Influence of double enzymic hydrolyses on gluten functionality

Influence of double enzymic hydrolyses ongluten functionalityBrahim Mimouni,† Jean Louis Azanza and Jacques Raymond*ISTAB, Laboratoire de Biochimie et Technologie des Aliments, Universite Bordeaux I, avenue des Facultes, 33405 Talence Cedex, France

Abstract: Functional properties of chemically deamidated and/or double enzymically-treated gluten

were studied. Performances of the modi®ed glutens were compared to those of egg white proteins,

casein, deamidated gluten and gluten submitted to deamidation plus single enzymic treatment. Higher

nitrogen solubility index (>90%) was obtained after treatment of the gluten with the sequence pepsin

�alcalase and deamidation�pepsin�papain. The sequence pepsin�papain has permitted the

production of a gluten with foaming properties close to those of egg white. Deamidation�papain

treatment leads to interesting emulsifying properties although different from those of casein. The

results show that modi®ed glutens present thresholds in the degree of enzymic hydrolysis (DH) for

foaming and emulsifying properties (respectively 1.3% and 2.0%). For higher DH the stability

decreases.

# 1999 Society of Chemical Industry

Keywords: gluten; functional properties; acid deamidation; enzymic hydrolysis

INTRODUCTIONOver the last 15 years the large increase in starch

demand has allowed production of increasing quan-

tities of wheat proteins. Gluten proteins possess typical

viscoelastic properties and their use remains limited to

bread-making industries. Finding a way to increase the

applications of this insoluble protein is economically

desirable to extend its range. As a side-effect of the

wheat starch industry, many researchers have devel-

oped methods of modifying the solubility and func-

tional properties of gluten. Among the methods

employed to improve solubility and surface properties,

chemical1 and enzymic treatments2±4 appear to

provide an effective way to improve the functional

properties of gluten. Previous studies5 have shown the

in¯uence of a double treatment (deamidation and

protease digestion) on the functionality of gluten.

In the present study, an attempt was made to

improve the functional properties of insoluble gluten

using double enzymic hydrolyses either alone or in

combination with acid deamidation.

MATERIAL AND METHODSMaterialGluten (75% (w/w) protein, dry basis), produced by

the Martin process, was provided by Amylum

(Bordeaux, France). The enzymes were purchased

from Novo (Alcalase 0.61: 0.6mAnsonmgÿ1,

Neutrase 0.50: 0.54mAnsonmgÿ1), Merck (Papain:

3.5mAnsonmgÿ1) and Serva (Pepsin: 15mAnson

mgÿ1). Casein and ovalbumin were supplied by

Merck.

DeamidationDeamidation was carried out as described by Popineau

et al.6 A dispertion of gluten (25mgmlÿ1) was stirred

in 0.1M HCl at 70°C for 1h (D1) or 2h (D2). The

reaction was stopped by cooling the samples quickly in

an ice bath followed by neutralisation (pH 7) with

NaOH.

Enzymic proteolysisGluten (25mgmlÿ1) was dispersed in different buffers

depending on the optimum pH for the enzymic

activities used.

Single treatments

For pepsin the sample (2.5g) was dispersed in 100ml

of 0-1M HCl at 30°C. The enzyme was dissolved in

0.5ml of distilled water before addition to the

substrate (E/S =1/33 (w/w)). The reaction time varied

according to the experiments (see results). The

reaction was stopped by neutralisation to pH 7.

For alcalase or papain, the sample (2.5g) was

dispersed in 100ml of 5mM Tris buffer, pH 8 at

30°C. The enzyme was solubilised in 0.5ml of distilled

water before addition to the substrate (E/S =1/33

(w/w)). The reaction time varied according to the

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1048±1053 (1999)

* Correspondence to: Jacques Raymond, ISTAB, Laboratoire de Biochimie et Technologie des Aliments, Universite Bordeaux I, avenue desFacultes, 33405. Talerce Cedex, France† Present address: Facultes des Sciences et Techniques, Department de Biologie, Av. A. El Khattabi, BP 618, Marrakech, Morocco(Received 8 May 1997; revised version 2 November 1998; accepted 17 December 1998)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1048

experiments (see results). The reaction was stopped by

boiling.

Double treatments

Pepsin hydrolysis preceeded a treatment with papain

or alcalase. Experimental conditions were the same as

above and, in the E/S ratio, E now represents the sum

(E1�E2) of the two enzymes (E/S =1/33 (w/w)).

The second enzyme was dissolved in 0.5ml distilled

water prior to its addition and the proportions between

the two enzymes were: pepsin/papain (or alcalase),

33/66, 50/50 or 66/33 (w/w). The reaction times

varied according to the tests and the reactions were

stopped by boiling.

Determination of the degree of deamidationAmmonia released by deamidation was determined by

a microdiffusion method.7 Deamidation of the differ-

ent samples is expressed as a proportion (%) of the

amount of ammonia released by complete deamida-

tion of gluten:

Per cent

deamidation��NH3� released by partial

deamidation of gluten

�NH3� released by

complete deamidation

of gluten

� 100

Ammonia determination was carried out directly on

aliquots of the reaction medium. Complete deamida-

tion of gluten was carried out according to Kato et al.8

Evaluation of the degree of enzymic hydrolysis (DH)The amount of released amino groups resulting from

gluten hydrolysis was estimated according to the

technique of Masson et al9 using reaction with

ninhydrin. Standard calibration curves were made

with leucine under the same experimental conditions.

Since the amount of leucine equivalents generated is

related to the hydrolysis equivalent, h, the curves

presented permit the number of peptide bonds cleaved

during the hydrolytic process to be determined when

reported asg of protein.

Deamidation plus single enzymic treatmentIf pepsin treatment follows the deamidation process,

the enzyme is added without pH correction. Con-

versely, before the use of papain or alcalase, the pH

must be adjusted to 8 and continuously checked and

re-adjusted until the end of the reaction. The experi-

mental conditions are the same as previously described

except for papain treatments where the ratio E/S is

1:66.

SolubilityGluten (native or treated) dispersions, at various pH

values, were centrifuged at 3000�g for 15min. The

nitrogen content of the various supernatants was

determined by the Kjeldahl method using the N to

protein conversion factor of 5.7 for wheat proteins.

Relative solubilities at various pH values, adjusted by

addition of 0.1M NaOH or HCl, were estimated as the

solubilized proportion (%) of the total nitrogen value

(determined on native gluten), ie as nitrogen solubility

index (NSI):

NSI �%� � amount of N in the supernatant

amount of total N� 100

Foaming propertiesGluten suspensions (1.75% (w/v) of protein, pH 7,

20ml) were introduced into the blender bowl of an

homogeniser (Virtis 60K) and mixed for 2min at

10000revminÿ1. The total volume of foam was

measured. The quantity of liquid that had drained

from the foam after 2 and 10min was used as the foam

stability indicator.10,11

Emulsifying activityEmulsifying activity was determined according to

Swift et al.12 The protein solutions were introduced

into a homogeniser (Virtis 60K) including a side arm

for addition of sun¯ower oil. An excess of oil causes

the emulsion to break as well as a substantial decrease

in its viscosity and, consequently, an increase in the

rotational velocity of the homogeniser. The emulsify-

ing activity is de®ned as the volume of sun¯ower oil

(ml) that can be emulsi®ed by 1g of protein. After

different tests, in which the rate of oil addition, the rate

of stirring and the protein concentration were varied

systematically,8 the following experimental conditions

were established: the gluten suspension (1% (w/w) of

protein, pH 7, 10ml) was mixed at 5000revminÿ1; the

¯ow rate for oil addition was 3.2mlminÿ1.

Emulsion stability (or flocculation-creaming kinetic)Emulsion stability was determined according to the

method of Dagorn-Scaviner et al.13 To prepare the

emulsion, dodecane (10ml) as apolar phase and

protein solution (15mg in 0.1M phosphate buffer,

pH 8, 30ml) were blended together at 20000

revminÿ1 for 30s at 25°C with an Ultra-Turrax

homogeniser. The ¯occulation-creaming phenomen-

on was followed as a function of time after the

emulsion was transferred into a 10ml measuring

cylinder. The volume of separated aqueous phase

and creamed phase was plotted as a function of time

over a period of 60min at 25°C. The kinetics were

analysed by plotting ln (Ve/VeÿVt)=k1t, where Vt is

the volume of separated aqueous phase at time t and Ve

the value of V after 24h (taken as the equilibrium limit

of the process). This enabled a determination of the

drainage rate constant, k1. All the results presented

concerning hydrolysis, deamidation and functionality

are the mean of triplicate experiments.

RESULTS AND DISCUSSIONSolubilityWhen compared to simply treated (deamidated or

J Sci Food Agric 79:1048±1053 (1999) 1049

Deamidation hydrolysis effects on gluten

hydrolysed) and doubly treated (deamidated�hydro-

lysed) glutens,5 the glutens treated either with a double

hydrolysis or a deamidation followed by a double

hydrolysis present a better solubility (Table 1). The

highest NSI (>90%) were obtained either after pepsin

�alcalase, (50/50, 10min �10min) treatment (NSI=

92.4) or with a deamidated gluten followed by a

double pepsin�papain hydrolysis (D2�pepsin�papain, 66/33, 5min�15min) (NSI=93.3%). In this

case, identical NSI were obtained with different DH

(2.1% and 1.6%, respectively). As demonstrated by

Thebaudin,3 different proteases do not improve the

solubility of gluten with the same ef®ciency. The

solubility is related not only to the DH but also to the

physico-chemical properties of the resulting peptides

(molecular weight, charge, surface hydrophobicity).

Proteases with the same speci®c activity may have

different speci®cities and cleavage sites on the poly-

peptide chain. This demonstrates that higher solubility

may be obtained with low DH avoiding the formation

of bitter peptides.14

The same observation may be made when deami-

dated gluten is then treated with alcalase (D2�alcalase, E/S =1/33). After 10min treatment the NSI

reached 88.5% (DH=1.5%) which is not signi®cantly

different from a 1h treated gluten whose NSI was

87.8% (DH=4.2%).5 This demonstrates that alcalase

produces soluble peptides in the ®rst period of

hydrolysis. This has been previously observed.15 Our

results con®rm those of Batey.16 Using different

experimental conditions, Adler-Nissen17 and Kim etal18 also observed the ef®ciency of alcalase treatment

on soya proteins.

Foaming propertiesWhen compared to our previous report,5 the foaming

properties (foaming capacity and foam stability) were

signi®cantly improved when gluten was submitted to a

double enzymic treatment (with or without a pre-

liminary deamidation process) (Fig 1). Modi®ed

glutens often displayed better foaming capacity than

egg white (reference), and equivalent foam stability,

particularly when gluten was hydrolysed by pepsin and

papain (66/33 (w/w), 5min�15min, DH=1.2%).

Whatever the treatment, it posessed better foaming

properties than casein.

Results mainly demonstrate that the improvement

of the foaming properties was strongly associated with

the double enzymic treatment (pepsin�papain).

According to our earlier results, the hydrolyses of

gluten using either pepsin or papain improved the

solubility and the foaming capacity but led to a low

foam stability. It appears now that the double enzymic

treatment (pepsin�papain) permits the production of

peptides with good foaming capacities able to adsorb

at the air±liquid interface, thus realising the three

necessary steps for development of foam struc-

ture.19,20

Glutens having the best foaming capacity are those

that have been deaminated prior to enzymic hydro-

lyses (Fig 1, numbers 8 and 9). Nevertheless the

stability remains low, showing that an improvement in

the solubility is not suf®cient to obtain stable foams.

The same observation has been observed when

deamidation was followed by a single enzymic hydro-

lysis.5 When the DH remains <10% the foaming

capacity increases21 but is accompanied by a decrease

in foam stability.22

Table 1. Effects of chemical and enzymic modifications on gluten solubility

Different treatments employed NSI (%)

1. pepsin (E/S =1/33, 10min) 81.1�5.0

2. D2a�alcalase (E/S =1/33, 10min) 88.5�6.5

3. pepsin �alcalase (50/50, 10min�10min) 92.4�5.5

4. D2�pepsin �alcalase (33/66, 5min�15min) 86.3�6.5

5. D2�pepsin�papain (66/33, 5min�15min) 93.3�7.2

6. D2�pepsin�papain (66/33, 5min�30min) 90.2�6.0

a D2: deamidated gluten (HCl 0.1M, 2h, 70°C).

Data are mean�SD of triplicate determinations.

Figure 1. (a) Volume and (b) stability of gluten foams after differenttreatments. Open and hatched boxes correspond to the released liquidafter 2 and 10min, respectively. Numbers correspond to the differenttreatments employed: 1, casein; 2, egg white (used as references);3, alcalase (E/S =1/33, 10min); 4, pepsin�papain (50/50, 5min�5min);5, pepsin�papain (50/50, 5min�10min); 6, pepsin�papain (66/33,5min�15min); 7, pepsin�papain (66/33, 5min�30min);8, D1*�pepsin�papain (66/33, 5min�15min); 9, D1�pepsin�papain(66/33, 5min�30min). *deamidated gluten (HCl 0.1M, 1h, 70°C). Barsrepresent standard deviations.

1050 J Sci Food Agric 79:1048±1053 (1999)

B Mimouni, JL Azanza, J Raymond

Emulsifying propertiesWe have improved the emulsifying capacity of gluten

but the stability remains in the same range when

compared to casein (Table 2). The majority of

modi®ed glutens having the best emulsifying capacity

have been ®rst deaminated and then hydrolysed with

only one enzyme. The sequence D2�papain (E/S =

1/66, 30min) gives the best result. The doubly treated

glutens possessing good emulsifying properties are

those submitted to the sequences: D2�pepsin�papain (50/50, 5min�30min) or D2�pepsin�papain (66/33, 5min�30min). Nevertheless all these

glutens display lower performances than casein,

particularly concerning the stability.

We observed that all these glutens were deaminated

prior to the enzymic treatment. This is in accordance

with our previous results showing that deamidation is a

prerequisite for the improvement of the emulsifying

properties. The sequence deamidation plus hydrolysis

increases the solubility and the net charge; both at

these are necessary to promote emulsifying properties.

It has been also shown that chemical deamidation of

proteins increases their interfacial activity.23,24 We

observe that, with the same hydrolytic conditions, and

when the deamidation time is increased, the emulsion

capacity is enhanced (Table 2, lines 5, 11). This

corresponds to previously reported data obtained with

different experimental conditions by Wu et al,25

Matsudomi et al23 and Popineau et al.6

When the enzyme concentration (Table 2, lines 4, 6

and 7, 11) or the hydrolysis time (Table 2, lines 4, 7

and 6, 11) is increased we also improve the emulsion

capacity. The enzymic treatment has mainly a role in

the increase of the nitrogen solubility2,3,5 except for

alcalase (Table 1, lines 1, 2).

The general observation resulting from these data is

the low stability of all the modi®ed glutens when

compared to casein. Increasing the emulsion capacity

without increasing the stability (Fig 2) shows the lack

of relation between these two functional properties.

This is particularly true for deaminated glutens either

simply treated (pepsin) or doubly treated (pep-

sin�papain) (Table 2, lines 8, 9, 10). Thebaudin3

has also reported contradictory results between emul-

sifying capacity and stability. These discrepancies may

also result from pH differences. The data of Thebau-

din3 on gluten and Elizalde et al26 on milk proteins

have shown that the stability of the emulsions is better

at pH 4 than at pH 7.

On the other hand, it has been shown that to avoid

coalescence and ¯occulation, the interfacial proteic

®lm must be as thick as possible, strongly hydrated and

carrying charges.13 Data on faba beans, soy beans and

casein have demonstrated that an extensive hydrolysis

has a negative effect on the emulsifying properties and

particularly on the stability giving birth to peptides

Table 2. Effects of chemical and enzymic modifications on the emulsifying properties of gluten

Different treatments employed Emulsion capacity (ml oil per g protein) Emulsion stability (k1�10ÿ4sÿ1)

1. Caseina 857�12.0 2.0�0.2

2. Gluten (D2)b�alcalase (E/S =1/33, 10min) 585�9.0 54.5�6.0

3. Gluten (D2)�alcalase (E/S =1/33, 5min) 595�9.0 52.5�6.0

4. Gluten (D2)�papain (E/S =1/100, 15min) 647�9.0 45.1�5.0




8. Gluten (D2)� (pepsin�papain) (66/33, 5min�30min) 715�10.0 72.0�7.5

9. Gluten (D2)�pepsin (E/S =1/33, 10min) 724�10.5 81.6�8.0

10. Gluten (D2)� (pepsin�papain) (50/50, 5min�30min) 747�10.5 85.2�8.0


a Casein (reference).b D1 and D2: deamidated gluten (HCl 0.1M, 1h and 2h, respectively, 70°C).

Data are mean�SD of triplicate determinations.

Figure 2. Relations between emulsifying capacity and emulsion stability indeamidated and hydrolysed glutens. Numbers correspond to the differenttreatments employed: 1, D2*�alcalase (E/S =1/33, 10min);2, D2�alcalase (E/S =1/33, 5min); 3, D2�papain (E/S =1/100, 15min);4, D1*�papain (E/S =1/66, 30min); 5, D2�papain (E/S =1/66, 15min);6, D2�papain (E/S =1/100, 30min); 7, D2� (pepsin�papain) (66/33,5min�30min); 8, D2�pepsin (E/S =1/33, 10min);9, D2� (pepsin�papain) (50/50, 5min�30min); 10, D2�papain(E/S =1/66, 30min). *D1 and D2, deamidated gluten (HCl 0.1M, 1h and 2h,respectively, 70°C). Bars represent standard deviations.

J Sci Food Agric 79:1048±1053 (1999) 1051


with low water adsorption capacity.26 Extensive

hydrolysis (as in our experiments with pepsin) (Fig

2, number 8, DH=3.1%) or with pepsin�papain (Fig

2, number 7, DH=3.45%) may generate charged

peptides unable to build thick ®lms and having a low

water binding capacity (because of their low Mr). In

these conditions, it is impossible to obtain a steric

stabilisation of the emulsion. The high emulsifying

capacity probably results from the amphiphylic nature

of the peptides (appearance of a surface hydrophobi-

city and increase of the net charge may result from the

deamidation process).

CONCLUSIONFoaming and emulsifying properties appear to be

different in nature. Good foaming properties are

obtained with the use of the sequence pepsin�papain

(with or without 1h deamidation). The sequence

pepsin�papain (66/33, 5min�15min) on gluten gave

results which may be compared to those of egg white

proteins. Good emulsifying properties were obtained

partly using the deamidation (D1 or D2) sequences�papain and partly the sequences D2�alcalase,

D2�pepsin or D2�pepsin�papain. When compared

to casein, the best data result from the sequence

D2�papain (E/S =1/66, 30min).

Glutens presenting good surface properties are

different from those having a good solubility. Our

results (including previous data from Mimouni et al5)

were submitted to statistical analyses (STAT-ITCF

program) which demonstrated that solubility was

strongly correlated with emulsifying properties and

less so with foaming properties (results not shown).

Different thresholds permit discrimination between

these two surface properties. The foaming properties

are better expressed in slightly modi®ed glutens, and,

conversely, emulsifying properties appear in more

deeply modi®ed glutens. Foaming and emulsifying

properties (particularly the stabilities) decrease when

DHs reach 1.3% and 2.0%, respectively. These results

are in accordance with those showing that for

DH>2.0%, modi®ed glutens present low surface

properties.27 Our results clearly demonstrate that

foaming properties of gluten were obtained after mild

(pepsin�papain) treatments and emulsifying proper-

ties of gluten result from more drastic treatments

(deamidation�pepsin�papain).

It is dif®cult to predict the behaviour of modi®ed

glutens but investigations are now under way in order

to identify the responsible peptides. To understand the

function of the proteins, their degradation products

and their denatured states would permit understand-

ing and further improvement of the stability of foams

and emulsions.

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Influence of double enzymic hydrolyses on gluten functionality