INTRODUCTION
Millets typically contain higher quantities of essential amino acids and are
higher in fat content than maize, rice and sorghum (Kamara
et al., 2009a). Millet contains 12.3% crude protein, 3.3% minerals
72% of carbohydrate which is thee main components of millet that include starch,
protein, lipid, vitamins and minerals (Kamara et al.,
2009a). Foxtail millet (Setaria italica L.) is also known as Italian
millet and is one of the world’s oldest cultivated crops. In the Northern
area of China it has been widely used as a nourishing gruel or soup for pregnant
and nursing women and has been applied in food therapy.
Functional properties of plant proteins have been exploited in a multitude
of applications (for example, solubility in beverages, foaming in whipped toppings
and emulsification in processed meat) resulting in an ever increasing demand
for plant protein ingredients with improved processing and functional characteristics
(Kamara et al., 2009b).
Enzymatic treatment is a particularly attractive technique to modify proteins
due to the milder process conditions required, the relative ease to control
the reaction and minimal formation of by-products (Mannheim
and Cheryan, 1992). It has been widely used to improve the functional properties
of proteins, such as solubility, emulsification, gelation, water and fat-holding
capacities and foaming ability and to tailor the functionality of certain proteins
to meet specific needs (Kim et al., 1990; Panyam
and Kilara, 1996).
Extensive hydrolysis could have a negative impact on the functional properties
(Kristinsson and Rasco, 2000; Qian
et al., 2010). However, enzymatic hydrolysis also introduces undesirable
attributes to the products. Among these, bitterness is one of the most objectionable
characters. Bitterness has been the major limitation in utilizing protein hydrolysates
in various applications, particularly in beverages. Enzymatic hydrolysis of
proteins at or above neutral pH releases hydronium ions (H3O+)
that cause a drop in pH, which if allowed to decline unabated may influence
the enzyme ionization properties and consequently, its catalytic ability leading
to denaturing. Likewise, the substrate susceptibility to enzyme hydrolysis is
influenced by the pH. There is, therefore, need for the pH to be regulated,
hence the wisdom of the pH-stat method (Adler-Nissen, 1986;
Jamel, 1992).
Protein purification is an art which has been refined over the last four decades
such that excellent techniques are now available that simplify or enhance the
recovery and homogeneity of protein products in relatively short period of time.
In protein purification, it is common to reach a desired purity acceptable for
product consumption. Various techniques have been used. Traditionally, desalting
of large biomolecules is performed using dialysis, which is slow besides requiring
large buffer volumes. Additionally, material loses have been reported as a result
of the protein adsorption to the dialysis membranes (Cuartas
et al., 2004).
Proteins have been desalted using either nanofiltration membranes or gel permeation
chromatography using the desalting Sephadex™ gels which are expensive (Cuartas
et al., 2004). Desalting and debittering of defatted foxtail millet
protein hydrolysate (DFMPH) enhances their value-added qualities as well as
processing safety into the product because of consumer sensitivity.
Macroporous Adsorption Resin (MAR) have been used for desalting biological
samples and protein hydrolysates with good hydrolysate recoveries. MAR is a
non-polar adsorbent resin used mainly for adsorption of organic substances and
decolourisation (Zhao et al., 2002; Wasswa
et al., 2007; Cheison et al., 2007).
It is important to select a desalting process which is simple and easy to operate.
While peptides bitterness is of both academic and technological interest, no
reports exist on desalting of defatted foxtail millet protein hydrolysate on
MAR, nor are there any reports of debittering with the same sample. Selective
extraction of the bitter fragments yields a product with acceptable sensory
properties making it easy to use in such applications as hypoallergenic infant
formulas, sport nutrition and functional foods (Meisel,
1997; Exl, 2001; Manninen, 2004;
Mahmoud, 1994; Clemente, 2000).
In this study, we examine the influence of MAR in simultaneous desalting and
debittering of defatted foxtail millet protein hydrolysate, analyzed their functional
properties, molecular weight distribution, amino acid content and organoleptic
properties.
MATERIALS AND METHODS
Foxtail millet was purchased from a local market in Wuxi, People’s Republic
of China. The seeds were milled using a laboratory scale hammer miller and the
resulting flour was sieved through a 60 mesh screen. The Foxtail Millet Flour
(FMF) was dispersed in hexane at flour to hexane ratio of 1:5 (w/v) and stirred
for 4 h at room temperature. The experiment was repeated twice as described
above. The hexane was decanted and the DFMF was air dry for 24 h under a vacuum
drier and stored at 5°C in sealed glass jars until used. This research was
conducted in the School of Food Science and Technology and State Key Laboratory
of Jiangnan University, Wuxi from November 2009 to January 2010.
Protein hydrolysates were made and evaluated using a range of food grade enzymes.
The enzymes tested (Novo Nordisk’s Enzyme Business, Beijing, China) were
Alcalase 2.4 L endonuclease from Bacillus subtilis with specific activity
of 2.4 AU g-1; Favourzyme from Aspergillus oryzae with activity
of 500 LAPU g-1; Neutrase from Bacillus subtilis strain with
activity of 1.5 AU g-1; Protamex, a Bacillus protease complex with
activity of 1.5 AU g-1 and Papain powder (Sigma, China) with 2.1
AU g-1 activity. The crude papain powder from papaya fruit was not
totally soluble. It was extracted (16 mg mL-1) with 0.05 M sodium
borate buffer (pH 8.3) and the insoluble material was removed by centrifugation
at 11.500x g for 10 min at 4°C with a ZOPR-52D refrigerated centrifuge (Hitachi
Koki Co Ltd, Tokyo, Japan). A styrene-based Macroporous Adsorption Resin (MAR),
branded DA201-C was got from Jiangsu Suqing Water Treatment Engineering Group
(Jiang-ying, Jiangsu, China). All other chemicals and reagents were obtained
from a local manufacturer (Sinopharm Chemical Reagent Co., Ltd. (SCRC) Shanghai,
China. Table 1 was available at the university chemical store
and all chemicals used in the experiments were of analytical grade.
Proteolysis with different enzymes: DFMF was hydrolyzed with five different
enzymes, under the conditions given in Table 2, based on optimum
hydrolysis conditions. One hundred grams of vessel immersed in a water bath
maintained at appropriate temperature and 700 mL; of distilled water was added
to make a suspension. The suspension was, for each enzyme, adjusted to appropriate
pH and preheated to appropriate temperature; then (1%) enzyme substrate ratio
was added with continuous stirring. Hydrolysis was carried out for 9 h. 75 mL
aliquots were taken after 60, 120, 180, 240, 300, 360, 420, 480 and 540 min
and each hydrolysate was centrifuged at 11500x g for 10 min at 4°C with
a D-3756 Osterode am Harz model 4515 Centrifuge (Sigma, Germany). The supernatant
was carefully decanted and immediately heated for 5 min in a boiling water bath
to inactivate the enzymes. Heat inactivation followed centrifugation to prevent
gelatinization of starch. The defatted foxtail millet hydrolysate was lyophilized
and stored at -20°C until used. All the experiments were performed in triplicate
and the results are the average of three values.
| Table 1: |
DA201-C macroporous adsorption resin properties |
 |
| This data is supplied with product in producer’s manual
manufactured from styrene based material |
| Table 2: |
Optimum conditions for hydrolysis of DFMF with different
proteases |
 |
| *AU (Anson units) is the amount of enzyme that under standard
conditions digests hemoglobin at an initial rate that produces an amount
of trichloroacetic acid-soluble product which gives the same color with
the Filon reagent as one milliequivalent of tyrosine released min-1.
†LAPU (Leucine aminopeptidase unit) is the amount of enzyme
that hydrolyzes 1 μmol of leucine-p-nitroanilide min-1 |
Degree of hydrolysis (pH-stat assay): The degree of hydrolysis (DH),
defined as the percent ratio of the number of peptide bonds broken (h) to the
total number of bonds per unit weight (htot), in each case, was calculated
from the amount of base consumed (Adler-Nissen, 1986),
as given below:
where, Vb is base consumption in mL; Nb is normality
of the base; α is average degree of dissociation of the α-NH2
groups; mP is mass of protein (Nx6.25) in g; and htot is total number
of peptide bonds in the protein substrate. Approximate value of 9.2 meqv g-1
was used. All the experiments were performed in triplicate and the results are
the average of three values.
Batch debittering and desalting in a beaker: The debittering and desalting
of the DFMPH was done in a beaker since this procedure is more efficient and
done within a short duration. The DFMPH was allowed to be absorbed onto the
MAR by stirring 1.0 L of the DFMPH supernatant liquid with 500 mL of MAR for
24 h using a mechanical stirrer. After the absorption, the content was allowed
to settle and the top layer skimmed off. The MAR was washed with five-bed volumes
of deionized water with stirring using a mechanical stirrer. After washing the
MAR with deionized water, it was further washed with three different concentrations
of alcohol in order to desorb the peptides.
Desorption with alcohol: Step-wise desorption was done by washing with
alcohol at different concentrations. The Alcohol Concentrations (ALC) varied
from 30, 55 and 70%, followed by deionised water. The collected fractions were
concentrated under vacuum and freeze-dried. The resin was regenerated by washing
it with 1 mol L-1 NaOH followed by 1 mol L-1 HCl and thoroughly
rinsed with deionized water until neutral pH. This was to ensure that the peptides
were properly washed of the resin.
Proximate analysis: The proximate analysis of defatted foxtail millet
protein hydrolysate (DFMPH) and the desalted and desorbed hydrolysates were
determined according to James (1995). The moisture content
was determined by drying in an oven at 105°C until a constant weight was
obtained. Ash was determined by weighing the incinerated residue obtained at
525°C after 4 h. The crude protein was determined by the micro-Kjeldahl
method and a Conversion factor of N x 6.25 was used to quantify the crude protein
content (Tkachuk, 1969).
Amino acid analysis: The dried samples were digested with HCl (6 M)
at 110°C for 24 h under nitrogen atmosphere. Reversed phase high performance
liquid chromatography (RP-HPLC) analysis was carried out in an Agilent 1100
(Agilent Technologies, Palo Alto, CA, USA) assembly system after precolumn derivatization
with o-phthaldialdehyde (OPA). Each sample (1 μL) was injected on a Zorbax
80 A C18 column (i.d. 4.6 x 180 mm, Agilent Technologies, Palo Alto, CA, USA)
at 40°C with detection at 338 nm. Mobile phase A was 7.35 mM L-1
sodium acetate/ triethylamine/tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted
to pH 7.2 with acetic acid, while mobile phase B (pH 7.2) was 7.35 mM L-1
sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). The amino acid composition
was expressed as g of amino acid per 100 g of protein.
Determination of Molecular Weight (MW): Molecular weight distributions
were determined by gel permeation chromatography (GPC) by using a high-performance
liquid chromatography (HPLC) system (waters 600, USA). A TSK gel 2000 SWXL
column (7.8 i.d.x300 mm, Tosoh, Tokyo, Japan) was equilibrated with 45%
acetonitrile (v/v) in the presence of 0.1% trifluoroacetic acid. The hydrolysates
(100 μg μL-1) were applied to the column and eluted at
a flow rate of 0.5 mL min-1 and monitored at 220 nm at room temperature.
A molecular weight calibration curve was prepared from the average retention
time of the following standards obtained from (Sigma, Germany: cytochrome C
(12500 Da), aprotinin (6500 Da), bacitracin (1450 Da) and tripeptide GGG.
Nitrogen solubility: Nitrogen solubility was determined according to
the procedure of Bera and Mukherjee (1989), with slight
modification. One hundred mg of the various samples were dispersed in 10 mL
of distilled deionized water. The suspensions were adjusted to pH 2.0 to 12.0
using either 0.1 M HCl or 0.1 M NaOH. These suspensions were shaken (Lab-Line
Environ-Shaker; Lab-Line Instrument, Inc., Melrose Park, Ill., USA) for 30 min
at room temperature (approximately 25°C) and centrifuged at 4000x g for
30 min. The protein content of the supernatant was determined by the Kjeldahl
method and percent protein solubility was calculated as follows:
Where:
| PS |
= |
Amount of protein in supernatant |
| PIS |
= |
Protein in initial sample |
All the experiments were performed in triplicate and the results are the average
of three values.
In vitro digestibility by trypsin: In vitro digestibility
was carried out according to the method described by Elkhalil
et al. (2001), with slight modification. Twenty milligram of protein
hydrolysate samples in triplicate were digested in 10 mL of trypsin (0.2 mg
L-1 in 100 mM Tris-HCl buffer, pH 7.6). The suspension was incubated
at 37°C for 2 h. Hydrolysis was stopped by adding 5 mL 50% Trichloroacetic
Acid (TCA). The mixture was allowed to stand for 30 min at 4°C and was then
centrifuged at 9500 x g for 30 min using a D-3756 Osterode am Harz model 4515
Centrifuge (Sigma, Germany). The resultant precipitate was dissolved in 5 mL
of NaOH and protein was measured using the Kjeldahl method. Digestibility was
calculated as follows.
Where:
| A |
= |
Total protein content (mg) in the sample |
| B |
= |
Total protein content (mg) in TCA precipitate |
All the experiments were performed in triplicate and the results are the average
of three values.
Colour measurements: The colour of the hydrolysate powder was evaluated
using the Hunter Lab colorimeter (WSC-S Colour Difference Meter, USA) and reported
as L*, a* and b* values, in which L* is a measure of lightness, a* represents
the chromatic scale from green to red and b* represents the chromatic scale
from blue to yellow. The instrument was standardized to measure the colour difference
with an L* value of 91.32, a* value of 0.03 and a b* value of 0.01. All the
experiments were performed in triplicate and the results are the average of
three values.
Viscosity: Apparent viscosity of aqueous solutions of the three products
got from the three levels of alcohol concentrations was estimated on a 30-40
mL of protein solution using NDJ-79 Viscometer (Shanghai, China). All the experiments
were performed in triplicate and the results are the average of three values.
Gelation properties: Gelation properties were determined by the method
of Obatolu and Cole (2000), with slight modifications.
The fractions and DFMPH were determined on a 5 mL test tube of each hydrolysate
sample suspension in deionised water at pH 7.0 and protein concentrations varying
from 2 to 20% (w/v) with increments for all the three products.
Sensory evaluation: In this study, the nine-point hedonic scale according
to the method of Sheppard (2006) was used to evaluate
the bitterness in defatted millet protein hydrolysates powder, was conducted
by 20 panelists.
Statistical analysis: Data and Statistical Analysis of Variance (ANOVA)
was performed and differences in mean values were evaluated by Tukey`s test
at p<0.05 using SPSS version 18.0 (SPSS Inc, Chicago, IL, USA).
RESULTS AND DISCUSSION
Enzymatic hydrolysis: The treatment of DFMF with Alcalase 2.4 L showed
the highest increase in protein content of DFMPH during the first 300 min of
hydrolysis 86.84%. The amount of proteins released decreased slightly to 76.21%
for the remaining hydrolyzing period up to 540 min (Fig. 1).
Flavourzyme solubilized 73.28% of protein during the first 300 min of hydrolysis
and the amount solubilized increased to 78.92% with longer hydrolyzing periods
up to 540 min. Neutrase showed an increase in protein solubilization during
the first 300 min of hydrolysis with 67.52% of the protein being solubilized.
The treatment of DFMF with Papain showed increased solubilization of protein
during the first 300 min of hydrolysis and reached 62.78% of protein. Later
the amount of protein released decreased moderately to 57.74%. After 300 min,
Protamex was able to solubilize 58.51% of the total protein and the amount solubilized
increased to 60.74% with longer hydrolyzing periods up to 540 min (Fig.
1). The results of our study exhibited a behavior that is, similar to Betancur-Ancona
et al. (2009). The high efficiency of Alcalase 2.4 L and Flavourzyme
may be a result of a high frequency of potential cleavable sites in DFMPH which
may have contributed to the high degree of solubilization. Alcalase 2.4 L was
selected for the current study because of its high yield under optimum conditions,
readily available, cost effectiveness and ease of handing.
Degree of hydrolysis (DH): The enzymatic hydrolysis DFMF processed with
Alcalase 2.4 L (Fig. 2), exhibited a behavior that is similar
to Adler-Nissen (1986). Hydrolysis with proteases at percentage
1% (enzyme to substrate ratio) developed rapidly in early reaction stage, as
shown in by the rise in DH and then decreased in the rise. The reaction was
asymptotic 60 min after hydrolysis began. In the first 240 min it reached 26,
15.5, 10.3, 10.8 and 7.4% DH for Alcalase, Flavourzyme, Neutrase, Protamex and
Papain respectively, indicating that enzymatic preparation reacted rapidly,
though hydrolysis increased only gradually during the remaining reaction, eventually
reaching 27, 17, 11, 11.5 and 8% DH, respectively when finished.
|
| Fig. 1: |
Amount of protein solubilized by enzymatic hydrolysis of defatted
foxtail millet flour (DFMF) by different proteases. Value represent the
Mean±SD of n = 3 duplicate assays |
|
| Fig. 2: |
Enzymatic progress curves of hydrolysis of defatted foxtail
millet protein hydrolysate (DFMPH) using different enzymes. Value represent
the Mean±SD of n = 3 duplicate assays |
Similar behavior was observed by Kim et al. (1990).
Desalting of DFMPH: The MAR properties are shown in Table
1. The peptides were desorbed from MAR using 30, 55 and 70% alcohol concentrations
(ALC) following desalting meaning the peptides hydrophobicities were different.
Desorption of DFMPH peptides from the MAR was achieved at all the three levels
of ALC after the resin was rinsed with deionised water. The result shows that
the interaction between the resin and the DFMPH is indeed hydrophobic in nature,
because even though alcohol has both hydrophobic and hydrophilic zones, the
hydrophobic zone was in greater part.
| Table 3: |
Summary of total amino acids composition of desorbed fractions
showing content of essential amino acid, hydrophobic and hydrophilic amino
acids composition (g/100 g protein) |
 |
| aSuggested profile of essential amino acid requirement
for infant and adult (WHO, 2007); bRequirements
for methionine + cysteine. CRequirements for phenylalanine +
tyrosine. dAspartic acid + asparagines. cCysteine
+ cystine. fGlutamin acid + glutamine; gTotal EAA
= Total essential amino acids. hHydrophobic amino acids (Alanine,
Isoleucine, Leucine, Methionine, Phenylalanine, Proline, Tyrosine and Valine).
iHydrophilic amino acids (Histidine, Lysine, Arganine, Glutamic acid,
Aspartic acid, Threonine and Serine) |
The non-polar amino acid residues had no contact with the water while the
polar side chains pointed out towards the water molecules (Cheison
et al., 2007). In that light, it is suffice to state that the DFMPH
interacted with the resins hydrophobically to achieve a favourable configuration
during the debittering, desalting and rinsing processes.
Proximate analysis: The proximate analysis data for the desorbed fractions
lyophilisates are shown in (Table 5), which shows significant
different (p<0.05) in moisture and ash contents of DFMPH and the desalted
fractions. Likewise, the protein contents in the desalted fractions were enriched
from 86.84% (DFMPH) to 96.77, 95.75 and 92.42% of 30, 55 and 70% ALC, respectively,
which were significantly different (p<0.05) from each other. The results
in Table 5 are within the values reported by Cheison
et al. (2007). The increase in the protein quantity could be attributed
to the mixing during the debittering and desalting process as it is likely that
more protein could have been released during desalting and debittering.
Total amino acids content of the desorbed fractions: The content of
amino acids in the fractions obtained from the alcohol fractionation, Table
3, showed slight different in their content of hydrophobic (and essential
including tryptophan) as well as hydrophilic amino acids. Thirty percent fraction
had the lowest while fraction 70% had the highest content of hydrophobic and
essential amino acids and our results corroboted with Zhang
et al. (2009) and Cheison et al. (2007).
The separation with various alcohol concentrations for desorption was achieved
owing to the different in the content of hydrophobic amino acid which make up
the peptides. The 70% Fraction contained the highest amount of hydrophobic amino
acids (Table 3) and hence required higher alcohol concentration
to disrupt the hydrophobic interactive forces between the hydrolysates and the
resin. Conversely, 30% fraction with the least hydrophobic amino acids and hence
poor interaction forces was desorbed with lower alcohol concentration, the results
are within the ranged reported by Wasswa et al. (2007)
and Zhang et al. (2009).
Molecular weight distributions: The molecular weight distributions of
the various fractions were determined by SE-HPLC. The molecular weights for
all samples were calculated according to the standard equation below:
Results in and Table 4, show that the molecular weight distribution
of different fractions (30, 55 and 70%), have similar molecular weight distributions
indicating that polyptides produced from the bittering and desalting have comparatively
smaller molecular weight distributions. There was significant influence of the
ALC on the fractions (Table 4). Similar observation was made
by Zhang et al. (2009). The MW distributions
are between 60 and 9000 Da for the various (Table 4).
Protein solubility: An increase in the extent of enzymatic hydrolysis
corresponded to an increase in the nitrogen solubility, over the pH range studied,
indicating a positive relationship (Fig. 3). It has been suggested
that an increase in the solubility of protein hydrolysates over that of the
original protein is due to the reduction of its secondary structure and also
to the enzymatic release of smaller polypeptide units from the protein (Adler-Nissen,
1986; Chobert et al., 1988).
| Table 4: |
Molecular weight distribution profile of the DFMPH and desalted
fractions |
 |
| ALC: Alcohol concentration; DFMPH: defatted foxtail millet
protein hydrolysate |
|
| Fig. 3: |
Effect of pH on nitrogen solubility of DFMPH and the various
alcohol concentrations fractions |
At pH 4.0, near the isoelectric point at which the net charge of the original
protein is minimized and consequently more protein-protein interactions and
fewer protein-water interactions occur (Adler-Nissen, 1976;
Chobert et al., 1988). Above pH 6.0, the nitrogen
solubilities increased rapidly with an increase in pH up to 12.0. These trends
in solubilities are in agreement with Tang et al.
(2003) and Chandi and Sogi (2007). At pH 12.0, the
solubility of 30, 55 and 70% fraction reached 97, 93 and 95%, respectively and
while solubility for DFMPH was 91% at pH 12.0 (Fig. 3).
In vitro protein digestibility: The in vitro digestibility
of DFMPH and the fractions were evaluated by TCA-soluble nitrogen release during
digestion of trypsin. Table 5 shows a typical profile of the
nitrogen release of DFMPH and ALC trypsin digestion. The fractions were more
easily digested than DFMPH. The fractions and DFMPH have digestibility values
with trypsin of 87.62, 85.76, 85.04 and 83.27% for 30, 55, 70% fractions and
DFMPH, respectively and they where significantly different (p<0.05). However,
our results are in agreement with Van der Plancken et
al. (2003) and Kamara et al. (2009a).
The unfolding of the native protein structure during the cause of hydrolysis
is yet another factor that likely facilitates digestibility (Van
der Plancken et al., 2003; Kamara et al.,
2009a).
| Table 5: |
Hunter colour parameter values of hydrolysate from the different
alcohol concentration, proximate analysis and in vitro protein digestibility |
 |
| Values are Mean±SD of three determinations; ALC: Alcohol
concentration; DFMPH: Defatted foxtail millet protein hydrolysate; L* Measure
of lightness, a* Chronic scale from green (-a) to red (+ a), b* Chronic
scale from blue (-b) to yellow (+ b) |
Colour measurement analysis: Colour influences the overall acceptability
of any food products (Papadakis et al., 2000).
Debittering with alcohol produced protein powders that were light yellow in
colour (Table 5). Thirty percent fraction was the darkest
(L* = 56.79) and most yellowish (b* = 29.59) whereas 55% fraction was the lightest
(L* = 60.47) and least yellowish (b* = 23.87). The L* value was significantly
different (p<0.05) for all fractions (Table 5).
Moreover, the results of this study corroboted with data reported by Wasswa
et al. (2007).
Viscosity: Viscosity is one of the most important functional properties
of food proteins. It is important for providing physical stability to emulsions
(Cho et al., 2004). The concentrations, molecular
weight’ polydispersity, hydrophobicity and conformation of each protein
species affect the viscosity of the solution. All of these factors tend to confound
the underlying inverse relationship of protein solubility and viscosity (Schenz
and Morr, 1996). Processing induced changes in proteins such as polymerization,
aggregation and hydrolysis affect the viscosity of food products. The apparent
viscosity of aqueous solutions of DFMPH with different alcohol concentration
as a function of protein is displayed in Fig. 4.
From the results, it is obvious that the various fractions were able to form
very low viscosity solutions even at high concentrations (Fig.
4). The low viscosity of protein even at high concentrations may be useful
in the development of high protein soft drinks and juice-based beverages without
suffering the adverse consequences of high viscosity (Frokjaer,
1994; Sekul and Ory, 1977).
Gelation properties: Gelation properties of the hydrolysates from the
four products were slightly different but they have some common trend Table
6. As shown in the results, the fraction from 30% fraction did not fall
from the inverted test tubes from 6 to 20% protein concentration. Similar observation
was made for 55% fraction; it started slipping out from the test tube at 18%
concentration. But a different scenario occurred for 70% fraction where in the
sample slipped out at the lowest concentration. Present results are contrary
to Yu et al. (2007). It could be attributed to
the enzyme used for the hydrolysis, as Alcalase 2.4 L is an endopeptidase with
a broad specificity to hydrophobic amino acids (Yu et
al., 2007).
|
| Fig. 4: |
Apparent viscosity of DFMPH and the various alcohol concentrations
fractions. Value represent the Mean±SD of n = 3 duplicate assays |
| Table 6: |
Gelation properties of the defatted foxtail millet protein
hydrolysate from different alcohol concentrations |
 |
| s: Slipped from inverted test tube; ss: Sample did not slip
from inverted test tube; st: Slight turbidity observed; ALC: Alcohol concentration;
DFMPH: Defatted foxtail millet protein hydrolysate |
Sensory evaluation: A general acknowledged problem encountered in the
use of enzymatic hydrolysis for modification of food proteins is the formation
of bitter taste. The bitter taste can be ascribed to hydrophobic peptides and
results from the degradation of the protein substrate.
The desorbtion of the hydrolysates from the MAR was done with 30, 55 and 70%
fractions but 30% of ALC was observed to have extracted the bitterness from
DFMPH and the final product was not bitter while 55% fraction was slightly bitter
and 70% fraction was completely bitter (Table 3). Nonetheless,
our results are similar to the data reported by Wasswa et
al. (2007) and Zhang et al. (2009). The
bitter taste in the DFMPH can be attributed to highly hydrophobic, short peptides
composed largely of a good supply of essential amino acids (Kanekanian
et al., 2000).
CONCLUSIONS
Understanding change of foxtail millet flour complex during enzymatic hydrolysis
can be useful for producing modified proteins with the desired functionality.
The present results showed that the bitter and salty taste can be removed with
adsorption of DFMPH on MAR followed by rinsing with deionised water to wash
out the salt during which instance the peptides remained adsorb onto the MAR
resins. It also provides an exciting technological manipulation to reduce bitter
and salty taste. MAR, therefore, present technological importance to remove
salt in protein hydrolysates. The alcohol used could be recovered and reduced
cutting down the process costs. From the results presented here, it is proposed
that excellent solubility of the protein hydrolysates could be attributed to
reduce size of the polypeptides obtained after debittering and desalting. The
DFMPH after desalting and debittering process generally had a lower molecular
weight but with no significant different between the fractions. There was also
an improvement in the functional properties studied. This could be incorporated
into the foods for human consumption making them potential competitors with
dairy based and plant based protein hydrolysates currently being used. The results
of this study could hold a prospecting future in the food industries.
ACKNOWLEDGMENTS
This research was financially supported by Governments of Sierra Leone and
People’s Republic of China; the authors wish to thank both Governments.
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