Stading e Hermansson, 1991

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Food Hydrocolloids Yol.5 no.4 pp.339-352,

Large deformation properties of 3 lactoglobulin gel structures

Mats Stading and Anne-Marie Hermansson

SIK The Swedish Institute for Food Research PO Box 5401 5 40229Goteborg Sweden

Abstract. Different gel structures formed by 13-lactoglobulin dissolved in distilled water (12 w/w atpH 3 .0-7.5 have been investigated using tensile measurements at large deformations. Gels formed atpH 4-6 were opaque, whereas at pH values below or above this range they were transparent. Thefracture properties showed large variations over the pH range studied . Gels formed at low pH werebrittle with low strain and stress at fracture , as opposed to those formed at high pH , which wererubber-like with high strain and stress at fracture . Gels formed at intermediate pH (pH 4-6 had anintermediate, near-constant, strain at fracture. The fracture stress was, however , higher at pH 5.56.0 than at pH 4 .0--5.2. The specific fracture energy resembled the stress at fracture, with amaximum of 6 J/m 2 at pH 6.0. Gels formed at pH 4.5, 5.5, 6.5 and 7.5 were all notch-sensitive. Theopaque gels were defined as aggregate gels and the transparent gels were defined as fine-strandedgels. The fracture properties clearly showed there are differences between the fine-stranded gelsformed at high pH and those formed at low pH. The fracture stress demonstrated that there arestructural differences within the pH range in which the aggregate gels are formed. The non-linearityof the stress-strain curve was the same for all fine-stranded gels, which had r-shaped stress-straincurves. The stress-strain curves of the aggregate gels were almost linear. The non-linearity did notinfluence the fracture properties. Young s modulus showed two peaks , at pH 3.5 and 6.0, coincidingwith the range where the structure changes between aggregated and fine-stranded. The stress atfracture also has a maximum at pH 6.0. The high elasticity and fracture stress may depend on strong,elastic areas in the network structure. Apart from the two peaks, Young s modulus shows the samebehaviour as the storage modulus, G measured at small deformations, but Young s modulus isslightly larger than 3G .

Introduction

Many food biopolymers are able to form gels and therefore have an importanteffect on the texture of food systems. The texture depends on the mechanicalproperties, which in turn depend largely on the structure of the gel network. Th emechanical propert ies can be divided into small deformation properties andlarge deformation properties. The small deformation properties are measuredusing non-destructive methods, whereas testing by large deformations includesfracture properties of the material. The large deformation measurements areusually performed in compression or , as in this paper, tension.

I3-Lactoglobulin was chosen as a model system in this study because it formsboth aggregated and fine-stranded gels according to the pH in question. Gelsformed at pH 4-6 in distilled water are opaque having a network formed byaggregates an d are defined as aggregate gels (1,2). Gels formed below or abovethis intermediate pH range are transparent and are defined as fine-strandedgels . Figure l a shows an example of an aggregate gel and Figure l b an

example of a fine-stranded gel. Both gels are formed by 13-lactoglobulin and thediffering property is the pH value . Th e aggregate gel is formed at pH 5.25 andthe micrograph is obtained by scanning electron microscopy (SEM). The finestranded gel is a 60 nm thick section of a 13-lactoglobulin gel at pH 7.5photographed in a transmission electron microscope (TEM). The experimental

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M.Stading and A.-M.Hermansson

Fig. 1. Electron micrographs of J3-lactoglobulin gels. a) 10 w/w) , pH 5.25, SEM); b) 12w/w), pH 7.5, TEM .

technique and full details of the ~ a c t o g l o b u l i ngel structure will be presented inanother paper M.Langton and A.-M.Hermansson, in preparation). In Figure 1the magnification factor is -25 times larger in b than in a.

We have earlier studied the small deformation behaviour of the different gelstructures formed by ~ a c t o g l o b u l i n3). The aggregate gels behaved differentlyfrom the fine-stranded gels in many ways: the aggregate gels had a higherstorage modulus, G lower critical gel concentration, were more frequencydependent and formed structures far below the denaturation temperature. Th efine-stranded gels formed a high pH behaved in the same way as those formed atlow pH , but the critical gel concentration was higher for the former.

The tensile strength of whey protein gels at pH 6.5 and 8.0 has been studied byLangley et l who used samples with varying contents of ~ l a c t o g l o b u l i nand lactalbumin 4). Their purest ~ a c t o g l o b u l i nsample contained 94 w/w ~ -

lactoglobulin and no a-lactalbumin. Th e whey powders were dissolved indeionized water to give 15 w/v) whey solutions, which were heated for 5 minat 80C. The stress at fracture and the elastic modulus were related by power lawrelations to the a-lactalbumin or ~ l a c t o g l o b u l i nfraction. The ~ l a c t o g l o b u l i ncontent was found to contribute more than the a-lactalbumin content to thetensile strength and to the clastic modulus.

Large deformation testing is a commonly used method for texture evaluation.Both compressive and tensile tests are utilized but, in the case of biopolymergels, large deformation testing has, to date, almost exclusively been synonymous

with compressive testing, even though tensile tests give a clearer picture of thestresses in the sample. The total stress in compressive testing is the sum of bothtensile stresses and shear stresses, whereas the shear stresses in tensile testingare negligible. Other advantages of tensile testing are: the energy used in atensile test is used only for deformation and not for friction, th e start of fracture

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Deformation properties of [3-lactoglobulin gels

can be determined more exactly and it is possible to study the notch sensitivity ofthe material 5 .

he main drawback of tensile measurements of biopolymer gels is theexperimental difficulties. The first difficulty encountered is to attach the sample

to the measuring system. Attempts to clamp the gel usually cause damage unlessit is a very firm gel. There are, however, results reported on clamped samples ofcross-linked alginate, but only a small strain strain


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Deformation propertie s of 13-lactoglobulin gels

stress at fracture

x

a

Fig. 2. Stress at fracture as a function of notch size for a material that is (a) notch-sensit ive and (b)not notch-sensitive . The size of the largest structure element is shown by x .

whey protein concentrate . a-Lactalbumin is removed by precipitation at itsisoelectric point followed by centrifugation. The supernatant is diafiltered andspray-dried, rendering the ~ a c t o g l o b u l i npowder.

The ~ l a c t o g l o b u l i npowder had the approximate composition: 95.8 drymatter, 84.7 protein, 3.5 ash. HPLC, FPLC and gel electrophoresis showedthat the ~ l c t o g l o b u l i nalso contained small amounts of a-lactalbumin.

The sodium, potassium and calcium contents of the ~ la c t o g l o b u l i npowderhave been measured using atomic absorption spectroscopy and X-ray fluorescence. Th e powder contained 1 w/w Na, 0.D3 w/w K and D.04 w/w Ca.

~ L a c t o g l o b u l i nfrom Sigma Chemical Co. (L-D13D, lot no. 106F 812D wasalso used as a comparison for small deformation measurements of the storagemodulus G These samples are denoted Sigma, as in a previous publication (3).

Sample preparation

The ~ a c t o g l o b u l i nwas dissolved in degassed, distilled water at 12 w/w andpH was adjusted with l mol/drrr HCl or l mol/dar KOH . At extreme pHand high concentration of ~ a c t o g l o b u l i n1 mol/drrr HCI or 1 rnol/dnr KOHwas used. Th e samples were then degassed . The ~ a c t o g l o b u l i nsolution was

poured into square moulds made of aluminium tubes, inner dimensions 13 x 13x 60 mm, sealed with heat-proof tape (Scotch 425) . The cylinders had beengreased with bearing grease to prevent the samples from sticking to the mould.Moulds made of glass and acrylic plastic were also tested but the ~ l a c t o g l o b u l i ngels stuck to the walls of the mould. Sigmacote was tested instead of the greasebut it promoted air bubble formation on the walls of the mould. The cylinderswere heated in a water-bath with the heating profile shown in Figure 3.

Tensile measurements

The ~ a c t o g l o b u l i ngels were taken out of the moulds and cut into D 3 mmlong test-pieces with a scalpel. A notch was cut along one side of the test-pieceperpendicular to the direction of elongation as illustrated in Figure 4(a). Thenotch was 1 mm deep unless otherwise stated. The samples that were fracturedwithout a notch were moulded in a separate mould , see Figure 4(b). Each test-

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lClmin

Temperature

M.Stading and A.-M.Hermansson

C 100

80

60

40

20

O ~o 20 40 60 80 100 120 140 160

time minutes

Fig. 3. Heat t rea tment of the 13-lactoglobulin samples.

Originalarea

Pneumat cgrip

Sample

otch

Sandpaper

Fig. 4. Exper imenta l set-up for the tensile tests a and sample shape for measurements withoutnotch b .

piece was attached to the measuring instrument using a method where the testpieces were glued to plates covered with sand paper, using a cyanoacrylate glue,Loctite 401 or 406. The sandpaper was applied to increase the gluing areabetween the sample and the plate. Th e plates were then clamped with twopneumatic grips. The samples were fractured in tension using an Instron 1122,and the force and the deflection were monitored on a personal computer. Aconstant cross-head speed of 10 mm/min was used which corresponds to aninitial strain rate of E = 8.3 X 10- 3 S for a 20 mm long sample.

alculations

Strain The strain is the dimensionless deformation and can be defined as:

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Deformation properties of J3-lactoglobulin gels

IEH = In ~ 7)

where is th e original length of the sample and I is the present length 17). EH is

called Hencky strain and is used in this study because it is additive. EH will bereferred to as E.An alterna tive to Hencky s tra in is Cauchy strain, E c :

L LoE c = fo

8)

At small strains, when approaches Lo Hencky and Cauchy strains ar eapproximately equal.

Stress The stress, rr is the force F per unit area

Fc r 9)

The eas ies t way to calculate the stress is to divide the measured force by th eoriginal area which is a sufficient measure for small s trains. The sample will,however, ge t narrower as the strain increases, since the volume of the sample isapproximately constant. is therefore more relevant to use the smallest a rea.T he shape of a sample at pH 4.5 an d 6.5 was photographed during a tensilemeasurement and th e area at fracture was calculated from the photographs.The samples at pH between 4.0 and 6.0 were assumed to narrow in the same wayas at pH 4.5 see Figure 5). The samples at pH >6 .0 were assumed to narrow inthe same way as the sample at pH 6.5, and the narrowing of the sample at pH

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Stading e Hermansson, 1991