This article was downloaded by: [Laurentian University]On: 04 October 2014, At: 04:47Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

International Journal of FoodPropertiesPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/ljfp20

Effect of water content, temperatureand storage on the glass transition,moisture sorption characteristics andstickiness of β‐caseinL. J. Mauer a , D. E. Smith b & T. P. Labuza aa Department of Food Science and Nutrition , University ofMinnesota , 1334 Eckles Ave., St. Paul, MN, 55108, USAb Department of Food Science and Nutrition , University ofMinnesota , 1334 Eckles Ave., St. Paul, MN, 55108, USA Phone:612‐624‐3260 Fax: 612‐624‐3260 E-mail:Published online: 02 Sep 2009.

To cite this article: L. J. Mauer , D. E. Smith & T. P. Labuza (2000) Effect of water content,temperature and storage on the glass transition, moisture sorption characteristics andstickiness of β‐casein , International Journal of Food Properties, 3:2, 233-248, DOI:10.1080/10942910009524630

To link to this article: http://dx.doi.org/10.1080/10942910009524630

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,

systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

INTERNATIONAL JOURNAL OF FOOD PROPERTIES, 3(2), 233-248 (2000)

EFFECT OF WATER CONTENT, TEMPERATURE AND STORAGE ON THEGLASS TRANSITION, MOISTURE SORPTION CHARACTERISTICS AND

STICKINESS OF β-CASEIN1

L. J. Mauer, D. E. Smith2,*, and T. P. Labuza

Department of Food Science and Nutrition, University of Minnesota,1334 Eckles Ave., St. Paul, MN 55108, USA

*Corresponding author (Tel.: 612-624-3260, Fax: 612-625-5272, and E-mail:dsmith@che.che.umn.edu )

ABSTRACT

Moisture sorption isotherms at +4 °C and +22.5 °C were obtained for β-casein afterisolation and after 9 months of storage at -29 °C and +22.5 °C. Glass transition statediagrams (Tg vs. moisture) were determined for β-casein after storage. The resultsshowed that effects of storage temperature on moisture sorption isotherms were varied;however, at any a w differences in moisture content were small (< 0.03g H2O/g solids athigh a w ). β-casein stored at -29°C had lower mo and T g values than that of β-caseinstored at +22.5°C. The glass transition temperatures for β-casein were above roomtemperature, even at a w = 0.76. Onset of stickiness occurred above a w = 0.76.

INTRODUCTION

In the ongoing effort to improve and expand the supply of nutritional and functionalfood ingredients, the unique properties of casein and individual casein proteins areattractive for many applications. The majority of caseins exist in milk as micelles.Individual caseins are relatively small (20,000-24,000 Daltons), amphipathic,conjugated, randomly coiled, hydrophobic, open molecules that are insoluble aroundtheir isoelectric points (pH 4.5-4.9) and have an uneven distribution of polar andhydrophobic residues (Fox, 1989). Caseinates, water soluble derivatives of casein, are

1 Published as paper number 99-1-18-0017 of the contribution series of the MinnesotaAgricultural Experiment Station based on research conducted under project 18-24 andsupported by the National Dairy and Promotion Board and the USDA National NeedsFellowship Program.

233

Copyright © 2000 by Marcel Dekker, Inc. www.dekker.com

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

234 MAUER, SMITH, AND LABUZA

added to many food products to utilize their water holding capacity, heat capacity, andsurface active properties. Casein and caseinates are ingredients used in foamed andwhipped foods, cream substitutes, infant foods, margarine, cheese products, instantbreakfasts, formulated meat products, and extruded snacks (Modler, 1985; Southward,1989). To increase the value of casein, one goal of the dairy industry is to obtainpurified a^-, a^-, p- and K-casein fractions which may have applications beyond those ofthe whole casein protein (Modler, 1985; Pearce, 1994).

Ward (1998) developed a method for isolating large quantities of β-casein fromsodium caseinate. β-casein constitutes 25-35 % of the casein in milk and is the mostamphipathic and hydrophobic of the casein fractions (Swaisgood, 1985). It has beenproposed that β-casein would be useful in new products as an emulsifier or foamstabilizer (Donnelly et al., 1991; Smithers et al., 1991), as a means of adjusting rennetcurd strength (Donnelly et al., 1991), as a nutritional part of infant formula (Murphy andFox, 1991), or as a starting ingredient for obtaining bioactive peptides (Maubois, 1984).In each of these applications, knowledge of the moisture binding characteristics of p-casein would be useful for product development.

The interactions between protein and water are important to most functionsperformed by milk proteins in food products. The information contained in a moisturesorption isotherm, the physico-chemical relationship between moisture content andwater activity, can be utilized to control chemical, physical, and quality attributes instored foods, to ensure microbial stability, and to select the most suitable packagingmaterials to minimize quality deterioration (Labuza, 1984). An excellent review ofwater sorption, hydration, and water activity of milk proteins at low moisture levels waspublished by Kinsella and Fox (1986); however, there are no moisture sorption dataavailable for β-casein in the literature.

The behavior of products during storage is an important consideration fornutritional formulations and functional ingredients. Karel et al. (1993) proposed that theeffects of moisture and temperature on the rates of changes that occur during storageare related to the physical state of the food, most notably above the glass transitiontemperature. The glass transition temperature (Tg) is the temperature at which anamorphous material undergoes a change from a highly viscous "glassy" state to a"rubbery" state in which deformation and flow are possible. At low moisture contents,proteins generally exist in the glassy state; however, as moisture content increases andwater acts as a plasticizer in the protein structure, the T g decreases, and the proteinchanges from a free flowing powder to a sticky cohesive mass (Roos and Karel, 1990;Peleg, 1993; Chuy and Labuza, 1994). Above the Tg, structural changes specific tomolecular weight and moisture content, including stickiness, caking, and collapse, maylead to deterioration of product quality (Saltmarch and Labuza, 1980; Roos and Karel,1990; Slade and Levine, 1991; Netto et al., 1998).

Chuy and Labuza (1994) showed that the T g may be used to predict the onset ofstickiness and caking in powders during long term storage. Stickiness occurs whenparticles stick together but may be broken apart by low shear. This generally appears at10-20 °C above the T g (Peleg, 1993; Roos, 1995). As moisture content increases or theproduct is stored longer, caking may be observed. Caking happens when the forcerequired to stir a powder increases dramatically (Aguilera et al., 1994) and can set inrapidly at 20-30 °C above the T g (Aguilera and Del Valle, 1993). The time,temperature, and moisture dependent viscous flow which results in a loss of structure attemperatures 40-50 °C above the T g is called collapse (Tsourouflis et al., 1976)Kalichevsky et al. (1993) suggested that casein is less plasticized by water than

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

P-CASEIN 235

amylopectin and gluten, and Netto et al. (1998) found that casein hydrolysates did notexhibit the caking and collapse observed for whey protein and fish protein hydrolysates.Therefore, β-casein could be more likely than other proteins to maintain product qualityduring processing and storage.

The objectives of this study were to determine the changes in moisture sorptionisotherms, glass transition behavior, and stickiness of β-casein immediately afterisolation and after storage for nine months at two temperatures, -29 °C and +22.5 °C,which simulate frozen and room temperature storage.

MATERIALS AND METHODS

Materials

Sodium caseinate (Alanate 180) was obtained from New Zealand Milk Products Inc.(Santa Rosa, CA). Calcium chloride, urea, sodium chloride, potassium chloride, sodiumnitrite, lithium chloride, magnesium nitrate, potassium carbonate, DrieRite, andmagnesium chloride were purchased from Fisher Scientific (Fair Lawn, NJ). Bis-Tris-propane and hydrochloric acid were purchased from Sigma Chemical Company (St.Louis, MO). A Mono Q anion exchange column was obtained from PharmaciaBiotechnology (Uppsala, Sweden).

P-Casein

p-Casein was isolated from sodium caseinate using the method developed by Ward(1998). The spray-dried isolated protein was divided into two lots, placed intocontainers, sealed using two layers of Para-Film® underneath screw-type lids, and thenstored in temperature controlled chambers (±1°C) at -29 °C and +22.5 °C for ninemonths. After storage, moisture content of protein samples was determined by theAmerican Dairy Products Institute (1991) vacuum oven method, 100 °C for 5 hours.-29 °C stored β-casein contained 3.6g H2O/100g sample, and +22.5 °C stored β-caseincontained 4.5g H2O/100g sample.

Fast Protein Liquid Chromatography

A^fast protein liquid chromatography (FPLC) method developed by Davies and Law(1987) was used to determine the purity of the isolated β-casein. Absorbance wasmonitored at 280 nm, a Mono Q anion exchange column was used, and the flow ratewas 1 ml/min. Buffer one contained 0.005 M bis-Tris-propane and 3.3 M urea adjustedto pH 7.0 with HC1, and buffer two consisted of 0.005 M bis-Tris-propane, 3.3 M urea,and 1 M NaCl also adjusted to pH 7.0. A representative chromatogram of the β-caseinsamples is shown in Figure 1. No differences were observed in the chromatograms ofinitial and stored β-casein. The two main peaks eluting at 18 to 20 minutes indicate thatthe isolated protein was mainly composed of a mixture of β-casein and mono-dephosphorylated β-casein. Smaller peaks indicate small quantities of a- and tc-caseins.

Moisture Sorption Isotherms

"Working" moisture sorption isotherms at two temperatures (+4 °C and +22.5 °C) weredetermined using the method proposed by Labuza (1980). Approximately 1 gram

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

236 MAUER, SMITH, AND LABUZA

5 10 15 20 25 30 35 40 45 50

Figure 1. FPLC chromatogram of β-casein

samples of β-casein were weighed into aluminum dishes and placed into a desiccatorwhich contained a saturated salt solution at a water activity between 0.01 and 0.92. Sixreplicates were used at each water activity. Saturated salt solutions used were: LiCl,MgCl2, K2CO3, Mg(NO3) 2, NaNO2, NaCl, and KC1. Each desiccator was evacuated toabout 30 mm Hg. The desiccators were then placed into a temperature controlledchamber (±1°C), and samples were allowed to equilibrate for 10 days. Preliminaryexperiments showed that β-casein powders reached equilibrium relative humidity at 22.5°C, whether by absorption or desorption, within 48 hours; however, to ensure a constantaw, β-casein samples were equilibrated for 10 days prior to use.

Moisture content was determined by three methods: gravimetric, vacuum oven ,and Karl Fischer. All six samples were weighed at day ten for the gravimetric moisturedetermined as weight difference from the initial weight. Initial moisture content wasdetermined by vacuum oven. The American Dairy Products Institute (1991) vacuumoven method was used to determine moisture by vacuum oven of three replicates bydrying at 100 °C for 5 hours. Moisture was determined as percent weight lost. Each ofthe remaining three replicates were sealed in a container with 25 mL of methanol, held at4 °C for 15 hours, then placed on a bench top for one hour. The methanol was decantedinto a vial, centrifuged at 5,000 rpm for 10 min, and then 0.5 mL was injected intriplicate into a Karl Fischer apparatus (Aquatest CMA, Photovolt, Indianapolis, IN).Moisture was determined from the quantity of electricity required to complete thewater-iodine reaction.

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

(3-CASEIN 237

Differential Scanning Calorimetry

Samples of β-casein were pressed into 3.3 mm diameter pellets weighing 10-15 mg eachand stored for 10 days at +22.5 °C in the same desiccators used for the isotherms. Afterequilibration, the pellets were placed into aluminum DSC pans, returned to thedesiccators for 2 days, hermetically sealed, and then weighed. The data on moisturecontent were obtained from the isotherms based on the vacuum method of moisturedetermination.

Glass transition temperatures were determined at least in duplicate using aPerkin-Elmer DSC 7 with a Perkin-Elmer TAC 7 Instrument Controller (Norwalk, CT)calibrated with HPLC-grade water (melting point 0 °C) and indium (melting point 156.6°C). The samples were scanned twice, from 0 to 160 °C, to eliminate the hysteresiseffect of thermal relaxation that is typical for a glass transition. A small peak between50 and 60 °C appeared in the first scan of each sample but was not present in the secondscan. This endothermic relaxation behavior was also observed for rennet casein byKalichevsky et al. (1993). The initial scanning rate was 5 °C/min, samples were cooledat 10 °C/min, and the glass transition temperatures were determined as the onset of theobserved change in heat capacity from the second scan at 5 °C/min.

Scanning Electron Microscopy

Samples of β-casein were placed on aluminum dishes in thin layers and allowed toequilibrate for 10 days in the same desiccators used for the moisture sorption isotherms.The protein samples were then sprinkled onto specimen stubs, freeze dried, and coatedwith a 60:40 mixture of gold and palladium at 25 °C and 2xl0'5 Torr pressure for 2-3minutes. The coated samples were viewed in a Hitachi 5450 at 10 kV using standardmicroscope operating procedures. Magnifications of 320, 640, and 2500 were used.

Statistical Analysis

The PC-SAS program (SAS, 1985) was used to determine p-values by the general linearmodel for comparison of moisture content and glass transition data. Student-Newman-Keuls' test was used to determine the significance of differences. Significance wasdefined as p < 0.05.

RESULTS AND DISCUSSION

Effect of Method on Moisture Content Determination

Moisture contents for initial and stored β-casein samples determined by gravimetric,vacuum oven, and Karl Fischer methods are shown in Tables 1, 2, and 3. The KarlFischer method produced significantly higher (p = 0.0001) moisture contents than theother two methods at each water activity, for each protein treatment, and for eachequilibration temperature. For samples equilibrated at +4 °C, moisture contentsdetermined by the gravimetric method were significantly lower (p < 0.05) than the othersat all water activities except a w = 0.46 and a w = 0.736. In the +22.5 °C equilibration thegravimetric method produced the significantly lowest moisture contents at aw ^ 0.33.

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

Table 1. Moisture contents (g water/g solids) of β-casein determined by gravimetric method

tooo

4 °C Equilibration "

unstorcd' -29 °C stored ° +22.5 °C stored' aw

22.5 °C Equilibration "

unstored -29 °C stored +22.5 °C stored

0.130 0.035 ±0.001 0.038 ±0.0005 0.047 ± 0.0008

0.355 0.068 ± 0.0007 0.069 ± 0.002 0.073 ± 0.0007

0.4 W) 0.0') I ±0.007 0.09210.004 0.09S 1 0.0003

0.587 0.11610.002 0.12410.003 0.13210.002

0.736 0.I3S1 0.001 0.153:1.0.002 0.1(.0 J. 0.0009

0.807 0.15910.002 0.16910.003 0.18410.002

0.938 0.21010.005 0.24010.005 0.25510.002

0.116 0.03010.0006 0.03710.001 0.03310.0005

0.332 0.0661 0.0008 0.074 ± 0.003 0.0571 0.001

0.445 0.085 ± 0.0004 0.10010.002 0.06710.002

0.541 0.1061 0.00! 0.12510.002 0.09510.002

0.667 0.130 J. 0.0006 0.1251 0.002 0.11110.005

0.767 0.15710.001 0.15810.001 0.14110.0006

0.861 0.195 1 O.(X)5 0.187 1 0.001 0.174 1 0.002

* P-cascin equilibrated in desiccators for 10 days at 4 °C prior to evaluationb 3-casein equilibrated in desiccators for 10 days at 22.5 °C prior to evaluationc (3-casein evaluated within one month after isolation

* P-cascin stored at -29 °C for 9 months prior to evaluatione β-casein stored at +22.5 °C for 9 months prior to evaluation

C/3

2

c!N>

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

Table 2. Moisture contents (g watcr/g solids) or P-cascin determined by vacuum method >

4 ° C Equil ibrat ion '

aw uns torcd ' -29 °C stored '' +22.5 °C stored '

22.5 °C Equilibration"

unslorcd -29 °C stored +22.5 °C stored

0.130 0.053 + 0.002 0.051 ± 0.0001 0.043 ± 0.002

0.355 0.082 ± 0.001 0.077 ± 0.0005 O.O7O+O.OOO4

0.460 0.09910.003 O.IO3+O.OO2 0.094+0.0004

0.587 0.12610.003 0.14110.004 0.13410.002

0.736 0.14910.0008 0.16010.002 0.15310.004

0.807 0.168+0.0002 0.185+0.004 0.19410.0008

0.938 0.22010.0005 0.25610.003 0.25910.011

0.116 0.038+0.002 0.03610.007 0.04910.0007

0.332 0.074+ 0.00.1 0.071 ± 0.004 0.071 ± 0.002

0.445 0.09010.002 0.09510.001 0.087+0.006

0.541 0.10810.001 0.122+0.002 0.10910.002

0.667 0.1301 0.002 0.1321 0.003 0.131 1 0.003

0.767 0.15610.0009 0.15310.003 0.16310.003

0.864 0.19510.003 0.18310.002 0.19210.001

' P-casein equilibrated in desiccators for 10 days at 4 °C prior to evaluationk P-cascin equilibrated in desiccators for 10 days at 22.5 °C prior to evaluation

' P-cascin evaluated within one month after isolation

' P-cascin stored at -29 °C for 9 months prior to evaluation

• P-cascin stored at +22.5 °C for 9 months prior to evaluation

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

Table 3. Moisture contents (g vvater/g solids) of P-cascin dctcrinincd by Karl Fischer inctliod

to©

4 °C E(|uilibration"

unslorcd c -29 "C stored * +22.5 °C stored *

22.5 °C Equilibration1

aw unstorcd -29 °C stored +22.5 °C stored

0.130 0.095 ±0.001 0.088 ± 0.003 0.082+ 0.002

0.355 0.121 ±0 .002 0.129 ± 0 . 0 0 2 0 .115+0.0004

0.460 0.152 ± 0.002 0.162+0.009 0.152 ± 0.000S

0.587 O.185± 0.002 0.202 ± 0.005 0.197 ± 0.0001

0.736 0.224 ±0.005 0.240 ± 0.005 0.230 ± 0.00<l

0.807 0.251 ±0.002 0.28f>± 0.001 0.259± 0.005

0.938 0.338 ±0.005 (l.3S5±(M)O7 0.375 ± 0.009

" P-cascin equilibrated in desiccators lor 10 days at 4 "C prior to evalualionb (5-cascin equilibrated in desiccators lor 10 days at 22.5 °C prior to evaluationc P-cascin evaluated within one month after isolationd P-cascin stored at -29 °C for 9 months prior to evaluation

' P-cascin stored at +22.5 °C for 9 months prior to evaluation

0.116 0.059 ± 0.001 0.066 ± 0.002 0.082 ± 0.001

0.332 0.113+ 0.0001 0.104 ±0.004 0.104 ±0.002

0.445 0.140 ± 0.003 0.138 ± 0.001 0.125 ±0.002

0.541 0.172 ± 0.001 0.188+0.004 0.161 ± 0.003

0.667 0.205 ± 0.005 0.182 ± 0.004 0.185 ±0.004

0.767 0.249 ± 0.005 0.224 ± (1.0006 0.231 ±0.025

0.864 0.317* 0.001 0.264 ±IM«)3 O.2/6± 0.001

135

Or>CO

N>

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

P-CASEIN 241

The Karl Fischer method is often preferred over oven drying methods becausehigh oven temperatures can decompose sugar and protein in food samples resulting in anover-estimation of moisture content. While the vacuum oven moisture contents weresomewhat higher than the gravimetric results, the Karl Fischer method consistentlyproduced the significantly highest moisture contents. When the Karl Fischer methodwas performed on β-casein samples that had been dried in the vacuum oven, theAquatest CMA indicated the presence of more water; however, combining the moisturecontents of the vacuum oven and Karl Fischer results for these samples did not add upto the original Karl Fischer moisture contents.

Either the Karl Fischer method is stripping off "bound" water not removed bythe other methods, refer to the classification of types of water by Kinsella and Fox(1984), or the β-casein is reacting with the vessel solution in such a way as to skew theresults. Whatever the case, the moisture contents determined by the Karl Fischermethod were not used for constructing isotherms or plotting glass transition behavior.The results obtained by the standard vacuum oven method were used for the remainderof this research.

Effect of Temperature on Moisture Sorption Isotherms

Moisture sorption isotherm data for each protein treatment (unstored, -29 °C stored,and +22.5 °C stored) and each equilibration temperature (+4 °C and +22.5 °C) art-shown in Table 2. When graphed, the type II sigmoidal isotherm behavior of GAB trendlines observed for β-casein is similar to the behavior of moisture sorption by other milkproteins. The water sorption by β-casein is lower than that of <xsl -casein and sodiumcasemate but in the same range as the casein micelle moisture sorption determined byBerlin (1981). Compared to moisture sorption isotherm data presented by Iglesias andChirife (1982), β-casein has the lowest moisture sorption of all milk proteins.

Water sorption data were analyzed using the BET (Brunauer-Emmett-Teller)and GAB (Guggenheim-Anderson-DeBoer) equations (Table 4). The BET isothermequation is adequate for proteins up to a w 0.5 while the GAB equation which takes intoaccount multilayer water sorption may be used to describe moisture binding at higherwater activities. Both BET (r2> 0.98) and GAB (P<0.06) equations provided goodmodels for moisture sorption by β-casein. Lomauro et al. (1985) determined that P <0.05 is an extremely good fit and that 43% of milk product moisture isotherms aredescribed well by the GAB equation. Monolayer values were higher for the GABanalysis than the BET analysis. Ozimek et al. (1992) observed similar differences inmonolayer values for ultrafiltered retentate skim milk powders and postulated that thedifference could be accounted for by the modified properties of adsorbed water in themultilayer region used in the GAB calculation.

Monolayer moisture contents determined by both BET and GAB methodsshowed the same trend. The β-casein stored at the higher temperature (+22.5 °C) hadthe lowest monolayer and moisture values while the frozen (-29 °C) β-casein had thehighest monolayer values. β-casein has an approximate molecular weight of 24,000Daltons (Swaisgood, 1992); however, β-casein polymerizes to self-limiting micellesthrough intermolecular hydrophobic interactions in the C-terminal region, and thepolymer size is dependent on conditions (Andrews et al., 1979; Buchenheim andSchmidt, 1979; Evans and Phillips, 1979). At 4 °C β-casein is a monomer withmolecular weight approximately 24,000 Daltons, at 22 °C and protein concentration

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

Table 4. BET and GAB parameters for β-casein based on vacuum oven moisture sorption isotherms -p.

unstorcd'

4 °C Equilibration "

-29 °C stored " +22.5 °C storedc

22.5 °C Equilibration"

unstoretl -29 °C stored +22.5 °C stored

BET Parameters

in. (p 11..O/g solids)aw al nionoljvnr1

e (.surface hc;it conlenl)Qs (J/molc 11,0)

GAB Parameters

in,, (g I I;O/g solids)aw at monolaycrP value (% avg. error)GAB constant (C)Qs (J/molc 11,0)kb

0.05550.151

o.yyys32.698544

0.07650.287

2.115.4667100.71

0.05710.177

0.9S7519.297288

0.0780.304

4.611.2859650.75

0.05340.198

0.990514.576170

0.07240.318

5.78.9450480.79

0.05330.1970.99315.096647

0.06190.248

0.99727.534969

0.05060.14

0.998837.888950

0.07370.332

0.89.51

55170.74

0.09990.472

3.66.3445460.6

0.06250.227

3.720.173880.81

>

rn

on

>

>68GN>

1 (3-cascin equilibrated in desiccators for 10 days at 4 °C prior to evaluationb P-casein equilibrated in desiccators for 10 days at 22.5 °C prior to evaluationc P-casein evaluated within one month after isolation

' P-casein stored at -29 °C for 9 months prior to evaluation

' P-cascin stored al +22.5 °C for 9 months prior to evaluation

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

(3-CASEIN 243

Table 5. Onset glass transition temperature (Tg in °C) and moisture content (Md,g/lOOg solids) at various water activities for β-casein stored at -29°C or +22.5°Cfor 9 months

-29 °C stored ' +22.5 °C stored"

a« Md T s Md T£

0.116

0.332

0.445

0.541

0.667

0.767

3.57

7.15

9.53

12.21

13.25

15.33

104 ± 3

87 ± 1

79 ± 2

63 ± 1

53 ± 1

41 ± 1

4.89

7.07

8.72

10.88

13.17

16.31

120 ±

95 ±

80 ±

73 ±

*

58 ±

3

1

1

2

2

2 3-casein stored at -29 °C for 9 months prior to evaluationh (3-casein stored at +22.5 °C for 9 months prior to evaluation* No T s observed

1.26 mg/ml β-casein is a polymer with a molecular weight of 45,000 Daltons, and at 22°C and 5.26 mg/ml the molecular weight of β-casein is 120,000 Daltons (Buchenheimand Schmidt, 1979; Evans and Phillips, 1979). While not in solution, the β-casein usedin these isotherms could have undergone some polymerization at 22.5 °C during storage,thereby decreasing the number of sights available for binding water and lowering themonolayer value.

The effects of storage temperature on moisture sorption isotherms were varied.No differences were observed in the moisture contents of unstored and +22.5 °C stored β-casein samples in the 22.5 °C isotherms. However, at +22.5 °C the frozen (-29 °Cstored) β-casein showed significantly higher moisture content than other samples at a w

=0.54 and significantly lower moisture content at a w £ 0.76. This behavior was notrepeated in the +4 °C isotherms. At +4 °C and a w ^ 0.59, the unstored β-casein had thelowest moisture content. β-casein stored at +22.5 °C had the lowest moisture contentat a w = 0.13 and highest moisture content at a w = 0.807 in the +4 °C isotherms.

Effect of Moisture Content on Tg

Onset glass transition temperatures determined by the second scan DSC heating rate at 5°C/min for β-casein stored for nine months at -29 °C and +22.5 °C and then equilibratedfor 10 days over salt solutions at 22.5 °C are shown in Table 5 and Figure 2. Significant

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

244 MAUER, SMITH, AND LABUZA

140

120

40

20

0

• -29 "C ft-casei

"100 ^ ^ ^ N. • *2 2 5 * c • " *° ^ ^ ^ « Power (.-22.5-C G-casein)o, o n ^ * ^ ^ ^ * " Linear (-29 "C G-casein)

I 60O

y = -53551 x + 125.75

20 R* = 09804

0 5 10 15 20Vacuum moisture content (g H2O/ 100g solids)

Figure 2. State diagrams from DSC analysis at a heating rate of 5 °C for β-caseinstored at two temperatures for nine months

differences between glass transitions of the β-casein storage treatments occur at both thelow and high moisture contents. This suggests that some protein aggregation occurredat the higher temperature (+22.5 °C) which raised the Tg. Aggregation increasesmolecular weight, and studies have shown that increasing molecular weight alsoincreases the T g (Fox and Flory, 1950). The effects of aggregation at 22.5 °C were alsoobserved in the lower monolayer values described in the previous section. Therelationship of onset T g and moisture content shown in Figure 2 was expected. Wateracts as a T g decreasing plasticizer (Roos, 1995); therefore, as moisture content isincreased, T g is decreased.

In polymer systems, the change in heat capacity at the glass transition point canbe small and therefore difficult to determine using a DSC method. More than 10 mg of β-casein had to be used to observe T g behavior, changes in heat capacity became smalleras water activity increased, and no glass transitions were observed for samples at a w >0.8. This behavior is consistent with that of the "strong" glasses described by Angell(1985, 1991). LeMeste and Duckworth (1988) were unable to detect any glasstransitions in hydrated caseinate using a DSC. T g values obtained for β-casein weresimilar to values for rennet casein and Sigma's casein determined by Kalichevsky et al.(1993). Matveev et al. (1997) calculated the T g at zero water activity for pure β-caseinbased on its amino acid content and reported it at 164 °C. This is beyond the a w rangeof this research but does support the relatively high T g values obtained for the β-caseinsamples. As expected, the glass transitions for β-casein were higher than those foundfor casein hydrolysates by Netto et al. (1998). Thus, it may be possible to add β-caseinto foods to increase their glass transition temperature or stability.

Visual Observation and SEM Analysis of the Samples

Immediately after isolation β-casein was a soft, light powder. After 10 days of storagein desiccators containing saturated salt solutions, little change in color, hardness, or

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

P-CASEIN 245

(a)

(b)

Figure 3. SEM micrographs of β-casein equilibrated at 0.11 a w (a) and 0.75 aw (b)after 10 days.

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

246 MAUER, SMITH, AND LABUZA

volume was observed in the low and intermediate a w samples. At aw's above 0.7, aslight surface hardening was observed, but no samples showed caking or collapse.Figure 3 shows SEM photos of β-casein at a w = 0.11 and at aw = 0.76. At a w = 0.76more particles were clustered in small groups than at the lower water activities. Nodifferences were noticed between the initial and the stored samples of β-casein. Similarobservations were recorded for casein hydrolysates (Netto et al., 1998) which showedless physical change at higher relative humidity than other protein hydrolysates. Netto etal. (1998) proposed that casein hydrolysates could be classified as noncohesive powderswith negligible interparticle interactions. The same can be said for β-casein powders.This free-flowing behavior is supported by the glass transition results. Even at a w =0.76 the glass transition temperature was above room temperature and β-casein was inthe glassy state. As described by Kalichevsky et al. (1993) for rennet casein, β-caseinhas the potential to inhibit the caking, stickiness, and structural collapse of powderedfood formulations and is probably a better structural stabilizer than starch in foodsexposed to intermediate or high humidity.

CONCLUSIONS

The glass transition temperature of β-casein is higher than room temperature up to a a w

of 0.76. Moisture content greater than 16g H2O/ lOOg solids is needed for the T g toapproach room temperature. Water sorption by β-casein is lower than that for othermilk proteins, even after the β-casein has been stored for nine months at - 29 °C or +22.5 °C. This behavior was expected since β-casein is the most hydrophobic protein inmilk. These results indicate that β-casein has the potential to be used as a functionalingredient for increasing the stability of food products.

REFERENCES

Aguilera, J. M., and Del Valle, J. M. 1993. Structural changes in low moisture foodpowders. In: The Glassy State in Foods. Blanshard, J. M. V. and Lillford, J. P.eds. Nottingham University Press, Loughborough, England, p. 675-691.

Aguilera, J. M., Levi, G., and Karel, M. 1993. Effect of water content on the glasstransition and caking of fish protein hydrolysate. Biotechnology Progress. 9:651-654.

American Dairy Products Institute. 1991. Determination of moisture by vacuum ovenmethod. In: Standards for Grades of Dry Milks Including Methods of Analysis:Bulletin 916. Chicago, IL. p. 24.

Andrews, A. L., Atkinson, K., Evans, M., Finer, E.G., Green, J.P., Phillips, M., and

Robertson, R. N. 1979. The conformation and aggregation of bovine β-caseinA. I. Molecular aspects of thermal aggregation. Biopolymers. 18: 1105-1121.

Angell, C. A. 1991. Relaxation in liquids, polymers, and plastic crystals - patterns andproblems. Journal of Non-Crystalline Solutions. 13: 131-133.

Angell, C. A. 1985. Strong and fragile liquids. In: Relaxations in Complex Systems.Ngai, K., and Wright, G.B. eds. National Technical Information Service, USDepartment of Commerce, Springfield, VA, USA. p. 1.

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

β-CASEIN 247

Berlin, E. 1981. Water Activity: Influence on Food Quality. Rockland, L., andStewart, G. eds. Academic Press, New York. p. 467.

Buchenheim, W., and Schmidt, D. G. 1979. On the size of monomers and polymers of

β-casein . Journal of Dairy Research. 46: 277-280.Chuy, L., and Labuza, T. P. 1994. Caking and stickiness of dairy-based food powders

as related to glass transition. Journal of Food Science. 5 (1): 43-46.Davies, D. T., and Law, A. J. R. 1987. Quantitative fractionation of casein mixtures by

fast protein liquid chromatography. Journal of Dairy Research. 54: 369-376.Donnelly, W. J., O'Callaghan, D. M., and Mehra, R. 1991. Modification of milk

protein functionality by new technologies for separation and hydrolysis. In. IDFspecial issue 9303; Int. Dairy Fed., Brussels, Belgium, p. 18-29.

Evans, M.T.A., and Phillips, M. C. 1979. The conformation and aggregation of bovine

β-casein A. II. Thermodynamics of thermal association and the effect of changesin polar and apolar interactions on micellization. Biopolymers. 18: 1123-1140.

Fox. P. F. 1989. The milk protein system. In: Developments in Dairy Chemistry - 4.Fox, P. F. ed. Elsevier Applied Science, London, p. 1-54.

Fox, T. G., and Flory, P. J. 1950. Second-order transition temperatures and relatedproperties of polystyrene. I. Influence of molecular weight. Journal of ApplliedPhysics. 21: 581-591.

Hermansson, A. M. 1977. Functional properties of proteins for foods-water vapoursorption. Journal of Food. Technology. 12: 177-187.

Iglesias, Η. Α., and Chirife, J. 1982. Handbook of food isotherms: water sorptionparameters for food and food components. Academic Press, New York.

Kalichevsky, M. T., Blanshard, J. M., and Tokarczuck, P. F. 1993. Effect of watercontent and sugars on the glass transition of casein and sodium caseinate.International Journal of Food Science and Technology. 28: 139-151.

Karel, M., Roos, Y., and Buera, M. P. 1993. Effects of glass transition of processingand storage. In: Glassy State in Foods. Blanshard, J. M., and Lilliford, P. J.eds. Nottingham University Press, Loughborough, England, p. 13-34.

Kinsella, J. E., and Fox, P. F. 1986. Water sorption by proteins: milk and whey proteins.CRC Critical Reviews of Food Science and Nutrition. 24: 91-139.

Labuza, T. P. 1984. Moisture sorption: Practical aspects of isotherm measurementand use. American Association of Cereal Chemists. St. Paul, MN.

Le Meste, M., and Duckworth, R. B. 1988. Effect of water content on the mobility ofsolute molecules and of protein side chains in caseinate preparationsInternational Journal of Food Science and Technology. 23: 457-466.

Lomauro, C. J., Bakshi, A.S., and Labuza, T. P. 1985. Evaluation of food moisturesorption isotherm equations part II: milk, coffee, tea, nuts, oilseeds, spices andstarchy foods. Lebensmittel Wissenschaft und Technologie. 18:118-124.

Matveev, Y. I., Grinberg, V. Y, Sochava, I. V., and Tolstoguzov, V. B. 1997. Glasstransition temperature of proteins. Calculation based on the additivecontribution method and experimental data. Food Hydrocolloids. 11(2): 125-133.

Maubois, J. L. 1984. Separation, extraction and fractionation of milk proteincomponents. Le Lait. 64: 485-495.

Modler, H. W. 1985. Functional properties of nonfat dairy ingredients - a review.Journal of Dairy Science. 68: 2195-2205.

Murphy, J. M., and Fox, P. F. 1991. Fractionation of sodium caseinate by ultrafiltration.Food Chemistry. 39:27-38.

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4

248 MAUER, SMITH, AND LABUZA

Netto, F. M., Desobry, S. A., and Labuza, T. P. 1998. Effect of water content on theglass transition, caking, and stickiness of protein hydrolysates. InternationalJournal of Food Properties. 1(2):141-161.

Ozimek, L., Switka, J., and Wolfe, F. 1992. Water sorption properties of ultrafitrationretentate skim milk powders. Milchwissenschaft. 47(12): 751-754.

Pearce, R. J. 1994. Food functionality: success or failure for dairy based ingredients.Australian Journal of Dairy Technology. 50:15.

Peleg, M. 1993. Glass transition and the physical stability of food powders. In: TheGlassy State in Foods. Blanshard, J. M. V. and Lilliford, J. P. eds. NottinghamUniversity Press, Loughborough, England, p. 435-451.

Roos, Y., and Karel, M. 1990. Differential scanning calorimetry study of phasetransition affection the quality of dehydrated materials. Biotechnology Progress.6:159-163.

Roos, Y. 1995. Phase Transition of Foods. Academic Press.Saltmarch, M., and Labuza, T. P. 1980. Influence of relative humidity on the

physicochemical state of lactose in spray dried sweet whey powders. Journal ofFoodScience. 45: 1231-1236.

SAS Institute. 1985. SAS User's Guide: Statistics. Version 5. SAS Inst., Inc. Cary,NC.

Slade, L., and Levine, H. 1991. Beyond water activity: recent advances based on analternative approach to the assessment of food quality and safety. CriticalReviews in Food Science and Nutrition. 30: 115-360.

Smithers, G. W., Bradford, R. S., Regester, G. O., and Pearce, R. J. 1991. New caseinprotein products for the food industry. Food Australia. 43(6): 252-254.

Southward, C. R. 1989. Uses of casein and caseinates. In: Developments in DairyChemistry - 4. Fox, P.F. ed. Elsevier Applied Science Publishers, London, p173-244.

Swaisgood, H. E. 1985. Characteristics of edible fluids of animal origin: milk. In:Food Chemistry, Fennema, O. R. ed. Marcel Dekker, New York, NY. p. 791-827.

Tsouflouris, S., Flink, J. M., and Karel, M. 1976. Loss of structure in freezecarbohydrate solutions: effect of the temperature, moisture content, andcomposition. Journal of the Science of Food and Agriculture. 27: 509-519.

Ward, L. 1998. Isolation of Casein Fractions by Manipulating Inherent Solubility andAssociation Properties of Casein. Ph. D. thesis, University of Minnesota, USA.

(Received June 28, 1999; revised August 24, 1999; accepted November 8, 1999)

Dow

nloa

ded

by [

Lau

rent

ian

Uni

vers

ity]

at 0

4:47

04

Oct

ober

201

4