Bohoua, Surfactants and dairy protein

Surface activity of surfactants and dairy proteins* B y G.L. BOHOUA~and Z.U. H A Q U E ~ 'Department of Food Science, Nutrition and Health Promotion, MAFES, Mississippi State University, Box 9805, Miss. State Univ., MS 39762. E-mail: [email protected] 2 Ecole des Sciences de la Nature, Centre Universitaire d'Abobo Adjame, Abidjan, Cote d'lvoire Approved for publication as Journal Article No. J-10989 of the Mississippi Agricultural and Forestry Experiment University. Funded by the Mississippi Agricultural and Forestry Experiment Station, Project NO. 347020.

Station, Mississippi State

Surface activity of some surfactants and dairy proteins were estimated by contact angle (8) measurements of sessile drops of their aqueous dispersions on a non-reactive planner hydrophobized surface. Data were statistically compared to traditional air-water surface tension (ST) and oil-water interfacial tension (IT) measurements that were determined using the ring method. Sessile drops of serially diluted surfactants (0.01 - 0.5%, wlv) and proteins (0.25 2.0%, wlv) in 5 mM sodium phosphate buffer at pH 7 (22°C) were placed on cleaned borosilicate glass covers that were hydrophobized by silination using y-methacryloxypropyltrimethoxysilane. In all protein dispersions, 8 was statistically comparable to ST and IT and showed the same concentration-dependant trends. The 8 of all surfactants was comparable to their ST and IT measurements above their apparent critical micelle concentrations. The 8 measurements of cationic sodium dodecyl sulfate and zwitterionic 3-[(3-cholamidopropyI)-dimethylammonio]-l-propanesulfonate were comparable to the other measurements at all concentrations studied. Data indicate that 8 measurements may be used as a rapid and inexpensive tool to estimate SA for quality assurance and control purposes. Oberflachenaktivitat von Tensiden und Milchproteinen Die Oberflachenaktivitat einiger Tenside und Milchproteine wurde durch Kontaktwinkel (8)-Messungen der fest haftenden Tropfen ihrer wassrigen Dispersionen auf einer nicht-reaktiven wasserabweisenden planen Oberflache festgestellt. Die Daten wurden mit der traditionellen Luft-Wasser-Oberflachenspannung (ST) und Ol-Wasser-Grenzflachenspannungs (IT)-Messungen mithilfe des Ring-Verfahrens verglichen. Sessile Tropfen seriell in 5 mM Natriumphosphatpuffer bei einem pH-Wert von 7 (22°C) geloster Tenside (0,Ol-03% wlv) und Proteine (0,25-2,0% W/V)wurden auf gereinigte Borosilikat-Glasdeckel, die durch Silierung mit y-Methacryloxypropyl-Methoxysilan wasserabweisend gemacht waren, platziert. Bei allen Proteindispersionen war 8 hinsichtlich ST und IT statistisch vergleichbar und zeigte die gleichen konzentrationsabhangigen Trends. Der 8 aller Tenside war vergleichbar bei ihren ST- und ITMessungen oberhalb ihrer apparenten kritischen Micellenkonzentrationen. Die 8-Messungen von kationischem Natrium-Dodecylsulfat und zwitterionischem 3- [3-Cholamidopropyl)-dimethylammonio]-l-propan-sulfonat waren vergleichbar mit den anderen Messungen bei allen untersuchten Konzentrationen. Die Daten zeigen, dass Messungen von 8 als schnelles und nicht-aufwendiges Verfahren zur Schatzung der Oberflachenaktivitat zur Qualitatssicherung und fur Kontrollzwecke eingesetzt werden konnen. 21 Surface activity (surfactants, milk proteins) 21 Oberflachenaktivitat (Tenside, Milchproteine)

1. Introduction The surface activity (SA) of amphiphiles is reflected by their tendency to reduce tension of the interface whether it is between air-water, i.e., surface tension (ST) (as in foams), or oil-water interfacial tension (IT) (as in emulsions). Reduced tension, on the one hand, facilitates emulsion making by reducing LaPlace pressure that counters disruption (1). On the other hand, the tendency of fat droplets to coalesce is facilitated as the IT is decreased (2). Therefore, tension at the interface is an important parameter that impacts functionality and stabilityof mixed food systems. Measurement of contact angle, 8, defined as the tangent of a sessile drop of liquid to a horizontal solid surface (Fig. I ) , is simple and is rapidly determined using a relatively inexpensive goniometer. In principal, the 9 is a reflection of a mechanical equilibrium of 3 interfaces; solid-vapor (SV), solid-liquid (SL), and liquid-vapor (LV). Therefore, in basic terms, the total energy of the system, E, may be expressed as, ...[ I ] E = A s v y s v + A S L y S L + A LYLV, V ............... where, A is the area and y is the surface energy (3). Based on this geometry (Fig. 1), Milchwissenschaft 62 (4) 2007

'ig. 1: A sessile drop on uniformly hydrophobized nonporous planar surface. ysv, ~ S and L ~ L represent V surface energy at the solid-vapor, solid-liquid and liquid-vapor interface, respectively. 0 is the tangent of the sessile drop of liquid to a horizontal planer surface at the three phase line. the Young equation (4) may be given as, cos 9 = (ysv - ysL)/yLv, . . . . .. . . . . ...... . . . . ...(2) where, 8 is the contact angle. If the solid surface is perfectly horizontal, non-porous, smooth, non-reactive, and uniformly hydrophobic and all other conditions are constant, then it is stipulated that the interfacial equilibrium at the three phase line, and consequently the 9 of the sessile drop will be impacted by the amphiphiles in

dohoua, Surfactants and dairy protein

/

Table 1: Surface activitv ofamuhiuhiles bv three different methods' . . Amphinhile

Method

Comarable

0.01

Concentration of amphiphile (%, wlv) 005 0.10 0.20 0.30

0.40

0.50

5.8' 19.0~

6.2' 20.0~

1

Fc

I

FT

(0 024) CHAPS

(0 22) TX-I00 (0.23)

-.LIL

(0.33)

IT 8

Y*

Y*

15.5a 33.0a

7.ab 5.7= ~ 3 . 6 ~ 16.3'

6.0' 20.0'

5.8' 19.3~

1.02

3.08

' ~ a t ameans ( ~ 3 ) Critical . micelle concentration (CMC,,,)(%.wlv) are in parentheses. Abbreviations : SDS is sodium dodecyl suifate. CHAPS is 3-[(3-cholamidopropyi)-dimethylammonio]- 1-propane-sulfonate, TDTM is tetradodecyi trimethyl ammonium bromide, TX-100 is triton X-IOOH, and 212 is N-dodecyl-N, N dimethyl 3 ammonio-I propane sulfonate (Zwittergent 3-12), ND is 'not done' based on concentration rage dependant on CMC, ST is surface tension (dynelcm), IT is interfacial tension (dynelcm), e is wntact angle in degree ("1, Y is statistically comparable across concentration range studied, Yf is statistically comparable above CMC, Fc is calculated F value for the interaction at wncentration range where comparable, and FT is the table value. Means with dissimilar superscripts in the same row are statistically different at c.05.

1

was less than the table value (FT)(3.08) making it not significant. Therefore, ST, IT, and 8 values were comparable across the concentration range studied. Anionic TDTM showed a close to one order higher CMC,, value (0.22% , wlv) compared to SDS (Table 1). where, Here the ST and IT varied slightly from 0.01 to 0.3 % df(MSE): degree of freedom associated with the error.. . (wlv). However, the 0 decreased considerably within the MSAB: . - mean sauare interaction. same range of concentration (Table 1). Two way CRD MSE. mean square error. FT= First, Ho: (ap),=(ap),=(~p)~~=O. If FA,> Fa(a-l)(b-I), 2.12). However, >CMC,, Fc was markedly lesser than df(MSEl. then Hnwas reiected at a=l% (a: the wrobabilFT (Fc=0.58< FT = 2.93) making 8 dataset comparable ity'to rekct Howhen Hahas true). ~hen'thetwb factors to ST and IT. The apparent lack of SA of TDTM at were not acting independently; therefore, no inferences Fa also means thatthere is no negativity of the silinated glass surface resulting in reparallelism). If FA, < Fa (a-l)(b-l),df(MSE), then FA, was not sig- pulsion of anionic TDTM until sufficient concentration (>CMC,) was reached. At the CMC, of surfactants, nificant. In this case. the factors were inde~endentand amphiph~le-interface interactions give way to amphithe main effects comparisons were permissible. phile-amphiphile interactions (13). Above the CMC,, surface or interfacial concentration of the monornerlc 3. Results a n d discussion amphiphiles that form the interfacial monolayer become The three sets of concentration dependant data; ST, constant (14) leading to a more stable interface (15). The ST of nonionic surfactant TX-100 decreased beIT and 8 measurements related to cationic SDS were tween 0.01 and 0.05 % (wlv), and then remained concompared (Table 1).SDS gave the lowest CMC,,,value of 0.024% (wiv) among all surfactants studied. SOKO- stant up to 0.5 % (wiv)(Table 1). The IT measurements showed the same trend (Table 2). The 8 values deLOWSKI et. a/. (12) reported that alkyl CH2 groups had close to two times greater thermodynamic drive to ad- creased with increasing concentration of TX-100. The Fc was 2.77 sorb at an interface compared to phenolic CH2 groups. Fc was significant. However, >CMC,, Therefore, the dodecyl hydrocarbon chain of SDS con- while the FT was 3.85 and the three data sets were tributes markedly to it association tendency and SA. comparable. Among the zwitterionic surfactants, CHAPS, which Comparison of means showed that the ST decreased significantly between 0.01 and 0.1 (% wlv) and then re- has a bulky cholic acid and zwitterionic sulfobetaine value of 0.18% mained constant at a=l%. The 8 decreased as the con- moiety in its structure, had a CMC, (wlv) and gave significant and continuous reduction of centration of SDS was increased, and then remained constant between 0.2 and 0.3 % wiv (Table 1). A de- measured values using all three methods as surfactant crease in ST and IT indicated that the concentration of concentration was increased (Table 1). This indicated increasing SA with increasing concenSDS at the interface increased with increase in total tration. The Fc across the full concentration range was concentration. The overall trend of the 8, ST and IT measurements were similar. Two way CRD showed 1.91 compared to 3.08 for F making the three datasets that the calculated F value (Fc)(1.91) for the interaction for this surfactant statisticaliy comparable. For the other

table was determined using SAS (Ver 9)(11) A test statistic for the hypothesis (Ha)that there was no interaction was: H,: F,,=MSABiMSE with (a-l)(b-I), df(MSE) [dl

Bohoua, Surfactants and dairy protern the liquid. Using p-casein as a model amphipathic protein, it was shown that the primary driving force for adsorption of proteins to hydrophobic surfaces is hydrophobic (5). Proteins populate hydrophilic-hydrophobic interfaces driven by the free energy advantage of reducIng hydrocarbon-aqueous interface. The oojuctlve of tti s stmy was to nvesl gare wnetner 0 measJremenrs of sess~leoroDs of scrtal d ,tons of a selection of anionic, cationic and zwitterionic surfactants and a selection of well studied food proteins determined on hydrophobized surface, are statistically comparable to their ST and IT measurements at concentrations of practical interest for food systems.

2. Materials and methods 2.1 Materials Sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)-dimethylammonio]- I-propane-sulfonate (CHAPS), tetradodecyl trimethyl ammonium bromide (TDTM), Triton X-100H (TX-loo), and N-dodecyl-N, N dimethyl 3 ammonio-I propane sulfonate (Zwittergent 3-12)(Z12) were obtained from Calbiochem, San Diego CA. Pinacyanol chloride (201715), y-ethacryloxypropyl-trimethoxysilane (silane), bovine serum albumin (BSA), Rlactoglobulin AB (R-Lg) from Sigma Chemical Co., St. Louis MO. a- (a-CN), P- (P-CN) and K-casein (K-CN) were purified as described elsewhere (6). Sodium caseinate (Na-CN) was prepared by acid (HCI) coagulation and neutralization ( I N NaOH) of fresh unpasteurized skim milk from the Mississippi State University Dairy Plant. Optical grade borosilicate square cover glasses (size 2 5 x 2 5 mm, thickness 0.13-0.17mm) (cat # 12-542C) were purchased from Fisher scientific, Fair Lawn, N J. All other reagents were analytical grade.

2.2 Methods Determination of critical micelle concentration (CMC,,,): The CMC, is the concentration atwhich surfactants form micelles. It was determined according to a dye binding method using pinacyanol chloride (7). A stock solution of each surfactant (initial solution of I % , wiv) was made with a 0.1% (wiv) solution of pinacyanol chloride (dye) in 5 mM phosphate buffer, pH 7 (22°C). A Milton Roy dual-beam (computerized) spectrophotometer (Spectronic 1201) was used to measure absorbance. An aliquot of the stock solution was diluted with an equal amount of the dye solution and vortexed to obtain serial dilutions. Optical density measurements were at 610 nm against the buffer (5mM sodium phosphate) at 22°C. The CMC, was determined by taking the inflection point of the absorbance vs. concentration curve (Table 1). Surfactant and protein dispersions: The concentration range of the surfactant tested was 0.01 to 0.5 % (wlv) in 5 mM sodium phosphate buffer at pH 7 (25°C) based on the CMC,, (Table 1). Proteins were also dispersed in the same buffer by vortexing and hydrating for Ih a t 22°C. Determination of surface and interfacial tensions: Surface tension (airiwater) and interfacial tension (oiliwater) of freshly made amphiphile dispersions were measured according to the ring method proposed by HARKINSand JORDAN(8). The maximum pull on a

Milchwissenschaft 62 (4) 2007

cleaned and flamed platinum ring at 0 degree angle of contact with the interface was determined using a SurfaceTensiomat (semi-automat~c,Fisher model 21). For IT measurements, the platinum ring was placed in the liquid, beneath the surface or interface so that the entlre ring would be wetted (3.175 mm immersion). These IT rnrasutements were de~ernined ~mrnedlately after caref~llv * .Dour rto - .u c a n ~ot I on rhn %rface of [lie aoucousdispersions. Determination o f contact angle: In order to produce solid surfaces with uniform hydrophobicity, new optical grade borosilicate based square cover glasses (25 mm X 25mrn) were cleaned by dipping for 16 h in freshly prepared chromic acid and immersing in five changes (30 min each) of freshly. .~ r e. ~ a r double ed alass-distilled deionized water. These clean slips were silinated by dipping for 16 h in silane, dried in vacuo in bell jars at 22°C and brought to uniform level of surface dehydration by storing in the evacuated jar over phosphorus pentoxide for 16 h prior to use. Surface hydration can be a source of error of €Imeasurements (9). A diamondground syringe needle (Kayeness,lnc., Honey Brook, PA.) was used to generate surfactant dispersion droplets (-50 pL) of uniform size on the silinated cover glasses. The 8 of the droplet of dispersions were determined immediately by taking the tangent of the sessile drop with the treated glass cover placed in the path of a beam of light from a tungsten source (Fig. 1). Accurate measurements were performed with a small telescope with cross-hairs attached to a goniometer (model D1060, Kayeness,lnc., Honey Brook, PA.) The projection system consisted of a 40 X magnifier, a semi-circular viewing screen, a rotatable protractor (360") for reading the contact angle and a focusing system. At least three readings were recorded for each concentration of a given amphiphile and means were tabulated. Statistical analysis: The 8, ST and IT data were statistically compared with each other. The two variables were: the method used to assess the surface activity (factor A at level a) and the concentration (factor B at level b). The experiment was a balanced two way randomized design: each factor combination (treatment) of the two factors cited above was randomly assigned to at least three homogeneous experimental units (10). The statistical model used was: Yilk = p + a +pi + (ap),, + eiik .......................PI where, i: level of the first factor (A); i= 1,2,....,a i: level of the second factor (€3);i=1,2,....., b k: level of replication number; 'k=1,2 ,........,r Y,: measure of the ithlevel of factor A, fh level of factor B in'the kl"eplication. p: Overall mean measure a:: added effect of the ithlevel A measured as a deviation from p, a,=O. 13, added effect of the i'vevel B measured as deviation o m, p,=o. added effect of the combination of the ifhlevel of A with the j'qevel of B, the A,xB, interaction effect,(ap),= (a13);;=0. e,,,: fandom error, e N(0, ) The sources of variability (identified and quantified) associated with data were: main effects (A,B) and interactive effect (AB). The analysis of variance (ANOVA)

,,,-

Bohoua, Surfactants and dairy protein amphipathic surface area, compared to the surfactants, which in turn potentially influences surface activity.

4. Conclusions The concentration dependent 9 measurement trends were comparable to those of the ST and IT measurecationicTDTM, nonionic ments above the CMC,,,ofthe TX-100, and zwitterionic Zq2.It was comparable across all the concentrations studied in case of the cationic SDS and zwitterionic CHAPS and at the concentrations studied for the milk proteins. Data show that 9 measurements may be used as rapid and inexpensive tools to determine surface activity of amphiphiles in colloidal food systems.

5. References

zwitterionic surfactant, ST and IT decreased significantly between 0.01 and 0.05% (wlv); while 8 decreased between 0.01 and 0.1 % (wlv) (Table 1). The FT=2.43) than F, while >CMCa,, Fc was smaller than FT (Fc=1.02 CMC,,. Protein dispersions: Among the globular proteins, for BSA, there was no significant difference in the ST, IT and 8 values in the full concentration range between 0.25 and 2 % (wlv) (Table 2). Fc of the globular proteins were 0.18 and 1.83 compared to F, of 2.34 and 2.34 for BSA and P-LG, respectively. Fcof the fractionated nonglobular proteins were 2.68,2.86, and 0.97 compared to F, of 3.67,3.67 and 3.60, a-, P-, and K-CN, respectively. Fc of the commercially important ingredient Na-CN was 0.22 compared to F, of 3.67. This made all three datasets comparable for all proteins studied. The markedly smaller effect of concentration of the proteins on SA compared to the surfactants tested may be explained by the facts that surfactants are more amphipathic and flexible. In addition, because of their much smaller size, small changes in the concentration (%, WIV) caused markedly greater increase (>230 fold more for SDS compared to BSA) in the number of surface active particles compared to the proteins. Furthermore, likesurfactants, the bulkier proteins also partake in self- and hetero-association (6, 16). These conceivably cause many fold greater reductions in effective

(1) WALSTRA,P.: Developm. in Dairy Chem. 2 119.158 (1983) (2) HAQUE, Z.U.: J. Dairy Sci. 76 (1) 31 1-320 (1993) M.D., DARVELL, B. W.: J. Physics D: AppI. (3) MURRAY, Physics 23 (9) 1150-1 155 (1990) (4) YOUNG,T.: Phil. Trans. Roy. Soc. London, 65-87 (18051 T., WAHLGREN, N.M.: Langmuir 13 (23) (5) NYLANOER, 6219-6225 (1997) Z.U.. Su. K.. ANSARI.R.R.: Food Science and (,6,) HAQUE. ~ e c h n o l keskarch, o~~ submitted (2007) (7) CORRIN,M.L., KLEVENS,H.B., HARKINS,W.D.: J. Chem. Phvsics 14480-486 (1946) W. D., JORDAN,H.F.: J. ;4m. Chem. Soc. 52 (8) HARKINS, 1751-1772 -~ - 11930) T., WIEGEL, D., NAJI, L., ARNOLD,K.: J. (9) ENGLAENDER, Colloid and Interface Sci. 179 (2) 635-636 (1996) (10) PETERSEN, R.G.: Design and Analysis of Experiments. Marcel Dekker, New York, NY (1985) (1 1) SAS Institute: SASISTAT Guide for personal computers. Version 6th edn. Cary, NC (1985) (12) SOKOLOWSKI,A., PIASECKI, A,, BURCZYK, B.: Langmuir, 8 (7) 1775-1778 (1992) (13) DICKINSON, E., WOSKETT, C.M.: Special Publ. Royal Soc. Chemistry 75 (Food Colloids) 74-96 (1989) (14) ISRAELICHVILI, J.N.: In Intermolecular -and Surface Forces with Applications to Colloid and Biological Systems. Academic Press, London, 229-245 (1985) (15) DICKINSON, E., WOSKETT,C.M., BEE, R.D., RICHJ.: In Food Colloids. Royal SOC. MOND, P., MINGINS, Chem., Cambridge, UK, 74-96 (1989) E.D.. FARRELL, (16) MALIN,E.L., BROWN,E.M., WICKHAM, H.M., JR.: J. Dairy Sci. 88 (7) 2318-2328 (2005) ~

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Milchwissenschaft 62 (4) 2007

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