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Materials and methodsExtraction of pigments. Pea samples (10 g) were homogenized with 5 g water at room temp. A 10 g sub-sample was suspended in 10 ml cold 100% acetone, homogenized, centrifuged and the resulting supernatant saved. The sample was re-extracted 3 times as described above, which ensured > 98% extraction of carotenoids and chlorophylls. The supernatants were pooled, diluted to 50 ml with 100% acetone and filtered directly into a 4 ml vial and analysed by analytical HPLC. The above method was developed after a series of initial experiments where e.g. the efficiency of extraction with 100% acetone with or without re-extractions was studied (Table 1).
Pigment stability during processing. For studies of pigment stability raw peas (cv. Bella) were divided into 3 sub-samples and each sub-sample then divided into a sample for direct extraction (raw sample) and a sample for cooking and then extraction (cooked sample). Peas were cooked for 3 min in boiling water, drained, cooled (25 °C) and then extracted.
Separation of pigments by reversed-phase HPLC. A Shimadzu HPLC system equipped with a SPD 10AV UV-vis detector was used. Separations were performed on a LiChrospher 100 RP-18 column. Mobile phases consisted of solvent A (80% MeOH–20% H2O) and solvent B (100% EtOAc). Separations were performed at 30 °C by the following solvent gradient: 0 min 20% B, 2.5 min 22.5% B, 20–22.5 min 50% B, 24–26 min 80% B, 31–34 min 100% B, 42–47 min 20% B. Flow rate: 1 ml/min. Injection volume: 25 l. Detektion: 440 nm.
Extraction and Quantification of Chlorophyll and Carotenoid Pigments in Processed Green Peas (Pisum sativum L.)
Merete Edelenbos*, Lars P. Christensen and Kai GrevsenDepartment of Horticulture, Danish Institute of Agricultural Sciences, Research Centre Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev. E-mail: [email protected]
Danish Institute of Agricultural Sciences
IntroductionGreen peas (Pisum sativum L. convar. medullare Alef.) for deep freezing is an important vegetable crop in Denmark. The market quality of this vegetable depends on factors such as flavor, texture, size and colour (1); parameters that are influenced by genotype, growing conditions, maturity at harvest, processing conditions and handling by consumers prior to consumption (2, 3). The work reported here is part of an extensive research program focusing on pea quality aspects in the total chain from plant breeding to consumption (1). The part of the program focusing on pea colour and pigment composition of pea cultivars included development of a method for the extraction and quantification of chlorophyll and carotenoid pigments in processed green pea cultivars.
AimThe objective of the present study was to develop an analytical method for the extraction and quantification of chlorophyll and carotenoid pigments in processed green peas.
Figure 1. The concentration of carotenoids and chlorophylls in raw and cooked peas of cv. Bella. Mean of three extractions. Pigments followed by different letters are significantly different at P = 0.05 by Duncan’s multiple range test.
Identification and quantification of pigments. Identification was based on chromatographic behavior on analytical HPLC, UV-spectra and their reaction with ethanolic 0.1 M HCl after isolation by flash CC and prep. HPLC (4). Acid catalyzes the specific isomerization of 5,6-epoxides to 5,8-furanoid oxides, resulting in a hypsochromic shift of approx. 20 nm for mono-epoxides and 40 nm for di-epoxides (4, 5). The carotenoids and chlorophylls were quantified using an external calibration method for (all-E)-lutein, (all-E)--carotene, chlorophyll a and b (5).
Results and discussionExtraction and quantification. A total of 17 pigments were identified and quantified in green peas (Table 1) including 8 xanthophylls, 4 chlorophyll b related compounds, 4 chlorophyll a related compounds and (all-E)--carotene. The efficiency of different extraction procedures using 100% acetone showed that initial extraction followed by three re-extractions without holding time in between resulted in a significant higher chlorophyll and carotenoid concentration than no re-extraction and 30 or 60 min holding time (Table 1).
References1. Juhl HJ, Bech AC, Kristensen K, Poulsen CS, Hansen M (1998) Consumer involvement and evaluation of green peas. J Sensory Studies 13: 1–11.2. Ottosson L (1958) Growth and Maturity of Peas for Canning and Freezing. Almqvist & Wiksells Boktryckeri AB: Uppsala, 112 pp. 3. Kidmose U, Grevsen K (1992) Quality of vining pea varieties for deep freezing. Danish J Plant Soil Sci 96: 353–360.4. Khachik F, Beecher GR, Whittaker NF (1986) Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J Agic Food Chem 34: 603–616.5. Edelenbos M, Christensen LP, Grevsen K (2001) HPLC determination of chlorophyll and carotenoid pigments in processed green pea cultivars (Pisum sativum L.). J Agric Food Chem 49: 4768–4774.
Pigment Stability during processing. The (all-E)-lutein concentration increased significantly with cooking from 1300 mg/100 fresh weight to 1800 mg/100 fresh weight (Fig. 1). However, this increase was not caused by loss of water during cooking. The water content was 78.0% in raw and 77.7% in cooked peas. Cooking had no significant effect on the total chlorophyll concentration but the chlorophyll a and b concentration decreased and the chlorophyll a’ and b’ and pheophytin a and b concentration increased, respectively (Figure 1).
CarotenoidsRawCooked
(all-E)-lutein (all-E)--carotene carotenoids
a
a
a a
b
b
0
500
1000
1500
2000
2500
3000
3500 3500
3000
2500
2000
1500
1000
500
0
Chlorophylls g/1
00 g
fre
sh w
eigh
t
1000
600
800
200
400
1200
14000
0
chl a'+chl b' pheophytin a+b chl a+b
1400
g chlorophyll a+b/100 g fresh w
eight
8000
10000
12000
4000
6000
2000
0
b
a
b
a
a
a
Table 1. The Pigment Concentration in Processed Peas (cv. Tristar) Using Different Extraction Techniques with 100% Acetonea.
number of re-extractions 0 0 1 3
holding time, min 30 60 10 0g/100 g fresh weight
(all-E)-neoxanthin 73a 7a 72a 71a
(9'Z)-neoxanthin 210c 200c 240b 260a
(all-E)-violaxanthinb 210c 210c 230b 240a
(all-E)-lutein epoxide 110b 110b 120a 130a
(all-E)-lutein 1480b 1490b 1650a 1670a
neolutein B 18ab 27a 13b 11b
neolutein A 64a 76a 67a 63a
(all-E)--carotene 490a 500a 440b 510a
chlorophyll b derivative 4a 15a 12a 5a
chlorophyll b 2140b 2240b 2350a 2360a
chlorophyll b’ 690b 720b 750b 760a
pheophytin b 130a 110b 140a 130a
chlorophyll a derivative 78b 65c 91a 73bc
chlorophyll a 7030c 7320b 7570ab 7690a
chlorophyll a’ 840c 910b 880b 950a
pheophytin a 90a 90a 100a 110a
aMean of triplicate analysis per method. Numbers within a row followed by different letters are significantly different at P = 0.05 by Duncan’s multiple range test. bApprox. 20% of the content was neochrome.
