MSc. Thesis
Evaluation of Enzymatic Hydrolysis of Wheat
Gluten at Different Protein Concentrations
María Cristina Sotomayor Grijalva
2013-03-08
MSc thesis title: Evaluation of Enzymatic Hydrolysis of Wheat Gluten at
Different Protein Concentrations
Course code:
FPE-80336
Author:
María Cristina Sotomayor Grijalva
Reg. nr:
840310786120
Date:
2013-03-08
Supervisors:
Nicolas Hardt MSc.
dr. Atze Jan Van Der Goot
Examiner:
dr.ir. Anja Janssen
2
Abstract
The effect of protein concentration and presence of salt, on wheat gluten hydrolysis at high
solid concentrations (40%) was investigated.
The aim of this project was to study the behaviour of 4 raw materials, with different protein
concentrations. The raw materials were wheat flour, shear cell gluten, washed gluten, and
commercial vital gluten. Shear cell gluten and washed gluten were obtained from native
wheat flour. They were all subjected to hydrolysis at 40% solid concentration using
Flavourzyme® at an enzyme to substrate ratio of 1:100. In order to evaluate the hydrolysis of
the different raw materials, the degree of hydrolysis (DH), the molecular weight distribution,
and the nitrogen solubility index (NSI) were evaluated. To investigate the factors that
determine the DH, experiments taking into account the protein content and water holding
capacity were performed.
Hydrolysis at high solid concentrations (40%) was possible for all the raw materials. Wheat
flour, which is the raw material with the lowest protein content in this study, presented the
highest DH. For materials with increasing protein content, the DH was decreasing. The
presence of salt during hydrolysis was found to have a negative effect on the DH at all the
concentrations evaluated. Furthermore, as the amount of salt in the material increased, its
negative effect was larger. The factor that was found to have a strong influence on the DH,
was the material’s protein content. Differences in the starch/water ratio did not show to have
an influence on hydrolysis.
Centrifugation after hydrolysis allowed a separation of wheat flour components. A postseparation of flour components could replace a pre-separation, which could lead to water
and energy savings.
3
Table of Contents
Abstract ................................................................................................................................. 3
Introduction ........................................................................................................................... 6
1
Theoretical Background.................................................................................................. 7
1.1
1.1.1
Wheat gluten proteins ...................................................................................... 7
1.1.2
Properties of wheat gluten ................................................................................ 8
1.1.3
Wheat gluten extraction.................................................................................... 9
1.2
2
3
Wheat gluten ........................................................................................................... 7
Hydrolysis ..............................................................................................................10
1.2.1
Types of hydrolysis .........................................................................................11
1.2.2
Hydrolysis at high concentrations ....................................................................13
1.2.3
Effect of hydrolysis on solubility .......................................................................13
1.2.4
Gluten hydrolysis .............................................................................................14
Materials and Methods ..................................................................................................15
2.1
Raw material ..........................................................................................................15
2.2
Methods .................................................................................................................16
2.2.1
Hydrolysis .......................................................................................................16
2.2.2
Degree of Hydrolysis .......................................................................................20
2.2.3
Solubility..........................................................................................................21
2.2.4
Peptide size distribution ..................................................................................22
Results and Discussion .................................................................................................23
3.1
DH at different protein concentrations ....................................................................23
3.2
Effect of NaCl on hydrolysis ...................................................................................26
3.3
DH and solubility ....................................................................................................29
3.4
Influence of water holding capacity and protein content on hydrolysis. ...................30
3.4.1
Influence of water holding capacity on hydrolysis ............................................30
3.4.2
Influence of protein content on hydrolysis ........................................................32
3.5
Effect of hydrolysis on protein-starch separation ....................................................33
4
4
Conclusions...................................................................................................................35
5
Recommendations ........................................................................................................36
6
Bibliography ..................................................................................................................37
7
Appendix .......................................................................................................................41
7.1
Wheat flour water absorption ..................................................................................41
7.2
Peptide molecular weight distribution considering the water holding capacity.........41
7.3
Peptide molecular weight distribution considering the protein content ....................42
5
Introduction
Wheat gluten comprises around 72% of the total protein in wheat flour. Gluten it is composed
of two proteins, glutenins and gliadins. Together they form a network with unique properties
that allow the entrapping of air bubbles in the leavening of dough.
Currently, wheat gluten is a relatively low cost by-product in wheat starch production. It is
utilized as a flour enhancer in bread production and as an additive in the baking industry.
However, due to its low water-solubility at neutral pH its further application in foods is limited.
The hydrolysis of gluten increases its water-solubility (Kong, et al., 2007).
Several treatments are available to accomplish protein hydrolysis. Solubilising with acid or
alkali is an option; nevertheless it can lead to undesirable side reactions during the
degradation of essential amino acids. Acid hydrolysis can also result in products with more
salt than needed (Kim, et al., 2004).
An alternative method is the hydrolysis of gluten using enzymes. The use of enzymes allows
more specific peptide bond cleavage. Also, enzymatic hydrolysis lacks the need of salt
removal which is necessary in chemical hydrolysis.
The application of gluten hydrolysates has already been studied. Gluten hydrolysates are
used for their emulsifying and foaming properties. Also, as it is a relatively cheap source of
protein it is used in fortified beverages (Bombara, et al., 1992). Moreover, gluten
hydrolysates can be used in savoury bases because they provide the umami flavour
(Chiagrupati, et al., 2001).
Generally, wheat gluten is hydrolysed after starch and gluten have been separated using
large quantities of water. Starch and gluten can also be separated under concentrated
conditions inducing shear flow (van der Zalm, et al., 2009). In this case, gluten is
concentrated up to 50% purity compared to a protein content of 75% in commercial wheat
gluten. However, the question arises, if extensive separation of starch and gluten is a
prerequisite for wheat gluten hydrolysis.
Therefore the following study has the purpose of analysing the behaviour of materials with
different protein contents subjected to hydrolysis. The aim is also to hydrolyse at high solid
concentrations as this implicates water and energy savings.
6
1
Theoretical Background
1.1
Wheat gluten
Gluten is the major protein present in wheat flour that allows the leavening in bakery
products. This protein-rich material has cohesive and viscoelastic properties which permit the
retention of gas bubbles in dough. It is obtained as a by-product during the separation of
starch from wheat flour (Day, et al., 2006).
According to Van Oort (2010), vital gluten, in dry state, is constituted by 70-85% of protein, 515% of carbohydrates, 3-10% of lipids and 1-2% of ash.
1.1.1
Wheat gluten proteins
The two major proteins of wheat gluten are glutenins and gliadins (Xu, et al., 2001). They
constitute 80% of the wheat flour proteins (Uthayakumaran, et al., 1999). Both proteins are
found in similar amounts in wheat flour (Goesaert, et al., 2005). Gluten has an amino acid
composition rich in glutamine and proline (Van der Borght, et al., 2005). In Figure 1, the
structures of gliadin and glutenin are depicted.
Figure 1: Structure of glutenin and gliadin
(Fasano, 2011)
7
1.1.1.1 Gliadins
Gliadins are monomeric proteins, which have molecular weights that range from 20,000 to
70,000 (Southan & MacRitchie, 1999). They have a role as plasticizer in dough formation;
therefore, they give extensibility and viscous flow to the dough. Furthermore, they become
sticky when moistened (Van der Borght, et al., 2005).
As a separate product, gliadin is utilised to improve the freeze-thaw and the microwave
stability of frozen dough. Other uses are natural chewing gum base replacer, texturizer in
pasta goods and pharmaceutical binder (Bassi, et al., 2008) .
1.1.1.2 Glutenins
Glutenins are proteins composed of glutenin subunits (GS) which are connected by
disulphide bridging (Goesaert, et al., 2005). The GS can be divided in low molecular weight
subunits (LMW-GS) and high molecular weight subunits (HMW-GS). The HMW-GS have a
molecular weight that ranges from 80,000 to 120,000. The LMW-GS can be divided in two
groups, one with molecular weight in between 40,000 and 55,000, and the other from 30,000
to 40,000 (Southan & MacRitchie, 1999).
Glutenin proteins provide the dough with resistance to extension (Van der Borght, et al.,
2005). Glutenin is used as a strengthening agent in the bakery industry, and also in meat
alternative goods (Bassi, et al., 2008).
1.1.2
Properties of wheat gluten
1.1.2.1 Water holding capacity (WHC)
WHC is defined as “the ability of a protein matrix to absorb and retain bound, hydrodynamic,
capillary, and physically entrapped water against gravity” (Traynham, et al., 2007). The
quantity of water that is absorbed by a protein depends on the protein´s amino acid
composition, the protein´s conformation, the number of polar surface groups, and the polarity
of its surface (Zhang, et al., 2010).
During hydration, gluten is capable to absorb an amount of water that represents
approximately two times the amount of dry gluten powder (Day, et al., 2006).
8
1.1.2.2 Solubility
Wheat gluten has poor solubility in water; this characteristic limits its application in many food
processes (Kanerva, et al., 2011). The low solubility of gluten can be explained by the strong
intermolecular interaction induced by hydrogen bonds and hydrophobic interactions, and the
large size of the proteins. Belton (2005) pointed out that the low solubility of gluten is not
because of weak water-protein interactions, but because of strong protein-protein
interactions. Also, the low solubility is affected by the high concentrations of proline and
leucine, which are non-polar amino acid residues (Cui, et al., 2011).
Gliadins can be solubilized in aqueous alcohols, and glutenins in dilute acetic acid, according
to the Osborne fractionation (Goesaert, et al., 2005).
1.1.3
Wheat gluten extraction
1.1.3.1 Traditional methods
The present methods used for the separation of wheat flour into starch and gluten involve
kneading and washing. These processes are not water or energy efficient (Van der Zalm, et
al., 2009). The methods that are frequently used in industry for starch-gluten separations are
the Martin process and the batter process.
The Martin process involves the washing of wheat dough with water while it goes through
tumbling cylindrical agitators. The starch leaves the dough, through small holes on the
cylinder’s wall, leading to a higher protein concentration in the remaining dough. The dough
receives continues washes until it reaches the end of the cylinder.
Another way of separation is the batter process. Here, a thick suspension of flour is made
and is subjected to constant stirring for several hours to provoke the separation of starch and
gluten. Afterwards, the suspension is filtered with a sieve, where the gluten curds are
retained and the starch goes through. The curds are finally washed as it is done in the Martin
Process (Day, et al., 2006).
9
1.1.3.2 Shear cell
A water and energy friendly process to induce the separation of starch and gluten is the
usage of the double cone shear cell. The components of the shear cell are shown in Figure
2. The upper cone is immobile, while the lower one rotates. A mixture of flour, water and salt
are added in the gap between the cones. The movement of the upper cone induces shear
forces that cause the aggregation of gluten, and its later migration to the apex of the cone
(Van der Zalm, et al., 2009).
Figure 2: Shear cell
(Habeych, et al., 2008)
The addition of salt favours the gluten separation. Gluten interactions are induced by the
presence of salt. An addition of 2-4% of salt promotes gluten interactions that allow the
formation of larger aggregates, and a faster migration to apex of the cone. Therefore, higher
protein contents are obtained in the centre of the cone. At lower salt concentrations the
gluten interactions are not strong enough to promote aggregation and migration of gluten. At
higher concentrations the interactions become too strong, the gluten aggregates are not
easily deformed during shearing. They break and are redispersed in the dough (van der
Zalm, et al., 2010).
1.2
Hydrolysis
The properties of proteins can limit their utilisation as food ingredients. A way to overcome
this problem is the production of smaller peptides by hydrolysing the protein. The reaction
mechanism for hydrolysis is presented in Figure 3.
10
Figure 3: Hydrolysis reaction
(Donohue & Osna, 2004)
An enzyme attaches to the protein to catalyse the reaction. Then, a water molecule attacks
the peptide bond and cleaves it. The water molecule splits into OH- and H+, which are added
to the remaining peptides. Hydrolysis is evaluated by calculating the degree of hydrolysis
(DH). This is equivalent to the number of peptides bonds cleaved over the total amount of
peptide bonds present in the native protein (Nielsen, 2002).
1.2.1
Types of hydrolysis
Hydrolysis can be performed with the utilisation of acid, alkali or enzymes (Batey, 1985).
Chemical and enzymatic hydrolysis have the objective of liberating a high number of amino
acids at low costs and at a high efficiency (Delest, et al., 2005). Chemical and enzymatic
hydrolysis result in an improvement of the functional properties of the native protein (Deeslie
& Cheryan, 1988).
1.2.1.1 Chemical hydrolysis
Acid hydrolysis:
The most common method of hydrolysis is the use of acid (Fountoulakis & Lahm, 1998). A
standard method for acid hydrolysis has been established with the use of HCl at 110 ° C for
11
more than 24h. The use of mixtures of strong acids, like concentrated hydrochloric acid and
trifluoroacetic acid, at higher temperatures, can reduce the time to 25 minutes and obtain the
same result as for the standard conditions (Tsugita & Scheffler, 1982).
Basic hydrolysis:
Sodium and potassium hydroxides are used to perform basic hydrolysis. Hydrolysis with
alkali is time consuming and inaccurate. Therefore, acid hydrolysis is preferred. However,
with acid hydrolysis an important compound, tryptophan, is destroyed. Consequently, basic
hydrolysis is used for tryptophan analysis (Oelshlegel, et al., 1970).
A complete hydrolysis can be achieved by using acid or basic reagents (Fountoulakis &
Lahm, 1998). However, acid and basic hydrolysis involves the destruction of essential amino
acids, and causes the formation of toxic compounds. (Aaslyng, et al., 1008). Also, acid
hydrolysis gives unwanted flavours to the product, and produces carcinogenic compounds
(Chiagrupati, et al., 2001).
1.2.1.2 Enzymatic Hydrolysis
Enzymatic hydrolysis of proteins is performed using specific proteases. This allows control
over the functionality of the resulting products (Kim, et al., 2004). According to Clemente
(2000), hydrolysis performed by enzymes is a process that utilises mild conditions, pH
ranging between 6-8, and temperatures in between 40-60 °C. Differently from acid and alkali
reactions, enzymatic reactions have no parallel degradation of separate components.
There exist several parameters that determine the hydrolysis reaction. According to Nielsen
(2002) and Apar & Özbek (2008), the most important are the protein content in the reaction
mixture, the enzyme/substrate ratio, pH, temperature, and reaction time. In contrast,
enzymatic hydrolysis comprises factors which make the reaction very complex. The more
remarkable are the diversity of the reactants, hydrolysis of products from a first hydrolysis
step, simultaneously cleavage of peptide bonds, complexity of the networks present in the
substrate, inhibition of the enzyme, and process conditions (pH, temperature and ionic
strength) (Qi & Zhiming, 2006).
Protein hydrolysis is performed by the use of endopeptidases and/or exopeptidases.
Endopeptidases are enzymes which separate the protein into peptides by allowing the
cleavage of central peptide bonds. An exopeptidase hydrolyses the terminal peptide bonds
by cleaving amino acids from the amino or carboxy terminus (Clemente, 2000). In order to
12
obtain the highest amino acid generation, mixtures of endopeptidases and exopeptidases are
available. The mixtures should contain various types of amino/carboxy exopeptidases. In this
way, the problem of hydrolysing only specific amino acids, or amino/carboxy terminus can be
solved (Edens, et al., 2002).
During the process the rate of hydrolysis decreases with time. According to Gonzalez-Tello
(1994), this phenomenon happens due to three reasons:

Decrease in the amount of peptide bonds that are prone to be cleaved

Enzyme inhibition by products

Denaturation of enzyme
1.2.2
Hydrolysis at high concentrations
Hydrolysis at high concentration of solids involves scarce amounts of water in the process.
Hydrolysis at high concentration of solids has several advantages. Greater amounts of
material can be processed using the same system. This leads in consequence to other
benefits. Less usage of water and energy are needed for heating and cooling the hydrolysis
mixture. In addition, less waste water is generated, and less energy is needed for drying.
However, processing at high solid concentrations brings also drawbacks. The enzyme
activity is hindered due to the presence of higher concentrations of end products and of
inhibitors. Also, the mixing is less efficient as the viscosity increases (Kristensen, 2009).
Furthermore, enzyme diffusion is obstructed by the compact substrate, which leads to a
decrease in the reaction rate (Cheng & Prud'homme, 2000).
1.2.3
Effect of hydrolysis on solubility
Hydrolysis increases the water-solubility of many proteins. It generates small molecular sized
peptides, which have more ionisable amino and carboxyl groups. This is believed to be the
reason of a higher solubility (Panyam & Kilara, 1996). The solubility of a protein depends on
the DH achieved with hydrolysis (Bombara, et al., 1992).
13
1.2.4
Gluten hydrolysis
Wheat gluten subjected to partial hydrolysis is more soluble than the intact protein. The
number of hydrophobic structures is reduced by this process (Mejri, et al., 2005). The usage
of enzymes shows that besides an increased solubility, an enhancement in foaming and
emulsifying characteristics is achieved (Kong, et al., 2007). In the work of Kong et al. (2007),
dispersions of 5% gluten were hydrolysed by different enzymes to obtain a DH from 0 to 15%
after 360 min of hydrolysis. The solubility of gluten was found to increase from 14% to 60%.
Bombara et al. (1992) worked on wheat flour hydrolysis. The hydrolysis reaction showed a
downward curvature, usual behaviour in proteolysis of many materials. In this investigation
the wheat protein solubilisation was found to be proportional to the DH. For a duration of 24
hours, a solubilisation of 80% the protein was achieved.
In a study about corn gluten hydrolysis performed by Apar & Özbek (2008), it was found that
there was no enzyme inactivation by the substrate. This could be explained as substrate
inhibition is caused by the binding of two substrate molecules to the enzyme. In the case of
substrates with low solubility a double binding is not expected (Kristensen, 2009). More
apparently, the decrease in the conversion rate was thought to be caused by saturation of
the enzyme with the substrate, a reduction in water activity, and limitations of mass transfer
(Apar & Özbek, 2008).
Wheat gluten hydrolysates have various uses in the food industry. They are used as foaming
and emulsifying improvers (Bombara, et al., 1992). For the production of savory bases wheat
gluten is desired as a raw material due to its large glutamic acid content that provides
peptides with less bitterness and sweeter flavor (Chiagrupati, et al., 2001).
14
2 Materials and Methods
2.1
Raw material
Five raw materials were used for hydrolysis experiments, wheat flour (Meneba Ibis),
commercial vital gluten (Roquette), self-washed gluten, shear cell gluten, and a mixture of
wheat flour with washed gluten.
Wheat flour:
The water absorption of wheat flour was analysed using a farinograph as it is explained by
the Wheat Marketing Center (Wheat Marketing Center, 2004), with some minor
modifications. First, 300g of wheat flour were placed inside the farinograph bowl tempered at
30 °C. The farinograph was connected to a computer where a torque curve, torque (Nm)
against time, was recorded. Then, the farinograph mixers premixed the dough for 5 minutes.
Afterwards, water was added to the flour in order to obtain the highest point of the torque
curve at 500Nm. The amount of water used was expressed in percentage.
Self-washed gluten:
In the case of self-washed gluten, a dough of 41.5 % water content (w/w) w.b. was subjected
to continuous rinses until a fibrous material was formed. This procedure was applied in order
to simulate the already known starch industry process for the separation of starch and gluten.
Self-washed gluten with 70-80% was obtained.
Shear cell gluten:
Shear cell gluten was obtained by following the procedure according to van der Zalm et al.
(2009), with some minor modifications.
Dough of 43% water content w.b. and 2% salt was prepared. Then, it was added to a shear
cell where a gluten concentration of about 35 % was achieved. The shear cell is a device
which consists of a double cone of stainless steel with a gap in between. The lower cone is
maintained still, while de upper one rotates at a constant speed. The material that is
contained in the gap is subjected to shear forces that induce the separation of starch and
gluten. The process was carried out for 1h at 30°C. During this period, the gluten aggregated
and migrated to the centre of the cone. The presence of salt facilitated the formation of
gluten aggregates during shearing.
15
Mixture of self-washed gluten and wheat flour:
The mixture of wheat flour and washed gluten was made with the aim of having a material
with the same protein concentration as the shear cell gluten but without the addition of salt; in
this way the influence of extra salt in the sample was evaluated.
A summary of the protein and moisture content of the used raw materials is presented in the
Table 1.
Table 1: Protein and moisture content of raw material
Raw material
Wheat flour (wf)
Shear cell gluten (scg)
Wheat flour + washed gluten (wf&wg)
Vital wheat gluten (vg)
Self-washed gluten (wg)
2.2
2.2.1
Protein content (%)
12.5
30-35
30-35
71
70-80
Moisture content (%)
12.75
6.25
2
7.8
2
Methods
Hydrolysis
Hydrolysis was performed using the raw materials, shown in Table 1, during 1 and 2 hours at
45 ⁰C. A mixture of endo-exopeptidases, called Flavourzyme, was used in a ratio of 1:100
(enzyme/substrate). The endopeptidase enzymes reacted with gluten by splitting the large
molecules into smaller pieces and the exopeptidases attacked the amino/carboxyl terminals.
The reaction was carried out in a double wall glass reactor.
After hydrolysis the samples were centrifuged at 4500 r.p.m. for 5 minutes. Subsequently,
they were inactivated at 95 ⁰C for 10 minutes. Afterwards, they were cooled down, frozen at 20 ⁰C, and finally freeze-dried and pulverised.
A scheme of the process is shown in Figure 4.
16
Protein content
72-80%
Washing
70%
Vital wheat gluten
Hydrolysis
(Flavourzyme)
35%
Wheat Flour
40% solid
concentration
Centrifugation
and
Inactivation
Shearing
13%
Figure 4: Hydrolysis
17
Freeze
Drying
Measure
%DH, MW,
and NSI
2.2.1.1 Hydrolysis at 40% of solid concentration
25g of sample were hydrolysed in a system containing a dry matter concentration of 40%.
The amounts of dry powder, water, and enzyme, used in hydrolysis for the different raw
materials at 40% of solid concentration and 1:100 of enzyme ratio are shown in Table 2.
Table 2: Components in hydrolysis at 40% solid concentration and 1:100 enzyme ratio (enzyme/substrate)
Sample
Dry powder (g)
Water (g)
Enzyme (ml)
11.51
10.67
10.2
10.85
10.2
13.49
14.33
14.8
14.15
14.8
15
40
38
83
80
Wheat flour
Wheat flour & washed gluten
Shear cell gluten
Vital gluten
Washed gluten
2.2.1.2 Hydrolysis considering the effect of WHC and protein content
2.2.1.2.1 Effect of WHC
The effect of WHC and protein content during hydrolysis was analysed. To achieve this, the
values of the solid/water concentration were adjusted, for each type of raw material, to obtain
the same WHC as in wheat flour. Therefore, the solid concentration during hydrolysis varied
for each sample.
The WHC of the different raw materials was obtained following several steps. First, 1.5g of
sample was suspended in 28.5ml of MQ-water in plastic centrifuge tubes. Then, the tubes
were shaken manually to improve the wetting of the sample. Later, the tubes were placed in
a rotatory mixer at 40 r.p.m. for 10 minutes. Next, the tubes were centrifuged at 5000 r.p.m.
for 30 minutes and the supernatant was decanted. Afterwards, the weight of the hydrated
sample was measured. Subsequently, the sample was dried in an oven at 105 ⁰C for 24h.
Finally, the weight of the dry sample was measured.
The WHC was calculated using Equation 1.
(
)
Equation 1
18
From the value of WHC, the amount of water not held by each material was obtained. Then,
the amount of water not held by each raw material was fixed, to achieve the same value as in
hydrolysis of wheat flour at 40% of solid concentration. Consequently, the solid concentration
in hydrolysis for the rest of raw materials varied. The enzyme ratio was maintained. In Table
3, the solid concentration and the amounts of dry powder, water, and enzyme, used in
hydrolysis for the different raw materials with a fixed value of water not held are shown.
Table 3: Components in hydrolysis for different raw materials with a fixed value of water not held
40
Dry powder
(g)
11.51
Water
(g)
13.49
35
9.33
15.67
35
36
33
9.18
8.42
15.82
16.58
34
73
Sample
Solid Concentration (%)
Wheat flour
Wheat flour & washed
gluten
Shear cell gluten
Washed gluten
Enzyme (ml)
15
2.2.1.2.2 Effect of protein content
The protein content was measured applying the Dumas method with the use of a Thermo
Scientific Flash EA 1112 Series Nitrogen and Protein Analyser. The used conversion factor
was Nx5.7.
The goal was to obtain a sample with the same protein content as in the wheat flour
hydrolysis sample. The protein content was calculated as follows in Equation 2.
Equation 2
Therefore, the solid/water ratio was adjusted to achieve 1.44g of protein for each raw
material. Table 4 shows the solid concentration and the amounts of dry powder, water and
enzyme used in hydrolysis, for each type of material, to achieve the same protein content as
in wheat flour hydrolysis.
Table 4: Components in hydrolysis for different raw materials with a fixed value of protein content
Sample
Wheat flour
Wheat flour & washed
gluten
Shear cell gluten
Solid Concentration (%)
40
Dry powder (g)
11.51
Water (g)
13.49
Enzyme (ml)
15
16
4.27
20.73
16
16
4.08
20.92
15
Washed gluten
7
1.79
23.21
15
19
2.2.2
Degree of Hydrolysis
The degree of hydrolysis (DH) was measured following the o-phtaldialdehyde procedure
(OPA method) stated by Nielsen (Nielsen, 2002).
For this method 4 main solutions were prepared.
1. 10% (w/w) SDS solution.
2. 15.625 mM Borax solution. The solution was stirred vigorously for 1-2 hours.
3. OPA-reagent. The solution was obtained in several steps. To start, 3.81g Borax and 1g
SDS were dissolved in 80ml of MQ-water for 1-2 hours. After this solution was made, 80
mg of OPA were dissolved in 2ml of ethanol. This latter was mixed with the SDS/Borax
solution. Next, 88mg of DTT were added to the last solution and mixed again. Later the
solution was filled up until 100 ml using a measuring cylinder. Then, the solution was
filtered using a syringe with a 0.45µl filter. Finally, the solution was stored in a dark glass
because the OPA is light sensitive.
4. Standard solution. This was prepared by the dissolution of 20mg of serine in 200ml of
MQ-water.
The hydrolysates were weighted in a way that the protein content would be similar for all the
samples analysed. Thus, for vital or washed gluten, shear cell gluten, and wheat flour, the
amounts of sample taken were 3.75±0.3mg, 7.5±0.3mg and 18.75±0.3mg respectively.
The samples were placed in plastic test tubes. Then 1ml of solution 1 and 4ml of solution 2
were added to the tubes. Afterwards, the tubes were mixed in the test tube shaker for 1h.
Later, the tubes were centrifuged at 4500 r.p.m. for 15min.
For the measurement, a spectrophotometer was used at an absorbance of 340 nm. The OPA
reagent was added to a cuvette and the blank absorbance was measured. The reagent, from
the cuvette, was then placed into an eppendorf tube, and 200µl of the supernatant from the
centrifuged tubes were added. Subsequently, the eppendorf tube was stirred, and the
content was returned to the cuvette. A second absorbance measurement was performed
after 3-5 minutes. The serine solution was also measured in the same way as the
hydrolysate samples.
For the Serine-NH2 calculation Equation 3 was applied.
[ ]
Equation 3
20
Where, Serine-NH2 is the meqv serine-NH2/g protein, Ahydr is the absorbance of the
hydrolysate mixed with the OPA reagent, AOPA,hydr is the absorbance of the OPA reagent
before adding the supernatant (similarly can be deducted from ASerine and AOPA,sernine), Protein
is the concentration of protein that is present in the sample, and mMserine is the molar
concentration of the serine solution.
Then, the DH was calculated as percentage by using Equation 4.
(
)
Equation 4
Where α and β are constants that depend on the raw material subjected to hydrolysis and the
htot is the total number of peptide bonds per protein equivalent; according to Nielsen (Nielsen,
2002), for vital gluten the values are 1, 0.15 and 8.3.
2.2.3
Solubility
The solubility was measured using the nitrogen solubility index (NSI) as described by Morr
(Morr, et al., 1985).
A suspension of 1.25% was obtained through the addition of 200mg of the pulverized
hydrolysate to 16ml of MQ water. The suspension was then mixed in plastic test tubes using
a test tube shaker for 1.5 hours. Subsequently, they were centrifuged at a speed of 4000 rpm
for 15 minutes. Afterwards, the supernatant and the pellet were separated for each
hydrolysate sample, frozen at -20 ⁰C, freeze dried and pulverised. Next, the protein content
for each phase was determined by applying the Dumas method. Finally, the NSI was
calculated as follows in Equation 5.
Equation 5
21
2.2.4
Peptide size distribution
The size distribution of the peptides after hydrolysis was measured using size exclusion
chromatography (SEC). The hydrolysate was suspended in a 2% (w/w) SDS solution, and
stirred overnight in eppendorf tubes.
The amount of sample used for the analysis depended on its protein content. Therefore, for
vital and washed gluten, for shear cell gluten and mixture of washed gluten and wheat flour,
and for wheat flour, the amounts of 3.75±0.3mg, 7.5±0.3mg and 18.75±0.3mg were
respectively weighted.
Then, the eppendorf tubes were centrifuged at 3900 g for 15 minutes. Afterwards, the
supernatants were injected into a TSKGel SWXL (300x7.8mm) column and eluted with a
solution of 1% (w/w) acetonitrile-trifluoroacetic acid (TFA) at a flow rate of 1.0ml/min and
detected at a wavelength of 214nm. The standards used as molecular mass markers were
carbonic anhydrase (29000 Da), α-lactalbumin (14100 Da), aprotinin (6510 Da), insulin (5700
Da), bacitracin (1420 Da), and phenylalanine (165 Da). Equation 6 was used for calculating
the molecular mass at every elution time.
Equation 6
Where, MM is the molecular mass, and t is the elution time. The peptide range was divided in
four sub-ranges, <2 kD, 2-10kD, 10-25kD, and >25kD.
22
3 Results and Discussion
The different protein concentration of the raw materials depended on the different process to
which they were subjected for concentration. The protein content of each material are
displayed in Table 1 in section 2.1. For wheat flour, the water absorption was 64%, the
farinograph result is presented in Appendix 7.1.
3.1
DH at different protein concentrations
In Figure 5, the results for DH against time are displayed for different starting materials.
14
12
DH (%)
10
wheat flour
shear cell gluten
8
wheat flour+vital gluten
6
wheat flour+washed gluten
vital gluten
4
washed gluten
2
0
0
1
2
t (h)
Figure 5: Degree of hydrolysis of raw materials.
There are three inferences that can be made from this graph. First, all the raw materials were
able to be hydrolysed, and the DH increased with time.
Second, the mixtures of wheat flour and vital gluten/washed gluten had the same protein
content as shear cell gluten. However, the DH for shear cell gluten was lower. The difference
was that shear cell gluten contained 2.4% salt. This suggests that the NaCl present in shear
cell gluten influenced the hydrolysis negatively.
23
Finally, wheat flour, which had the lowest protein content in this study, presented the highest
DH. When the protein concentration was higher, the DH decreased. In a study about
hydrolysis of whey proteins performed by Butré et al. (2012) it was found that by increasing
the substrate concentration the DH decreased. The same was found for hydrolysis of casein
with Alcalase (Camacho Rubio, et al., 1993). The results from Figure 5 were done at 40% of
solid concentration; however, the protein concentration varied for each reaction mixture. It is
important to stress that the degree of hydrolysis was not determined by taking a sample at 1
hour and later at 2 hours. Each data point corresponds to a separate experiment, which was
only done once. Therefore, a clear difference between vital, washed gluten and their
combinations with wheat flour could not be drawn from Figure 5. In order to better analyse
the effect of substrate concentration, an average of the value of DH for the experiments was
made for materials that contained the same protein content, but no salt. The averaged
results are presented with discontinued lines in Figure 6. The average was done for each
experiment with duration of 1 and 2 hours.
14
12
DH (%)
10
wheat flour
8
shear cell gluten
6
vital/washed gluten
+ wheat flour
washed/vital gluten
4
2
0
0
1
2
3
t (h)
Figure 6: Degree of hydrolysis of raw materials (Simplified)
In the graph above a clearer influence is appreciated. It can be observed that the DH
decreased with a higher concentration for experiments with duration of 1 hour. At 2 hours the
24
DH was similar. Nonetheless, we expect to obtain a sharper DH decrease with an increase in
protein concentration. This expectation is due to the results obtained at after 1 hour, and the
behaviour or wheat flour and vital gluten after 2 hours.
The results for the molecular weight distribution of hydrolysates of the different raw materials
after 2 hours are presented in Figure 7. For all raw materials the peptide distribution was very
similar with the exception of shear cell gluten. For shear cell gluten, a higher peak is
displayed at the beginning of the elution. This indicates that larger hydrolysates were present
in higher amounts in the hydrolysed shear cell gluten sample.
DH
120
100
mAU
80
12.8
wheat flour
5.24
shear cell gluten
8.19
wheat flour+washed gluten
10.23
washed gluten
7.82
vital gluten
60
40
20
0
2
4
6
8
time (min)
10
12
14
Figure 7: Molecular size distribution of the different raw materials at 2 hours of hydrolysis.
The molecular weight distribution was divided into classes to show the difference of the
quantities of peptides in a certain range of molecular weight. This is presented in Figure 8.
For all the raw materials, except for shear cell gluten, the amount of hydrolysates within all
molecular weight classes was very similar. In contrast, the shear cell gluten sample
contained a larger amount of peptides with a molecular weight higher than 25 kDa, and lower
amounts of peptides of lower molecular weights. This is explained by the low DH achieved by
shear cell gluten. A lower DH would imply that less peptide bonds were cleaved during
hydrolysis. Thus larger peptides are found in the hydrolysed material.
25
DH
12.18
5.24
8.19
10.23
7.82
100
Peptide distribution (%)
80
>25 kD
60
10-25 kD
2-10 kD
<2 kD
40
20
0
wf
scg
wf+wg
wg
vg
Material
Figure 8: Molecular weight distribution (%)
3.2
Effect of NaCl on hydrolysis
From the shear cell gluten experiments, presented in section 3.1, it was suggested that NaCl
has a negative effect on hydrolysis. Therefore, the effect of NaCl was investigated for
materials of different protein concentrations. The amount of salt present in shear cell gluten
was determined, using the method proposed by Ukai et al. (2008), and was found to be
2.4%.
Wheat flour, vital gluten, and a mixture of wheat flour and washed gluten (to obtain the same
protein content as in shear cell gluten), were used to analyse the effect of the salt content at
different protein concentrations. The amount of salt was added in 2% and 4% with respect to
dry matter. Figure 9 shows the effect of 2% salt on the raw materials with different protein
content. The pictures were taken after centrifugation, after hydrolysis.
26
For wheat flour the addition of salt did not result in a distinct change in appearance. In the
case of the other materials, with higher protein concentration, the amount of supernatant
decreased dramatically on salt addition.
a)
0%
b)
2%
0%
c)
2.4%
0%
2%
Figure 9: Hydrolysed materials with and without the addition of salt, a)wheat flour, b) wheat flour + washed
gluten, and shear cell gluten, and c) vital gluten
The DH for the different materials with and without salt addition were analysed. The results
are presented in Figure 10. The percentage values represent the decrease of DH with
respect to the sample without salt. The DH decreased when salt was added. Moreover, with
the increase of the amount of salt added, the effect of DH reduction is larger.
Two factors have been taken into account to explain the negative influence of salt on the DH.
On one hand, the enzyme activity could be affected by the presence of salt. In a study by
Warren et al. (1966) it was seen that enzyme activity was hindered by the presence of
neutral salts. It was suggested that the presence of salts could change the organization of
27
the enzyme’s structure causing its inhibition. However, in the same work, it was found that
certain salts could induce a higher activity of the enzymes. This was thought to be caused by
the same effect, though in this case buried active sites were exposed and facilitated the
enzyme’s action. This implies that the activity of an enzyme is determined by the salts
present in the solution.
12
wheat flour
vital gluten
10
-16%
shear cell gluten
DH (%)
8
-43%
6
-24%
4
-42%
2
-63%
0
0
2
4
Salt content (%)
Figure 10: DH for materials of different protein concentration with and without salt addition
On the other hand, salt could affect the protein (substrate) in a way that is not available
anymore for the enzyme. In a study performed by Ukai (2008) on wheat gluten, the presence
of NaCl induced the aggregation of gliadins. When gliadins were treated with other salts the
aggregation results were different. Hence, aggregation is specie-dependent and could be
related to an interaction of protein residues with certain ions. I was also shown that gluten
aggregation was not caused by an increase in ionic strength. Also, proteins showed more
crosslinking when salt was present. This facts suggest that the protein would have a more
entangled structure. In turn, these effects on the protein could lead to a lower enzyme
activity.
28
The concentration of gluten using the shear cell is promoted by the presence of NaCl as
explained in section 1.1.3.2, because it induces higher interactions within gluten. A way to
overcome this problem could be to find a salt that can provide the interactions during
concentration, but that stimulates the enzyme activity during hydrolysis. In this way the
negative effect of salt could be worked out.
3.3
DH and solubility
The protein solubility is enhanced on hydrolysis as explained in section 1.2.3. The same was
found when hydrolysing the different raw materials in this study. The results for the relation of
nitrogen solubility index (NSI) with DH are shown in Figure 11.
70
60
NSI (%)
50
wheat flour
shear cell gluten
40
wheat flour+vital gluten
30
wheat flour+ washed gluten
washed gluten
20
vital gluten
10
0
0
2
4
6
8
10
12
14
DH (%)
Figure 11: Relation of DH an NSI
From the graph it can be seen that the NSI increases with the increase of DH. In a study in
gluten hydrolysis by Bombara (1992), it was demonstrated that the solubility was proportional
to the DH. The same can be withdrawn from Figure 11. An interesting observation is that this
effect can be seen for materials that were subjected to different concentration processes, or
29
even no concentration at all. Wheat flour was hydrolysed in its native form. From wheat flour
washed gluten and shear cell gluten were obtained. The same applies to materials from
different protein source. Wheat flour and vital gluten were obtained from different companies,
Meneba and Roquette respectively. In addition, vital gluten was concentrated through an
industrial process. This implies that the origin of gluten does not affect the NSI, the only
variable that influences it, would be the DH.
3.4
Influence of water holding capacity and protein content on hydrolysis.
As shown above, the protein and the salt concentration at a constant solid concentration, of
40%, influenced the hydrolysis. In the following, the influence of the protein concentration will
be approached with respect to the water holding capacity and varying solid concentrations.
3.4.1
Influence of water holding capacity on hydrolysis
Each of the used raw materials had a different protein content. Protein absorbs water as
explained in section 1.1.2.1. The water absorption could have an effect on hydrolysis. In
order to evaluate the influence of the water holding capacity (WHC) on hydrolysis, the
amount of water not held by the native raw material was set to be the same for all materials.
Consequently, the solid concentration varied for each sample. Also, it is important to be
noted that the enzyme/protein ratio (1:100) was the same as in experiments at 40% solid
concentration.
For each material, the values of WHC, water not held by the native raw material, and
resulting solid concentration are presented in Table 5.
Table 5: WHC, water not held, and recalculated solid concentration for different raw materials
Sample
Wheat flour
Shear cell gluten
Mixture wheat flour &
washed gluten
Washed gluten
Protein content
dry material (%)
12.5
35
WHC
(gwater/gdry sample)
1.04
1.24
35
1.28
80
1.44
Water not
held (g)
4.6
Solid
Concentration (%)
40
36
35
33
30
From the table above it is observed that, increasing protein concentrations resulted in
increasing WHC. Wheat flour with a protein content of 12.5 % had a WHC of 1.04 g water/gsolid.
This increased to 1.44 gwater/gsolid for washed gluten with a protein content of 80 %. Thus, to
obtain the same amount of water not held for all raw materials, the needed solid
concentration decreases with an increase in protein content.
The influence of the amount of water held, on hydrolysis of the different raw materials, is
presented in Figure 12.
12
10
DH (%)
8
wheat flour
shear cell gluten
6
washed gluten
wheat flour + washed gluten
4
2
0
25
35
45
Solid content (%)
Figure 12: Influence of amount of water not held on hydrolysis
If the water not held by the native raw material was the only reason for the differences in DH
for different raw materials at 40% solids, then, the materials in Figure 12 should have
obtained the same DH. It can be observed from the graph that the materials achieved
different DH, when the amount of water not held by the native raw material was the same.
The lower solid concentrations for washed gluten, washed gluten and wheat flour, and shear
cell gluten, did not show a large difference from the initial value (40%). Thus, a big change
on the DH was not likely.
In the case of the washed gluten sample, the value is much higher than expected, and
probably an outlier.
31
The DH of the mixture of wheat flour and washed gluten, and of shear cell gluten, did give
expected values, similar to those shown in Figure 5. For the mixture of wheat flour and
washed gluten the DH was about 6%, for hydrolysis at solid concentration of 40%. The DH of
shear cell gluten is, as expected, lower than the DH of the other raw materials. For shear cell
gluten, the DH was about 4, at 40% solid concentration.
The peptide molecular weight distribution gave a similar result as in Figure 7. The results are
presented in the Appendix 7.2.
3.4.2
Influence of protein content on hydrolysis
In the work of Nielsen (2002) and Apar & Özbek (2008) it is stated that, among several
factors, protein concentration in the reaction mixture influences hydrolysis. Therefore, the
solid concentration was adjusted for all the raw materials to obtain the same protein content
as in wheat flour. The values for the recalculated solid concentrations are shown in Table 6.
Table 6: Protein content, fixed protein content and recalculated solid concentration for different raw materials
Sample
Wheat flour
Shear cell gluten
Mixture wheat flour &
washed gluten
Washed gluten
Protein content dry
material (%)
Fixed protein
content (%)
12.5
35
35
80
Solid Concentration (%)
40
16
12.5
16
7
From the table above, it is observed that the adjusted solid concentrations for the different
raw materials differed considerably. Figure 13 shows the influence of the protein content on
hydrolysis of the different raw materials.
It is visible that the same DH was achieved for the different raw materials after 1h. This result
suggests that the protein content is the determining factor on the DH. Furthermore, it implies
that the other components present in the reaction mixture did not interfere in hydrolysis.
The major component, present in the raw materials, which could influence hydrolysis is
starch. Starch in solution increases the viscosity. In a study about whey protein hydrolysis,
performed by Butré et al. (2012), it was found that an increase of viscosity (caused by
increasing protein concentration) did not show mass transfer limitations that influence the
hydrolysis rate. In this case the viscosity was increased by starch, because the protein
content is the same. Nevertheless, when analysing the effect of viscosity it was observed
32
that it did not influenced hydrolysis either. A reason for this is that the temperature at which
the hydrolysis was performed, 45 ⁰C, is lower than the gelatinisation temperature. The
gelatinisation temperature of wheat starch is over 52 ⁰ C (Gough & Pybus, 1971). Therefore,
the viscosity does not reach a high value for being an obstacle during hydrolysis.
% Protein
12
10
DH (%)
8
wheat flour
6
shear cell gluten
4
wheat flour + washed gluten
washed gluten
2
0
0
10
20
30
40
Solid content (%)
Figure 13: Influence of protein content on hydrolysis
In these experiments, the adjustment of the solid concentration lead to variation on the
amount of water present in the reaction mixture. The protein content was the same, but the
starch/water ratio was different . This means that, the replacement of starch by water did not
have an effect on hydrolysis. Such finding is very interesting because apparently there is no
literature about it.
The peptide molecular weight distribution gave a similar result as in Figure 7. The results are
presented in the Appendix 7.3.
3.5
Effect of hydrolysis on protein-starch separation
33
During hydrolysis starch was dispersed in the water from the reaction mixtures. Therefore, a
process similar to the batter process, explained in section 1.1.3.1, was carried out. Figure 14
shows the phase separation of hydrolysed wheat flour, after centrifugation. Protein
quantification was obtained for each phase.
Gluten was mainly present in the supernatant and in the gluten-rich phase, whereas the
starch-rich phase was depleted in gluten. Towards a more sustainable gluten hydrolysate
production, this would suggest that a pre-separation step could be avoided before hydrolysis.
Instead, wheat flour could be hydrolysed and separated afterwards. In addition, section 3.1
shows that the DH is higher for wheat flour than for the materials of higher protein
concentration after 1h and at constant enzyme-to-substrate ratio. Therefore, post-separation
of the components of wheat flour could be an interesting alternative to pre-separation.
Phase : Protein content (%)
Supernatant : 46
Gluten-rich phase: 22
Starch-rich phase: 7
Figure 14: Phase separation of hydrolysed wheat flour after centrifugation
Even so, there are some drawbacks. With the use of Flavourzyme it is not possible to
solubilize 100% of protein. Still, if a solubility of 100% could be achieved, the result would be
mainly small peptides and amino acids. In the work of Kong et al. (2007), it was found that
functional properties of hydrolysed gluten were enhanced for a DH of about 5%.
Consequently, improvements should be done to find the right conditions for the process.
34
4 Conclusions
The present work investigates the enzymatic hydrolysis of different raw materials derived
from wheat flour, with different protein contents. The materials used were commercial vital
wheat gluten, self-washed wheat gluten, shear-separated shear cell gluten, and wheat flour.
The influence of the protein content, the water holding capacity, as well as the role of NaCl
was evaluated.
It was possible to hydrolyse all the materials at high solid concentrations (40%) using the
enzyme Flavourzyme at 45 ⁰C for 1 and 2 hours. Wheat flour, the material with the lowest
protein content in this study, showed the highest DH. Shear cell gluten, which contained salt
by nature, was found to have the lowest DH. Also, with exception of the shear cell gluten, it
was seen that all the samples presented a similar peptide molecular weight distribution.
Hydrolysed shear cell gluten showed to have a greater amount of larger peptides than the
other hydrolysed raw materials due to the lower DH obtained compared to the other raw
materials.
As NaCl is suggested to have a negative effect on hydrolysis, its effect on the other raw
materials was investigated for 1 hour of reaction time. It was found that the presence of salt
had negative effect on hydrolysis for all the materials. Furthermore, the DH decreased as the
salt concentration increased. This outcome could be attributed to a negative effect of salt on
enzyme activity. Also, in the presence of NaCl, gluten exhibits stronger inner interactions, so
it may not be available anymore for the enzyme.
To analyse which material property determined the DH, experiments taking into account the
WHC and the protein content were performed. For evaluating the influence of the WHC, the
amount of water not held by the raw material, was set the same. Hence, the solid
concentration was adjusted. However, the DH obtained differed between the raw materials.
The protein content in the reaction mixture was also considered to be a possible influencing
factor in hydrolysis. Thus, the influence of protein content was analysed by setting fixed the
amount of protein present in the reaction mixture. Also here, the solid concentration was
adjusted. In this case, the DH obtained by the different raw materials was found to be the
similar, suggesting that the protein content is a major influencing factor. In addition, it means
that the other components present during hydrolysis, mainly starch, did not interfere during
the hydrolysis reaction.
35
5 Recommendations
In this investigation many variables were analysed. Hence, duplicates were only performed
for some experiments.. Even though clear tendencies were shown from the comparison
between the different experiments, it is advised to make more repetitions in order to have a
more accurate value of DH.
The only functional property, which was evaluated in this study was solubility of the
hydrolysates at neutral pH. This property is very important, as the aim is to add gluten to food
products (e.g. protein beverages), and many of them have a pH around 7. However, other
properties like emulsifying and also foaming capacity should also be evaluated for the raw
materials investigated in this study. An enhancement of these properties, due to hydrolysis,
would allow a more diverse application of gluten hydrolysates.
During this research it was found that a post-separation of wheat flour components could be
an alternative to their pre-separation. The concentration of wheat gluten, as it is currently
done, is a high energy and water consuming process. If the purpose is to hydrolyse wheat
gluten it was shown that it can be done using native wheat flour. However, the postseparation process was not studied in this project. For this matter, the methods of separation
as well as the processing conditions should be further analysed.
Finally, according to Apar & Özbek
(2008), water activity is another parameter that
influences hydrolysis. It was not studied in this project, but it should also be further
investigated. This would give information about the effect of free water during hydrolysis.
36
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7 Appendix
7.1
Wheat flour water absorption
6
Torque (Nm)
5
4
3
Series1
2
1
0
0
5
10
15
20
25
30
time (min)
Figure 15: Wheat flour (Meneba Ibis) water absorption curve.
7.2
Peptide molecular weight distribution considering the water holding
capacity
160
140
120
mAU
100
wf
80
wf + wg
60
scg
40
wg
20
0
0
2
4
6
8
10
12
14
16
t (min)
Figure 16: Peptide size distribution considering the water holding capacity
41
7.3
Peptide molecular weight distribution considering the protein content
120
100
mAU
80
wf
60
wf + wg
scg
40
wg
20
0
4
6
8
10
12
14
16
t (min)
Figure 17: Peptide size distribution considering the protein content
42