Effect of Germination Time and Type of Illumination on Proximate
Composition of Chickpea Seed (Cicer arietinum L.)
Amal Badshah Khattak
Mohammad Saeed Khattak
Impact of germination time and type of illumination on proximate composition
of chickpea seed was investigated. Germination time and type of illumination
had highly significant influence (p<0.001) on the level of moisture,
protein, fat, fiber, ash and Nitrogen Free Extract (NFE) contents. Increase
in germination time was associated with increase in moisture, protein,
ash and fat contents and decrease in fiber and NFE contents. Moisture
accumulation increased significantly (p<0.001) with dark, fluorescent
light and γ-irradiated seed sprouts, while green, blue and yellow
lights have significant (p<0.001) promotional effects on protein and
fiber contents. Germination of γ-irradiated chickpea seed had significant
(p<0.001) promotional effect on ash and fat contents, while dark, fluorescent
and yellow lights on NFE content. Interaction of the treatments (germination
time X type of illumination) on all the parameters studied was also highly
Chickpea (Cicer arietinum L.) is a source of dietary protein in
general and particularly for poor/vegetarian segments of the world population.
It is also used as a protein supplement in the European countries (Viveros
et al., 2001). Chickpea is an ancient crop and has been grown and
consumed in tropical, sub-tropical and temperate regions for centuries.
It is valued for its nutritive seeds with high protein content. Chickpea
is used exclusively as food in many countries (Muehlbauer and Singh, 1987;
Malhotra et al., 1987) and its traditional uses include boiling,
roasting, canning or processing into humus (a traditional dish in the
Sprouting (the practice of soaking then draining and leaving seeds until
they begin to sprout) has been identified as an inexpensive and effective
technology for improving the nutritional quality of cereals and grain
legumes. It is reported to be associated with improvements in the nutritive
value of seeds (Badshah et al., 1991; Sattar et al., 1995;
Zanabria et al., 2006; Khattak et al., 2007a). As the seed
imbibe water the enzymes are activated and the biochemical changes take
place. Proteins break into amino acids. Water-soluble vitamins such as
B complex and vitamin C are created. Fats and carbohydrates are converted
into simple sugars. Weight increases as the seed absorbs water and minerals.
At the same time there are reports that germination is effective in reducing
phytic acid, flatulence causing oligosaccharides (namely stachyose and
raffinose) and polyphenols thereby increasing protein digestibility and
improving sensory properties (Lintschinger et al., 1997; Zanabria
et al., 2006; Khattak et al., 2007). It is reported that
sprouting improved the protein/amino acid digestibility by decreasing
anti-nutritional factors and increasing the true/apparent protein/amino
acid digestibility (Schulze et al., 1997; Rubio et al.,
2002). According to Lorenz (1980) the practice of sprouting can be used
in many different foods including breakfast items, salads, soups, casseroles,
pasta and baked products. It has been recently reported that germination
under different type of illumination has significant effect on biosynthesis
of ascorbic acid and sprout yield of soybean and chickpea (Mao et al.,
2005; Khattak et al., 2007a).
The present research, being part of a study “Nutritional enhancement
of chickpea seed through germination techniques”, was undertaken
to investigate the impact of germination time and type of illumination
on proximate composition of chickpea sprouts.
MATERIALS AND METHODS
The present research is a continuation of previous studies (Khattak et
al., 2007a, b). It was started in the Nuclear Institute for Food and
Agriculture (NIFA), Tarnab, during March 2006. A brief account of methodology
used was as follows:
Chickpea seeds of desi type variety NIFA-2005, developed at the Nuclear
Institute for Food and Agriculture (NIFA), Peshawar were cleaned from
all impurities including broken and diseased seeds. Part of the un-soaked
sample was ground in a stainless steel grinder to pass through a 40-mesh
screen. The ground samples were kept in plastic bags, stored at 4°C
for chemical determinations.
Soaking of Chickpea Seeds
The seeds were soaked by submerging in tap water in glass containers
for 24 h at room temperature. After pouring off the soaking water, the
seeds were rinsed with water, spread evenly on a tray lined with absorbent
paper and then placed in a controlled environment chamber at 28°C.
Wooden chambers each with 91x91x60 cm (LxHxW) dimensions were used
for germination of seeds. There were 5 chambers used for five types of
illumination, i.e., fluorescent, yellow, blue, green and red and two for
dark and gamma irradiated samples. The light source in the illuminated
chambers was fitted on the ceiling of the chamber. The temperature of
the chambers was maintained at 28±3°C.
Gamma Irradiation Treatment
The seed samples were irradiated at a dose of 30 krad in Co-60 gamma
radiation source (Isseldovatel, Konhpobba, USSR). Soaking and sprouting
was then carried out in dark conditions.
Sprouting was started in triplicate for each treatment (illumination
i.e., dark, red, blue, tungsten, green and fluorescent and length of time
i.e., 0, 24, 48, 72 and 96 h) in trays lined with absorbent paper (blotting
paper). Seed/sprouts were washed twice a day to avoid microbial growth.
Tap water was sprayed throughout the germination period at 9 am, 1 and
6 pm daily.
Fluorescent tubes (40 W, Philips, Lahore, Pakistan) were used as a
white light source. Respective colored bulb (40 W, Philips, Lahore, Pakistan)
were used as per illumination treatments. The trays were distributed under
the light so as to give uniform flux density to each tray. The same flux
density were obtained by turning on the fixed number of light sources
and by adjusting fixed distances between the lamps and the test materials.
Germination under all types of illuminations was repeated four times.
Air oven method was (AOAC, 1984, method # 14.004) used to determine
moisture content of the sprouted and un-sprouted chickpea samples. Protein
content (%) was determined using Micro Kjeldahl (AOAC, 1984 method # 14.067).
Crude fat was determined by soxhlet method using soxtec (labconco) apparatus
(AOAC, 1984-method # 14.066).
For ash determination, 5 g samples were ignited in broad ashing dish
that has been previously ignited, cooled in desiccator and weighed soon
after reaching room temperature. The charred samples were placed in furnace
at 550°C until light gray ash results, or to constant weight. The
samples were then cooled in desiccator and weighed soon after reaching
room temperature (AOAC, 1984, method # 14.006). Crude fiber was determined
using AOAC method #7.070 (AOAC, 1984). Nitrogen Free Extracts (NFE) was
measured by difference i. e; 100 - (moisture+protein+Crude fat+ash) =
percent nitrogen free extract. Determination of each of the trait was
repeated three times and the values reported are on moisture free basis.
Statistical analysis was conducted for each of the measured traits
by analysis of variance (ANOVA- using CRD factorial design) and the means
were separated by Duncan Multiple Range test (DMR) using Mstat-C software
RESULTS AND DISCUSSION
Moisture contents of the sprouts were significantly (p<0.001) influenced
by germination time, type of illumination as well as their interaction
(Table 1). Mean value for maximum moisture percent was noted in samples
of 120 h germination, while in case of type of illumination, maximum mean
values were noted for germination under dark, fluorescent light and germination
of gamma irradiated seed. Lowest mean value for percent moisture was observed
in samples germinated under red light (49.98%) followed by germination
under blue (50.28%), yellow (50.35%) and green (50.37%) lights. Germination
under dark after 120 h has the highest moisture content (62.57%) followed
by germination under fluorescent light after 120 h (62.40%) and germination
in dark after 96 h (62.00). The impact of time of germination was almost
linear on moisture content under all types of illuminations up to 24 h,
beyond which time the moisture of almost all samples leveled off with
very little increase during the rest of germination time (Fig. 1).
||Effect germination time and type of illumination on moisture content
Highly significant (p<0.001) variations in protein content were observed
due to time of germination, types of illuminations and their interaction.
The average protein content of chickpea was 19.84% which increased to
a maximum level of 21.97% after 96 h germination. The increase in protein
content as a function of germination time was linear for all types of
illuminations. Highest mean value for protein % was observed in germination
under blue light (21.40%) followed by germination under red (21.38%) and
green (21.31%) lights. Lowest mean protein content (21.10%) was observed
in germination in dark condition. Maximum increase in protein concentration
due to interaction of germination time and type of illumination was investigated
in germination in yellow light after 96 h (22.67%) followed by germination
in red light after 120 h (22.41%) and green light after 96 h (22.23%).
In general, blue, green and red lights seem to have a promoting effect
on protein concentration of chickpea sprouts (Fig. 2).
All the factors (sprouting time, light type and sprouting time x light
type) have a highly significant effect (p<0.001) on ether extract (fat
%). As for moisture and protein content, there was a linear increase in
mean values of fat % (4.24 to 6.03%) with increase in time of germination.
Highest mean value for fat % was observed in irradiated chickpea sprouts
(6.09%), while blue light ranked 2nd in this respect. Sprouts under red
light have the lowest fat % (5.19%). Forty eight hours sprouting of irradiated
seed has the maximum content of fat percent (7.42%) followed by 72 h germination
under blue light (6.98%).
||Analysis of variance showing mean sum of the squares (and F-values
|*: p<0.05, **: p<0.01, ***: p<0.001
||Effect of germination time and illumination on protein content of
||Effect of germination time and type of illumination on fat content
Highest values for fat% in germination in dark, fluorescent and red light
conditions was observed after 120 h, while in blue, green, yellow and gamma
irradiation were 96, 72, 96 and 48 h, respectively (Fig. 3). Germination
of irradiated seed, blue light and to some extent green light have promotional
effect on fat content of chickpea sprouts as maximum fat content were observed
in these sprouts.
Fiber content of chickpea sprout decreased significantly (p<0.001)
with the advancement of germination time (Table 1). The average content
of fiber in chickpea was 7.90%. The mean value decreased to 5.55% after
120 h germination. While, on the other hand, mean values of fiber content
were statistically the same for all the illumination types except for
the fluorescent light which has significantly (5.96%) lowest mean vale
for fiber content. There was a linear decrease in fiber content in sprouts
of all types of illuminations with the increase in germination time. The
control have minimum and 120 h sprouts maximum fiber content for sprouts
under all conditions (Fig. 4).
Ash contents of the sprouts were significantly (p<0.001) influenced
by germination time, type of illumination as well as their interaction
(Table 1). It increased significantly (p<0.001) with the advancement
of germination. The mean value of ash content of chickpea control was
3.76% which increased to 4.69% after 120 h germination. Highest mean value
for ash content was observed in sprouts of irradiated chickpea seeds (4.44%)
followed by red (4.37%) and green (4.36%) illuminations. In general, fiber
content increased with the increase in germination time in sprouts under
all types of lightings (Fig. 5).
The values for nitrogen free extract varied significantly (p<0.001)
with germination time and type of illumination. The mean value of control
samples was 64.26%, which decreased to 61.88% after 120 h germination.
Sprouts under green and blue lights have lower value for NFE while sprouts
under all other lights have higher values (Fig. 6).
Although, reports on the effects of sprouting on nutrient contents in
various cereals and legumes are well documented (Lintschinger et al.,
1997; Badshah et al., 1991; Sattar et al., 1995), evidence
on the effect of sprouting under different illuminations on nutrient content
is lacking. In fact fairly contrasting effects have been observed for
the effects of sprouting under different types of illuminations in seeds
of chickpea and soybean (Mao et al., 2005; Khattak et al.,
2007a, b). Biochemical changes in the germinating seeds have also been
reported to be significantly different in different varieties.
||Effect of germination time and type of illumination on crude fiber
content of chickpea
||Effect of germination time and type of illumination on ash content
Khalil et al. (2007) while working on Kabuli and desi type chickpea
varieties, reported significant increase in moisture, protein, ash and there
is no period after ether extract contents. and decrease in fiber and NFE
contents. According to their investigations, changes in macro nutrients
among desi and Kabuli types were significant. These findings are in agreement
to our present investigations. El-Mahdy et al. (1985) studied the
effect of germination on the nutritional quality of two varieties of lentil
seeds. They reported changes in nutritional quality due to germination as
well as genotypic differences. Effect of sprouting on inter-varietal differences
in water-uptake and biochemical traits of legumes were also reported by
Jood et al. (1997), Mulimani et al. (1996), Obizoba (1991)
and Hoene et al. (1987). They noted differences in water uptake during
4 days germination of wheat, chickpeas and mung beans. Increases in the
content of polyunsaturated fatty acids in wheat and of dietary fiber in
wheat and mung beans were noted. Similarly Chung et al. (1998) reported
that in barley (but not in canola), sprouting was associated with significant
increase in crude fiber. These findings are contradictory to present results
in chickpea. In canola, there were significant losses in lipid content (Badshah
et al., 1991). These results again confirm the existence of inter-specific
differences in the biochemical changed during sprouting. Jimenez et al.
(1985) noted an increase in the content of protein and fiber in soybean
||Effect of germination time and type of illumination on nitrogen
free extract (NFE) of chickpea
Ether extract increased until the third day and then decreased on the fifth.
Lorenz (1980) found that the increase in nutrients during sprouting is not
true increases. They simply reflect the loss of dry matter, mainly in the
form of carbohydrates (NFE), due to respiration during sprouting. As total
carbohydrates decrease, the percent ratio of other nutrients increases.
Parameswaran et al. (1994) noted increase in the percent protein
in germinated grains of porso millet as a result of dry matter loss during
germination (Mao et al., 2005; Khattak et al., 2007), while
working on soybean and chickpea, respectively, reported significant effects
of germination time and type of illumination on biosynthesis of ascorbic
acid and sprout yield. The earlier one found a promotional effect of ultra
violet illumination on biosynthesis of ascorbic acid while the same type
of illumination depressed the sprout growth. According to Khattak et
al. (2007), green illumination was effective in promoting synthesis
of ascorbic acid and retarding the growth of sprouts. Khattak et al.
(2007) reported in another study significant impact of illumination on degradation/synthesis
of phytic acid and polyphenols. These reports as well as the present one
indicate that lights with different wavelengths influence differently the
enzyme system responsible for biosynthesis of different nutrients and hence
result in differing concentration of the compound in the germinating seeds.
In the present study, higher mean values of protein and fiber contents of
sprouts under blue green and red illuminations, of moisture content under
dark, fluorescent and gamma irradiated chickpea sprouts, of ash content
of irradiated chickpea sprouts and that of NFE content under dark, red yellow
illumination, can be attributed to the wavelengths influence. Hence, it
can be inferred that different biochemical pathways are differently influenced
by different wavelengths of light. The mechanism as to how the illumination
types influenced the nutrients hydrolysis or biosynthesis is still to be
investigated; however, the present findings suggest that different light
types have differing effects on the biochemical reactions at various stages
of the germinating chickpea seeds.
It is inferred from this study that germination time and type of illumination
have highly significant (p<0.01) effect on proximate composition of
chickpea sprouts. Protein, moisture, fat and ash contents increased with
the increase in germination time while the NFE and fiber content decreased.
Red, green, blue and yellow lights have promotional effect on protein
and fiber contents, irradiation on ash and fat content and dark, fluorescent
and yellow on NFE content.
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