Vol. 1 Issue 4, pp: (64-78), July 2016. Available online at: http://www.prudentjournals.org/IRJAFS

Full Length Research Paper

Effects of Enzymatic Hydrolysis on the Foaming Properties of Whey Proteins

Griselda Ballerini1, 2 *, Marta Ortega1, and Virginia Giordanengo1 1Centro de Investigación y Desarrollo en Tecnología de Alimentos CIDTA. Facultad Regional

Rosario, Universidad Tecnológica Nacional. E. Zeballos 1341. Rosario 2000, Santa Fe, Argentina.

2Área de Tecnología de los Alimentos. Departamento de Tecnología. Facultad de Ciencias. Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario. Suipacha 530. Rosario. 2000.

Santa Fe. Argentina.

*Corresponding author. E-mail: griballerini@yahoo.com.ar

Received 29 February, 2016; Accepted 22 June, 2016.

ABSTRACT

Changes in the foaming properties of whey proteins concentrate (WPC) subjected to enzymatic

hydrolysis were studied. Activity and stability of the foam generated from the enzymatic hydrolysis of

WPC with two proteases: Trypsin from porcine pancreas (analytical quality) and Alcalase® 2.4 L FG

(Alcalase) (commercial food grade) were analyzed. WPC solution at concentration 100 mg/mL in 50

mM phosphate buffer, pH 8,0 were hydrolyzed with Trypsin and Alcalase at the ratio of

enzyme/substrate 1:100 and 1:250, respectively. The reactions were stopped by heating. The

relationship between the concentrations of different species with hydrolysis time was studied by

means of electrophoretic techniques and it was found that Alcalase hydrolyzed more efficiently than

Trypsin. For Alcalase hydrolysates of WPC the degree of hydrolysis, for 60 minutes of treatment,

was about 45% while for Trypsin hydrolysates, with the same treatment time, was around 7%.

Foams were obtained by bubbling. We could observe little variation on the activity of the foams

obtained with both enzymatic treatments for all times of hydrolysis. Foams obtained from Trypsin

hydrolysates were more stable than the ones obtained from Alcalase hydrolysates. Although foams

formed with WPC hydrolysates using Alcalase were less stable than their counterparts obtained with

Trypsin, faster hydrolysis, availability and lower cost make Alcalase a very good choice when you

want to work with additives obtained by hydrolysis of WPC having improved foam stability.

Keywords: Alcalase, Trypsin, enzymatic hydrolysis, foaming properties, hydrolysis degree,

proteases, whey protein concentrate (WPC).

INTRODUCTION

The increasing preference of consumers for

tastier and healthier foodstuff has meant a

unique opportunity for the dairy industry to

develop and provide protein ingredients to

improve the nutritional and functional properties

of foods [51].

Proteins are one of the main constituents of

food due to their structural and functional

properties and they determine the sensorial

characteristics thereof. The study of the

International Research Journal of Agricultural and Food Sciences Article Number: PRJA54259933 Copyright ©2016 Author(s) retain the copyright of this article Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution

4.0 International License.

relationship between protein structure and

function is the basis for the rational design of

protein ingredients with specific functionality to

be applied to the development of foods with

certain organoleptic and textural characteristics,

to the formulation of low-fat and low-cholesterol

foods, or simply as stabilizers, emulsifiers,

gelling agents or foaming agents [7].

Whey is obtained as a by-product when the

casein fraction of the milk coagulates for

obtaining cheese or casein. Besides the

minerals and lactose, whey contains about

20 % of proteins originally present in the milk

[51]. Its concentration depends on the type of

whey, stage of lactation, health and nutritional

status of the animals, climate and processing

conditions [25]. Whey was for a long time, more

a problem than a product of interest [51]. Its use

as fertilizer or livestock food is of little

commercial interest, and its disposal is

problematic because of its highly polluting

effects [60].

The processes for obtaining whey proteins are

diverse, depending on the available raw

material, the degree of purity and desired

performance. The most common processes are:

casein fraction separation by acid precipitation,

by enzymatic coagulation or by centrifugation,

followed by purification, ultrafiltration or electro-

dialysis to remove the lactose and minerals.

The membrane separation techniques and

drying have contributed to the isolation of whey

proteins and the commercial production of whey

protein concentrates (WPC). The WPC are

presented in cream colored powder form, with a

characteristic smell. Except for moisture, the

proportion of liquid whey constituents remains

the same in the WPC. The usual protein content

of the WPC ranges between 40 % and 80 %

W/W [25].

The dairy companies located in the so called

"Argentinian dairy area" (in the provinces of

Santa Fe and Buenos Aires) produce WPC

regularly in the production chain of their

facilities. Besides a high nutritional value, WPC

exhibit functional characteristics (such as gelling

agents, and to a lesser extent, emulsifiers and

foaming agents) that have led to their use as

additives in the food industry for dairy, meat and

bakery products. They can be used to modify

the surface activity of food, as well as its

organoleptic, structural, textural, rheological and

hydration properties, allowing greater

commercial acceptance of products [60].

Attempts to make better use of this by-product

must inevitably go through maximizing the use

of potentially valuable components, especially

protein [25]. Recently, there has been a marked

improvement in the detailed study of the

structure of the major milk proteins, which has

led to the description and explanation of many

of their functional properties, even at the

molecular level [60].

In most of the foodstuffs that involve the

presence of foam, the proteins are the agents

that manage the formation and stabilization of

the dispersed gas phase, forming an elastic

protective barrier between gas bubbles trapped

into aqueous phase.

In general, for the formation and stabilization of

foam, the protein has to be quickly adsorbed at

the interface, so it must spread, penetrate and

be rearranged at the interface. This requires,

first of all, being soluble and flexible, having low

molecular weight or being dissociated and

possessing adequate lipophilic-hydrophilic

balance, given by the ratio (surface

hydrophobicity)/(surface charge). On the other

hand, to increase the stability of the foam, a

stable film should be formed surrounding the

bubble, with specific rheological characteristics

for which the adsorbed protein molecules must

be associated with each other (whether by

disulfide bridges or hydrophobic interactions) to

form large aggregates of low surface charge

and high water absorption capacity. To stabilize

the foams more effectively, the protein must

have both surfactant and film-forming effects.

The foam stabilizing capacity of a protein is

modified by environmental factors (pH, ionic

strength and temperature) and by factors

associated with the protein itself (protein

concentration, solubility, surface hydrophobicity,

net charge and superficial, molecular size and

flexibility) [21].

We have proved that structural modifications of

proteins can lead to the improvement of some

of its functional properties; these modifications

can be achieved by thermal treatments [34],

[35], [36] chemical or enzymatic hydrolysis [2], [4],

65 Int. Res. J. Agric. Food Sci.

Maillard reaction [3], transglutaminase action [2],

and others.

Protein hydrolysis is a powerful tool in the

modification of the functional properties of

proteins in food systems, including solubility,

gelification, emulsifying and foaming

characteristics. Proteolysis, besides decreasing

the molecular weight, also increases the

number of ionizable groups and can expose

hydrophobic groups, which can change physical

or chemical environmental interactions [54]

During hydrolysis, proteins are broken down

into peptides of different sizes and free amino

acids, as a result of cleavage of peptide bonds.

Enzymes, acids or alkali can carry out this

degradation. Both, acid and alkaline, hydrolysis

tends to be difficult to control and yields

products with reduced nutritional qualities.

Chemical hydrolysis can form toxic substances

like lysino-alanine [30]. On the other hand,

enzymatic hydrolysis developed under mild

conditions of pH (6–8) and temperature (40–60

ºC) may also lead to the development of

biologically active nutritional components to

promote health-giving opportunities for the use

of dairy ingredients [50]. For the rapid and

complete hydrolysis of whey proteins with

Trypsin, a modification of the conformational

structure is necessary [23]. In the change of

conformation the proteins unfold and become

sensitive to the action of enzymes [44]; [52].

The main reason for unfolding is the cleavage of

one or more disulfide bonds, which hold the

protein molecule in its characteristic structure

and shape. Usually, enzymatic processes avoid

side reactions and do not decrease the

nutritional value of the protein source.

Additionally, enzymes present substrate

specificity which permits the development of

protein hydrolysates with better defined

chemical and nutritional characteristics. The

preservation of the properties of the generated

products, peptides and amino acids, is

especially desirable, thus, the hydrolysis

process must be carefully controlled [54].

Many studies have demonstrated that selective

enzymatic hydrolysis of salmon protein [18],

[26], [27], [48], herring [31], [47], capelin [49],

soy protein [5], [14], [22], [56], and whey protein

[2], [3], [12], [20], [23], [28], [34], [35], [36], [40],

[41], [42], [50] improved their functional

properties, including solubility, water holding, oil

holding, emulsifying and foaming

characteristics.

The aim of this work was to study the surface

properties of the foams (foam stability and

foaming capacity) obtained from WPC solutions

hydrolyzed with Trypsin and Alcalase, to

compare the results obtained with both

enzymes and to suggest new options for

obtaining additives from WPC, with improved

foaming properties.

MATERIALS AND METHODS

Materials

For this study we used WPC 80 % W/W from

Arla Food Ingredients SA, (Argentina),

Trinitrobenzene sulfonic acid (TNBS),

Coomassie Brilliant Blue R250, L-Leucine,

Trypsin from porcine pancreas (Type II-S,

declared activity 1,000-2,000 U/mg dry solid),

supplied by Sigma Aldrich, (USA) and

Alcalase® 2.4 L FG (declared activity 2.4 AU/g)

from Novo Nordisk, (Denmark). All other drugs

were of analytical quality.

Phosphate buffer (PB) (50 mM, pH 8,0) was

prepared by mixing 50 mL of 100 mM KH2PO4

solution with 47.6 mL of 100 mM Na (OH)

solution and adding distilled water to 100 mL.

Methods

Enzymatic hydrolysis with Trypsin at pH 8.0

Trypsin (at concentration 3% W/V dissolved in

1mM HCl) was added to WPC solutions (at

concentration 100 mg/mL in PB) to obtain an

enzyme/substrate ratio of 1:100 [4]. The

hydrolysis was carried out at 37ºC and stopped

after 30, 60, 120, 180, 240 and 300 minutes by

heating the respective solution at 80ºC for 5

minutes and subsequent cooling to room

temperature [4]. The samples were reserved for

further analysis.

Enzymatic hydrolysis with Alcalase

Alcalase was added to WPC solutions (at

concentration 100 mg/mL in PB) to obtain an

enzyme substrate ratio of 1:250 [12], [63]. The

hydrolysis was carried out at 50ºC and stopped

after 5, 10, 15, 30, 60 minutes by heating the

respective solution at 90ºC for 15 minutes and

Ballerini et al 66

subsequent cooling to room temperature [63].

The samples were stored for further analysis.

Determination of hydrolysis degree (HD) -

TNBS method

This method is a spectrophotometric test of the

chromophore formed by the reaction of

trinitrobenzene sulfonic acid (TNBS) with the

primary amine groups. TNBS also reacts slowly

with hydroxyl ions, through which the reading of

the blank increases and this increase is

stimulated by light. It is an exact and

reproducible method for determining hydrolysis

degree of food protein hydrolysates [1].

Standard curve of L-Leucine

The standard curve of L-Leucine (L-Leu) was

generated with ordered pairs (mg L-Leu;

Absorbance at 340 nm) obtained by applying

the method of TNBS [1] to samples of 0.088

mM L Leu solution in 1% SDS. Samples were

taken with L-Leu content from 0.01 mg to 0.05

mg.

Determination of 100% WPC hydrolysis

Four milliliters of HCl 6 N were added to 10 mg

of WPC. The mixture was kept at 110°C for 24 h

and let reach the room temperature, then pH

was adjusted to 8.2 with 3N Na(OH) solution.

The TNBS method was applied to a sample of

this solution and the absorbance at 340 nm was

read against the reagent blank. The amount of

L-Leu corresponding to this absorbance value,

according to the standard curve (2.2.3.1), was

taken as reference for 100% of hydrolysis.

TNBS method for hydrolyzed samples

The TNBS method was applied to samples

containing the same mass of hydrolysates as

the mass of L-Leu taken as reference for 100%

hydrolysis.

Electrophoresis

Electrophoresis was performed in polyacrylamide

gels (SDS-PAGE) following the method described

by Laemmli [29] using a stacking gel of 10 % and a

separating gel of 15 %. Six microliters of each

sample were seeded and proteins of known

molecular weight were used as standards (data not

shown). The electrophoretic runs were carried out

in denaturing and non-reducing conditions at

constant voltage of 150 V for 90 minutes.

Afterwards, gels were stained with Coomassie

Brilliant Blue R250 and scanned using a

Hewlett-Packard Scanjet 5p connected to a

computer. To quantify the concentration of the

different species, the relative intensity of the

colored bands in the scanned images of the

gels was analyzed using specific non-

commercial software developed by our group

[36], [39].

Foaming properties

Foams were formed using a bubbling apparatus

[2], [3], [4], [10], [19], [34], [35], [36]. Both,

untreated control sample and hydrolyzed WPC

were diluted to 5 % (W/V) in 20 mM phosphate

buffer, pH 6.8. Determinations were made in a

transparent acrylic tube (3.5 cm x 20.0 cm)

equipped with a pair of electrodes and a porous

disk located at the base of the tube. Ten

milliliters (Vinit) of dilution were placed in the

tube and air was forced to pass through the disk

at a constant flow rate of 5 mL/s, creating foam.

Bubbling was stopped when the foam reached a

set volume of 115 ml (Vf). During the test, the

conductivity and the volume of foam were

recorded by a computer and a digital camera

Fujifilm Finepix S8100. Conductivity

measurements at different times (Ct) and the

initial conductivity (Cinit), were used to calculate

the volume of liquid incorporated in the foam

(VLF) (equation 2-1) [10], [58].

VLF = Vinit∙ [1-Ct

Cinit] 2-1

The maximum foam density (FD) was taken as

a measure of foaming activity. FD is defined as

the ratio between the maximum amount of liquid

incorporated into the foam (VLFmax) and the foam

volume reached at the end of the sparging

period (VFmax).

DF =VFLmax

VFmax 2-2

where VFmax = Vf − (Vinit − VLFmax)

In terms of volume variation with time, the

longer it takes the foam to collapse, the more

stable the foam is [15]. To quantify the stability

of the foams it was measured the time interval

that takes to collapse the foam to half of the

maximum volume (T1/2). It is expressed as T1/2 =

t1/2 – t0, where t1/2 is the time at which the

volume of foam is reduced to half of the

67 Int. Res. J. Agric. Food Sci.

maximum volume and t0 is the time at the end of

bubbling [2], [3], [4], [34], [35], [36].

RESULTS AND DISCUSSION

Electrophoresis

The electrophoretic gels showed well-defined

bands for β-LG and α-LA, a broad band for

species of molecular weights between 30 and

60 kDa and another diffuse band for species of

molecular weights above 60 kDa. The analysis

of these gels (Figures 1 and 2) showed

decreasing concentrations of β-LG and α-LA

with hydrolysis time. The same was observed

for species of molecular weights between 30

and 60 kDa and those of molecular weights

above 60 kDa. Some of the latter remained in

the stacking gel when treatment times were less

than 15 minutes when hydrolyzed with Alcalase

and less than 60 minutes when hydrolyzed with

Trypsin. In the same figures it can be observed

that the concentration of β-LG dropped more

abruptly in Alcalase hydrolysates than in their

counterparts hydrolyzed with Trypsin. Similar

concentrations were reached for 10 min of

treatment with Alcalase, and 150 min of

treatment with Trypsin. These results show that

Alcalase has a greater hydrolyzing capacity

than Trypsin. Regarding this, Davis et al. [12]

informed that Alcalase is a serine alkaline

protease produced by a selected strain of

Bacillus licheniformis. Its main enzyme

component, subtilisin Carlsberg, has broad

specificity, hydrolyzing most peptide bonds,

preferentially those containing aromatic amino

acid residues. Within whey proteins, Alcalase

was observed to have a high specificity for not

only aromatic amino acid residues (Phe, Trp,

and Tyr) but also for acidic (Glu), sulfur-

containing (Met), aliphatic (Leu and Ala),

hydroxyl (Ser), and basic (Lys) residues [15].

The high HD obtained with this enzyme

supports this hypothesis. Trypsin specifically

cleaves at lysine (Lys) and arginine (Arg)

residues for which there are 18 total sites in the

β-LG primary sequence [4], explaining its

relatively less HD [12].

Fig 1. Concentration of WPC species by densitometry

Ballerini et al 68

Fig 2. Concentration of WPC species by densitometry

Fig 3. Hydrolysis degree for WPC hydrolized with Trypsin

69 Int. Res. J. Agric. Food Sci.

Fig 4. Hydrolysis degree for WPC hydrolyzed with Alcalase

Hydrolysis degree

The corresponding data for both treatments, which

were obtained in triplicate, (Figures 3 and 4) were

statistically analyzed using SigmaPlot version 10

software. Good fits (p < 0.05) were obtained for two

parameters hyperbolas asymptotic to 21.7 %

(hydrolysis with Trypsin) and 58.3 % (hydrolysis

with Alcalase). The HD reached for maximum

hydrolysis time tested was around 14 % for Trypsin

hydrolysates (300 min) and around 45 %, for

Alcalase hydrolysates (60 min). It can be noted,

from the HD values obtained for hydrolysates

treated for 60 min with Alcalase, the presence of

very low molecular weight structures that are not

suitable as food additives because of their

unpleasant tastes. In this regard, Tavano et al. [54]

indicate that the hydrolysis conditions need to be

controlled to avoid excessive protein hydrolysis that

can impair functionality and cause unfavorable

effects, such as production of bitter-flavored

peptides.

In recent research, Xiangzhen et al. [62] proved

that, when wheat gluten proteins were treated with

various enzymes, the Alcalase hydrolysis produced

more polypeptides of low molecular weight than

treatments with Pepsin, Pancreatin and Neutrase.

Wróblewska and Troszyñska [61] studied the

hydrolysis of whey protein with Papain, Lactozime

and Alcalase and obtained the highest values of

HD with Alcalase. Smyth and Fitzgerald [53]

hydrolyzed a WPC preparation applying Alcalase

0.6 L. After 30 min of the process they observed

the presence of protein fractions of molecular

weight below 30 kDa. Further hydrolysis increased

the percentage of low-molecular weight peptides

even more. Electrophoregrams of hydrolyzed

proteins obtained after 8 hours of hydrolysis

revealed only the presence of fractions below 14

kDa. The application of Neutrase, a neutral

proteinase from Bacillus subtilis, caused similar

results. According to Wróblewska and Troszyñska

[61], β-LG is a protein which due to a high lysine

content is capable of forming complexes with

lactose. In regarding this, Morgan et al. [33],

studied the vulnerability of lactose β-LG present in

a commercial preparation of β-LG to the action of

β-galactosidase isolated from Kluyveromyces lactis

yeast. No hydrolytic action of the enzyme on

lactose bound to complexes with proteins was

observed even following long (6-h) hydrolysis.

Although Alcalase and Trypsin are serine

proteases, they differ in the type and amount of

Ballerini et al 70

peptide bonds that can break [12], [15]. This fact,

coupled with the possibility of lactose to form

complexes with β-LG and α-LA could influence the

hydrolyzing capability of both enzymes and

therefore the HD.

Foaming properties

Foaming activity

Figures 5 and 6 illustrate the variations of the

foaming activities expressed by FD/FD0 relative

parameter, where FD0 is the maximum foam

density, obtained by foams formed from untreated

control sample. The same figures show that there

is very little influence of the kind and time of

treatment on the FD values. In the case of Trypsin

hydrolysates, it was observed that the maximum

foaming activity was obtained at 120 minutes of

treatment, which represents an increase of around

15 %, relative to the maximum volume of liquid

incorporated in the foam, compared to the

untreated control sample and it was obtained with

a HD of around 11 % (Figures 3 and 5). With

respect to the WPC hydrolyzed with Alcalase, the

maximum foaming activity was obtained for 30

minutes of hydrolysis with an increase of

approximately 12 %, relative to the maximum

volume of liquid incorporated in the foam,

compared to the untreated control sample and it is

obtained with 35 % of HD approximately (Figures 4

and 6). These results are consistent with Kirara

and Panyam [24], who reported that it is well

known that a limited treatment with proteases (at

low hydrolysis degree) usually improves interfacial

properties of globular proteins, mainly due to an

increase in exposed hydrophobic areas. Pérez et

al. [41] observed the effect of HD (in the range of

0.0–5.0%) on b-LG foam formation and concluded

that α-chymotrypsin treatment produced a

significant increase in foaming activity. This

behavior would suggest that a limited hydrolysis

could improve the β-LG foaming power promoting

the formation of smaller and denser bubbles, and

increasing the liquid retention in foams. Moreover,

these results are in accordance with data about

enzymatic treatment of other proteins such as

soybean [5], [32], wheat [6], rapeseed [57] and

sunflower [46]. Proteins, due to their amphiphilic

nature, are distinguished by their good interfacial

and foaming properties. Thus, foam formation is

influenced by protein adsorption at the air–water

interface and its ability to reduce the system

interfacial tension [34], [35], 36], [40], [41]. In fact,

for foam formation, proteins must be adsorbed at

the interface in order to form a protective film

around gas bubbles. Furthermore, foam

stabilization against liquid drainage (gravitational

drainage and marginal regeneration),

disproportionation (gas diffusion from smaller to

larger bubbles), and coalescence (bubble rupture)

requires an adequate control of the bulk and

interfacial properties [8], [13], [17], [34], [35], [36],

[37], [45], [46], [59].

Sinha et al. [50] hydrolyzed whey proteins and

demonstrated that the foam volume of the control

sample was less than that of the treated samples.

Enzymatic hydrolysis of whey proteins caused an

increase in the foam volume initially and then a

decrease with time. In the fungal protease treated

sample the foam volume was found to be similar at

both 20 and 40 min of hydrolysis, with a significant

decrease after 60 min.

On the other hand, foaming capacity of a globular

protein could be linked with its diffusion rate toward

the air–water interface [9] and with the rheological

properties of protein interfacial films at short

adsorption times [40]. Relating the foaming with the

apparent diffusion rate constant and the initial

dilatational elasticity of the adsorbed films for β-LG

and its hydrolysates, Pérez et al. [41] suggested

that increment of β-LG foaming power with the

increase in diffusion rate could be associated to: (i)

reduction of β-LG molecular size, and/or (ii)

increment in its surface activity due to an increased

exposure of hydrophobic areas on the protein. In

our case, both could be a direct result of enzymatic

treatment and could act together in order to

increase the diffusion rate and consequently to

increase the protein foaming activity. According to

our results it would seem that the limited increased

foaming capacity obtained with both enzymes

would be linked to poor modification of the surface

hydrophobicity of the hydrolysates with respect to

the control sample, justified through the formation

of new disulfide bonds, intra and intermolecular,

capable of stabilizing the structures resulting from

the hydrolysis process without exposing highly

hydrophobic groups. It could be inferred that these

hydrolysates have a slower rate of diffusion to the

interface air/water and less reorganization capacity

therein. The small variation of the foaming activity

for all samples and all hydrolysis times, would

71 Int. Res. J. Agric. Food Sci.

indicate that hydrolysis did not produce changes

involving a structural modification in the flexibility of

the species formed during treatments.

Fig 5. Foaming activity of foams from WPC solutions hydrolyzed with Trypsin, expressed as relative density. VLFmax0 = 6.51 ± 0.34 mL

Fig 6. Foaming activity of foams from WPC solutions hydrolyzed with Alcalase,

expressed as relative density. VLFmax0 = 6.82 ± 0.35 mL

Ballerini et al 72

Fig 7. Stability of foams from WPC solutions hydrolyzed with Trypsin.

expressed as relative T1/2. T1/2(0) = 300 ± 60 s

Fig 8. Stability of foams from WPC solutions hydrolyzed with Alcalase.

expressed as relative T1/2. T1/2(0) = 300 ± 60 s

73 Int. Res. J. Agric. Food Sci.

Foam stability

Figures 7 and 8 illustrate the variation of the T1/2

relative parameter, T1/2 relative parameter is

defined as the relation T1/2/T1/2(0), where T1/2(0) is

the half-life of the untreated control sample foam.

The stability of the foams obtained from Trypsin

hydrolysates rapidly increases with the time of

treatment until reaching the maximum (60 min of

treatment with a HD of around 6.9 %) (Figures 7

and 3). This value indicates that the foam stability

was improved up to 3 times compared to the

untreated control sample. The same figures show

that maximum stability of the foams was obtained

at relatively low HD, which ensures the absence

of low molecular compounds that would produce

undesirable flavors if they were incorporated into

food. These results agree with those obtained by

Mutilangi et al. [38], who reported that the foams

generated by tryptic hydrolysates of WPC had

higher foaming activity and foaming stability than

those resulting from unhydrolyzed WPC solutions.

Figures 5 and 7 show that greater HD favors

activity more than foaming stability, considering, in

both cases, the treatment times and HD required

for obtaining the corresponding maximum. These

results are in agreement with those reported by

Wilde and Clark [43] and Foegeding et al. [17],

who evaluated the effects of controlled enzymatic

hydrolysis of WPC proteins on the foaming activity

and stability. They observed that increasing the

HD, the foaming stability and viscosity decreased

in relation to the untreated control sample;

besides, they showed that hydrolysates of WPC

protein, designed for foams, are generally

hydrolyzed in a low percentage. The fact that the

peptides are smaller than proteins allows a faster

adsorption, which would generate a better foam

production. They suggested that small peptides

do not interact molecularly as the proteins do,

making the interfacial net less stable. However,

the foaming activity is improved for HD between

5.4 % and 15.9 %. In our case, working with HD

not higher than 15 %, the stability of the foams

was improved significantly, being higher than that

of the untreated sample for all times of hydrolysis,

but the foaming activity was improved only to a

limited extent. Figure 7 shows that stability of

foams is favored mainly for low HD, reaching a

maximum for 60 min of treatment (approximately

6.9% HD) and then decreasing with time of

treatment. Nevertheless, even for 300 minutes of

treatment, foams from hydrolysates are more

stable than foams from untreated control sample.

These results agree with those obtained by

Mutilangi et al. [38] and Tosi et al. [55], who

reported that, for the range of HD analyzed, tryptic

hydrolysates of WPC had better foaming stability

than the untreated sample.

Figure 8 shows that the stability of the foams

obtained from Alcalase hydrolysates increased

until about 15 minutes of treatment where it

reached a maximum of about 1.8 times the value

for the untreated control sample. This maximum

value occurred with a HD of around 27 % (Figure

4). For hydrolysis times longer than 15 minutes,

stability of the foams decreased, being, for 60

minutes of treatment, about 40% of the foams

stability value obtained with untreated control

sample. Increasing HD, negatively influenced the

stability of foams, from a HD of 38.8 %

approximately, the stability of the foams was less

than values obtained with the untreated sample,

which would indicate the presence of structures of

very low molecular weight. Although these

structures diffuse rapidly to the interface, do not

have good viscoelastic properties [11], [16], [17],

[34], [35], [36].

Comparing the stability, foams generated from

Trypsin hydrolysates of WPC were more stable

than the foams from Alcalase hydrolysates

(Figures 7 and 8).

Our results agree with those reported by Mutilangi

et al. [38], who analyzed foams from Trypsin and

Alcalase hydrolysates of WPC. They concluded

that the Trypsin hydrolysates showed greater

stability than their counterparts obtained with

Alcalase and that in both cases the maximum

foaming activities were higher than foaming

activity of untreated control samples.

As shown in Figures 7 and 8, the maximum foam

stability, obtained for Trypsin hydrolysates, was

reached for 60 minutes of treatment with a HD of

about 6.9%, while for the samples hydrolyzed with

Alcalase, the maximum stability was reached for

15 minutes of treatment with a HD of about 27 %

(Figures 4 and 7). Considering that we worked on

hydrolysis with Alcalase with less enzyme-

substrate ratio than on hydrolysis with Trypsin,

and a higher HD was obtained in a shorter

Ballerini et al 74

treatment, the results show that Alcalase enzyme

hydrolyzed WPC to a greater extent than Trypsin.

Moreover, Sinha et al. [50], hydrolyzing whey

proteins with fungal proteases, showed that foam

stability of the control was greater than that of the

treated samples. Samples treated with fungal

protease and papain showed gradual decrease in

stability with an increase in proteolysis. The foam

stability at 60 min was almost negligible.

According to Ipsen et al. [20], the initial interfacial

viscosity of β-LG samples with limited hydrolysis

(19–26% degradation of β-LG) was increased

compared with untreated β-LG. More severe

hydrolysis, however, resulted in a much slower

initial increase in interfacial viscosity and lower

maximum value.

An increase in HD seemed to promote a more

rapid adsorption at the air/water interface. So,

increasing the HD should 1) yield a higher

percentage of smaller peptides and 2) increase

the potential for exposing previously buried

hydrophobic residues, both of which should

promote a more rapid adsorption [43]. However,

due to the different specificities of each enzyme

and hence different distributions of peptides within

each hydrolysate, a definitive statement relating

HD and adsorption rates is not possible [4], [12].

Foamability and foam stability are closely linked

with structural changes of the protein. The

increase in surface hydrophobicity is considered a

decisive factor in the improved foamability in spite

of

the presence of very low molecular weight

aggregates. Besides, hydrophobic interactions

improve foam stability trough rapid formation of a

viscoelastic film [2], [3], [4], [34], [35], [36].

CONCLUSION

Through the enzymatic hydrolysis controlled

with both proteases, the foam stability increased

during the treatment until the maximum was

reached.

Time of treatment did not produce a very

marked effect on the foaming activity, neither for

Tripsin hydrolysates nor for Alcalase

hydrolysates.

In neither case, the time in which the best

foaming stability was obtained coincided with

the maximum foaming activity.

In a comparative analysis, it can be inferred that

Alcalase enzyme hydrolyzed to a greater extent

than Trypsin. Trypsin and Alcalase hydrolysates

showed higher foaming stability than

unhydrolyzed WPC solutions, except when the

degree of hydrolysis of the Alcalase

hydrolysates was approximately 38.8%. Trypsin

hydrolysates showed better foaming stability

than those obtained with Alcalase.

Although foams formed with WPC hydrolysates

using Alcalase are less stable than their

counterparts obtained with Trypsin, faster

hydrolysis, availability and lower cost make

Alcalase a very good choice when you want to

work with additives obtained by hydrolysis of

WPC having improved foam stability.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

REFERENCES

[1]. Adler-Nissen J (1979) “Determination of the

degree of hydrolysis of food protein

hydrolysates by trinitrobenzenesulfonic acid”. J.

Agric. Food Chem., Vol. 27, pp 1256-1262.

[2]. Báez GD, Moro A, Ballerini GA, Busti PA,

Delorenzi NJ. (2011). “Comparison between

structural changes of heat-treated and

transglutaminase cross-linked beta-

lactoglobulin and their effects on foaming

properties”. Food Hydrocolloids, Vol. 25, pp

1758-1765

[3]. Báez GD, Busti PA, Verdini R, Delorenzi NJ

(2013). “Glycation of heat-treated β-

lactoglobulin: Effects on foaming properties”.

Food Res. Int., Vol. 54, pp 902–909

[4]. Ballerini G (2011). “Desarrollo de un bio-reactor

con proteasas inmovilizadas para la hidrólisis

limitada de proteínas del lactosuero y posterior

análisis de las propiedades funcionales

superficiales de los hidrolizados obtenidos”.

Tesis Doctoral Facultad de Ciencias

Bioquímicas y Farmacéuticas. Universidad

Nacional de Rosario. Argentina

[5]. Bernardi LS, Pilosof AMR, Bartholomai GB

(1991). “Enzymatic modification of soy protein

concentrates by fungal and bacterial

proteases”. J. Am. Oil Chem. Soc., Vol. 68, pp

102–105

[6]. Bombara N, Añón MC, Pilosof AMR (1997).

“Functional properties of protease modified

wheat flours”. Lebensmittel Wissenschaft und

Technologie, Vol. 30, pp 441–447.

75 Int. Res. J. Agric. Food Sci.

[7]. Borraccetti M (2007) “Propiedades

fisicoquímicas y estructurales de estados no.

nativos de k-caseína bovina y su relación con

propiedades de estabilización de emulsiones”.

Tesis Doctoral Facultad de Ciencias

Bioquímicas y Farmacéuticas. Universidad

Nacional de Rosario. Argentina

[8]. Bos MA, van Vliet T (2001). “Interfacial

rheological properties of adsorbed protein

layers and surfactants: a review”. Adv. Colloid

Interface Sci., Vol. 91, no. 3, pp 437–471

[9]. Carrera Sánchez C, Rodríguez Patino. JM

(2005). “Interfacial, foaming and emulsifying

characteristics of sodium caseinate as

influenced by protein concentration in solution”.

Food Hydrocolloids, Vol. 19, no. 3, pp 407–416.

[10]. Chevalier F, Chobet JM, Popineau Y, Nicolas

MG, Haertle T (2001). “Improvement of

functional properties of β-lactoglobulin glycated

though the Maillard reaction is related to the

nature of the sugar”. Int. Dairy J. Vol. 11, no. 3,

pp 145-152

[11]. Croguennec T, Renault A, Bouhallab S,

Pezennec S (2006). “Interfacial properties and

foaming properties of sulfhydryl-modified bovine

β-lactoglobulin”. J. Colloid Interface Sci., Vol.

302, no. 1, pp 32-39.

[12]. Davis JP, Doucet D,Foegeding EA (2005).

“Foaming and interfacial properties of

hydrolyzed β-lactoglobulin”. J. Colloid Interface

Sci., Vol. 288, pp 412–422

[13]. Dickinson E (2003). “Hydrocolloids at interfaces

and the influence on the properties of dispersed

systems”. Food Hydrocolloids, Vol. 17, no. 1, pp

25–39.

[14]. Don BLS, Pilosof AMR, Bartholomai GB (1991).

“Enzymatic modification of soy protein

concentrates by fungal and bacterial proteases. J. Am. Oil Chem. Soc., Vol. 68, pp 102–105

[15]. Doucet D, Otter DE, Gauthier SF, Foegeding EA (2003). “Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity”. J. Agric. Food. Chem.,Vol. 51, no. 21, pp 6300-6308.

[16]. Foegeding EA, Davis JP, Doucet D, McGuffey

MK (2002). “Advances in modifying and

understanding whey protein functionality”.

Trends Food Sci. Technol., vol. 13, pp 151-159.

[17]. Foegeding EA, Luck PJ, Davis JP (2006).

“Factors determining the physical properties of

protein foams”. Food Hydrocolloids, Vol. 20, no.

2–3, pp 284–292

[18]. Gbogouri GA, Linder M, Fanni J, Parmentier M

(2004). “Influence of hydrolysis degree on the

functional properties of salmon byproducts

hydrolysates”. J. Food Sci., Vol. 69, no 8, pp

615–622.

[19]. Hagolle N, Relkin P, Popineau Y, Bertrand D

(2000). “Study of the stability of egg white

protein-based foams: effect of heating protein

solution.” J. Sci. Food Agric., Vol. 80, no. 8, pp

1245-1252

[20]. Ipsen R, Otte J, Sharma R, Nielsen A, Gram

Hansen L, Bruun Qvist K (2001). “Effect of

limited hydrolysis on the interfacial rheology

and foaming properties of β-lactoglobulin A”.

Colloids Surf., B, Vol. 21, pp 173–178

[21]. JR Wagner (2000). Propiedades superficiales,

en Caracterización Funcional y Estructural de

Proteínas; Pilosof AMR, Bartholomai GB.

(editores). Programa Iberoamericano. de

Ciencia y Tecno.logía para el Desarrollo.

Editorial Universitaria de Bueno.s Aires

(Eudeba), chapter 3, pp. 41-70.

[22]. Jung S, Murphy PA, Johnson LA (2005).

“Physicochemical and functional properties of

soy protein substrates modified by low levels of

protease hydrolysis”. J. Food Sci., Vol. 70, no 2,

pp 180–187.

[23]. Kananen A, Savolainen J, Mäkinen J, Perttilä

U, Myllykoski L, Pihlanto-Leppälä A (2000).

“Influence of chemical modification of whey

protein conformation on hydrolysis with pepsin

and trypsin”. Int. Dairy J., Vol. 10, pp 691-697

[24]. Kilara A, Panyam D (2003). “Peptides from milk

proteins and their applications”. Crit. Rev. Food

Sci. Nutr., vol. 43, no. 6, pp 607–633.

[25]. Korhonen H, Pihlanto-Leppälä A, Ramantamäki

P, Tupasela T (1998) “The functional and

biological properties of whey proteins:

prospects for the development of functional

foods”. Agric. Res.Centre of Finland, Vol. 7, pp

283-296.

[26]. Kristinsson HG, Rasco BA (2000a).

“Biochemical and functional properties of

Atlantic salmon (Salmo salar) muscle

hydrolyzed with various alkaline proteases”. J.

Agric. Food. Chem., Vol. 48, pp 657–666.

[27]. Kristinsson HG, Rasco BA (2000b). “Fish

protein hydrolysates: Production, biochemical

and functional properties”. Crit. Rev. Food Sci.

Nutr., Vol. 40, no. 1, pp 43–81...

[28]. Kuehler CA, Stine CM (1974). “Effect of

enzymatic hydrolysis on some functional

properties of whey proteins”. J. Food Sci., Vol.

39, pp 379-382.

[29]. Laemmli UK (1970). “Cleavage of structural

proteins during the assembly of the head of

bacteriophage T4”. Nature, Vol. 227, pp 680-

685.

Ballerini et al 76

[30]. Lahl WJ, Windstaff DA (1989). “Spices and

seasonings: hydrolysed proteins. In

Proceedings of the 6th SIFST symposium on

food ingredients – applications, status and

safety. Singapore: Singapore Institute of Food

Sci. and Tech., 27–29 April, pp. 51–65.

[31]. Liceaga-Gesualdo AM, Li-Chan ECY (1999).

“Functional properties of fish protein

hydrolysate from herring (Clupea harengus)”. J.

Food Sci., Vol. 64, no. 6, pp 1000–1004.

[32]. Martínez KD, Carrera Sánchez C, Rodríguez

Patino. JM, Pilosof AMR (2009). “Interfacial and

foaming properties of soy protein and their

hydrolysates”. Food Hydrocolloids, Vol. 23, no.

8, pp 2149–2157.

[33]. Morgan F, Henry G, Le Graet Y, Molle D, Leonil

J, Bouhallab S (1999). “Resistence of β-

lactoglobulin bound lactose to the hydrolysis by

β-galactosidase”. Int. Dairy J., Vol. 9, pp 813-

816.

[34]. Moro A, Báez G, Busti P, Ballerini G, Delorenzi

N (2011). “Effects of heat-treated β-

lactoglobulin and its aggregates on foaming

properties”. Food Hydrocolloids, Vol. 25, pp

1009-1015.

[35]. Moro A, Báez GD, Ballerini GA, Busti PA,

Delorenzi N (2013). “Emulsifying and foaming

properties of β-lactoglobulin modified by heat

treatmen”. Food Res. Int., Vol. 51, pp 1–7

[36]. Moro A, Báez GD, Busti PA, Ballerini GA,

Delorenzi NJ (2011). “Effects of heat-treated β-

lactoglobulin and its aggregates on foaming

properties”. Food Hydrocolloids, Vol. 25, pp

1009-1015

[37]. Murray BS (2007). “Stabilization of bubbles and

foams”. Curr. Opin. Colloid Interface Sci., Vol.

12, no. 4–5, pp 232–241.

[38]. Mutilangi WAM, Panyam D, Kilara A (1996).

“Functional properties of hydrolysates from

proteolysis of heat-denatured whey protein

isolate”. J. Food Sci., Vol. 61, pp 270-275.

[39]. Palazolo G, Rodriguez F, Farruggia B, Picó G,

Delorenzi NJ (2000). “Heat treatment of β-

lactoglobulin: structural changes studies by

partitioning and fluorescence”. J. Agric. Food.

Chem., Vol. 48, no. 9, pp 3817-3822.

[40]. Pérez AA, Carrara CR, Carrera Sánchez C,

Santiago LG, Rodríguez Patino. JM (2010ª).

“Interfacial and foaming characteristics of milk

whey protein and polysaccharide mixed

systems”. AIChE J., Vol. 56, no. 4, pp 1107–

1117

[41]. Pérez AA, Carrera Sánchez C, Rodríguez

Patino. JM, Rubiolo AC, Santiago.LG (2012).

“Foaming characteristics of β-lactoglobulin as

affected by enzymatic hydrolysis and

polysaccharide addition: Relationships with the

bulk and interfacial properties”. J. Food Eng.,

Vol. 113, pp 53–60

[42]. Pérez AA, Carrera Sánchez C, Rodríguez

Patino. JM, Rubiolo AC, Santiago LG (2012).

“Effect of enzymatic hydrolysis and

polysaccharide addition on the β-lactoglobulin

adsorption at the air–water interface”. J. Food

Eng., Vol. 109, no. 4, pp 712–720

[43]. PJ Wilde, DC Clark. “Foams formation and

stability”. In G. M. Hall (Eds). Methods of testing

protein functionality. London: Blakie

Academic.1996, pp 110-152.

[44]. Reddy IM, Kella NKD, Kinsella JE (1988).

“Structural and conformational basis of the

resistance of b-lactoglobulin to peptic and

chymotryptic digestion”. J. Agric. Food. Chem.,

Vol. 36, pp 737-741.

[45]. Rodríguez Patino. JM, Miñones Conde J, Millán

Linares H, Pedroche Jiménez JJ, Carrera

Sánchez C, Pizones V, Millán Rodríguez F

(2007). “Interfacial and foaming properties of

enzyme-induced hydrolysis of sunflower protein

isolate”. Food Hydrocolloids, Vol. 21, no. 5–6,

pp 782–793.

[46]. Rodríguez Patino. JM, Rodríguez Niño MR,

Carrera Sánchez C (2008). “Implications of

interfacial characteristics of food emulsifiers in

foam formulations”. Adv. Colloid Interface Sci.,

Vol. 140, no. 2, pp 95–113.

[47]. Sathivel S, Bechtel JP, Babbitt J, Smiley S,

Crapo C, Reppond KDI (2003). “Biochemical

and functional properties of herring (Clupea

harengus) byproduct hydrolysates”. J. Food

Sci., Vol. 68, pp 2196–2200.

[48]. Sathivel S, Smiley S, Prinyawiwatkul W,

Bechtel PJ (2005). “Functional and nutritional

properties of red salmon (oncorhynchusnerka)

enzymatic hydrolysates”. J. Food Sci., Vol. 70,

no. 6, pp 401–406.

[49]. Shahidi F, Han XQ, Synowiecki J (1995).

“Production and characteristics of protein

hydrolysates from capelin (Mallotus villosus)”.

Food Chemistry, Vol. 53, pp 285–293

[50]. Sinha R, Radha C, Prakash J, Kaul P (2007).

“Whey protein hydrolysate: Functional

properties, nutritional quality and utilization in

beverage formulation”. Food Chemistry, Vol.

101, pp 1484–1491

[51]. Smithers GW, Ballard FJ, Copeland AD, De

Silva KJ, Dionysius DA, Francis GL, Goddard

C, Grieve PA, McIntosh GH, Mitchell IR, Pearce

RJ, Regester GO (1996). “New opportunities

from the isolation and utilization of whey

proteins”. J. Dairy Sci., Vol. 179, pp 1454-1559.

77 Int. Res. J. Agric. Food Sci.

[52]. Schmidt DG, van Markwijk BW (1993).

“Enzymatic hydrolysis of whey proteins.

Influence of heat treatment of α-lactalbumin and

β-lactoglobulin on their proteolysis by pepsin

and papain. Netherlands Milk and Dairy J., Vol.

47, pp 15-22.

[53]. Smyth M, Fitzgerald RJ (1998). ”Relationship

between some characteristics of WPC

hydrolysates and the enzyme complement in

commercially available protease preparations”.

Int. Dairy J., Vol. 8, pp 819–827.

[54]. Tavano OL (2013). “Protein hydrolysis using

proteases: An important tool for food

biotechnology”. J. Mol. Catal. B: Enzym. Vol.

90, pp 1–11

[55]. Tosi E, Canna L, Lucero E, Ré E (2007).

“Foaming properties of sweet whey solutions as

modified by thermal treatment”. Food

Chemistry, Vol. 100, pp 794-799.

[56]. Tsumura K, Saito T, Tsugea K, Ashida H,

Kugimiya W, Inouye K (2005). “Functional

properties of soy protein hydrolysates obtained

by selective proteolysis”. Lebensmittel-

Wissenschaft und-Technologie, Vol. 38, pp

255–261.

[57]. Vioque J, Sánchez-Vioque R, Clemente A,

Pedroche J, Millán F (2000). “Partially

hydrolyzed rapeseed protein isolates with

improved functional properties”. J. Am. Oil

Chem. Soc., Vol. 77, pp 1–4.

[58]. W Loisel, J Guéguen and Y Popineau. “A new

apparatus for analyzing foaming properties of

proteins”. In KD Schwenke, YR Mothes (Eds.).

Food proteins: Structure and functionality. New

York: VCH Publishers, 1993, pp 320-323.

[59]. Wierenga PA, Gruppen H (2010). “New views

on foams from protein solutions”. Curr. Opin.

Colloid Interface Sci., Vol. 15, no. 5, pp 365–

373.

[60]. Wong DWS, Camirand WM, Pavlath AE (1996).

“Structures and functionalities of milk proteins”.

CRC Crit. Rev. Food Sci. Nut., Vol. 36, no. 8,

pp 807-844.

[61]. Wróblewska B, Troszyñska A (2005).

“Enzymatic hydrolysis of cow’s whey milk

proteins in the aspect of their utilization for the

production of hypoallergenic formulas”. Pol. J.

Food Nutr. Sci., Vol. 4, pp 349–357

[62]. Xiangzhen K, Huiming Z,Haifeng Q (2007).

“Enzymatic hydrolysis of wheat gluten by

proteases and properties of the resulting

hydrolysates”. Food Chemistry, Vol. 102, pp

759–763

[63]. Zheng H, Shen X, Bu G, Luo Y (2008). “Effects

of pH, temperature and enzyme-to-substrate

ratio on the antigenicity of whey protein

hydrolysates prepared by Alcalase”. Int. Dairy

J., Vol. 18, pp 1028–1033.

Ballerini et al 78