Journal of Food Engineering 90 (2009) 161–170

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Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

In situ microstructure evaluation during gelation of b-lactoglobulin

Sanghoon Ko a, Sundaram Gunasekaran b,*

a Department of Food Science and Technology, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Koreab Department of Biological Systems Engineering, University of Wisconsin-Madison, 460 Henry Mall, Madison, WI 53706, USA

a r t i c l e i n f o

Article history:Received 7 December 2007Received in revised form 6 June 2008Accepted 20 June 2008Available online 29 June 2008

Keywords:Gelationb-lactoglobulinImage processingMicrostructureConfocal laser scanning microscopy

0260-8774/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2008.06.030

* Corresponding author. Tel.: +1 608 262 1019; faxE-mail address: guna@wisc.edu (S. Gunasekaran).

a b s t r a c t

In situ microstructure evolution of heat-induced b-lactoglobulin (BLG) gels were investigated using con-focal laser scanning microscopy (CLSM) at various gel preparation conditions: pH (2, 5, and 7), proteinconcentration (5%, 10%, and 15%), and salt concentration (NaCl 0, 0.1, and 0.3 M). Temperature-inducedCLSM micrographs were used to measure several morphological features of the evolving protein clusters.The cluster area, perimeter, and circularity served as useful microstructural indices of BLG gels. Varyinggelation conditions yielded BLG gels of different microstructures during heating. At pH 2 and 7, averagearea, and perimeter of protein clusters increased with protein content. Larger electrostatic repulsion pro-vided microstructure with stranded clusters, whereas less electrostatic repulsion resulted in particulateclusters. The cluster size and shape were greatly affected by gelation pH; at pH 2 and 7, the average area,and perimeter increased with increasing salt content which provided particulate features to the gelmicrostructure. The combined effect of protein and salt content and pH is critical in imparting the gelits overall microstructure.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Globular proteins such as egg white, soy, and whey proteins arecommonly used as functional ingredients in foods. The primaryfunctionalities of the globular proteins are gelation, emulsifica-tion, water-holding capability, and stabilizing (Huffman, 1996;Burrington, 1998; DeWit, 1998; Szczesniak, 1998). Among these,the ability to form heat-induced gels is perhaps the most importantfunctionality of the proteins as it can be used to create desirablefood textures. The physical properties of heat-induced protein gelsare strongly influenced by the gel microstructure (Emmons et al.,1980; Holcomb, 1991). Thus, control of food properties for variousapplications such as heat-induced protein gels requires a betterunderstanding of structure-function relationships of proteins infoods. Studying the microstructure of a protein system during gela-tion could help understand how the structure changes affect gelproperties.

The study of morphological evolution in microstructure can pro-vide further understanding of the material behavior and properties(Olsson et al., 2002). Investigations of microstructural changes infoods are numerous and continue to grow with the advent andincreasing availability of new microscopic techniques. Heat-inducedwhey protein gelation has been widely investigated in the past usingtransmission electron microscopy (TEM) (Clark et al., 1981; IreneBoye et al., 2000; Kavanagh et al., 2000b; Olsson et al., 2002),

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: +1 608 262 1228.

scanning electron microscopy (SEM) (Relkin et al., 1998; Dumay etal., 1999), and atomic force microscopy (Ikeda and Morris, 2002;Ikeda, 2003). However, both TEM and SEM often introduce artifactsin the sample microstructure as well as deform and/or dehydrate inthe microstructure during sample preparation. AFM can examinerelatively small window of a sample. Additionally, AMF is not properfor studying the gel microstructure since it scan only the surface ofthe sample microstructure.

Confocal laser scanning microscopy (CLSM) is a recently devel-oped microscopic technique for obtaining high quality, opticalsection images free from out-of-focus blur or fluorescence flare.CLSM has several advantages for studying gel microstructurestudies because (1) dehydration is usually unnecessary; (2) opti-cal sectioning can enable imaging without disturbance in gelmicrostructure; and (3) gel microstructure can be continuouslymonitored. Thus, CLSM can probe in situ the thermally-inducedchanges in microscopic features of protein clusters and gels with-out disturbing the sample (Everett et al., 1993; Heinemann et al.,2002). However, CLSM suffers from limited resolution (approxi-mately 250 nm) whereas the resolution of SEM, TEM, and AFMis less than 100 nm (Merchant et al., 2005). This limitation pre-cludes studying the gel assembly until the cluster/strand lengthis sufficiently large. Thus, CLSM images show the large BLG clus-ters resulting from thermal gelation above the CLSM resolution.CLSM is still enough to investigate BLG gelation since protein con-tent should be over a critical concentration (�5%). It means that aprotein gel is a dense network of a number of protein moleculeswhich can be shown under the CLSM.

Table 1Chemical composition of b-lactoglobulin (BLG)

Component Valuea

Protein (% dry Basis) 97.4b-lactoglobulin (% of protein) 95.0Fat (%) 0.1Ash (%) 2.4Moisture (%) 5.8pH of 10% BLG at 20 �C 7.3

a The composition data were provided by Davisco Foods International, Inc.

162 S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170

The b-lactoglobulin (BLG) is the major component and the pri-mary gelling agent of whey proteins. The BLG solution can begelled under heat, which results in an irreversible gel from theunfolding of protein molecules to the formation of a three dimen-sional (3D) network (Aguilera, 1995). This irreversible gelationprocess involves different types of interactions such as van derWaals interactions, hydrogen bonds, hydrophobic effects, depend-ing on pH, and, covalent links (i.e., disulfide) (Kinsella andWhitehead, 1989; Ziegler and Foegeding, 1990; Aguilera, 1995).The sulfhydryl-disulfide exchanges also depend on pH. Althoughdisulfide bonds were not essential to heat-induced BLG gelation,they contributed to the BLG gelation (Errington and Foegeding,1998). Since intermolecular sulfhydryl-disulfide exchange isabsent or short at pH 2, a weak gel is formed (Aymard et al., 1999).

The formation and properties of BLG gels are affected mainly bypH, ionic strength, and protein concentration. The effects of thesefactors on BLG gels have been widely investigated so that it is pos-sible to produce a transparent gel or a turbid gel (Stading andHermansson, 1990, 1991; Ikeda and Morris, 2002). The transparentgel, known also as fine-stranded gel, consists of a network of linearaggregates of denatured protein molecules. It is formed under con-ditions of large electrostatic repulsions between the protein mole-cules, which occur at low ionic strength and at pH values far fromthe protein isoelectric point (pI, �5.2). The turbid or opaque gel,also called particulate gel, is made up of random aggregates ofheat-denatured protein molecules. This type of gel is formed underconditions of high ionic strength and at pH values close to pI (pHrange 4–6). BLG gels are weak at low protein concentration butthey become stronger with increasing protein concentration as itprovides an increase in density of protein strands, which easilytends to aggregate (Qi et al., 1995). Gelation conditions such aselectrostatic repulsion in the systems seem to affect the role of pro-tein (Ziegler and Foegeding, 1990; Verheul and Roefs, 1998).

Studying in situ changes in the microstructure using the CLSMprovides a better understanding of structure-function relationshipsof BLG in foods. The effects of different gelation conditions on insitu gel formation and microstructural properties can be investi-gated by quantifying the morphological characteristics on theCLSM micrographs. The morphological parameters of the CLSMimages represent the evolution of gel microstructure during heat-ing. However, practical difficulties in studying CLSM images arisefrom errors and distortions in the acquired images, especially un-der dynamic conditions. These include: aberrations in the opticalpath of specimen (Carlsson, 1991; Hell et al., 1993; White et al.,1996), uneven distribution of intensity, fluorescence signal attenu-ation with depth (Liljeborg et al., 1994), and the Z-axis misalign-ment of successive image slices (Durr et al., 1989; Baba et al.,1993). Recently, we have developed several algorithms to correctimage alignment, compensate light attenuation with depth, and re-move noise and correct uneven intensity on the images to correctCLSM images for any associated errors (Ko and Gunasekaran,2007).

The objectives of this study were to: (1) study in situ, tempera-ture-induced microstructural characteristics of BLG system at var-ious protein concentrations, pHs, and ionic strengths using CLSMand (2) evaluate morphological parameters of the CLSM micro-graphs as a function of gelation conditions.

2. Materials and methods

2.1. Sample preparation

Purified bovine milk BLG powder containing genetic variants Aand B (BioPURE b-lactoglobulin, Davisco Foods International Inc.,Eden Prairie, MN) was used without further purification. Thechemical composition of the BLG powder is listed in Table 1.

Protein solutions, at various concentrations (5%, 10%, or 15% w/v)were prepared by adding appropriate amounts of BLG powder toa portion of distilled water containing 0, 0.1, or 0.3 M NaCl, andthen stirring for 2 h at room temperature. Solutions were storedat 5 �C overnight for complete hydration of the protein molecules.The pH of protein solutions was adjusted at room temperature tothe following pH values: 2.0, 5.0, and 7.0 (below and above pI ofBLG) using 1 M HCl or NaOH.

2.2. In situ image acquisition

Rhodamine B solution (0.025%, laser grade 99+%, excitation540 nm; emission 625 nm, ACROS Organics, Morris Plains, NJ,USA) was added to the BLG solutions to facilitate easy visualizationof the protein phase during imaging. Rhodamine B is a cationic dyewhich forms amide bonds with the N-terminus of amino groups inthe protein. The temperature sensitivity of Rhodamine B fluores-cence emission excited at various wavelengths is at best about�2% per �C, i.e., the fluorescence intensity decreases about 2% forevery 1 �C increase in temperature (Hu et al., 2006). Due to thetemperature instability of the Rhodamine B, the micrographs ob-tained were processed using the image processing algorithms wedeveloped previously. Rhodamine B specifically attaches on theprotein molecules with fluorescence at excitation/emission 543/565 nm, respectively. Numerous protein staining experimentshave been performed to investigate protein microstructure(Merchant et al., 2005; Ko and Gunasekaran, 2007). In addition,Rhodamine B is proper to study the effect of pH on microstructuredue to its pH-insensitivity and photostability. Rhodamine B can at-tach on the protein molecules minimizing its disturbance duringprotein aggregation and network formation (Kerstens et al.,2005). A drop of 0.3 mL BLG solution prepared at each conditionwas poured into a 0.5-mm deep hole concave slide glass which isan enough volume for the BLG gelation. CLSM can examine thinoptical sections from thick specimens. Thus, the CLSM images rep-resent the BLG aggregates and matrix even though there is possiblea glass-protein interaction. After placing a cover slip on the slideglass, the specimen slide was fixed on the specimen stage of CLSM.

A CLSM (Biorad MRC 1024, Bio-Rad Inc., UK) attached to aninverted camera (Eclipse TE300, Nikon Inc., Japan) was used forinvestigating BLG microstructure in situ. CLSM operated a kryp-ton/argon laser with excitation at 488, 568, and 647 nm andemission collected above 522, 605 and 680 nm, respectively, withoil-coupled differential-interference contrast objective lens on60� magnification. An optical plane of a specimen was scannedtwice using Kalman filter to reduce noise during imaging.

The BLG specimen was heated at 1 �C/min from 25 to 95 �Cusing a specimen temperature control device described below.CLSM images of 512 � 512 pixel resolution were obtained contin-ually at each 5 �C of sample temperature increment from 50to 95 �C. The examination area of each layer was 160.4 �160.4 lm2; with 256 gray level intensities each pixel representedan area of 0.313 � 0.313 lm2. CLSM imaging was repeated threetimes to obtain micrographs of BLG which were analyzed to deter-mine their morphological characteristics.

S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170 163

2.3. Specimen temperature control device

Temperature control is essential for in situ microscopy duringgelation. However, most CLSM systems have neither a high-tem-perature heating stage (>90 �C) nor a heating rate control system.Generally, a temperature control system for microscopic imagingrequires accuracy, fast heating and cooling capacity, mountabilityon the microscope stage. Recently, a rapid heating/cooling devicecalled Peltier is commercialized. The Peltier is a thermoelectricmodule which converts directly electric voltage to thermal differ-entials. We developed a specimen temperature control deviceand a Peltier-type heating stage. The specimen temperature con-trol device can heat up and cool down at a given heating/coolingrate. They consisted of a Peltier-installed specimen stage with a10 mm hole, a temperature controller (5C7-362, McShane, Inc.,Medina, OH, USA), a thermocouple, and a data logger. The overallCLSM system with the temperature controller and the heatingstage is shown in Fig. 1.

The Peltier-installed specimen stage, shown in Fig. 2, consistedof a Peltier heating/cooling element with a hole (thermoelectricmodule CH-119-1.0-1.5, TE Technology, Inc., Traverse City, MI,USA) to generate heat, a water cooler (CPU-200G, Koolance USA,Federal Way, WA, USA) as a heat sink, and a specimen slide. Thedetails of the Peltier-installed specimen stage are shown in Fig. 2.A thin thermal conductive tape was applied to the both surfacesof the Peltier heating/cooling element for effective heat exchange.One side of the Peltier heating/cooling element was placed on thespecimen slide and the other on the heat sink. The water cooler cir-culated coolant effectively. A 10-mm hole was drilled in the centerof the water cooler to allow the CLSM laser beam to pass throughthe specimen without any interference.

The PID temperature controller provided automatic bi-phaseheating and cooling control for the Peltier module. It was capableof maintaining the set temperature (±0.1 �C) or ramping tempera-ture at various rates. Thermocouple was used to continuouslymonitor the specimen temperature by inserting it into the BLGsolution during in situ imaging on CLSM. The data logger collectedand stored the temperature data.

2.4. Image processing and analysis

The CLSM images were processed using a library of algorithmswritten in MatLab software (MatLab R14, The MathWorks, Inc.,Natick, MA, USA), details of which have been previously published(Ko and Gunasekaran, 2007). These algorithms facilitated correc-tion for image alignment, compensation for light attenuation withimage layer depth, noise removal noise and correction for uneven

Data logging

Data loggerCLSM

Sample stage

PID control Data

logging

Data logger

Temperature controller

CLSM

PC

Data store

Image acquisition

Fig. 1. The schematic diagram of the specimen temperature control device for insitu imaging configuration.

image intensity. The processed images were stored as tagged im-age file format (TIFF) files for further analyses.

The protein phase in the processed CLSM images was seg-mented using the IMAQ vision builder software (v. 6.1, Nationalinstruments, Austin, TX, USA) and the morphological features ofthe segmented objects were analyzed. Fig. 3 shows simple mor-phological measurement from the CLSM image. The morphologicalparameters were used for representing the evolution of gel micro-structure during heating. Several parameters such as number, area,perimeter, and circularity of the protein clusters change during gelevolution. These shape features were calculated as follows:

Average cluster area

¼P

bright pixelsNumber of clusters

; ð1Þ

Average cluster perimeter

¼Pof clusters

Length of outer contour of a clusterNumber of clusters

; ð2Þ

Average cluster circularity

¼Pof clusters

Perimeter of a clusterPerimeter of circle with same area as the cluster

Number of clusters: ð3Þ

These parameters could be used as indices to explain micro-structural aspects of BLG gels.

2.5. Statistical analysis

The morphological image data were subjected to ANOVA usingthe general linear models of the analysis tool from the MicrosoftExcel (v. 2003, Microsoft Corporation, Redmond, WA, USA). Thestatistical model employed was:

Yijk ¼ aBþX3

i¼1

X3

j¼1

bijEij þ eijk; ð4Þ

where Y, morphological properties, B, effect of BLG content (5%, 10%,and 15%), a, coefficient of the effect of BLG content, E, effect of theinteraction between pH and salt content, b, coefficient of the effectof the interaction between pH and salt content, and e, residual var-iation. The subscripts i, j, and k refer to different levels of pH, salt,and BLG, respectively. The effect of the BLG content was usuallylinearly related to the parameters from the image analysis, butthe effect of pH (2, 5, and 7) and/or NaCl (0, 0.1, and 0.3 M) wasnot related linearly to them. Individual term for salt content orpH was not considered since they affect each other to provideelectrostatic attraction or repulsion condition. pH adds or removescharges on the R group of amino acids in BLG depending on NaClcontent, and NaCl affects the charges on the amino acids by ionicshielding depending on pH. Therefore, the B variable for the effectof the BLG content was defined as a continuous variable, whereasthe variable E was defined as a dummy variable to categorize theeffect of both pH and NaCl contents.

3. Results and discussion

3.1. In situ BLG gelation at various conditions

Typical microstructure evolution with temperature during BLGgelation is shown in Fig. 4. Protein clusters in the BLG gels arewhite since they are stained using fluorescent dyes. The figuresare the CLSM images of 5% BLG with 0.3 M NaCl at pH 2, 5, and 7as the specimens were heated at 1 �C/min. Due to the salt content,the BLG systems formed particulate clusters at all pHs. Individualprotein clusters are seen at 50 �C which is below the gelationtemperature. The clusters grew in size as temperature increased,

Cover slip

Specimen drop

Concave slide

Thermal conductive tape

with a hole

Peltier device with a hole

Thermal conductive tape

with a hole

Water cooler

Cover glass

Thermocouple

Concave slide glass

Peltier

Hole

Specimen

(a) Top view

(b) Side view

Water cooler with a hole

Water pump

Fig. 2. The details of the specimen stage for in situ imaging of the BLG system on CLSM.

(a) CLSM raw image (b) Thresholded image (c) Individual clusters (area,

perimeter, circularity)

Fig. 3. Morphological measurement of CLSM image (85 �C, 10% BLG, 0.1 M NaCl, and pH 7).

164 S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170

and after reaching the gelation temperature, the clusters coalescedand formed a network structure. Thus, during gelation there aremarked changes in the morphological features of the gel micro-structure. The pH condition for the gel formation affected the gelmicrostructure. Our CLSM images look similar to the previouslystudied confocal micrographs of final BLG gels heated at 85 �C for

30 min (Kerstens et al., 2005) and a temperature-induced BLGsystem during heating from a dispersion at 20 �C to a gel at 90 �C(Olsson et al., 2002). However, we obtained the images in situand in the real time.

The network of the clusters formed at pH 2 and 5 appearedrelatively at lower temperature than those at pH 7. The micro-

Fig. 4. Microstructure evolution at different pHs with temperature and time elapsed (5% BLG, 0.3 M NaCl, and 1 �C/min heating rate).

S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170 165

graphs imply that the gelation started later at pH 7 than at pH 2or 5. However, it is hard to describe the differences either quali-

tatively or quantatively. The analysis of the morphological param-eters on the micrograph permits to examine the evolution of BLG

Fig. 4 (continued)

166 S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170

gel structure and to compare the structures at different gelationconditions.

The BLG gels are fine-stranded or particulate depending on thegelation conditions. The morphologies of protein clusters of thesegels observed on CLSM micrographs are different. These can poten-

tially reveal structure evolution during gelation as well as help dis-tinguish structural differences among the gels prepared at variousgelation conditions. The morphological parameters of the proteinclusters calculated from typical CLSM micrographs at 65 �C arelisted in Figs. 5–7. The in situ change of structure with temperature

S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170 167

at various gelation conditions was compared using theseparameters.

3.2. Effect of protein concentration

The ANOVA analyses of morphological parameters from theCLSM images are listed in Table 2. Average area and perimeter ofthe BLG clusters increased with increasing protein content, i.e.,the coefficient a of the variable B is positive as listed in Table 2.

Fig. 5. Average area of the protein clusters of BLG system at 65 �C as a

Fig. 6. Average perimeter of the protein clusters of BLG system at 65 �C a

Especially, at pH 2 and 7, the cluster size increased considerablywith protein content approaching a plateau value. Increase of pro-tein content offered more chances for all the interactions whichcontributed to the irreversible aggregation of BLG. In addition,the protein molecules also have more chances to undergo electro-static and hydrophobic interactions. On the contrary, averagearea and perimeter were independent of protein content atpH 5. At pH 5, the hydrophobic interactions are more dominantthan the sulfhydryl-disulfide exchange interactions which can

function of BLG concentration and NaCl content at different pHs.

s a function of BLG concentration and NaCl content at different pHs.

Fig. 7. Average circularity of the protein clusters of BLG system at 65 �C as a function of BLG concentration and NaCl content at different pHs.

Table 2The ANOVA analysis of morphological parameters of the protein clusters calculated from CLSM micrographs of BLG system at 65 �C

Source of variation Coefficients of ANOVAa

Average area Average perimeter Average circularity

BLG (Bb) 0.88**d 0.94*** 0.02***

pH NaCl

2 0 (E11c) 10.4* 18.8*** 1.58***

0.1 (E12) 21.9*** 27.1*** 1.62***

0.3 (E13) 37.0*** 31.8*** 1.47***

5 0 (E21) 30.9*** 24.1*** 1.25***

0.1 (E22) 31.5*** 23.6*** 1.22***

0.3 (E23) 31.1*** 24.1*** 1.25***

7 0 (E31) 0.6NS 7.3* 1.30***

0.1 (E32) 7.8NS 13.1** 1.29***

0.3 (E33) 12.6** 12.7** 1.09***

R2 0.99 0.99 1.00

a The statistical model employed was Yijk ¼ aBþP3

i¼1P3

j¼1bijEij þ eijk . The B variable for the effect of the BLG content was defined as a continuous variable, whereas thevariable E was defined as a dummy variable to categorize the effect of both pH and NaCl contents.

b B = effect of BLG content (5%, 10%, and 15%).c E = effect of the interaction between pH (2, 5, and 7) and NaCl content (0, 0.1, and 0.3 M).d Significantly different at p-value < 0.05 (*); p-value < 0.01 (**); p-value < 0.001 (***); not significantly different (NS).

168 S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170

produce inter- and intramolecular covalent cross-links (Langtonand Hermansson, 1992). In a solution with a pH close to the pI ofthe protein, the protein phase separates at low concentrationsdriven by the increased hydrophobic interactions between theprotein molecules (Olsson et al., 2002). These interactions formlarge clusters which are less sensitive to protein concentration.

Average area of the protein clusters of BLG system at 65 �C as afunction of BLG concentration and NaCl content at different pHs ispresented in Fig. 5. At pH 2, the average area was 12–52 lm2, theincrease coming from increased protein concentration. The clusterarea was larger at pH 5 (36–44 lm2) but the lowest at pH 7 (6–27 lm2). The pH 7 condition also produced linear-stranded clus-ters; few of the strands are seen at 65 �C. The average perimeterof the protein clusters of BLG system at 65 �C is shown in Fig. 6.The trend of the average perimeter of BLG clusters as a functionof BLG concentration and NaCl content at different pHs was similar

to that of the average area (Fig. 7). Fig. 7 shows the average circu-larity of the protein clusters of BLG system at 65 �C. Average circu-larity at pH 2 was relatively larger, i.e., the coefficient b of thevariable E (interaction term of pH and salt content) is the largestat all NaCl conditions, and increased with protein content (Table2). At pH 7, average circularity was the smallest and increased withincreasing protein content due to the appearance of the smallquantity of the protein clusters on the CLSM images at 65 �C, whichresulted in small average circularity. At pH 5, average circularitywas relatively small and independent of protein content.

3.3. Effect of pH

During gelation, the network structure in heat-induced BLG gelis strongly dependent on the balance between attractive and repul-sive conditions in BLG systems. BLG has a net negative charge at

S. Ko, S. Gunasekaran / Journal of Food Engineering 90 (2009) 161–170 169

pH 7, while at pH 2 the protein is positively charged (Aymard et al.,1999). When the pH is far from the pI of BLG and its ionic strengthis sufficiently low, electrostatic repulsion among protein moleculesis abundant and concomitantly the molecules form a continuousnetwork with fine-stranded aggregates (Clark et al., 1981; Stadingand Hermansson, 1990; Langton and Hermansson, 1992; Stading etal., 1992, 1993; Kavanagh et al., 2000a). When pH is shifted towardpI and/or the ionic strength is increased, gel networks with partic-ulate aggregates are formed.

The BLG systems of 10% or 15% protein content at pH 2 with0.3 M NaCl produced the largest BLG clusters, whereas BLG sys-tems at pH 7 without salt showed the smallest (Fig. 5). BLG sys-tems at pH 2 without salt had relatively small average area ofclusters. With the addition of salt in the systems, the cluster areaincreased drastically. Average cluster area was much smaller(about 10–20 lm2) at pH 7 than that at any other pH regardlessof protein content or salt concentration. At 65 �C, the gelationkinetics of systems at pH 2 and 7 were different. At pH 2 a gelstarted to form at 65 �C, i.e., the coefficient b of the variable Ewas larger than that at pH 7 (Table 2). The effect of interaction be-tween pH and added NaCl is substantial at pH 2 compared with atpH 7 as shown in Fig. 5. The BLG clusters were relatively larger andaffected greatly at pH 2 since they began aggregating at 65 �C. Theformation of BLG clusters at pH 7 was tenuous at 65 �C but vigor-ous at higher temperatures. Thus, gelation temperature can beused as a critical index to determine the gelation properties undercertain conditions (Hagolle et al., 1997; Tobitani and Ross-Murphy,1997; Verheul and Roefs, 1998).

The BLG systems at pH 2 evolved their structure at low tem-perature, whereas those at pH 7 started forming clusters withsize above CLSM resolution at relatively high temperature. Differ-ence between the BLG gels at pH 2 or 7 is due to the sulfhydryl-disulfide exchanges. When a BLG gel forms under a propercondition, sulfhydryl groups are replaced with covalent disulfidebonds which enhance gel strength. The weak/brittle texture ingels formed at low pH was due to the prevention of intermolec-ular disulfide bonding (Foegeding et al., 1995). The BLG gelsformed at pH 7 were experienced the formation of initial junc-tion zones by intermolecular disulfide bonds (Hoffmann andvan Mil, 1999) and subsequent strengthening by other interac-tions including hydrophobic interactions, hydrogen-bond forma-tion, and electrostatic interactions (Shimada and Cheftel, 1988).However, the intermolecular sulfhydryl-disulfide exchange reac-tion is short at pH 2 (Aymard et al., 1999). Since the intermolec-ular disulfide bonds are relatively smaller at pH 2, hydrophobicinteractions, hydrogen-bond formation, and electrostatic interac-tions are mainly contributed to the formation of the gel. Thedisulfide bonds were not essential to heat-induced BLG gelationbut contributed greatly to the textural properties (Erringtonand Foegeding, 1998). Lowering the pH from 7 toward acid in-duces brittle gels due to the inhibition of sulfhydryl/disulfidereactions.

At pH 5, the average area of BLG clusters was almost uniformregardless of salt content, i.e., the b values were almost the same.At pH 5, the protein molecules have a low net charge, and so inter-molecular attraction is stronger than electrostatic repulsion, fur-ther favoring aggregation (Langton and Hermansson, 1992, 1996;Stading et al., 1993; Verheul and Roefs, 1998; Marangoni et al.,2000). The strong hydrophobic interactions formed large clustersof protein molecules with a particulate shape. Average perimeterdata showed a pattern similar to that of the area. Average circular-ity at pH 2 with 0.3 M NaCl was the largest whereas that at pH 7with 0.3 M NaCl was the smallest indicating that the shape of iso-lated BLG clusters was close to circle (Fig. 7). The reason why theBLG systems at pH 7 had the smallest average circularity is thattheir isolated clusters just started to appear at 65 �C, whereas the

BLG clusters at other pHs evolved earlier. At pH 5, average circular-ity was uniform and relatively small regardless of other factors.The pH 5 condition offered large clusters even at low temperature.

3.4. Effect of salt concentration

The in situ change in morphology of protein clusters as a func-tion of temperature was influenced by the presence of salt andits concentration. The BLG gels prepared at pH 2 and 7 without saltwere translucent, but they became turbid with increasing salt con-tent. Salt is responsible for shielding the surface charge of proteinmolecules by altering electrostatic properties on protein mole-cules. The change in salt concentration induces different gelationconditions and different gelation properties.

At pH 2 and 7, the average area and perimeter increased withincreasing salt content, i.e., the b values increased with NaCl con-tent as shown in Figs. 4 and 5. These results agree with a previousstudy that BLG molecules are particulated with increasing ionicstrength at neutral pH by kinetic effects without accompanyingfundamental changes in aggregation mechanisms (Ikeda andMorris, 2002). Since salt shields the surface charges on the proteinmolecules, the hydrophobic protein–protein interactions are pro-moted. The effect of salt on the change of average area and perim-eter was the most sensitive at pH 2. The small addition of salthighly affected the increase in average area and perimeter of theclusters at pH 2. Hydrophobic interactions are promoted suddenlywith addition of salt at pH 2.

At pH 5, average area and perimeter were unaffected by salt(the b values were almost same for all NaCl conditions). Averagecircularity is also a good parameter to describe the effect of salton the structure during gelation. At pH 2 and 7, the average circu-larity was not linearly related to salt content at 65 �C. The averagecircularity was the largest at 0.1 M NaCl. With addition of saltabove 0.1 M, the circularity decreased dramatically, i.e., clustersbecame round (Fig. 7). The effect of salt addition up to 0.1 M onthe change in average circularity was weak while that above0.1 M was very effective. Consequently, the average circularity ofBLG systems with 0.3 M NaCl was the smallest for all conditionsat pH 2 and 7.

4. Conclusions

In situ evaluation of dynamic changes in BLG microstructurewas studied using confocal laser scanning microscopy. The mor-phological parameters such as area, perimeter, and circularity ofprotein clusters during heating were analyzed to determine evolv-ing BLG gel microstructure and properties. Quantitative analyses ofchanges in these morphological parameters significantly help im-prove our understanding of various structure-function relation-ships in heat-induced BLG gelation. The methods presentedherein are extendable to analyzing in situ microstructure evolutionand end-use properties of other food gels.

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