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. 2010 Nov 18;47(6):656–661. doi: 10.1007/s13197-010-0111-4

Amino acid composition of Lagenaria siceraria seed flour and protein fractions

Moriyike Esther Ogunbusola 1, Tayo Nathaniel Fagbemi 2,✉, Oluwatooyin Faramade Osundahunsi 2
PMCID: PMC3551139  PMID: 23572701

Abstract

Defatted seed flours of Lagenaria siceraria (calabash and bottle gourd) were fractionated into their major protein fractions. The amino acid composition of seed flours and their protein fractions were determined and the protein quality was evaluated. Glutamic acid (139–168 mg/g protein) was the most abundant amino acid followed by aspartic acid (89.0–116 mg/g protein) in both the seed flours and their protein fractions. The total essential amino acid ranged from 45.8 to 51.5%. The predicted protein efficiency ratio and the predicted biological value ranged from 2.4 to 2.9 and 8.7 to 44.0, respectively. Lysine and sulphur amino acids were mostly concentrated in the globulin fractions. The first and second limiting amino acids in seed flours and protein fractions were methionine and valine or threonine. The seed flours contained adequate essential amino acids required by growing school children and adults. The seed has potential as protein supplement in cereal based complementary diets or in the replacement of animal proteins in conventional foods.

Keywords: Lagenaria siceraria, Calabash, Bottle gourd Protein fractions, Amino acid composition

Introduction

Studies on the utilization of vegetable proteins continue to attract attention globally due to the increasing demand for cheap and affordable dietary proteins, particularly among the low income group. Projections based on the current trends indicate a gap between human population and protein supply (Vijayakumari et al. 1997). Hence, the need to examine unconventional legumes and oilseeds as alternative protein sources for the future (Egbe and Akinyele 1990; Onweluzo et al. 1994; Chau and Cheung 1998; Fagbemi 2007). This development has stimulated research on the utilization of Lagenaria siceraria, an indigenous underutilized oil rich seed as alternate protein source.

Lagenaria siceraria (calabash and bottle gourd) belongs to Cucurbitaceae family. The plants are annual, herbaceous, and monoecious with creeping stems. The seeds are edible and used in the preparation of local soups, fermented food product (ogiri), fried cake (robo) and pudding (igbalo or ugbaotiri). The seeds of Lagenaria siceraria are rich in dietary proteins (Fokou et al. 2004).

Researchers have worked extensively on other species of Cucurbitaceae such as Colocynthis citrullus, Citrullus vulgaris and Telfairia occidentalis (Akobundu et al. 1982; Sathe et al. 1982; Ige et al. 1984; Fagbemi and Oshodi 1991; Fagbemi et al. 2005, 2006; Fagbemi 2007). Report on Lagenaria siceraria has been limited to its proximate composition and functional properties (Fokou et al. 2004). The knowledge of its amino acid composition and amino acid profile is necessary in food product formulations. This investigation was aimed at fractionation of Lagenaria siceraria seed flour into their major protein fractions and determination of their amino acid composition to further exploit the seeds potential use in food system.

Materials and methods

Two varieties of Lagenaria siceraria (calabash seed LS1 and bottle gourd seed LS2) were bought from farmers in Irele Ekiti, Ekiti State, Nigeria. The seeds were manually shelled, washed and later dried in a hot air oven at 50 °C. The seeds were pulverized using a Brabender blender, defatted by refluxing continuously for 8 h using n-hexane. Defatted meals were dried, pulverized and sieved to pass through a 500 μm sieve.

Protein fractions were extracted according to their solubility in different solvents as described by Wisal et al. (2003). Defatted Lagenaria siceraria seed flour (3.5 g) was extracted twice with 50 ml of distilled water for 30 min at room temperature (28 ± 2 °C). The extract was centrifuged at 4,500 rpm for 20 min and the supernatant was used for the determination of water soluble albumin. The residue was then extracted successively in a similar manner with 1 M NaCl, or 1 M NaOH solution, extract was collected separately and used to estimate the salt soluble (globulin) or alkali soluble (glutelin) fractions.

The amino acid profiles of the seed flours and protein fractions were determined using ion exchange chromatography. The samples were defatted, hydrolyzed and evaporated in a rotatory evaporator and then injected into the Technicon sequential multisampling Amino Acid Analyzer (Technicon Instrument Co. Ltd., United Kingdom), (Adeyeye and Afolabi 2004). Tryptophan content of the seed flours was determined using the method of Concon (1975) as modified by Ogunsua (1988). The amino acids obtained were used to evaluate the protein quality of seed flour. Predicted biological value (BV) was calculated using the regression equation of Morup and Olesen (1976) as reported by Chavan et al. (2001).

graphic file with name M1.gif

Where,

  • Inline graphic for ai sample ≤ ai reference

    or

  • Inline graphic for ai sample ≥ ai reference

  • ai = mg of the amino acid per g of total essential amino acids.

The predicted protein efficiency ratio (PER) was calculated using one of the equations developed by Alsmeyer et al. (1974) as stated below.

graphic file with name M4.gif

Isoelectric point (IP) was estimated from the amino acids using the equation of the form given by Olaofe and Akintayo (2000).

graphic file with name M5.gif

where, IP is the isoelectric point of the ith amino acid in the mixture, Xi is the mass or mole fraction of the ith amino acid in the mixture and IPm is the isoelectric point of the mixture.

Determinations were carried out in triplicate, along with standard deviations. Data were subjected to analysis of variance using SPSS 15 computer programme.

Results and discussion

Variety and fractionation have significant (p < 0.05) effect on the amino acid composition of Lagenaria siceraria seed flours (Table 1). Glutamic acid was the most abundant amino acid in both seed flours and all protein fractions. The values ranged from 140 to 168 mg/g protein with minimum value in LS2 globulin fraction and maximum in LS1 albumin fraction. The second most abundant amino acid in all the seed flours was aspartic acid ranging from 89.0 to 116 mg/g protein in LS2 albumin and seed flour, respectively. Mora-Escobedo et al. (1990) reported similar observation for the amino acids of the albumin and globulin fractions of amaranth. Oshodi et al. (1998) reported tryptophan to be the most concentrated amino acid in legumes. The most concentrated essential amino acid in all seed flours and their protein fractions was leucine with values ranging from 60.0 to 72.1 mg/g protein (LS2 albumin and the seed flour, respectively).

Table 1.

Amino acid composition of total seed flours and protein fractions of Lagenaria siceraria (mg/g protein) variety

Amino acid LS1 LS2
Seed flour Albumin Globulin Glutelin Seed flour Albumin Globulin Glutelin
Cystine * 12.3 ± 0.08c 10.6 ± 0.08d 13.9 ± 0.08a 10.6 ± 0.08d 12.5 ± 0.08b 9.9 ± 0.08e 12.6 ± 0.08b 10.5 ± 0.08d
Methionine* 10.4 ± 0.16c 7.0 ± 0.08a 11.2 ± 0.08b 8.3 ± 0.08f 12.5 ± 0.08a 8.8 ± 0.08e 12.6 ± 0.08a 9.1 ± 0.08d
Aspartic acid 109 ± 0.08d 99.7 ± 0.16f 112 ± 0.33c 108 ± 0.24e 116 ± 0.16a 89.0 ± 0.33 g 109 ± 0.33d 113 ± 0.33b
Threonine* 29.1 ± 0.08e 21.6 ± 0.08 h 25.0 ± 0.08 g 30.1 ± 0.08d 32.6 ± 0.16c 25.9 ± 0.08f 36.2 ± 0.08b 41.1 ± 0.08a
Serine 40.4 ± 0.04c 31.7 ± 0.16 h 39.1 ± 0.08d 32.0 ± 0.08 g 50.4 ± 0.08a 32.8 ± 0.08f 46.6 ± 0.16b 38.0 ± 0.08e
Glutamic acid 166 ± 0.24b 168 ± 0.24a 152 ± 0.41d 161 ± 0.41c 143 ± 0.16f 149 ± 0.16e 140 ± 0.41 g 148 ± 0.24e
Proline 37.5 ± 0.08a 30.8 ± 0.08e 34.2 ± 0.08b 32.0 ± 0.08d 32.5 ± 0.08c 29.0 ± 0.08 g 30.1 ± 0.08f 28.0 ± 0.08 h
Glycine 31.7 ± 0.08 g 40.6 ± 0.16b 40.1 ± 0.08c 38.9 ± 0.08d 32.4 ± 0.04f 40.3 ± 0.08c 35.0 ± 0.08e 47.0 ± 0.16a
Alanine 40.6 ± 0.08ab 40.1 ± 0.08b 36.3 ± 0.08e 37.2 ± 0.08d 32.6 ± 0.08 g 34.7 ± 0.08f 40.9 ± 0.08a 37.9 ± 0.08c
Valine* 40.6 ± 0.08b 30.2 ± 0.08 g 43.6 ± 0.08a 39.2 ± 0.16d 40.0 ± 0.08c 31.1 ± 0.08f 40.2 ± 0.08c 32.5 ± 0.08e
Isoleucine* 33.0 ± 0.08d 35.1 ± 0.08b 35.5 ± 0.08a 30.4 ± 0.08f 35.6 ± 0.08a 28.2 ± 0.08 g 31.4 ± 0.08e 34.5 ± 0.08c
Phenylalanine* 46.3 ± 0.08b 32.9 ± 0.08 g 41.4 ± 0.16d 36.3 ± 0.08f 48.8 ± 0.08a 37.2 ± 0.08e 45.6 ± 0.16c 37.2 ± 0.08e
Lysine* 56.2 ± 0.16c 42.4 ± 0.16 g 60.1 ± 0.24a 57.2 ± 0.016b 50.8 ± 0.16e 37.5 ± 0.08 h 52.6 ± 0.16d 48.9 ± 0.16f
Arginine* 58.6 ± 0.16d 50.2 ± 0.16 g 62.9 ± 0.24a 60.3 ± 0.24b 55.2 ± 0.16e 49.4 ± 0.08 h 54.4 ± 0.16f 59.6 ± 0.16c
Histidine* 26.1 ± 0.08a 16.3 ± 0.08 g 24.4 ± 0.08b 22.4 ± 0.08e 23.0 ± 0.08e 16.9 ± 0.08f 22.6 ± 0.08d 24.5 ± 0.08b
Leucine* 65.8 ± 0.016e 65.0 ± 0.24f 71.1 ± 0.16b 66.2 ± 0.16d 72.1 ± 0.16a 60.0 ± 0.16 h 68.3 ± 0.08c 61.5 ± 0.21 g
Tyrosine* 34.9 ± 0.08b 22.5 ± 0.08 g 35.6 ± 0.08a 22.5 ± 0.08 g 30.1 ± 0.04d 27.4 ± 0.08f 30.5 ± 0.08c 29.0 ± 0.08e
Tryptophan* 11.9 ± 0.16c 13.0 ± 0.16b 13.9 ± 0.24a 10.6 ± 0.08d 9.9 ± 0.08e 8.1 ± 0.08f 10.8 ± 0.16d 10.7 ± 0.16d
Cal. Isoelectric point 5.1 4.5 5.1 4.9 4.9 4.3 4.9 4.9
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*Essential amino acids; values followed by different letters in the same row are significantly different (p < 0.05); LS1: Calabash seed flours; LS2: Bottle gourd seed flours

The lysine content of Lagenaria siceraria seed flours and their protein fractions ranged between 37.5 and 60.1 mg/g protein, this is similar to the lysine content of fluted pumpkin (37.5–66.6 mg/g cp) reported by Fagbemi (2007). The lysine content of seed flour is comparable with that of reference egg protein (63 mg/g crude protein, FAO/WHO/UNU 1985). Hence, Lagenaria siceraria seed flours and their protein fractions could be mixed with cereals like maize in weaning food formulation (Chavan et al. 2001). Globulins were however, richer in lysine than the water-soluble albumin or alkali soluble glutelin. This indicates that globulin fraction may be a better supplement in cereal based diet preparation. Tryptophan content of seed flours ranged from 8.1 to 13.9 mg/g protein. The calculated isoelectric point varied from 4.3 to 5.1 in LS2 albumin and LS1 seed flour respectively. This will serve as useful guide in quick precipitation of proteins from biological samples (Olaofe and Akintayo 2000).

The total amino acid content ranged from 715 to 851 mg/g protein (LS2 albumin and LS1 seed flour, Table 2). The total essential amino acid of Lagenaria siceraria flours ranged between 340 and 439 mg/g protein. This is lower than 566 mg/g protein reported for egg reference protein (Paul et al. 1980). It is, however comparable with values (190–503 mg/g protein) reported for some oilseeds such as Colocynthis citrullus, peanut meal and soybean flours (Lusas 1979; Akobundu et al. 1982; Sosulski 1983; Kuri et al. 1991). The range of percentage total essential amino acid (45.8–51.5) obtained for Lagenaria siceraria seed flour and their protein fractions is well above 36%, which is considered adequate for an ideal protein (FAO/WHO 1973). This suggests that seed flours and their protein fractions may find use as a food supplement. Globulin fraction had the highest percentage of total essential amino acid (51.0 and 51.5%) in all the protein fractions. The total sulphur amino acid content ranged from 17.6 to 25.2 mg/g protein with cystine ranging from 50.0 to 60.2%. The range of total neutral, acidic and basic amino acids were 49.1–53.8%, 30.4–35.4% and 14.4–17.4%, respectively, which showed that protein in seed flours and their protein fractions may be acidic in nature. Similar observation was reported by Aremu et al. (2006) for some Nigerian underutilized oilseeds.

Table 2.

Summary of amino acid composition of the total seed flour and protein fractions of Lagenaria siceraria (mg/g protein) variety

LS1 LS2
Seed flour Albumin Globulin Glutelin Seed flour Albumin Globulin Glutelin
Total amino acids (TAA) 851 758 852 803 830 715 819 811
Total essential amino acids (TEAA) 426 347 439 394 423 340 418 399
TEAA/TAA (%) 49.9 45.8 51.5 49.1 51.0 47.6 51.0 49.2
Total non essential amino acids (TNEAA) 425 411 413 409 407 375 402 412
Total sulphur amino acids (TSAA) 22.7 17.6 25.1 18.9 25.0 18.7 25.2 19.6
Cystine (%) in TSAA 54.2 60.2 55.4 56.1 50.0 52.9 50.0 53.6
Total aromatic essential amino acids phe.+tyr. (ArEAA) 81.2 54.4 77.0 58.8 78.9 64.6 76.1 66.2
Total acidic amino acids (TAAA)% Glu. + Asp. 32.4 35.4 30.9 33.5 31.2 33.3 30.4 32.2
Total basic amino acids (TBAA)% Lys. + Arg. + His. 16.6 14.4 17.3 17.4 15.5 14.5 15.8 16.4
Total neutral amino acids (TNAA)% 50.0 50.2 51.8 49.1 53.3 52.2 53.8 51.4
Ratio of TEAA:TNEAA 1.0 0.8 1.1 1.0 1.0 0.9 1.0 1.0
Predicted protein efficiency ratio (PER) 2.5 2.7 2.8 2.7 2.9 2.4 2.7 2.4
Predicted biological value (BV) 27.1 8.7 21.0 23.4 31.4 12.3 40.2 44.0
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LS1 Calabash seed flours; LS2 Bottle gourd seed flours

The predicted PER of Lagenaria siceraria seed flours and their protein fractions ranged from 2.4 (LS2 albumin and glutelin) to 2.9 (LS2 seed flour). The predicted PER values were higher than seed proteins of cowpea (1.21), pigeon pea (1.82) and L. sativus (negative value to 0.03) (Salunkhe and Kadam 1989). These values are also higher than the ranges of 0.66–1.24 and 0.63–2.21 for fluted pumpkin seed flours and cotton seed, respectively (Fagbemi 2007). The predicted BV of Lagenaria siceraria protein ranged between 8.7 and 44.0. Fractionation affected BV. Globulin and glutelin fractions have higher BV than their albumin fraction. The predicted BV of Lagenaria siceraria compared well with the range of 36.5–40.13 reported for beach pea protein isolates (Chavan et al. 2001)

The chemical score ranged from 21.9 (LS1 albumin) to 39.4 (LS2 globulin) (Table 3). The first limiting amino acid in both raw seed flours and their protein fractions was methionine (7.0–12.6 mg/g protein) followed by valine (30.2–43.6 mg/g protein) except in LS1 globulin fraction, where threonine (25.0 mg/g protein) was the second limiting amino acid. The recommended sulphur amino acid for infants, growing preschool children and growing school children are 42, 25 and 22 mg/g crude protein respectively (FAO/WHO/UNU 1985). The seed flours on this basis provide 41.9–60%; 70.4–100.8% and 80—over 100% of the recommended sulphur amino acid for infant, a growing preschool child and growing school child, respectively. Salt soluble proteins are found to be more concentrated in sulphur amino acid. In general, Lagenaria siceraria seed flours and their protein fractions contained adequate amounts of most of the essential amino acids required by preschool children and all amino acids essential for school children and adults.

Table 3.

Amino acid scores of the total seed flour and protein fractions of Lagenaria siceraria variety

LS1 LS2
Essential amino acid Referencea Seed flour Albumin Globulin Glutelin Seed flour Albumin Globulin Glutelin
EAAC AAS(%) EAAC AAS(%) EAAC AAS(%) EAAC AAS(%) EAAC AAS(%) EAAC AAS(%) EAAC AAS(%) EAAC AAS(%)
Cys. 18.0 12.3 68.3 10.6 58.9 13.9 77.2 10.6 58.9 12.5 69.4 9.9 55.0 12.6 70.0 10.5 58.3
Met. 32.0 10.4 32.5 7.0 21.9 11.2 35.0 8.3 25.9 12.5 39.1 8.8 27.5 12.6 39.4 9.1 28.4
Thre. 51.0 29.1 57.1 21.6 42.4 25.0 49.0 30.1 59.0 32.6 63.9 25.9 50.8 36.2 70.9 41.1 80.6
Val. 76.0 40.6 53.4 30.2 39.7 43.6 57.4 39.2 51.6 40.0 52.6 31.1 40.9 40.2 52.9 32.5 42.8
Iso. 56.0 33.0 58.9 35.1 62.7 35.5 63.4 30.4 54.3 35.6 63.6 28.2 50.4 31.4 56.1 34.5 61.6
Phe. 51.0 46.3 90.8 32.9 64.5 41.4 81.2 36.3 71.2 48.8 95.7 37.2 72.9 45.6 89.4 37.2 72.9
Lys. 63.0 56.2 89.2 42.4 67.3 60.1 95.4 57.2 90.8 50.8 80.6 37.5 59.5 52.6 83.5 48.9 77.6
Arg. 61.0 58.6 96.1 50.2 82.3 62.9 103 60.3 98.9 55.2 90.5 49.4 80.9 54.4 89.2 59.6 97.7
His. 24.0 26.1 109 16.3 67.9 24.4 102 22.4 93.3 23.0 95.8 16.9 70.4 22.6 94.2 24.5 102
Leu. 83.0 65.8 79.3 65.0 78.3 71.1 85.7 66.2 79.8 72.1 86.9 60.0 72.3 68.3 82.3 61.5 74.1
Tyr. 40.0 34.9 87.3 22.5 56.3 35.6 89.0 22.5 56.3 30.1 75.3 27.4 68.5 30.5 76.3 29.0 72.5
Trp. 18.0 11.9 66.1 13.0 72.2 13.9 77.2 10.6 58.9 9.9 55.0 8.1 45.0 10.8 60.0 10.7 59.4
Chemical score 32.5 21.9 35.0 25.0 39.1 27.5 39.4 28.4
1st limiting a. a Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine
2nd limiting a. a Valine Valine Threonine Valine Valine Valine Valine Valine
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aProvisional amino acid pattern egg as reference (FAO/WHO/UNU 1985). EAAC Essential amino acid composition (mg/g protein)

AAS Amino acid score; a.a amino acid; LS1 Calabash seed flours; LS2 Bottle gourd seed flours

Conclusion

Amino acid profile of Lagenaria siceraria seed flours and their water, salt and alkali (albumin, globulin and glutelin, respectively) soluble protein fractions are of high quality and very high in lysine. The percentage total essential amino acids in all flour samples were well above the recommended values. Lagenaria siceraria seed flours and their protein fractions may be incorporated into cereals for the formulation of a wide range of cereal based weaning foods and other complementary diets because of their high lysine content.

Contributor Information

Moriyike Esther Ogunbusola, Email: riikebusola@yahoo.com.

Tayo Nathaniel Fagbemi, Email: tnfagbemi55@yahoo.co.uk.

Oluwatooyin Faramade Osundahunsi, Email: tosundahunsi@yahoo.com.

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