Genet Resour Crop Evol (2012) 59:999–1014 DOI 10.1007/s10722-011-9739-9
RESEARCH ARTICLE
Storage protein and amino acid contents of Tunisian and Chinese pomegranate (Punica granatum L.) cultivars Walid Elfalleh • He´dia Hannachi • Arbi Guetat Nizar Tlili • Ferdaous Guasmi • Ali Ferchichi • Ma Ying
•
Received: 3 February 2011 / Accepted: 21 July 2011 / Published online: 11 August 2011 Ó Springer Science+Business Media B.V. 2011
Abstract In the present study, free amino acids and seed protein contents of pomegranate germplasm from Tunisia and China were conducted using Hitachi L8800 amino acid analyzer, Bradford and Kjeldahl methods. The work offers more consideration to protein pomegranate seeds which can be with great importance regarding the nutritive seed value of this crop. In other hand, the protein and amino acids contents were used to compare the Tunisian and Chinese pomegranate cultivars based on multivariate analyses (principal component and clustering analyses). Results show that the Tunisian and Chinese W. Elfalleh (&) F. Guasmi A. Ferchichi Laboratoire d’Aridoculture et Cultures Oasiennes, Institut des Re´gions Arides de Me´denine, 4119 Medenine, Tunisia e-mail:
[email protected] H. Hannachi Institut Supe´rieur des Sciences Applique´es et de Technologie, 6072 Gabe`s, Tunisia A. Guetat Laboratory of Plant Biotechnology, National Institute of Applied Sciences and Technology, B.P. 676, 1080 Tunis Cedex, Tunisia N. Tlili Laboratoire de Biochimie, De´partement de Biologie, Faculte´ des Sciences de Tunis, Universite´ Tunis El-Manar, 2092 Tunis, Tunisia M. Ying School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China
pomegranate seeds were rich in storage proteins independently of organoleptic taste as sweet, sour sweet or sour. Qualitatively, the storage protein and free amino acids of pomegranate seeds were identical. Besides, the segregation of Chinese and Tunisian pomegranate cultivars into three groups was reveled based on high, low or moderate amino acids contents. Statistically, the differences between organoleptic groups (sweet, sour sweet and sour) were not significant (P \ 0.05). However, the differences between pomegranate cultivars were statistically significant within groups. The separation of cultivars is independent to the organoleptic taste neither the geographical origin. Keywords Clustering analysis Free Amino acids PCA Pomegranate seed Punica granatum L. Storage protein
Introduction It is well known that the most of the commerciallyimportant fruits belong to the citrus Rutaceae and the rose Rosaceae families. Fruits of the citrus family share a characteristic form, a kind of berry called a hesperidium with a succulent glandular hairy endocarp. In contrast the fruits of Rosaceae are very diverse including pomes, drupes, drupecetum and pseudocarp (Vaughan and Geissler 2009). Compared with their wild ancestors, cultivated fruits are fleshier,
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sweeter, with fewer tannins and glycosides, and frequently less seedy. However it’s not the case for pomegranate fruit which belong to the Punicaceae family. Punica granatum L. seems to maintain comparable characters to the wild ancestors. During the domestication process, the seeds number per fruit is one of the most conserved characters. Barone et al. (2000), in their study focusing on domesticated pomegranate varieties from Sicilia, counted 359 to 761 seeds per fruit. There are more than 600 seeds in the wild variety of pomegranate fruit (Seeram et al. 2006). Morphologically, the pomegranate fruit is described to be with a ripe up to five inches wide with a deep red, leathery skin, grenade-shaped, and crowned by the pointed calyx. The fruit contains many seeds (arils) separated by white, membranous pericarp, and each is surrounded by small amounts of tart, red juice. (Julie-Jurenka 2008). Pomegranate fruit is characterized by its high phenolic contents and antioxidant capacities (Elfalleh et al. 2009, 2011a). Particularly, Pomegranate peel is a natural source of phenolic coumpounds: anthocyanins, ellagic acid glycosides, free ellagic acid, ellagitannins, punicalin and gallotannins (Seeram et al. 2006). In Several studies, the pomegranate is valuated for its nutritional and medicinal benefits, such as antioxidant (Elfalleh et al. 2009), antiallergic (Damiani et al. 2009), antimicrobial (Al-Zoreky 2009), antiplasmodial (Dell’Agli et al. 2009), antidiabetic (McFarlin et al. 2009), and anticarcinogenic activities (Khan 2009). Pomegranate is known as important ingredient in daily food; in addition many uses in therapeutic purposes are well-known, P. granatum fruit contains considerable amount of protein, sugar and mineral. In pomegranate, storage protein consists about 17% of seed dry weight (Elfalleh et al. 2010). The seed storage proteins are important because they ensure feeding the germinating embryo which enables it to attain the autotrophy. They are also important for the human and animal nutrition providing more than the half of daily protein requirement (Cheftel et al. 1985). These proteins were initially classified by Osborne (1924) according to their solubility properties into albumins (water soluble), globulins (saline soluble), prolamins (alcohol soluble) and glutelins (residue). Most of the physiologically active proteins (enzymes) are found
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in the albumin or globulin groups. Nutritionally, the albumins and globulins have important amino acid balance due to their higher lysine, tryptophan, and methionine contents. The prolamins were among the earliest proteins to be studied; with the first description of wheat gluten (Yildiz 2010). The difference in concentration of these various protein fractions often illustrates the food value of seeds. The protein quality of cereal grain is determined by the quantity and composition of the prolamin, major storage proteins as hordeins in barley (Takaiwa et al. 1986) and glutelin in rice (Carbonero et al. 2000). Furthermore, there is considerable interest in the production of mutants with increased protein content or increased amount of essential amino acids (Yildiz 2010). Amino acid synthesis is the set of biochemical processes which build the amino acids from carbon sources like glucose. Amino acids are the monomers and are polymerized to produce proteins. Generally, plant proteins have lower content of some essential amino acids such as lysine and methionine. Amino acids are the basic components of hormones that are essential chemical signaling messengers of the body (Yildiz 2010). Good quality proteins are readily digestible and contain the essential amino acids in quantities corresponding to the human requirements. Numerous studies concerning storage proteins structure and their biosynthesis have, therefore, been published during the last few years. Storage protein composition and amino acids sequences were investigated in several species such as Pinus pinea L. (Nasri and Triki 2007), genus Arachis (Bertozo and Valls 2001) and grain legumes (Malviya et al. 2008). Consequently, seed storage protein and amino acid profiling have comparable importance to other molecular and biochemical markers in the studies of plant genetic diversity. Worldwide there are many pomegranate cultivars, broadly divided into sweet sour sweet and sour taste. In current study we focus for possible structure of 21 pomegranates from sweet sour sweet and sour cultivars (15 Tunisian and 6 Chinese) based on 18 amino acids content and seed protein quantified by Bradford and Kjeldahl methods. Multivariate analyses were applied on pomegranate cultivars based on protein and amino acids content. Furthermore we offer more consideration to protein from pomegranate seed which can be with great importance approximating seed oil value.
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Materials and methods
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Storage proteins extraction from delipided pomegranate seeds
Chemicals and reagents All solvents were of reagent grade without any further purification. Coomassie Brilliant Blue G-250 was obtained from Sigma-Adrich Co. (USA). BSA purchased from Equitech-Bio, Inc., Kerriville, TX (USA). The water used in free amino acid analyzer and sampling was prepared with Millipore Simplicity (Millipore SAS, Molsheim, French). All other chemicals used were of analytical grade. Sample preparation Punica granatum seeds are originated from 21 pomegranates cultivars (15 cultivars from Tunisia and 6 from China). The cultivars implicated in this study are subdivided into sweet, sour sweet and sour pomegranate groups based on their organoleptic characteristics and chemical compositions. In the same way earlier pomegranate accessions were classified by several authors as sweet, sour sweet and sour accessions (Martı´nez et al. 2006; Melgarejo et al. 2000). Pomegranate trees were maintained in the Tunisian national germplasm collection of pomegranate located at Zerkin (33°450 N, 10°160 E), and cultivated under homogenous conditions, without any special management (no fertilizers, no irrigation except natural rainfall). Pomegranates were randomly picked from three trees of each cultivar. Pomegranate fruits were sampled at ripe stage, i.e. ready for fresh consumption. The seeds were manually separated from pulps and dried at room temperature. Table 1 shows more information about geographical cultivated area in Tunisia and protein contents of cultivars. Kjeldahl assays The amount of protein present in dry pomegranate pulp was determined by the analysis of total nitrogen protein (TNP) using the Kjeldahl method (AOAC 1995). This method was demonstrated to be efficient for pomegranate seed protein content as described by Elfalleh et al. (2009). The nitrogen content of protein was expressed as g per 100 g dry weight (DW). All measurement was performed in triplicate.
In order to extract all classes of storage proteins of pomegranate, we adopted a fractionation protocol of the various proteins categories basing on their difference in solubility as described previously by Nasri and Triki (2007) and later by Elfalleh et al. (2010). The extraction procedure is based on solubility differences of proteins in various solvents (Osborne 1924). Therefore, these protein fractions were classified by Osborne (1924) according to their solubility properties into albumins (water soluble), globulins (saline soluble), prolamins (alcohol soluble) and glutelins (residue fraction). Firstly, a milled sample (500 mg) was extracted with distilled water (10 ml). The suspension was stirred at laboratory temperature for 20 min and then centrifuged (10,000 rpm for 15 min). The filtrated supernatant was used as the extract 1 (albumin fraction). The remaining insoluble sample was mixed with 10 ml of aqueous NaCl 5% (w/v) solution. The extraction procedure was repeated, and the second extract was collected (globulin fraction). After following extractions with aqueous 70% (v/v) ethanol and aqueous 0.2% NaOH solution, the third extract (prolamin fraction) and the fourth extract (glutelin fraction) were obtained. Storage protein determination using Bradford assay The protein content of each sample was quantified using the method described by Bradford (1976). 100 mg Coomassie Brilliant Blue G-250 (SigmaAdrich Co) was dissolved in 50 ml ethanol (95%) and 100 ml of phosphoric acid (85%) was added. The solution was diluted, filtered, and used as the color reagent for protein quantification. Standard solutions of reagent grade BSA (Equitech-Bio, Inc., Kerriville, TX) were prepared containing 0–400 lg protein. Samples were covered with parafilm mixed, and then incubated for 5 min before absorbance measurement at 595 nm in spectrophotometer (Anthelie Advanced, Microbeam, SA). Standard solutions and sample unknowns possessed the same solution matrix. The protein content of each sample was determined by fitting a least squares regression curve of the quantity of standard protein concentration versus photometric absorbance.
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Table 1 Protein content and geographical cultivated area of the 21 pomegranate cultivars used for population profiling Name
Code
Country
Latitude
Longitude
Protein content (%)
Kalaii
KL1
Tunisia
35°570 N
10°310 E
24.31 ± 4.47 A,B,C
Zaghouani Gabsi 3
ZG1 GB3
Tunisia Tunisia
36°240 N 33°400 N
10°080 E 10°150 E
22.98 ± 5.10 A,B,C 25.37 ± 2.42 A,B,C
Chelfi 4
CH4
Tunisia
35°570 N
10°270 E
22.32 ± 2.77 B,C
Sweet
0
Tounsi 3
TN3
Tunisia
36°33 N
9°250 E
25.61 ± 3.26 A,B,C
Gabsi 2
GB2
Tunisia
33°450 N
10°110 E
20.86 ± 4.19 C
Jebali3
JB3
Tunisia
37°100 N
10°010 E
23.89 ± 4.06 A,B,C
0
0
Sichuan 2
SCH 2
Sichuan, China
30°36 N
104°04 E
Mengzi
MYN
Yunnan, China
23°210 N
103°230 E
Mean
28.54 ± 1.89 A 27.82 ± 2.26 A,B 24.63 ± 2.50
Sour Mezzi 2
MZ2
Tunisia
33°540 N
8°080 E
22.05 ± 4.28 B,C
Garci 2
GA2
Tunisia
33°540 N
8°080 E
22.60 ± 2.05 B,C
0
0
Nabli1
NB1
Tunisia
36°31 N
9°25 E
23.35 ± 0.67 A,B,C
Rafrafi
RF1
Tunisia
33°400 N
10°150 E
25.57 ± 2.63 A,B,C
Garoussi
GR1
Tunisia
33°370 N
10°170 E
20.70 ± 3.45 C
Shanxi
SHA
Shanxi, China
36°460 N
112°370 E
25.32 ± 0.81 A,B,C
DYN1
Yunnan, China
25°350 N
100°160 E
21.48 ± 2.50 C 23.01 ± 1.86
CT1
Tunisia
37°100 N
10°010 E
25.40 ± 1.71 A,B,C
Dali 1 Mean Sour sweet Chetoui Djerbi
JR1
Tunisia
33°49 N
10°550 E
25.61 ± 0.83 A,B,C
Zehri 2
ZH2
Tunisia
36°330 N
9°250 E
21.37 ± 4.07 C
Sichuan 1
SCH 1
Sichuan, China
30°360 N
104°040 E
22.15 ± 0.60 B,C
Dali 2
DYN2
Yunnan, China
0
0
25°35 N
100°160 E
24.64 ± 1.39 A,B,C
Mean
23.83 ± 1.95
Fobs (within-groups)
1.088
P value (within-groups)
0.358 (NS)
Protein contents were determined by Kjeldahl method Values are average of three individual samples each analyzed in duplicate ±SD Different letters indicate significant difference (P \ 0.05) analyzed by Duncan’s multiple range test F Snedecor-Fisher Factor. P values were determined by Fisher’s exact test, significantly different (P \ 0.05); NS not significant
Free amino acid analysis The Amino acid analyzer (Hitachi L8800, Tokyo, Japan) was used for the detection of each free amino acid (FAA) in pomegranate seed. Acid hydrolysis of the samples (100 mg) was performed in 6 M HCl under vacuum for 22 h at 110°C. After acid hydrolysis, the hydrolyzed samples were mixed in 9 ml of 6 M NaOH and adjusted to 100 ml with 0.02 M HCl.
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Samples were filtered twice through a micropore filter (0.45 lm). 20 ll of the filtrate was injected into an amino acid analyzer (Hitachi L8800, Tokyo, Japan). The analyses for determination of amino acid compositions were performed in triplicate. Tryptophan could not be analyzed since it is degraded upon acid hydrolysis. By summing the amounts of the analyzed amino acids, it was possible to estimate the protein content for each sample. The determination was
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carried out at 38°C. Flow rate 1.0 ml per minute and detection wavelength were respectively 570 nm for most amino acids residues and 440 nm for the ninhydrin–proline derivative. Final results of free amino acid composition were reported as mg per 100 g protein. Statistical and chemometric methods Cultivar values for each compound were compared to the mean of all cultivars by calculating a confidence interval. An analysis of variance (ANOVA) was used to compare populations grouped according to their geographical origin and thus identify possible regional trends. In addition, to evaluate the information contained in experimental data, principal component analysis (PCA) was applied. PCA is a chemometric method to visualize information contained in experimental data and to find the true dimensions of a dataset (Miloun et al. 1992). The clustering analysis was conducted on free amino acids contents according to the Euclidean distance using dissimilarities algorithm. These analyses were computed on the data using XLSTAT software (www.xlstat.com).
Results and discussion Protein contents The protein contents expressed as g per 100 g dry weight (% DW) and determined following Kjeldahl method (AOAC 1995) were reported in Table 1. The protein contents varied significantly between Tunisian and Chinese cultivars. It ranged from 20.70 (GR1, Tunisian cultivars) to 28.54% (SCH2, Chinese cultivars). In sweet pomegranate, the Chinese cultivars have higher protein content compared to the Tunisian ones. In contrast, in the two others groups (sweet and sour sweet) the protein contents differs between cultivars including Tunisian and Chinese samples. Statistically, the difference between organoleptic groups (sweet, sour and sour sweet) were not significant (P = 0.358). However, within groups, the differences between pomegranate cultivars were statistically significant. Generally, we noted that pomegranate pulp of Tunisian and Chinese cultivars were rich in protein
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content. For sweet pomegranate, the protein content ranged between 20.86 (Gabsi 2) and 25.61% (Gabsi 3) for Tunisian cultivars, whereas it varied from 27.82 to 28.54% in Chinese ones. The protein contents in sour pomegranate ranged between 20.70 and 25.57% (in Tunisian cultivars Garoussi and Rafrafi, respectively). This content is 21.48 and 25.32% in Shanxi and Dali1, correspondingly, for the Chinese sour cultivars. The protein extracted from sour sweet pomegranate varied from 21.37 (Zehri2) to 25.40% (Chetoui) in Tunisian cultivars and it is 22.15 and 24.64% in respective Sichuan1 and Dali2, Chinese cultivars. This result confirms a previous study reporting protein content from dry pulp ranged from 20.38 to 26.25% (Elfalleh et al. 2009). Storage protein content of Tunisian and Chinese pomegranate seeds Seeds represent a special organ with respect to amino acid metabolism and hence also to metabolic engineering. The vast majority of the amino acids in the seeds are stored in specific types of proteins, familiar known as storage proteins. These latter are deposited in protein located either within the endoplasmic reticulum or inside a special class of vacuoles, named protein storage vacuoles (Galili and Ho¨fgen 2002). The storage protein of pomegranate seeds was determined using Bradford assay (Table 2). This results show that the Tunisian and Chinese pomegranate seeds were rich in storage proteins independently of their organoleptic groups (sweet, sour sweet or sour). Total storage protein ranged between 144.34 (CH, Tunisian cultivar) and 210.95 mg/g DW (DYN2, Chinese cultivar). The mean percentage of storage proteins is 16.9, 17.8 and 18.8% DW in sweet, sour sweet and sour groups, respectively. Statistically, the difference between the three groups was not significant. But, we noted significant differences between pomegranate cultivars inside each group (Table 2). The storage protein of sweet pomegranates varied from 144.34 (Chelfi4) to 177.24 mg/g DW (Kalaii) in Tunisian cultivars seeds (Table 2). However, for Chinese ones it is 187.68 and 188.8 mg/g DW seed, respectively for Mengzi and Sichuan2 cultivars. This protein average in sweet pomegranate (both for cultivars from Tunisia and China) is 16.9% DW. We noted a significant difference between cultivars.
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123 51.02 ± 5.77 E,F,G 66.75 ± 5.53 A,B 58.27 ± 7.97 C,D,E
JB3
SCH 2
MYN
Jebali 3
Sichuan 2
64.12 ± 2.08 A,B,C 62.40 ± 3.09 A,B,C,D 69.78 ± 0.14 A
ZH2
SCH 1
DYN2
Zehri 2
Sichuan 1 62.90 ± 5.00 33.54 3.149
Mean mg/g DW
Mean (%)
Fobs (within-groups)
Dali 2
55.78 ± 6.75 C,D,E,F
CT1
JR1
Chetoui
Djerbi
62.42 ± 5.43 A,B,C,D
33.93
Sour sweet
60.33 ± 6.96
Mean (%)
69.94 ± 0.00 A
61.59 ± 8.34 A,B,C,D
47.41 ± 0.88 G
Mean mg/g DW
SHA
DYN1
Shanxi
Dali 1
62.24 ± 2.01 A,B,C,D
RF1
GR1
Rafrafi
Garoussi
GA2
NB1
Garci 2
Nabli 1
62.45 ± 0.00 A,B,C,D 62.45 ± 0.00 A,B,C,D
MZ2
Mezzi 2
56.22 ± 2.51 C,D,E
32.19
Mean (%)
Sour
54.69 ± 6.38
46.77 ± 2.30 G
Mean mg/g DW
Mengzi
48.04 ± 5.48 F,G
TN3
GB2
Tounsi 3
Gabsi 2
54.28 ± 2.01 D,E,F,G 50.50 ± 5.77 E,F,G
GB3
CH4
Gabsi 3
Chelfi 4
59.64 ± 2.46 B,C,D
KL1
ZG1
56.95 ± 4.58 C,D,E
Albumin
Kalaii
Code
Zaghouani
Sweet
Name
0.421
39.82
74.67 ± 12.60
92.16 ± 18.82 A
77.47 ± 14.50 A,B,C,D,E
60.88 ± 7.21 E,F
64.00 ± 6.75 D,E,F
78.84 ± 10.03 A,B,C,D,E
41.54
73.87 ± 10.51
70.43 ± 0.00 B,C,D,E,F
85.80 ± 15.05 A,B,C
68.03 ± 4.84 B,C,D,E,F
88.36 ± 6.28 A,B
62.36 ± 0.00 D,E,F
62.88 ± 0.00 D,E,F
78.74 ± 4.04 A,B,C,D,E
41.14
69.90 ± 9.84
83.16 ± 6.64 A,B,C,D
77.22 ± 11.77 A,B,C,D,E
52.70 ± 2.32 F
65.09 ± 15.93 C,D,E,F
82.55 ± 0.01 A,B,C,D
63.57 ± 2.52 D,E,F
69.10 ± 0.01 B,C,D,E,F
64.91 ± 23.45 C,D,E,F
70.81 ± 20.36 B,C,D,E,F
Globulin
2.087
10.54
19.77 ± 1.91
18.49 ± 3.74 B,C,D,E
22.87 ± 4.22 A,B
18.72 ± 4.50 B,C,D,E
18.39 ± 2.95 B,C,D,E
20.38 ± 5.17 B,C,D
10.48
18.64 ± 5.06
15.02 ± 0.01 D,E,F
21.95 ± 7.25 A,B,C
13.39 ± 1.78 E,F
18.39 ± 2.09 B,C,D,E
26.75 ± 5.78 A
13.39 ± 0.01 E,F
21.60 ± 1.72 A,B,C
9.31
15.82 ± 3.31
16.38 ± 2.23 C,D,E,F
20.65 ± 5.13 B,C,D
16.73 ± 1.15 B,C,D,E,F
13.60 ± 2.23 E,F
10.93 ± 1.16 F
11.42 ± 0.01 F
19.46 ± 0.01 B,C,D,E
16.06 ± 0.01 C,D,E,F
17.18 ± 1.96 B,C,D,E,F
Prolamin
1.262
16.10
30.20 ± 8.31
30.52 ± 9.52 A,B
36.54 ± 5.76 A
15.88 ± 2.88 D
33.21 ± 8.51 A
34.85 ± 3.85 A
14.05
24.98 ± 6.89
19.68 ± 0.01 C,D
33.16 ± 5.87 A
24.32 ± 1.33 B,C
31.58 ± 1.73 A,B
17.58 ± 0.01 C,D
17.55 ± 0.01 C,D
30.99 ± 2.31 A,B
17.35
29.49 ± 5.07
29.86 ± 4.03 A,B
24.18 ± 7.84 B,C
35.22 ± 2.90 A
31.23 ± 2.48 A,B
28.60 ± 0.01 A,B
18.84 ± 0.01 C,D
32.60 ± 0.44 A
32.56 ± 1.71 A
32.30 ± 1.55 A
Glutelin
Table 2 Contents of seeds storage proteins (mg/g DW seed) and compositions in proteinic classes from 21 pomegranate cultivars
1.004
18.8% DW
187.54 ± 21.26
210.95 ± 31.29 A
199.27 ± 15.59 A,B,C,D
159.60 ± 2.07 F,G,H
171.38 ± 3.96 D,E,F,G,H
196.50 ± 5.57 A,B,C,D,E
17.8% DW
177.82 ± 19.87
175.07 ± 0.02 B,C,D,E,F,G
202.49 ± 35.22 A,B
153.16 ± 7.75 G,H
200.57 ± 6.60 A,B,C
169.66 ± 5.79 E,F,G,H
156.27 ± 0.02 G,H
187.55 ± 4.13 A,B,C,D,E,F
16.9% DW
169.91 ± 14.87
187.68 ± 20.06 A,B,C,D,E,F
188.80 ± 16.02 A,B,C,D,E
155.66 ± 9.62 G,H
157.97 ± 9.31 G,H
168.86 ± 2.97 E,F,G,H
144.34 ± 4.06 H
175.44 ± 1.62 B,C,D,E,F,G
173.16 ± 24.02 C,D,E,F,G
177.24 ± 22.81 B,C,D,E,F,G
R SSP
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0.352 (NS)
Different letters indicate significant difference between cultivars (P \ 0.05) analyzed by Duncan’s multiple range test
F Snedecor-Fisher Factor. P values were determined by Fisher’s exact test, significantly different (P \ 0.05); NS not significant
Values are average of three individual samples each analyzed in duplicate ±SD
P value (withingroups)
Protein contents expressed as mg per g DW were determined by Bradford method
0.306 (NS) 0.153 (NS) 0.662 (NS) 0.067 (NS)
Prolamin Name
Table 2 continued
Code
Albumin
Globulin
Glutelin
R SSP
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In seeds from sour pomegranate, the storage protein were 175.07 (Dali1) and 202.49 mg/g DW (Shanxi) in Chinese cultivars, and varied from 153.16 (Garousssi) to 200.57 mg/g DW (Rafrafi) in Tunisian pomegranate seeds. For the third organoleptic category including sour sweet pomegranate, the cultivar ‘Chetoui’ demonstrate the highest value of storage protein content (196.50) and ‘Zehri2’ the lesser value (159.60). For both ‘Sichuan1’ and ‘Dali2’ Chinese pomegranates, the storage protein of seeds is respectively 199.27 and 210.95 mg/g of dry weight. Statistically, the differences between cultivars were significant; in contrast it is not significant between organoleptic groups. In this case, under homogenous conditions in the same germplasm collection, the cultivar seems to be the major factor affecting the storage protein content in pomegranate seeds. Seeds of pomegranate store a higher proportion of protein than species from Graminaeae family such as the cereals: wheat present about 7–18%, corn present 7–12% and rice with 7.5–9%. But it is steel less than leguminous plants such as colza (15–30%), lupin (30–50%) and lentils (23–32%) (Daulau and The´baudin 1998). Tunisian and Chinese pomegranate seeds seem to be an interesting edible protein source. In previous study we reported that the storage proteins content of Tunisian pomegranate seeds ranged from 15.4 to 20.1% (Elfalleh et al. 2010). Storage protein fractionation The fractionation of storage protein was made following the protocol based on the difference solubility in various solvents (Osborne 1924): Albumins (water soluble), globulins (saline soluble), prolamins (alcohol soluble) and glutelins (residue fraction). Upon protein fractionation, it was found that the globulin and albumin are the major protein constituent, followed by glutelin and prolamin fractions (Table 2). This result is in agreement with a previous study on Tunisian pomegranate cultivars (Elfalleh et al. 2010). The Table 2 shows that the globulin content varied significantly according to the cultivars in each groups (sweet, sour sweet and sour), whereas, non-significant differences were observed between this three groups. The globulin content ranged between 52.70 (JB3, Tunisian cultivar) and 92.16 mg/g DW (DYN2,
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Chinese cultivar). The second most frequent storage protein in pomegranate seeds is the water soluble, albumin fraction. The Chinese cultivar DYN1 shows the highest content of albumin fraction with the value of 69.94 mg/g DW, whereas the Tunisian cultivar TN3 (Tounsi3) shows the lower content with the value of 46.77 mg/g DW. The glutelin protein fractions have an average of 17.35, 14.05 and 16.10% for sweet, sour sweet and sour groups, respectively. The prolamin fraction did not exceed 11% in three organoleptic groups. Globulins, prolamins and glutelins as storage reserves, are not present systematically in the seeds of all plant species (Bewley and Black 1983). In the present study, we noted that the globulin is the major protein fraction constituent for all Tunisian and Chinese pomegranate seeds (sweet, sour sweet and sour) also the presence of others proteins fractions with lesser proportion (albumin, prolamin and glutelin) (Table 2). The storage protein fractions varied significantly between cultivars; whereas, the variation is not significant between organoleptic groups. Therefore, it seems that the cultivars are the major factors to influence the protein fractions constituents which are in agreement with those for most legumes (Vasconcelos et al. 2010). In contrast, it has been reported that protein fractions varied according to several factors as in vignera genus, the extracting method, the cultivars and also the genetic and environment factors (Adsule et al. 1986). Free amino acids composition The free amino acids compositions of storage protein from Tunisian and Chinese pomegranate seeds are shown in Table 3. The results showed that pomegranate seeds contain eighteen familiar free amino acids, including all of the essential amino acids (EAAs), sulfuric-containing and aromatic amino acids (Fig. 1). The percentage of EAAs in pomegranate seeds was approximating 30% of total amino acid. The sulfuric-containing amino acids were higher than 15% and the aromatic amino acids percentage was lower than 10% (Fig. 1). As shown in Table 3, the differences of the amino acids composition expressed as mg/100 g DW seed, were significant within pomegranate cultivars (P \ 0.001) and not significant within groups (sweet,
123
Genet Resour Crop Evol (2012) 59:999–1014
sour sweet and sour). The glutamic acid, the arginine and the aspartate acid were the major amino acids followed by glycine, leucine, serine and proline. In sweet pomegranates, the free amino acids, as percentage of dry weight, ranged between 9.06 (Chelfi4) and 14.06% (Kalaii). For the sour taste, the lower amino acids percentage is 4.48 (Shanxi, Chinese cultivar) and the highest is 9.95% (Garoussi, Tunisian cultivars). The amino acid percentage varied from 4.19 (Dali2, Chinese cultivar) to 11.30% (Zehri2, Tunisian cultivars) for the sour sweet taste. Free amino acid analysis has an important role in the study of the proteins composition and foods. Amino acids are found in the free state or as linear chains in peptides and proteins. Both glutamine and asparagine which are formed from glutamic and aspartic acids, respectively, constitute important reservoirs of amino acid groups for the body. Furthermore, glutamine has received attention as primer fuel source for the intestinal tract, especially controlling glycogen synthesis and protein degradation (Mahon and Escott-Stump 1996). Although, the glutamine transformed on carbanyl-P has a capital role in several biosynthetic, especially, as the NH2 donor role. Lysine is synthesized in plants from aspartate family pathway. Another branch of this pathway leads to the synthesis of two others important essential amino acids, methionine and threonine. For the human being, lysine catabolism is apparently a major pathway of generating glutamate in brain tissues, in order to regulate nerval signal transmission via glutamate receptors (Galili et al. 2001). Several authors showed that total amino acids contents varied between grapevine cultivars and these variations might be due to the cultivars effect, climate and nitrogen fertilization factors (Asensio et al. 2002). Amino acids represent important targets for metabolic engineering for a number of reasons, some amino acids such as proline, arginine, methionine and glutamate are directly involved in the regulation of plant responses to various environmental signals, including light and mineral availability as well as biotic and abiotic stresses (Ho¨fgen et al. 2001). Others amino acids (lysine, threonine, methionine and tryptophane) contribute significantly to the mineral quality of plant-based food (Hesse et al. 2001). These essential amino acids cannot be synthesized by humans and have to be supplied to them in the diet
1155.58 ± 88.82
Sichuan 2
Mean
267.72 ± 120.20
774.71 ± 347.69
319.02 ± 10.20
325.55 ± 15.99
268.35 ± 87.93
Kalaii
Zaghouani
Gabsi 3
Sweet
Ile
P value (within-groups)
Name
1.477
0.255
Fobs (within-groups)
859.87 ± 273.68
1000.84 ± 67.57
1020.19 ± 20.90
Leu
0.141
2.187
20.461 \0.0001
16.988
\0.0001
P value
Fobs (Within-cultivars)
747.49 ± 47.91
0.154
2.080
\0.0001
16.876
335.36 ± 100.64
384.12 ± 30.95
0.120
2.386
\0.0001
16.957
476.75 ± 155.70
570.90 ± 26.78
193.43 ± 75.30
228.85 ± 15.79
177.21 ± 56.73
192.52 ± 5.68
275.76 ± 78.59
346.56 ± 25.53
1464.16 ± 479.07
1718.25 ± 122.75
447.09 ± 155.92
508.16 ± 54.33
11.70
13.62
14.06
R AA %
0.411
\0.0001
16.585
124.19 ± 65.90
16.36 ± 1.67
104.18 ± 13.85
172.24 ± 23.26
18.79 ± 1.02
140.42 ± 5.94
79.60 ± 52.88
513.53 ± 38.50
Pro
0.110
2.505
\0.0001
18.831
272.90 ± 61.11
202.05 ± 15.28
227.11 ± 57.96
326.03 ± 24.88
393.30 ± 1.67
302.38 ± 0.94
298.05 ± 117.45
102.72 ± 21.33
17.48 ± 0.46 203.93 ± 23.81
162.77 ± 19.38
314.86 ± 3.08
165.76 ± 0.57
109.92 ± 29.46
139.26 ± 23.92
136.16 ± 0.81
123.78 ± 42.53
44.37 ± 27.37
92.38 ± 61.05
146.88 ± 13.34
174.33 ± 32.00
171.41 ± 0.89
149.55 ± 10.89
60.25 ± 24.46
67.62 ± 38.60
99.39 ± 6.76
Met
208.58 ± 2.19
311.10 ± 2.89
213.68 ± 74.29
301.01 ± 14.11
308.22 ± 35.26
341.28 ± 76.80
168.74 ± 17.26
499.34 ± 0.08
305.96 ± 10.83
349.38 ± 18.85
309.70 ± 40.83
295.27 ± 15.56
366.08 ± 108.31
443.14 ± 29.03
472.50 ± 2.42
Val
1670.47 ± 9.13
Arg
0.062
3.265
\0.0001
15.493
323.63 ± 64.11
255.84 ± 2.65
277.89 ± 16.28
390.68 ± 35.95
371.70 ± 0.92
349.58 ± 9.97
323.17 ± 80.58
271.00 ± 23.88
241.86 ± 3.11
346.83 ± 21.65
367.76 ± 1.04
275.31 ± 67.92
326.74 ± 12.70
349.11 ± 44.86
389.43 ± 70.79
222.55 ± 8.49
460.92 ± 25.30
323.65 ± 39.06
393.28 ± 5.58
444.50 ± 26.96
360.77 ± 17.97
394.07 ± 91.84
455.79 ± 44.62
505.34 ± 27.05
Cys
349.97 ± 1.22
His
0.153
2.090
\0.0001
14.834
353.54 ± 112.68
189.63 ± 36.57
284.49 ± 103.39
475.77 ± 74.87
471.24 ± 1.57
389.44 ± 1.86
362.52 ± 157.13
266.56 ± 60.66
206.41 ± 2.35
423.25 ± 14.87
395.69 ± 10.39
258.23 ± 116.25
418.70 ± 1.15
417.44 ± 29.30
447.44 ± 109.56
181.75 ± 0.43
600.63 ± 41.90
408.01 ± 28.42
448.88 ± 17.55
389.26 ± 54.50
386.41 ± 54.36
495.39 ± 158.47
594.72 ± 35.98
630.35 ± 6.25
Ala
207.08 ± 5.02
NH3
0.227
1.611
\0.0001
14.532
575.53 ± 209.05
288.46 ± 40.25
437.87 ± 139.65
772.75 ± 131.01
755.01 ± 4.93
646.16 ± 2.51
562.34 ± 234.34
411.76 ± 79.86
296.77 ± 0.09
723.46 ± 30.12
672.94 ± 13.20
435.65 ± 191.19
689.99 ± 1.35
687.69 ± 43.08
726.20 ± 163.72
249.72 ± 0.77
829.86 ± 74.04
656.74 ± 44.16
750.73 ± 26.76
650.93 ± 81.94
702.79 ± 107.60
778.70 ± 264.26
906.32 ± 57.88
1005.58 ± 7.19
Gly
314.92 ± 1.20
Lys
1909.94 ± 661.91
989.12 ± 173.29
1494.45 ± 540.70
2650.46 ± 431.05
2576.69 ± 8.53
1806.09 ± 23.87
1911.55 ± 882.46
1408.03 ± 321.91
1075.77 ± 0.04
2406.51 ± 85.88
2008.95 ± 54.32
1408.96 ± 623.84
2023.28 ± 40.09
2235.64 ± 142.94
2433.91 ± 701.26
857.76 ± 6.35
3289.44 ± 151.79
2129.55 ± 148.55
2208.62 ± 106.62
2174.67 ± 257.93
2168.81 ± 250.87
3068.07 ± 956.09
3421.73 ± 222.97
3433.18 ± 43.64
Glu
550.19 ± 25.75
Phe
451.84 ± 158.85
233.05 ± 32.35
366.67 ± 125.69
617.60 ± 103.92
600.64 ± 4.07
485.69 ± 1.69
454.98 ± 200.63
337.97 ± 85.35
235.84 ± 1.74
547.36 ± 20.68
520.44 ± 13.05
335.67 ± 143.61
526.26 ± 5.29
532.17 ± 39.11
578.85 ± 145.20
209.60 ± 0.24
428.20 ± 7.56
Tyr
262.25 ± 93.16
133.51 ± 19.07
206.67 ± 67.56
352.67 ± 48.08
766.90 ± 272.18
414.76 ± 87.01
296.43 ± 3.99 358.55 ± 1.85
Mean
581.88 ± 209.19
Dali 2
1094.54 ± 40.46
Zehri 2
Sichuan 1
767.36 ± 8.06
1236.06 ± 6.28
Chetoui
Djerbi
Sour sweet
Mean
188.12 ± 36.17
551.45 ± 123.69
Dali1
317.10 ± 6.30 138.64 ± 2.20
947.22 ± 31.71
437.24 ± 3.76
Garoussi
Shanxi
314.74 ± 3.36
199.61 ± 83.06
521.54 ± 231.95
804.07 ± 21.29
Nabli1
796.72 ± 4.46
Rafrafi
312.08 ± 6.55
915.38 ± 76.54
308.92 ± 29.10
345.50 ± 92.75
Garci 2
918.08 ± 205.71
441.59 ± 36.77 119.77 ± 2.49
510.73 ± 28.44
571.60 ± 22.84
306.36 ± 10.02
516.27 ± 57.44
305.93 ± 39.41
509.95 ± 65.45
654.85 ± 196.87
778.00 ± 51.99
815.86 ± 7.02
Ser
344.02 ± 17.06
Mezzi 2
Sour
382.93 ± 6.24
939.49 ± 64.03
Jebali 3
Mengzi
832.69 ± 98.89
919.06 ± 33.63
Tounsi 3
794.50 ± 101.50
Gabsi 2
382.00 ± 113.09
1005.19 ± 320.30
Gabsi 3
Chelfi 4
294.75 ± 27.37
475.23 ± 33.46
1222.37 ± 11.38
1200.21 ± 85.23
498.13 ± 0.67
Thr
Kalaii
Asp
Zaghouani
Sweet
Name
Table 3 Amino acid composition (mg/100 g DW seed) of pomegranate seeds from 21 cultivars
Genet Resour Crop Evol (2012) 59:999–1014 1007
123
123 570.23 ± 270.16
69.23 ± 1.40
95.64 ± 20.14
156.83 ± 97.36
Shanxi
Dali1
0.106
P value (withingroups)
0.116
0.131
2.284
\0.0001
18.423
226.55 ± 73.25
128.77 ± 19.16
160.43 ± 50.93
314.25 ± 35.74
310.42 ± 1.89
239.10 ± 4.37
224.62 ± 93.25
167.46 ± 41.82
137.49 ± 0.74
272.88 ± 19.89
240.70 ± 3.40
162.00 ± 67.69
252.79 ± 11.82
250.05 ± 19.95
287.18 ± 83.05
111.49 ± 0.47
356.79 ± 32.29
229.49 ± 22.07
285.33 ± 13.57
277.53 ± 16.34
245.07 ± 26.82
Tyr
141.25 ± 3.33
0.152
2.092
\0.0001
17.973
288.00 ± 117.73
115.73 ± 25.51
205.94 ± 88.14
420.57 ± 87.75
399.40 ± 1.92
298.22 ± 7.50
293.63 ± 158.31
190.50 ± 52.82
Values are average of three individual samples each analyzed in duplicate ±SD
F Snedecor-Fisher Factor. P values were determined by Fisher’s exact test, significantly different (P \ 0.05)
0.719
0.336
\0.0001
12.225
185.04 ± 65.93
91.05 ± 12.53
171.63 ± 62.07
251.44 ± 29.04
259.45 ± 1.02
187.18 ± 5.71
169.21 ± 66.80
145.68 ± 31.52
90.48 ± 0.54
208.61 ± 3.31
228.99 ± 7.50
361.01 ± 8.48
135.17 ± 58.99
203.32 ± 98.22
201.46 ± 0.95
215.29 ± 17.77
208.63 ± 48.55
83.52 ± 3.18
198.52 ± 32.89
238.01 ± 17.81
205.34 ± 10.93
145.66 ± 15.49
192.68 ± 20.57
Lys
326.66 ± 5.91
351.90 ± 1.96
342.39 ± 17.46
380.35 ± 124.12
106.78 ± 5.19
535.88 ± 71.83
327.76 ± 33.16
362.31 ± 21.91
324.14 ± 42.42
306.94 ± 40.05
Phe
Amino acid contents expressed as mg per 100 g DW of seed were determined by amino acid analyzer
2.543
2.438
\0.0001
\0.0001
Fobs (within-groups)
P value
18.657
24.174
554.93 ± 194.73
144.89 ± 58.73
Fobs (withincultivars)
283.90 ± 36.62
Mean
54.62 ± 15.22
Dali 2
428.19 ± 141.47
761.90 ± 131.44
203.00 ± 24.35
114.37 ± 54.08
Zehri 2
215.32 ± 2.08
Sichuan 1
731.56 ± 3.75
153.50 ± 7.80
Djerbi
594.96 ± 6.43
298.90 ± 5.77
671.14 ± 19.42
Chetoui
Sour sweet
Mean
404.01 ± 82.20
182.79 ± 4.15
Garoussi
417.58 ± 174.88 638.70 ± 13.38
100.19 ± 48.47
173.78 ± 0.69
Nabli 1
646.00 ± 9.04
651.36 ± 47.80
723.49 ± 197.49
Rafrafi
177.99 ± 14.30
169.75 ± 8.08
Mezzi 2
Garci 2
Sour
205.72 ± 78.12
251.78 ± 2.83
53.97 ± 8.00
Mean
1009.42 ± 70.34
325.94 ± 31.70
Sichuan 2
Mengzi
700.79 ± 33.60 624.13 ± 30.82
191.00 ± 16.30
168.33 ± 8.29
Gabsi 2
Jebali 3
621.07 ± 67.31 661.38 ± 63.42
153.19 ± 5.81
158.17 ± 34.38
Leu
Chelfi 4
Ile
Tounsi 3
Name
Table 3 continued
0.353
1.104
\0.0001
12.387
142.84 ± 45.71
81.84 ± 11.79
119.64 ± 38.07
188.96 ± 32.47
198.10 ± 5.15
142.65 ± 2.75
144.69 ± 60.66
109.43 ± 27.16
81.07 ± 0.23
174.30 ± 10.91
160.45 ± 0.08
99.95 ± 38.40
165.32 ± 6.18
177.85 ± 14.76
166.53 ± 25.85
68.25 ± 3.96
221.51 ± 3.33
156.36 ± 0.10
172.11 ± 6.13
163.56 ± 28.19
167.59 ± 19.27
NH3
0.182
1.873
\0.0001
20.004
207.93 ± 67.78
108.83 ± 12.51
172.89 ± 42.18
287.89 ± 20.71
298.21 ± 1.20
210.64 ± 4.57
203.95 ± 84.72
156.14 ± 23.92
120.99 ± 2.27
234.88 ± 8.18
239.77 ± 8.06
159.41 ± 60.60
231.46 ± 12.91
232.31 ± 21.80
259.94 ± 57.85
94.96 ± 5.91
302.50 ± 34.15
236.98 ± 3.78
246.47 ± 9.80
249.34 ± 17.32
250.88 ± 20.91
His
0.225
1.624
\0.0001
16.181
969.79 ± 407.38
416.75 ± 103.93
706.80 ± 280.98
1484.28 ± 266.72
1384.75 ± 3.81
884.72 ± 42.33
937.59 ± 472.43
641.54 ± 195.21
483.21 ± 10.16
1188.99 ± 44.12
1050.93 ± 25.64
672.28 ± 321.61
1024.96 ± 5.27
1102.98 ± 43.69
1183.00 ± 352.89
364.75 ± 17.13
1596.66 ± 211.54
1003.24 ± 56.74
1112.83 ± 51.15
1066.31 ± 143.54
1020.27 ± 115.36
Arg
8.25
379.93 ± 141.85
0.464
0.802
\0.0001
11.624
4.19
6.36
11.30
11.11
8.30
7.70
5.92
4.48
9.95
9.05
5.99
9.04
9.49
10.41
3.74
13.21
9.14
9.91
9.29
9.06
R AA %
182.40 ± 28.58
295.88 ± 109.62
535.83 ± 103.45
530.41 ± 0.23
406.62 ± 2.43
372.82 ± 162.08
271.88 ± 66.55
194.30 ± 9.86
465.43 ± 5.03
432.21 ± 13.62
276.72 ± 119.23
465.58 ± 1.23
444.62 ± 31.69
439.72 ± 64.58
164.10 ± 2.40
545.91 ± 3.60
428.92 ± 34.33
474.98 ± 19.06
445.18 ± 48.43
437.11 ± 56.11
Pro
1008 Genet Resour Crop Evol (2012) 59:999–1014
Genet Resour Crop Evol (2012) 59:999–1014
1009
which is largely derived from plants. In this, study we noted that the amino acids extracted from pomegranate seeds would to be a source of essential amino acids (Table 3 and Fig. 1). Among the essential amino acids, lysine and methionine are considered important
ones because they are the most limiting amino acids in cereal and legume seeds (Galili and Ho¨fgen 2002). Table 4 shows that all amino acids contents were in agreement with FAO/WHO scoring pattern requirements for adults. In contrast, for Pre-School
Fig. 1 Essential, sulfuric and aromatic amino acid composition (%) of pomegranate seeds from 21 cultivars
Table 4 Comparison of AA composition of pomegranate seed to suggested patterns of AA requirement (mg per g of protein) By taste
R Essential AA mg/g proteinb
FAO/WHO patterna
By country
Sweet
Sour sweet
Sour
China
Tunisia
Pre-school child
Adults 122.00
288.22
287.64
290.19
295.62
285.99
328.00
Met ? Cys
20.03
17.37
17.64
16.96
19.26
28.00
13.00
Phe ? Tyr
71.35
68.02
68.98
68.99
70.08
66.00
19.00
Val
19.65
23.38
22.66
21.82
21.43
58.00
16.00
Ile
51.67
53.82
56.89
59.91
51.53
25.00
17.00
Leu
65.24*
61.93
62.78
60.79
64.77
63.00
19.00
Lys
35.01
36.93
35.50
41.33*
33.35
35.00
13.00
His
25.28
26.18
25.74
25.82
25.58
19.00
16.00
a
Reproduced from FAO/WHO (1991)
b
Total essential amino acids excluding Trp. (Tryptophan could not be analyzed since it is degraded upon acid hydrolysis)
*Values are significantly higher than the mean at p = 0.05
123
1010
Child, the limiting amino acids were methionine, cystine, valine and histidine which have contents lower than the FAO/WHO. It has been reported that in cowpea cultivars, the methionine and cystine are the limiting amino acids (Vasconcelos et al. 2010). The deficiency of sulfuric-containing amino acids was common in the great majority of leguminous seeds (Laurena et al. 1991) and among cowpea cultivars (Onwuliri and Obu 2002). The sweet Tunisian pomegranate has the higher leucine content once compared to FAO/WHO pattern for both adults and Pre-School Child. For lysine and excepting Tunisian cultivars, the Chinese ones from the three groups (sweet, sour sweet and sour) present a higher content than FAO/WHO pattern for both adults and Pre-School Child (Table 4). It well known that the nutritive value of a protein depends primarily on its capacity to satisfy the needs for nitrogen and essential amino acids. The knowledge about the amino acids is basic for the evaluation of the nutritional significance of dietary protein quality. Pomegranate has been also considered a natural source of phenolic compounds, sugar, protein and minerals (Elfalleh et al. 2009). Twenty amino acids were recognized as standards and constitute proteins of different kinds of life. Several amino acids exist in plant and were specific according species. Most animal protein are known to be poor in cystine, in contrast, many vegetable Fig. 2 Dispersion of Tunisian and Chinese pomegranate cultivars in plane formed by axes F1 and F2 of the Principal component analysis based on protein and amino acids data
123
Genet Resour Crop Evol (2012) 59:999–1014
proteins contain substantially more cystine than methionine. Thus, for animal protein diets or mixed diets containing animal protein, cystine is unlikely to contribute more than 50% of the total sulfuriccontaining amino acids (Adeyge and Afolabi 2004; FAO/WHO 1991). Principal component analysis In order to elaborate synthetic vision regarding the structuration of pomegranate germplasm, from Tunisia and China, and on the basis of amino acids richness, PCA has been performed (Fig. 2). Inertia percentage and correlated variables with axes 1 and 2 are displayed in Table 5. Axis 1 explained 71.90% of the inertia and was mainly made by the whole amino acids contents excepting the methionine. Axis 2 explained 9.77% of the inertia and is made positively methionine and negatively by Kjeldhal protein, albumin, globulin, prolamin, and glutelin. The dispersion of pomegranate cultivars on the plan defined by axes 1 and 2 showed three clusters of cultivars (A, B and C). Tunisian and Chinese pomegranate cultivars were clustered independently of both geographic origin and taste. The first cluster (cluster A) includes four Tunisian and one Chinese cultivar. Only one of them was sour sweet (cultivar JR1) and the others were sweet pomegranate cultivars. The cluster B comprises ten Tunisian cultivars. Four out of them were
Genet Resour Crop Evol (2012) 59:999–1014
1011
Table 5 Discriminate variables factors of principal components analysis based on pomegranate storage protein and free amino acid composition Axis
F1
F2
Proper Value
16.54
2.25
Variability (%)
71.90
9.77
Cumulated %
71.90
81.76
Kjeldahl content (%)
–
19.30
Albumin
–
-13.09
Globulin
–
-12.39
Prolamin
–
-12.66
Glutelin
–
-9.97
Asp
?5.73
–
Thr
?5.96
–
Ser Glu
?6.00 ?5.83
– –
Gly
?5.92
–
Ala
?5.96
–
Cys
?5.33
–
Val
?5.52
–
Met
–
?22.83
Ile
?5.60
–
Leu
?5.90
–
Tyr
?5.86
–
Phe
?5.87
–
Lys
?4.58
–
NH3
?5.71
–
His
?5.93
–
Arg
?5.87
–
Pro
?5.49
–
sour; three were sweet and the others cultivars were sour sweet. The C cluster grouped NB1 cultivar (from Tunisia) and five Chinese cultivars (‘MYN, DYN1, SCH1, SHA and DYN2). PCA shows discrimination between the Tunisian and Chinese cultivars especially based on free amino acids profiles which were more correlated with axis 1 explaining the highest percentage of the inertia (Fig. 2). This discrimination clusters pomegranate cultivars which have high (A), moderate (B) or lower (C) amino acids contents. Even though Chinese and Tunisian pomegranate cultivars have both been grouped based on free amino acids profile and not based on organoleptic classification (sweet, sour sweet and sour,) and/or the geographical sites (Tunisia, Chinese). In previous study we reported the storage protein profiling of 8 Tunisian
pomegranate cultivars. The SDS–PAGE pattern of albumin, globulin, prolamin and glutelin, was comparable in seed of all cultivars (Elfalleh et al. 2010). For plant characterization, several criteria were used to found more information to classify any plant species. Particularly in pomegranate profiling, several characters where used starting with morphologic, pomologic, (Mars and Marrakchi 1999) molecular marker; AFLP (Jbir et al. 2008) RAPD (Sarkhosh et al. 2006) and biochemical markers such as anthocyanin and sugar (Hasnaoui et al. 2011), fatty acid (Elfalleh et al. 2011b). Clustering analysis Clustering analysis using Dissimilarities method and based on Euclidean Distance was applied on pomegranate data. These latter were the amino acids contents which correlated positively with the axis 1 of PCA analysis explaining the highest percentage of the inertia (71.90%) (Table 5). The clustering analysis shows three groups (Fig. 3). The group A clusters six Tunisian pomegranate cultivars, four of them were sweet and two sour sweet. Excepting the cultivars ZH2, the others were grouped in the A cluster defined by PCA analysis and have the higher amino acids contents. The group B clusters nine pomegranate cultivars. Four were sour (RF1, MZ2, GA2, GR1), four were sweet (CH4, GB2, JB4, TN3) and one is sour sweet pomegranate (CT1). This group clusters nine out of ten pomegranate cultivars defined previously in B cluster of PCA analysis, the centered groups having moderate amino acids contents (Fig. 2). The group C clusters five Chinese (MYN, DYN1, SCH1, SHA and DYN2) and NB1 Tunisian pomegranate cultivars. This group is identical of the C group defined by PCA analysis having the lowest amino acids contents (Fig. 2). These results show that the groups defined in PCA and clustering analyses were clustered independently of their geographic origins and their taste (sweet, sour sweet or sour). The clusters were defined especially on free amino acids contents; theses parameters were characterized by high discriminate potentiality between pomegranate cultivars. Huang and Ough (1991) used the proline/arginine ratio as an instrument for the grapevine cultivars characterization. In this study we tested this ratio for possible characterization of pomegranate cultivars.
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Fig. 3 Dendrogram of 21 pomegranate cultivars constructed according to Euclidean distance estimated on seed amino acid composition
The first group in clustering analysis (is the same upper left group in PCA) characterized by high amino acid contents and the Pro/Arg ratio ranged from 0.40 to 0.45. In contrast the group3 was characterized by lower level of amino acids contents and the Pro/Arg ratio ranged from 0.30 to 0.38. The pomegranate clustering in the group 2 has a moderate level of amino acids compared to group 1 and 2; the Pro/Arg ratio ranged from 0.39 to 0.46. In our study this ratio cannot be used for complete clusters characterization. However, we noted that cultivars which were closed in dendrogram have appreciatively the same ratio value. In cluster1, the NB1 (Tunisian cultivar) close with DYN1 (Chinese cultivar) have ratios 0.41 and 0.42, respectively. The two Chinese cultivars DYN2 and SHA were closed in this cluster and have ratios 0.44 and 0.40, respectively. In cluster 2, three cultivars couples were closed: GB2 with MZ2 (0.43 and 0.44 ratios), GA2 with RF1 (0.45 and 0.41) and CH4 with TN3 (0.43 and 0.42). In cluster 3 the cultivar KL1 close with ZG1 and have 0.31 and 0.30 ratios, respectively. The two cultivars JR1 and ZH2 were closed and have 0.38 and 0.36 ratios, respectively.
Conclusion Pomegranate has been reported as a natural source of phenolic compounds, sugar, protein and minerals source (Elfalleh et al. 2009). The study confirms also
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that Tunisian and Chinese pomegranate cultivars would be a source of protein and amino acids. Qualitatively, free amino acids and storage proteins of pomegranate seeds were identical. Besides, quantitatively, we noted the clear segregation of Chinese and Tunisian pomegranate cultivars into three groups independently of their geographical origins and the organoleptic taste subdivision. These groups were separated based on high, low or moderate amino acids contents. Furthermore this work offers more consideration to protein from pomegranate seed which can be with great importance approximating seed oil value. By the end of this study, the new high quality traits of plants will have to meet agronomical requirements and should be safe and provide benefits to both farmers and consumers.
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