successful, easy to implement, and inexpensive. 

ACKNOWLEDGEMENTS

This research was supported by a grant program (PJ907121022012) funded by Rural Development Administration(RDA), Republic Korea in 2012. 

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