Extraction and Fractionation of Insoluble Fibers from Foxtail Millet (Setaria italica (L.) P. Beauv)
Mohamed Lamine Bangoura,
Zhou Hui Ming,
John Nsor- Atindana,
Zhu Ke Xue,
Michel Bano Tolno
The study was carried out to investigate the extraction and fractionation of insoluble fibers from two varieties of foxtail millets. Moreover, the glucose uptake of the purified extracts was assayed. The results showed that Neutral Detergent Insoluble Fibers (NDIF) were as high as 54.59 and 55.37% for white and the yellow foxtail millets, respectively. Moreover, while the white samples yielded 15.44 11.56 and 27.00% as the respective insoluble, soluble, total dietary fibers that of the yellow offered 97, 15.91 and 27.88% as the insoluble, soluble and total dietary fibers in that order. White foxtail millet had the highest total hemicellulose (52.8%) content followed by yellow foxtail (50.34%). Cellulose and lignin were (32.41 and 31.34%) and (2.89 and 3.07%), respectively. The pectin substances were estimated to be lowest in both samples (0.806 and 0.906%). Glucose uptake was varied from 12.43 to 98.22% and those for PIM-Y from 14.36 to 98.57% and the increase was dependent on both glucose and sample concentrations. This founding fractionation procedure suggested that these purified insoluble fibers could be incorporate as low calorie bulk ingredients in high fiber foods production to reduce calorie level and help to control blood glucose levels.
July 29, 2011; Accepted: October 14, 2011;
Published: December 12, 2011
Foxtail millet (Setaria italica L. Beauve) is used mainly as animal
feed especially in North America and Europe. However, the consumption of millet
as human diet is gaining acceptance in recent times particularly, among people
seeking gluten-free alternatives and healthier diets (FAO,
1995; Jiaju and Yuzhi, 1993). Millet is commonly
used for preparation of many foods and beverages, such as steamed meal, porridge,
bread and beer (Brink, 2006; FAO, 1995).
Due to its nutritional quality, several researchers have demonstrated that foxtail
millet contains high amount of bioactive compound that possess many health benefits
(Pawar and Machewad, 2006; Ushakumari
et al., 2004), whereas study on foxtail millet have mainly reported
on the treatment of diabetes by improving cholesterol-metabolism (Choi
et al., 2005).
Though previous studies have attempted to determine the soluble and insoluble
fibers of foxtail millet as chemical components (Malleshi
and Hadimani, 1994; Rooney et al., 1982; Shaohua
et al., 2009), no specific research has reported on its insoluble
dietary fibers isolation and/or fractionation from its seeds up to date. Several
methods regarding the insoluble dietary fibers isolation and/or fractionation
been developed and categorized: (1) gravimetric methods, (2) gravimetric-enzymatic
methods, (3) calorimetric methods and (4) chromatographic methods (Claye
et al., 1996). Even though the gravimetric techniques have been employed
to estimate and quantify crude fiber underestimated yields leading to adoption
of newer and more accurate methods. Fractionation is one such method used to
quantify fiber constituents and isolate interest fractions as well as eliminate
The techniques for fractionation of dietary fibers into their individual components
are limited in number (Anderson and Ydesdale, 1980;
Dreher, 1987; Furda, 1977; Graham
et al., 1988; Monte and Maga, 1980; Southgate,
1969, 1977). Lawther et al.
(1995) fractionated wheat straw polysaccharides into water soluble, pectic,
80% ethanol-soluble, sodium chlorite-soluble, hemicellulosic and cellulose fractions,
using essentially, gravimetric methods. The hemicellulosic material was further
separated into a DMSO-soluble fraction, hemicellulose types A, B and C. Claye
et al. (1996) reported on fraction of different fibers after studying
the previous attempts. Using cold and hot water extraction, enzymatic and chemical
treatment, they obtained four fractions (cellulose, hemicellulose A and B and
lignin). Sun et al. (1996) extracted six hemicellulose
fractions from wheat straw and reported their chemical composition. In fact,
fractionation methods are varied and developed according to the raw materials.
Therefore, there is no standard method used to fractionate the fiber components.
Some approaches permitted refined separation of compounds, enabling determination
of molecular structure (Mukhiddinov et al., 2000;
Zou et al., 2010).
Fractionation of foxtail dietary fiber may be an ideal, since the technique by its scope provides unique way to quantify and isolate interest fractions with limited undesirable components. Moreover, the understanding of functionality (physicochemical effects) of the individual dietary fiber components in foxtail millet in relation to human health would depend on their isolation and quantification. However, to the best of our knowledge, no data on the extraction of insoluble dietary fibers from foxtail millet their fractionation has been reported so far. Consequently, the present study was conducted to isolate and fractionate insoluble fibers from white and yellow foxtail millets. Also and the Purified Insoluble Material (PIM) was assayed on glucose uptake.
MATERIALS AND METHODS
Source of the samples and preparation of the fiber sources: Foxtail millets (Setaria italica L. Beauv), white and Yellow seeds were purchased from millet research institute in DongBei (Liao Ning Province, China).
The Foxtail millets were sorted and cleaned to remove foreign materials. The
cleaned seeds were washed using tap water, drained and dried in an air oven
at 35°C for 1 h. The grains were milled and sieved through a 100 mesh to
obtain flour. Parts of the flours (500 g) were defatted overnight with n-hexane
(w/v) according the ratio (1:5) at room temperature (25°C). The defatted
materials were dried in an air oven at 25°C for 5 min and before being packed
into polyethylene bags and stored in refrigerator at 4°C until used for
study. The amyloglucosidase (Code: NS-22035) an trypsin (Code: HS35079010) were
obtained from (Novozymes Co.Ltd, China). All chemicals used in this experiment
were of analytical grade.
Chemical composition of fiber sources: The defatted flours were processed
for analysis of protein, moisture, ash and Neutral Detergent Fiber (NDF) using
the methods previously described by James (1995). Glucose
content was estimated by the method of the glucose assay kit (Megazyme K-GLUC,
Wicklow, Ireland). Reducing sugar, total carbohydrate and starch were analyzed
with the methods cited by Sadasivam and Manickam (1992).
Insoluble, soluble and total dietary fibers: According to the method
of Prosky et al. (1988), 1 g of defatted flours
(triplicate) were subjected to sequential enzymatic digestion by heat stable
α-amylase (95-100°C), protease and amyloglucosidase (60°C). The
Insoluble Dietary Fiber (IDF) was filtered and washed with hot distilled water
(70°C), 95% ethanol and acetone. The IDF are dried in vacuum at 50°C
overnight. Combined solution of filtrate and hot water washings was precipited
with 95% ethanol for Soluble Dietary Fibers (SDF). Precipitates were then filtrated
and residues washed with 78% ethanol, 95% ethanol and acetone, dried in vacuum
at 50°C overnight. Combining the values for IDFs and SDFs put out the Total
Dietary Fibers (TDFs).
Fraction procedure: Figure 1 shows the extraction
and fractionation procedure described by Claye et al.
(1996) with minor modification. Prior to the optimization of yield of components,
cold and hot water extractions of the fibers were used to partially remove soluble
polysaccharides and proteins before enzyme treatment. This procedure has been
found to reduce these components by about 20% (Anderson
and Ydesdale, 1980; Claye et al., 1996).
Potassium hydroxide (under nitrogen atmosphere) was used in place of the detergent
technique for extracting lignocellulose. Hemicellulose A and B were extracted
according to the procedure described by Monte and Maga (1980).
A triplicate extraction was carried to increase sample sizes as recommended
by Rasper (1981).
Removal of soluble complex carbohydrates and proteins: Using the procedure
previously described by Claye et al. (1996) with
minor changes, defatted millet flours were extracted at 20°C for 2 h using
slightly alkaline water (1:10 W/V ratio, pH 7.0-7.5). The mixtures were centrifuged
(300 gx15 min at 25°C). The supernatants were discarded and the procedure
repeated three times. The residues were extracted with 0.01 M EDTA for 2 h to
bind cations and solubilize more pectic substances (Furda,
1977). The mixtures were filtered and the extraction repeated twice. The
residues were washed twice with 80% ethanol and three times with distilled water,
lyophilized and kept for further analysis. These residues represent the non-purified
insoluble materials (N-PIMs).
Enzymatic extraction of non-purified insoluble materials: According
to the method of Southgate (1991), the N-PIMs were enzymatically
treated. The 25 g of N-PIM of both samples were weight into two liters beakers
to which 0.1 M acetate buffer (50 mL-1 pH 4.8) was added. The triplicate
treatments were carried out under controlled pH. Amyloglucosidase (0.15 mL-1)
was added, the beakers were incubated at 55°C for 3 h with continuous agitation.
After cooling, the pH was adjusted to eight and trypsin (5 mg) was added. The
beakers were incubated at 37°C for 18 h stirring slowly. The record mixtures
were filtered washed three times with 80% ethanol, one 95% ethanol and three
times of distilled water and freeze-dried.
|| Flow diagram of extraction and fractionation of Setaria
italica insoluble dietary fibers
These residues represent the Purified Insoluble Material (PIM). The presence
of starch in the enzyme treated fiber fractions was checked using an iodine
Insoluble pectic substances: As previously described by Monte
and Maga (1980), triplicate 1 g PIMs of each samples were extracted three
times with 0.5% ammonium oxalate (10 mL) at 85°C for 2 h. The fiber residues
were filtered and washed with ethanol, distilled water and then dried in vacuum
at 35°C. These residues were called as Depectinated Insoluble Materials
(DIMs). The loss in weight on drying was the yield of insoluble pectin present
in the sample.
Cellulose: The method described by Robertson and Van
Soest (1981) was adopted for crude cellulose determination. Briefly, 2 g
of lignocellulose were extracted with 20 mL-1 of combined reagent
(KMnO4/lignin buffer, ratio 2:1, v/v) in sintered glass crucible
and allowed to stand for 90 min for 22°C with periodic stirring. The reagent
in crucible was made to remain purple by changing frequently by duration of
the extraction process. The combined reagent was drawn out by suction and the
crucibles were transferred to clean pan. Demineralized solution (20 mL) was
added to each crucible and allowed to stand for 5 min, refilling as necessary
and then remove by suction. The completion of demineralization was indicated
by removal of black manganese from white/gray cellulose.
The extraction lasted for an average of 30 min. Crude cellulose was washed with 80% ethanol, distilled water then lyophilized and store in refrigerator.
Extraction of Hemicellulose A and B: The method previously reported
by Monte and Maga (1980) was adopted to extract hemicellulose
A and B. Briefly, 5 g of kepectinated knsoluble Material (DIM) were weighed
(triplicate) into 250 mL plastic stoppered centrifuge bottles and 100 mL of
(5%, w/v) potassium hydroxide was added. The bottles were flushed with nitrogen
and shaken for 24 h, then centrifuged (1500 gx10 min at 37°C); the supernatant
was decanted, saved for analysis. After extracting two times, these residues
could be lignocellulose. Lignocellulose was dried and at 35°C overnight
and kept for further analysis. The filtrate was combined by adding 5% acetic
acid and pH was adjusted to 5-5.5 and then centrifuged (4000 gx15 min). The
hemicellulose A (HC-A) fraction was washed and freeze-dried. Collected supernatant
from HC-A was diluted with four volumes of 95% ethanol to precipitate hemicellulose
B (HC-B) fraction.
Lignin: The Klason lignin method Robertson and Van Soest
(1981) was performed for the extraction of the lignin. Triplicate samples
of lignocellulose (5 g) were extracted with cold 72% sulfuric acid solution
(1 g, w/v) at 4°C for 30 h. Cold distilled water was added (150 mL) and
the residue allowed to precipitate. The residue was washed with warm distilled
water until no acid was detectable. The Crude Lignin Material (CLM) was then
air dried and kept in refrigerator for further studies.
Assay of PIMs effect on glucose uptake: In accordance with the previous
method described by Cirillo (1962), the commercial yeast
for bakery was washed repeatedly with distilled water and centrifuged (3000xg;
5 min at 25-37°C) until the supernatant was clear and 10% (v/v) of suspension
was prepared in distilled water. The amount of PIMs (1-5 mg), were added to
1 mL at different concentration of glucose solution (5-25 mmoL-1)
and incubated for 10 min at 37°C. The suspension of yeast (100 μL-1)
was added and mixed well before incubation at 37°C for 1 h. After incubation,
the tubes were cooled at room temperature (25-37°C) and centrifuged (2500xg,
5 min) and glucose was measured in the supernatant (Cirillo,
1962). The increased of glucose uptake by yeast was estimated using the
following Eq. 1:
Abs. control: is the absorbance of the control reaction (containing all reagents
except the test sample),
Abs. sample: is the absorbance of the test sample.
Statistical analysis: One-way analysis of variance (ANOVA) was carried out on each of the variables and the Least Significant Difference (LSD) test at α level of 0.05 was performed using SAS software (SAS 8.1 for Windows, SAS Inc., Cary, NC, USA) to compare the difference between treatment means. Results were expressed as the Mean±standard deviation of three separate determinations.
RESULTS AND DISCUSSION
Chemical composition of fiber sources: The chemical composition of both
defatted fiber sources is presented in Table 1. The values
of the crude protein varied from 12.02% to 13.81%. Significant difference (p<0.05)
was found between the two determinations and these results were slightly comparable
to those reported by Wankhede et al. (1979).
For the moisture content no significant difference (p<0.05) was observed
in both white and yellow fiber sources. This was in a good agreement to that
reported on moisture in foxtail millet by Shaohua et
al. (2009). The ash values (0.79-0.87%), respectively were found to
be low than that reported in previous study, this variation depends of the agricultural
and/or cultivation conditions (NAS, 1996). Total starch,
carbohydrate and reducing sugar were significantly different (p<0.05) in
both the fiber source samples. Results for reducing sugar (115.72-17.33 mg g-1)
fell slightly comparing to those reported by Raghavendra Rao
et al. (2011).
|| Chemical composition of defatted foxtail milletsa
|aMeans of triplicates, values in the same rows
with different letters are significantly different (p<0.05). Standard
curves and R2 of different determinations were: Starch: y = 0.699x
+ 0.081, R2 = 0.9924. Carbohydrate: y = 0.853x + 0.009, R2
= 0.9979. Reducing sugar: y = 0.706x-0.102 R2 = 0.9914. Glucose:
y = 0.1964x-0.0948, R2 = 0.9989
This variation could be due to the factors such as agricultural conditions
as reported by NAS 1996. Perhaps, it could also be related
to milling-separation procedures and/or varietal/environmental conditions of
the fiber source samples. The total starch and carbohydrate values (57.57-52.44
and 53.26-67.68 mg g-1), respectively were high and close to those
reported by Wankhede et al. (1979). Glucose content
in the both samples were estimated to be high (70.67 and 72.20 mg g-1)
and significantly different (p<0.05). The NDF content in both fiber source
samples were significantly different (p<0.05) and seated within the ranges
of those reported by Aniola et al. (2009) and
Smith and Kallenbach (2006).
Insoluble, soluble and total dietary fibers: The high values of fiber
were found in the both defatted samples and no significant difference (p<0.05)
was recorded between them Table 2. However, high value of
NDF was found in yellow seed and significantly different (p<0.05) compared
to the values obtained for white seed Table 1. Anju
and Sarita (2010) reported on foxtail millet flour for IDF (15.88%), SDF
(11.04%) and TDF (26.92%). These values are similar to those reported in this
investigation, but a slight difference was observed due to the quality of the
flour; perhaps contained amount of other components (i.e, insoluble polysaccharides)
and could not be easily hydrolyzed by enzymes.
Insoluble pectic substances: The pectic substances play an important role in plant life, their primary function is the cementing together of the individual cells that compose the plant.
The insoluble pectins removed for white (0.806%) and yellow (0.906%) foxtail
millet fiber fractions were presented in Table 3 and significantly
different (p<0.05) to each other. As schematically shown in Fig.
1, the pure insoluble materials were obtained after removing the pectin
substances. In our study, the lowest values have been found when compared to
those reported by Claye et al. (1996) where ammonium
oxalate utilization led to the complete extraction of pectin from the various
fiber sources and Lawther et al. (1995) when
fractionated wheat straw. These founding results are not in good agreement to
those reported during our investigation.
Cellulose: The major component in the rigid cell walls in plants, cellulose
is a linear polysaccharide polymer with many glucose monosaccharide units. The
crude cellulose content of both PIMs (white and yellow fibers) were elevated
(32.41 and 31.34%), respectively in Table 3 and significantly
different (p<0.05). These values were significantly higher to that reported
in foxtail millet by Wankhede et al. (1979),
but they did not process for crude cellulose by fractionation procedure.
|| Insoluble, soluble and total dietary fiber of defatted fiber
sources (g/100 g)a
|aMeans of triplicates, values in the same column
with different letters are significantly different (p<0.05)
|| Defatted fiber sources and hemicellulose, cellulose, lignin
and insoluble pectin (%) in fractionated PIMa
|aMeans of triplicates, values in the same column
with different letters are significantly different (p<0.05)
However, the values reported in our study were within the range to those reported
by Lawther et al. (1995) in wheat straw polysaccharides.
Claye et al. (1996) also brotherly reported in
insoluble fiber from five fiber sources and Gaspar et
al. (2005) reported on fractionation and utilization of corn fiber carbohydrates.
Except the result given by Wankhede et al. (1979),
no cellulose data was available for foxtail millet fiber for comparison.
Hemicellulose A and B: Hemicellulose (also a polysaccharide) consists
of shorter chains sugar units as opposed to glucose molecules per polymer in
cellulose. It is a branched polymer, while cellulose is unbranched. Hemicellulose
A and B extracted from DIM for white foxtail and yellow foxtail millets are
shown in Table 3. Hemicellulose A ranged for white fox (19.41%)
and yellow fox (18.69%) were significantly different (p<0.05) and lower than
that reported by Wankhede et al. (1979) in setaria,
while hemicellulose B were (33.39 and 31.65%), respectively for the both white
and yellow Setaria italica and lower than founding results.
These differences may be due to the procedures used to the pretreatment of
setaria seeds and/or perhaps to the method used for extracting the HC-A and
HC-B. In addition, the results reported by Wankhede et
al. (1979) were most important for comparison in our research, even
though they did not provide their results using fractionation procedures. Nevertheless,
other searchers reported on HC-A and HC-B when fractionated insoluble fiber
from five sources (Claye et al., 1996). Lawther
et al. (1995) fractionated wheat straw and Gaspar
et al. (2005) studied fractionation and utilization of corn fiber
carbohydrates. The results given in our study are within the range provided
by above statements. Now-a-days, no reports found on HC-A and HC-B extraction
in Setaria italica for comparison, except those given by Wankhede
et al. (1979).
Lignin: Crude lignins content in lignocellulose from both extracted
fiber sources are shown in Table 3. Significant difference
(p<0.05) was found between white (2.89%) and yellow (3.07%). An appropriate
length of time for acid hydrolysis is still controversial. Wankhede
et al. (1979) brotherly reported crude lignin value of 0.04% for
Setaria italica. This value as well as others in the literature is not
consistent, probably because of differences in methodology and length of time
of hydrolysis, whereas, other studies reported (14.13-25.83%) in wheat straw
(Lawther et al., 1995) and (12.2%) in corn (Gaspar
et al., 2005). Moreover, these differences may be due to some carbohydrates
monomer loss during the fractionation.
Assay effect on PIMs on glucose uptake: The effects of PIM-W and PIM-Y on the rate of glucose uptake cross-cell membrane are mostly studied an in vitro system comprising yeast cell suspended in glucose solution in various concentrations (5-25 mmol L-1) (Fig. 2). This study was a simple assay after purification of the extracted insoluble (white and yellow setaria italica) fibers by fractionation procedure. The amount of glucose remaining in the medium after specific time serves as indicator of glucose uptake by yeast cells.
Results from our study showed that PIM-W and PIM-Y increased significantly
(p<0.05) different of the glucose uptake in yeast cells. The values for PIM-W
were varied from 12.43 to 98.22% and those for PIM-Y from 14.36 to 98.57% and
the increase was dependent on both glucose and sample concentrations. However,
the percentage increased in glucose uptake by yeast cells was inversely proportional
to glucose concentration and found to decrease with increase in the molar concentration
of the glucose solution.
||Effect of PIM-W and PIM-Y on glucose by yeast uptake by yeast
cells at different glucose concentrations
This was in good agreement to those reported by Ahmed and
Urooj (2010) when they studied in vitro hypoglycemic potential of
Ficus racemosa Stem bark. This mechanism suggested that glucose transportation
across the yeast cells membrane could be more attractive form of an in vitro
screening method for hypoglycemic effect of various compound/medicinal plants
(Teusink et al., 1998). In addition, this research
suggests and reports the characteristics of glucose transport in the presence/absence
of PIMs from Setaria italica and projected abilities of glucose transport
across the yeast cell membrane function of external glucose concentration as
well as sample concentration. This relationship will cease to set out the yeast
cells reach saturation point (Ahmed and Urooj, 2010).
The present investigation demonstrated the advantage of fractionation procedure and revealed that the major component of Setaria italica fiber-fractions could be fractionated and therefore, provides information for further research such as physiological effects of the glucose uptake that we processed by assay. This point needs to be completed by glucose-transport across yeast cell membrane in further study. The amount of fiber components in our study showed slight difference between fiber sources. Insoluble fibers have been implicated to bind bile acids and reduce availability of minerals. A good proportion of hemicellulose, cellulose and glucose up-take of both fiber sources could be food ingredient (bulk agent) in food product formulations which could be satisfactory for the market demand of high-fiber foods and will increase more supporting data become available on their disease prevention ability.
The Guinea-China cooperation has been very instrumental in the realization of our research. Through the ministry of high school education and scientific research of Guinea, the Guinea-China cooperation supported us financially to carry out this research project. Our special gratitude goes to ministry of education of China, State Key Laboratory of Food Science and Safety and Convenient Food and Quality Control Laboratory in Jiangnan University where the experiments were conducted.
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