Δευτέρα 26 Οκτωβρίου 2015

Nutritional evaluation and antioxidant activity of sesame sprouts


doi:10.1016/j.foodchem.2011.05.024
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Abstract

Sesame sprouts are consumed as vegetables in Asian folk. In this study, the nutritional evaluation and antioxidant activity of sesame sprouts were investigated. As seeding days progressed, the free amino acids, γ-aminobutyric acid and total phenolic compounds in the sprouts were rapidly increased while sesamin was reduced. Although a fatty acid composition analysis showed that sesame sprouts were abundant in unsaturated fatty acids, the crude fat content was gradually reduced during sprout growth. In the antioxidant assays, it was found that the DPPH radical scavenging activity and the reducing power of the sprouts increased as the seeding days progressed, which was positively related to the total phenolic content. Sesame sprouts can be recommended for functional ingredients, as well as being an excellent dietary source of natural antioxidants.

Highlights

► Most of the free amino acid contents were increased as seeding days progressed. ► The crude fat content significantly reduced during seeding. ► γ-Amino-n-butyric acid and total phenolic compounds were rapidly increased. ► Sesamin in sprouts was significantly reduced during seeding. ► Antioxidant activity of sprouts was positively related to the total phenolic content.

Keywords

  • Sesame sprout;
  • Functional food;
  • Phenolic compound;
  • Antioxidant

1. Introduction

Sesame (Sesamum indicum L.) is an important oilseed crop in the world and provides a good source of edible gourmet oil. Sesame serves as a nutritious food for humans and is used widely in bakery and confectionery products ( Shahidi, Liyana-Pathirana, & Wall, 2006). Sesame is cultivated on a worldwide basis; the seed is composed of 55% lipid and 20% protein ( Abou-Gharbia, Shahidi, Shehata, & Youssef, 1997). Some nutraceutical characteristics of sesame seeds have been identified, including antioxidant, hypocholesterolaemic, and hepatoprotective effects, and have also been associated with prevention of hypertension ( Chen et al., 2005 and Lazarou et al., 2007).
Bean sprouts, rich in dietary fibres, various nutrients, and bioactive components, are important vegetables consumed in Asian countries, and, nowadays, they have become more popular in the United States and European countries (Liu, Chen, Yang, & Chiang, 2008). Although the most popular bean sprouts are cultivated from mungbean and soybean, sesame seeds are also a good source of bean sprouts. Sesame sprouts have been consumed as vegetables in China for hundreds of years. However, little is known about sesame sprouts. The objective of this study was to investigate the nutritional value and antioxidant capacity as the seeding days progressed.

2. Materials and methods

2.1. Materials and chemicals

Sesame seeds (cv. Yuzhi 4) for this study were provided by Henan Academy of Agricultural Sciences (Zhengzhou, China). Fatty acid methyl esters used as standards, sesamin, γ-aminobutyric acid, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemicals Co. (USA). All other chemicals used were of analytical grade.

2.2. Preparation of sesame sprouts

Soaked sesame seeds were put into an artificial climate incubator (PQX-330B-22H, Ningbo Scientific Co., Ltd., Ningbo, China) at a temperature and humidity of 25 °C and 70%, respectively, without sunlight and sprayed with water at intervals of 8 h everyday. The water used to grow the sesame seeds was de-ionised water. The general characteristics of the sprouts were measured on various days after seeding (DAS). Randomly collected sprouts were immediately frozen at −40 °C in a refrigerator (DWF-L362, Zhongke Meiling Cryogenics Limited Company, Hefei, China). After freeze-drying (Alpha 1-4, Christ, Germany), the sprouts were finely ground in order to prepare the samples for the following analysis.

2.3. Crude fat and protein analyses

The crude fat and crude protein contents of the dried samples were analysed using the standard AOAC methods (Firestone, 1998), and the compositions were denoted by percentage on dry basis.

2.4. Analysis of fatty acid composition

The fatty acid composition of the sample was analysed according to the IUPAC method 2.302 (Paquot & Hauntfenne, 1987). The analysis of the fatty acid methyl esters was performed on a gas chromatograph (GC) (Agilent 6890 N) equipped with a flame ionization detector (FID) and a DB-FFAP capillary column (30 m × 0.32 mm, 0.25 μm of film thickness) (Agilent Technologies Co., Ltd.). The column, injector, and detector temperatures were set at 180, 230, and 230 °C, respectively. The flow rate of the carrier gas N2, with a split ratio of 1:20, was set at 70 ml/min. The fatty acids were identified based on the retention times of the standard fatty acid methyl esters at the same conditions.

2.5. Analysis of free amino acids

The contents of free amino acids were determined by a published method (Wang, Tang, Chen, & Yang, 2009). The free amino acid composition of the protein isolate samples was determined with an Amino Acid Analyzer (Waters M510, USA). 0.5 g of the sample was soaked in 10 ml of de-ionised water, and then sonicated for 15 min. The resulting mixture was then centrifuged at 10,000 rpm for 15 min. Determination was achieved by pre-column derivation of the sample with phenyl isothiocyanate. The separation of the corresponding derivatives was made on a PICO.TAG NH2 analytical column at 38 °C. The buffer system used for the separation consisted of sodium acetate pH 6.4 as buffer A and 60% acetonitrile as buffer B. A mobile-phase gradient was used as follows: 0–10 min, 0–46% B; 10–12 min, 46–100% B. After each run the chromatographic system was equilibrated with 100% A for 10 min. The injection volume was 10 μl. The flow rate was 1 ml/min and the wavelength for detection was set at 254 nm. The different amino acids recovered were presented as g/100 g on dry basis.

2.6. Analysis of γ-aminobutyric acid

The content of γ-amino butyric acid was determined by a published method (Zhang, Wu, & Yao, 2003) using an Agilent 1100 HPLC (USA). 1.0 g of sprouts was extracted with 25 ml of 5% trichloroacetic acid solution, and then sonicated for 15 min. The resulting mixture was centrifuged at 10,000 rpm for 10 min. The HPLC analysis included a pre-column derivation of the sample with OPA and FMOC-C. A Hypersil C18 column (4.6 × 250 mm; 5 μm particle size) was then employed for the separation of the corresponding derivatives. The buffer system used for the separation consisted of 10 mM sodium acetate pH 7.2 as buffer A, and a mixture of methanol, acetonitrile and 10 mM sodium acetate pH 7.2 (4:4:2) as buffer B. A mobile-phase gradient was used as follows: 0–27.5 min, 8–60% B. After each run the chromatographic system was equilibrated with 100% B for 10 min. The injection volume was 10 μl. The flow rate was 1 ml/min and the wavelength for detection was set at 338 nm.

2.7. Analysis of sesamin

The sesamin content of the sample was determined by using high performance liquid chromatography; the procedure was standardised and was approved by the Ministry of Agriculture, PR China for national implementation in 2008 (NY/T 1595-2008, 2008). 1.0 g of powdered sprouts was soaked in 50 ml of 80% ethanol, and then sonicated for 15 min. The resulting mixture was centrifuged at 5000 rpm for 20 min. The sample was separated in a reversed phase column, SunFireTM C18 column (4.6 × 250 mm; 5 μm particle size) made by Waters (Milford, MA, USA). The mobile phase consisted of water and methanol (3:7) with a flow rate of 1.0 ml/min. The HPLC analysis of sesamin was performed on a Waters Alliance HPLC system (Milford, MA, USA), which consisted of a Waters 2695 separations module and a Waters 2487 dual wavelength detector. The injection volume was 10 μl and the wavelength for detection was set at 287 nm. The quantitative analysis of sesamin in the sample was based on an external standard. The chromatographic data were recorded and processed by Empower 2 software.

2.8. Determination of total phenolic content

According to a published method (Randhir, Kwon, & Shetty, 2008), the total phenolic content was measured as gallic acid equivalents (GAEs) from a gallic acid standard curve (20–180 μg range). The freeze-dried sprout powder (0.5 g) was extracted and sonicated twice with 50 ml of 80% ethanol each time for 30 min, followed by centrifugation (5000 rpm, 20 min) and the supernatant was used for the estimation. 0.5 ml of the sample extract was transferred into a test tube, and 3.75 ml of the Folin-Ciocalteu phenol reagent (20%) was added. After an incubation period of 5 min, 3.75 ml of 5% Na2CO3 was added, mixed well and kept in the dark for 90 min. Then, the sample was vortexed and the absorbance was measured at 725 nm using a spectrophotometer.

2.9. Preparation of the ethanol extract from sesame sprouts

The freeze-dried sprout powder (3 g) was extracted and sonicated twice with 50 ml of 80% ethanol each time for 30 min. The ethanol solution was filtered and then ethanol was removed in an evaporator at a temperature lower than 40 °C. The residue was freeze-dried (Alpha 1-4, Christ, Germany) and stored at −40 °C until use. The freeze-dried residue served as the sample for subsequent antioxidant tests.

2.10. DPPH radical scavenging assay

The DPPH radical scavenging assay was done according to a published method (Sun & Ho, 2005). Briefly, 2 ml of the DPPH solution (0.2 mM, in ethanol) was incubated with different concentrations of the ethanol extract. The reaction mixture was shaken and incubated in the dark for 30 min, at room temperature. The absorbance was read at 517 nm against ethanol. Controls containing ethanol instead of the antioxidant solution, and blanks containing ethanol instead of the DPPH solution were also made. The inhibition of the DPPH radical by the samples was calculated according to the following formula:
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2.11. Reducing power assay

The reducing power of the sample was determined according to the method of Strivastava, Harish, and Shivanandappa (2006). 0.5 ml of the extract in ethanol was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%). The mixture was incubated at 50 °C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. An increased absorbance of the reaction mixture indicated reducing power.

2.12. Statistical analysis

The data obtained in this study were expressed as the mean of triplicate determinations and standard deviation (SD). Statistical comparisons were made with Student’s t-test. p values of <0.05 were considered to be significant.

3. Result and discussion

3.1. Crude fat content and fatty acid composition

In this study, it was found that the crude fat content significantly decreased during seeding (p < 0.05). The crude fat contents of seed, 1 DAS (days after seeding), 3 DAS and 5 DAS were determined as 57%, 54%, 44% and 20%, respectively. The GC analysis of the samples is shown in Table 1. The sesame seeds were abundant in unsaturated fatty acids, with only lower amounts containing saturated fatty acids. The compositions of unsaturated fatty acids, including oleic acid (18:1) and linoleic acid (18:2) were 39.32% and 44.68%, respectively. Meanwhile, the major saturated fatty acid of the seeds was palmitic acid (16:0), which comprised approximately 8.99% of the total fatty acids. Only small quantities of stearic acid (18:0), arachidic acid (20:0), and behenic acid (22:0) were observed. As the seeding days progressed, among the unsaturated fatty acids, linoleic acid gradually decreased, while oleic acid and linolenic acid (18:3) increased. During seeding, the composition of the total unsaturated fatty acid was greater than 80%. It could be concluded that sesame sprouts were rich in polyunsaturated fatty acids with oleic acid and linoleic acid being the major fatty acids.
Table 1. Fatty acid composition of sesame sprouts according to days after seeding (%).
Fatty acidsSeedDays after seeding
135
C16:08.99 ± 0.229.17 ± 0.348.89 ± 0.289.82 ± 0.25
C16:10.15 ± 0.010.15 ± 0.010.17 ± 0.01–
C18:05.50 ± 0.235.47 ± 0.165.40 ± 0.215.41 ± 0.18
C18:139.32 ± 2.1239.09 ± 1.6239.80 ± 1.4142.41 ± 1.82
C18:244.68 ± 2.3444.69 ± 1.7243.36 ± 2.5740.05 ± 1.39
C18:30.32 ± 0.010.41 ± 0.021.16 ± 0.051.43 ± 0.04
C20:00.66 ± 0.020.64 ± 0.030.72 ± 0.010.77 ± 0.02
C20:10.21 ± 0.010.21 ± 0.01––
C22:00.14 ± 0.010.14 ± 0.010.24 ± 0.01–
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3.2. Crude protein and free amino acids

The crude protein contents of seed, 1 DAS, 3 DAS and 5 DAS were 23.33%, 23.66%, 23.52%, and 23.15%, respectively. The crude protein content nearly remained unchanged during seeding (p < 0.05). However, HPLC analysis showed that most of the free amino acids increased as seeding days progressed ( Table 2). The total free amino acid content in 5 DAS sprouts was almost 11-times higher than that of seeds. The increment in the free amino acid content is favourable as the protein quality of the vegetable depends not only on its amino acids but also on the availability of these amino acids ( Kim, Kim, & Park, 2004). It was considered that the high contents of necessary amino acids, such as threonine, valine, methionine, isoleucine, leucine, tryptophane and phenylalanine, would provide high nutritional value.
Table 2. Free amino acid contents of sesame sprouts according to days after seeding (mg/100 g dry basis).
Free amino acidsSeedDays after seeding
135
Aspartic acid5.7374.5654.88109.03
Glutamic acid1.18132.05196.01125.17
Serine3.8561.55325.98547.23
Glycine7.0022.1131.1435.80
Hlstidine41.7332.2571.5473.38
Argnine25.3481.07248.45238.55
Threonine5.1139.1088.7173.48
Alanine2.9637.30123.5878.46
Proline5.9029.9553.3237.62
Tyrosine4.7451.2998.3363.68
Valine0.7125.7870.7165.49
Methionine0.0311.4020.3916.97
Cysteine2.221.691.781.99
Isoleucine6.8221.0338.2233.03
Leucine4.4138.7761.6945.58
Tryptophane3.9714.1932.6839.30
Phenylalanine4.1128.6230.6824.22
Lysine16.3927.1220.4316.35

Total142.22729.831568.511625.33
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3.3. γ-Aminobutyric acid

γ-Aminobutyric acid (GABA) is an important ubiquitous non-protein amino acid in both prokaryotic and eukaryotic organisms. It is a representative depressive neurotransmitter in the sympathetic nervous system and has been proved to be effective for lowering the blood pressure of experimental animals and humans (Zhang, Yao, & Chen, 2006). Besides improving hypertension, GABA could also be used as a dietary supplement and/or nutraceutical to help treat sleeplessness, depression and autonomic disorders, chronic alcohol-related symptoms and to stimulate immune cells (Oh, Soh, & Cha, 2003). As germination days progressed, an increase of GABA was found in this study (Fig. 1). The GABA content in the seeds was only 24.13 μg/g while the GABA content was 95.28 μg/g at 5 DAS, almost 3 times higher than that of seeds.
GABA content of sesame sprouts on various days after seeding.
Fig. 1. 
GABA content of sesame sprouts on various days after seeding.
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3.4. Sesamin

Sesamin is a compound (known as lignan) found in sesame seeds, which has multiple physiological functions, such as decreasing blood lipids and arachidonic acid levels, increasing antioxidant ability, providing anti-inflammatory function and oestrogenic activity (Wu, 2007). The contents of Sesamin of seed, 1 DAS, 3 DAS and 5 DAS were 0.1413%, 0.1694%, 0.0122% and 0.0037%, respectively. The content of sesamin in the sprouts decreased rapidly, which coincided with the crude fat content.

3.5. Total phenolic content

Oxidative damage is thought to be one of the major mechanisms involved in chronic human diseases, such as cancer and heart disease. Phenolic compounds are ubiquitous phytochemicals present in plant foods with numerous biological activities including antioxidant properties. It has been shown that antioxidant-rich diets can reduce oxidative damage to DNA, thus preventing a critical step at the onset of carcinogenesis (Zhang, Seeram, Lee, Feng, & Heber, 2008). In this study, it was found that the total phenolic content increased rapidly as seeding days progressed (Fig. 2); the total phenolic content in the seeds was only 0.51 mg GAE/g while the content at 5 DAS was as high as 13.42 mg GAE/g.
Total phenolic content of sesame sprouts on various days after seeding.
Fig. 2. 
Total phenolic content of sesame sprouts on various days after seeding.
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3.6. DPPH radical scavenging activity

The DPPH radical is a stable organic free radical with an adsorption peak at 517 nm. It loses this adsorption when accepting an electron or a free radical species, which results in a visually noticeable discolouration from purple to yellow (Sánchez-Moreno, 2002). As shown in Fig. 3, the DPPH radical scavenging activities of the extracts were influenced by concentration. The radical scavenging activities during plant growth at 3 and 5 DAS increased significantly. The DPPH scavenging activity of the extracts followed the following order: 5 DAS > 3 DAS > 1 DAS > seed.
DPPH radical scavenging activity of sesame sprouts on various days after ...
Fig. 3. 
DPPH radical scavenging activity of sesame sprouts on various days after seeding, as a function of concentration (of ethanol extract from sesame sprouts).
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3.7. Reducing power

The reducing power of the complex, which may serve as a significant reflection of antioxidant activity, was determined using a modified Fe (III) to Fe (II) reduction assay; the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of the samples. The presence of antioxidants in the samples causes the reduction of the Fe3+/Ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring of the formation of Perl’s Prussian blue at 700 nm (Zou, Lu, & Wei, 2004). Fig. 4 shows the reducing power of the extracts. All samples showed some degree of reducing power. The reducing power of the samples increased linearly with increasing concentration. The extract of 5 DAS showed the highest reducing power. The reducing power of the samples followed the following order: 5 DAS > 3 DAS > 1 DAS > seed.
Reducing power of sesame sprouts at various days after seeding.
Fig. 4. 
Reducing power of sesame sprouts at various days after seeding.
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4. Conclusions

As seeding days progressed, the free amino acids, γ-amino-n-butyric acid and the total phenolic compounds in the sprouts rapidly increased while sesamin and crude fat decreased. The ethanol extracts of the sprouts showed strong DPPH radical scavenging activity and reducing power, which were positively related to the total phenolic contents. Due to its high contents of free amino acids, GABA and phenolic compounds, sesame sprouts can be recommended as a functional food.

Acknowledgements

The financial supports provided by the Foundation of Henan Educational Committee (No. 2009A550005) and the Science and Technology Plan of the Henan Institute of Science and Technology (No. 7040) are greatly appreciated.

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