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Bound water measurements for aqueous protein solutions and food gels
Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140 www.elsevier.com/locate/colsurfb
Bound water measurements for aqueous protein solutions and food gels Mikhail Vorob’ev Institute of Elementoorganic Compounds, Russian Academy of Science, 28 ul. Vavilova, Moscow 117813, Russia Received 21 August 2002; received in revised form 22 October 2002; accepted 14 January 2003
Abstract The role of aqueous media in the stabilization of globular proteins and formation of gels was studied by absorption millimeter spectroscopy. This method allowed to measure bound water, the fraction of water which had decreased rotational mobility owing to the presence of solute. Hydration data for globular proteins were compared with data obtained previously for low-weight molecules and groups. It was found that rotational mobility of water molecules in the hydration shells of various kinds of solutes (groups) decreased in the following order: water structure breaking compounds /polar groups /unfolded proteins /globular proteins /non-polar groups. Time courses of the storage modulus were determined for the chemical acidification by glucono-d-lactone (GDL) of milk samples prepared from skimmed milk powder (SMP). Gelation of unheated milk was a monotonous process that started at pH 4.9. Heattreated milk from SMP (16 and 14 g per 100 ml) acidified by GDL (3 g per 100 ml) at 43 8C gave non-monotonous kinetics of gelation with two phases corresponding to the mechanisms induced by denatured whey proteins at pH /5 and by casein /casein interactions at pH 4.8 /4.9. For heat-treated milk, measurement of bound water gave two stages of decrease in water mobility. Additional hydration of SMP during acidification gave 0.15 /0.2 g and 0.8 g bound H2O per gram of SMP for unheated and heat-treated milk, respectively. # 2003 Elsevier B.V. All rights reserved. Keywords: Water; Protein hydration; Millimeter spectroscopy; Milk gels; GDL
1. Introduction Presence of other molecules in water leads to modification of water structure. An important problem is to estimate the amount of modified water in the hydration shell of solute. This water known as bound water exhibits properties which are different from those of bulk water. A decrease
E-mail address: [email protected] (M. Vorob’ev).
of water mobility is one of the most significant properties of bound water, which can be determined by several experimental techniques. It was shown by various relaxation methods including dielectric relaxation spectroscopy and NMR that the reorientational mobility of water molecules in a hydration shell is significantly lower than mobility of bulk water [1,2]. A change of rotational mobility of H2O molecules influenced by solute can be studied by far-infrared and millimeter spectroscopies [3]. In the millimeter
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00050-X
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M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
range of electromagnetic waves (1 /10 mm wavelength, 30 /300 GHz), the absorption of solute is negligible and, consequently, the absorption of water solution is almost completely determined by the fraction of freely rotating water molecules [3,4]. In this range of electromagnetic waves, a coefficient of absorption is significantly lower for ‘‘bound’’ water than for bulk water. Therefore, the deviation (absorption deficit) of the experimental absorption of solution from the theoretical contribution to the absorption of the aqueous phase is determined by the solute-induced alteration in adjacent water. Absorption millimeter spectroscopy (AMS) is able to display formation of Hbonds between water and polar groups of solute or between water molecules themselves (hydrophobic hydration) because the lifetime of corresponding water associates is comparable with the characteristic time of AMS method (10 ps) [4,5]. A lot of physicochemical principles were used for the studies of water structure. Meanwhile, a problem of the selection of appropriate methods that are able to measure hydration effects in various aqueous systems including low-molecular solutes, proteins, protein gels, etc. still exists. The aim of this work was to measure bound water by AMS for different systems from protein solutions to milk gels and to compare hydration of these systems to the data obtained previously for amino acids [5].
(DL30; Haake, Germany) for 20 min. Heat-treated samples were rapidly cooled by immersing vessels containing samples in cold water. Glucono-dlactone (GDL) was supplied by Sigma (St. Louis). 2.2. Acidification and characterization of milk gels
2. Materials and methods
Small deformation oscillatory measurements of storage modulus (G ?) and loss modulus (G ??) were made using cup-and-bob geometry (inner diameter 25 mm; outer diameter 28 mm) on a straincontrolled Bohlin VOR rheometer with 17.52 g cm torsion bar. For characterization of the time course of gel formation, the rheometer was preset at the gelation temperature of 43 8C. Reconstituted milk (15 ml) was incubated at 43 8C in closed vessel for 30 min, then 450 mg of GDL (for experiments with the concentration of 3%, 3 g per 100 ml) was introduced into this vessel. GDL was mixed intensively for 1 min with a magnetic stirrer. The mixture was kept at rest for 1 min before a 13 ml sample was loaded into the rheometer cup. After loading, the exposed surface was covered by silicon oil to prevent evaporation. A further 3 min was allowed for sample equilibration before the test was started. G ? and G ?? were recorded over time at a fixed frequency of 1 rad s 1 and at a strain of 0.5% for 3 h of gelation with interval 1 /5 min. Loss angle (d) was determined as arctg(G ??/ G ?). Acidification dependencies were monitored using a pH electrode which was inserted into the acidified milk samples (GDL concentrations from 1.8 to 3.0%, w/v) by a digital pH-meter (HI32200).
2.1. Materials
2.3. Bound water measurements
Globular proteins were supplied by Sigma (St. Louis, MO) and used without additional purification. Protein concentrations were determined by microbiuret method. Reconstituted milk was prepared by dissolving skimmed milk powder (SMP), kindly supplied by Glanbia (Dungarvan, Ireland), in the demineralized water with addition of NaN3 (0.02 g per 100 ml). Aqueous solutions of SMP (16 and 14%, 16 and 14 g per 100 ml) were stirred overnight at room temperature. Heat treatments were performed at 80 8C in a circulating water bath
Electromagnetic radiation in the millimeter range (10 mm wavelength, 31 GHz) was generated by a stabilized generator G4-156 (RAS, Russia, Moscow). The waveguides were used for canalization of radiation into measuring line with a waveguide resonator at the end. Introducing the protein solutions or acidified milk into waveguide resonator leads to change of the parameters of dielectric resonance including amplitude and resonance frequency [4]. Absorption of electromagnetic radiation a is /log(I/I0), where I0 is the intensity of the initial radiation and I the intensity
M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
of radiation after passing through the solution. The hydration effect was calculated as the difference (da ) between the theoretical contribution of the aqueous component k1C1 and absorption of the solution aexp using the equation: da k1 C1 aexp ;
(1)
where C1 and k1 are the molar concentration of water component and the extinction coefficient of pure water, respectively [4]. The validity of the Lambert /Beer law in the millimeter range allows the hydration number to be calculated: N
da k1 C2
;
(2)
where C2 is the molar concentration of solute. The quantity of bound water in grams of bound H2O (N /18) was determined for milk gels as a function of gelation time.
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3. Results and discussion Positive hydration as a surface phenomenon implies an increase in hydration parameter with increase of accessible surface area (ASA) of molecules. Bound water, the number (hydration number, N ) of water molecules which lose their rotational mobility, was measured by AMS method for 1% (w/v) aqueous solutions of following globular proteins: bovine serum albumin, ovalbumin, pepsin, chymotrypsinogen, chymotrypsin, trypsin, soybean trypsin inhibitor, blactoglobulin, a-lactalbumin, lysozyme and ribo˚ 2) nuclease. For these globular proteins, ASA (A was calculated by empirical formula, ASA / 11.12M2/3, where M is the molecular mass of protein in Da and 11.12 is an empirical coefficient according to Janin [6]. Dependence of N on ASA for globular proteins (Fig. 1) can be treated as a proportional one with a slope in the interval of ˚ 2. This parameter represents bound 0.04 /0.06 A ˚ 2 of ASA (specific hydration water per 1 A
˚ 2) of globular proteins: (1) bovine serum albumin, (2) ovalbumin, (3) Fig. 1. Relationship between hydration numbers N and ASAs (A pepsin, (4) chymotrypsinogen, (5) chymotrypsin, (6) trypsin, (7) soybean trypsin inhibitor, (8) b-lactoglobulin, (9) a-lactalbumin, (10) lysozyme and (11) ribonuclease.
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number, n /N /ASA) characterizing average hydration which takes place at the surface of globular proteins. Previously, the values of N were determined for protein a-amino acids, that allowed to build up a hydrophobicity scale on the basis of water rotational mobility in the hydration shell [5]. These values were shown to decrease in the following order: Ile (8.2), Leu (7.4), Trp (7.1), Phe (6.5), Val (6.2), Tyr (5.4), Met (3.9), Lys (3.4), Arg (2.5), Thr (2.2), Pro (2.2), Ala (1.9), Gln (1.9), His (1.9), Asp (1.4), Glu (1.4), Ser (0.8), Asn (0.8), and Gly (/1.3). Hydrophobic amino acids Ile, Leu, Trp, Phe, Val and Tyr have highest N [5]. Thus, AMS is sensitive to hydrophobic hydration characterizing relatively weak additional stabilization of H-bonds in the water near non-polar groups. Hydrophobic hydration can also be depicted as an increase of the degree of water /water hydrogen bonding [7] or an increase of the size of water clathrates around hydrophobic groups [8]. An increase of water vibrations is accompanied by decrease in water rotational mobility. Use of AMS method makes it convenient to evaluate hydrophobic hydration in terms of the rotational mobility and thereafter hydration number. For low-weight amphiphilic molecules like alcohols and amino acids, hydration number N was represented as an additive sum of DNnp and DNp, the contributions of non-polar and polar groups, respectively. Hydrophobic part of hydration is linear function of ASA of aliphatic groups with ˚ 2 [5]. slope nnp /DNnp/ASAnp /0.0819/0.003 A Contribution of methylene group to hydration (hydrophobic hydration) is DNnp /nnp ASAnp / 2.6, where ASAnp for the CH2 group is accepted ˚ 2. This constitutes 2.6 /18 g bound to be 32 A water per 16 g of solute (weight of CH2 group), i.e. 2.9 g of bound H2O per 1 g of non-polar groups. The proportionality of hydrophobic hydration to ASA of non-polar groups, which we used for calculations, can be registered not only by millimeter spectroscopy method but also by other methods including calorimetric, ultrasonic, etc. Contributions of single hydrophilic groups (OH, CONH2, COOH) to the total hydration of small amphiphilic molecules, as determined by AMS, were lower than hydrophobic hydration of CH2
group [4]. Meanwhile, it is difficult to evaluate contributions of hydrophobic and hydrophilic parts to the total hydration of polymers because of the presence of intramolecular interactions, which make polymer hydration to be a nonadditive phenomenon in principle. Normalization of the hydration parameters either to 1 union of surface area or weight depends on the solute and task. We concluded that hydration effects of organic molecules and proteins with known ASA can be compared between themselves by means of n [4]. Oppositely, hydration effects of gels with unknown ASA are easy to evaluate and compare in grams of bound water per gram of dry matter. Estimation of this parameter for globular proteins gave 0.3 g bound water per gram of protein [3,4]. Hydration data for proteins (Fig. 1) and our previous studies show that rotational mobility of water molecules in the hydration shells for the various kinds of solutes (groups) decreases in the following order: water structure breaking compounds (negative values of n )B/polar groups ˚ 2) B/ (/0) B/unfolded proteins (0.015 /0.025 A 2 ˚ globular proteins (0.04/0.06 A )B/non-polar ˚ 2). groups (0.081 A According to the structural data for globular proteins, ASA of non-polar groups constitutes about 50% of total ASA of groups located at the protein surface. The same parameter for unfolded polypeptide chain in the assumption of the absence of intramolecular association and for natural occurring of amino acid residues was estimated to be approximately 50% of total surface. Specific hydration of these non-polar groups constitutes ˚ 2. Thus, hydrophobic 0.081 /0.5 :/0.04 A groups of globular proteins give main contribution to the hindering of water rotational mobility. Time courses of the storage modulus (G ?), pH and bound water were determined during milk gelation at slow acidification by GDL. Time courses of G ? during chemical acidification by GDL at 43 8C are presented in Fig. 2 for both unheated and heat-treated milk prepared from SMP. Dependencies of G ? on pH are shown in Fig. 3 for heat-treated milk (16% SMP; 3, 2.4 and 1.8% of GDL) and for unheated milk (16% SMP; 3% GDL). As can be seen from Fig. 2, the heat
M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
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Fig. 2. Time courses of storage modulus (G ?) for milk acidification with GDL (3%). Unheated milk was made from (k) 14% and (I) 16% SMP. Heat-treated milk was made from (m) 14% SMP and (j) 16% SMP by heating at 85 8C for 20 min.
Fig. 3. Graph showing storage modulus (G ?) as a function of pH change during acidification of milk. Unheated milk was acidified by (k) 3% GDL. Heat-treated milk (20 min at 85 8C) was acidified by (') 3%, (m) 2.4% and (") 1.8% GDL. The pH interval 4.8 /4.9 corresponds to the onset of gelation for unheated milk and to the beginning of the basic gelation process for heat-treated milk.
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treatment promotes the gelation process faster by shortening gel-point time. Gelation of unheated milk is a monotonous process that starts at pH 4.9 (Fig. 3). Heat-treated milk gives non-monotonous kinetics of gelation with two phases, first of which is induced by denatured whey proteins at pH/5 including, probably, an interaction of heated blactoglobulin with k-casein (Figs. 2 and 3). After first stage of gelation (pre-gelation), some retardation of the process takes place (Fig. 2) producing a characteristic shoulder on the kinetic curves which is dependent on the temperature of heat treatment (these data are not presented here). Gel-point time and height of the shoulder were found to exhibit sharp change in the temperature interval of denaturation of whey proteins (70 /80 8C). The remarkable observation for heat-treated milk is that the pH interval of the onset of basic gelation is approximately the same for all samples in spite of different concentrations of GDL used (Fig. 3). This interval is 4.8 /4.9, which also coincides with the pH of gelation onset for unheated milk (Fig. 3), and is in the area of
isoelectric point of a- and b-caseins. Thus, this finding confirms our suggestion that after the shoulder appearance, the gelation is controlled by casein /casein interactions (basic gelation process). Specific value of hydration, the quantity of bound water in grams of H2O per 1 g of SMP, is presented together with G ? and loss angle d (Fig. 4 for unheated milk and Fig. 5 for heat-treated milk). Before gelation, hydration of SMP constitutes around 0.6 g bound H2O per 1 g of SMP. Additional hydration of SMP on gel formation gives 0.15 /0.2 g and 0.8 g bound H2O per gram of SMP for unheated and heat-treated milk, respectively. For unheated milk, as shown in Fig. 4, the onset of the increase of bound water is accompanied by the onset of decrease in d . For heat-treated milk, in which pre-gelation and basic gelation were observed by rheology, two stages of the alteration of water state were also found (Fig. 5). In this case, the change of bound water corresponding to the secondary increase in G ? and to the simultaneous
Fig. 4. Time courses of (m) bound water (g H2O per 100 g SMP), (I) storage modulus, G ? (Pa) and (') loss angle, d (8) for the acidification of unheated milk. Concentration of SMP was 16%. The acidification was performed with 3% GDL for all treatments. Increase of bound water as a result of gelation is indicated by arrows.
M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
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Fig. 5. Time courses of (m) bound water (g H2O per 100 g SMP), (I) storage modulus, G ? (Pa) and (') loss angle, d (8) for the acidification of heat-treated milk. Concentration of SMP was 16%. The acidification was performed with 3% GDL. Increase of bound water as a result of gelation is indicated by arrows.
decrease in d is significantly high (0.8 g H2O). For example, this value is higher than bound water for globular proteins (0.3 g bound H2O per gram or proteins). Thus, water molecules with additional stabilization of H-bonds and respective loss in rotational mobility are involved in the interactions of denatured whey proteins with other proteins of the system. Possible mechanism of the stabilization of hydrogen bonds could be similar to the hydrophobic hydration mechanism. This could be if an additional number of non-polar amino acid residues become more acceptable for water as a result of the destabilization of casein micelle, but an interaction was not enough to minimize contact with water of these non-polar groups. The increase of bound water during both gelation of unheated milk and pre-gelation of heat-treated milk by 0.15 /0.2 g bound H2O per gram of SMP is comparable by magnitude with the contribution of hydrophobic hydration to the total hydration of proteins, as estimated by AMS. The increase of bound water by 0.8 g bound H2O per gram of SMP, as determined for the overall acidification of
heat-treated milk, is too high to be explained by the mechanism of hydrophobic hydration. Other possible mechanism of the water structure stabilization includes the cooperative H-bonding of water to the various H-bonding sites of proteins. Water bridges can be stabilized owing to the limited mobility of the milk proteins in gel. Horne [9] suggested that the positive drive to association of caseins comes from interaction between hydrophobic sequences of the molecules. This hypothesis is supported by Dalgleish and Law [10,11]. They reported that colloidal calcium phosphate is almost totally solubilized at low pH (below /5.3), but casein micelles remain intact. Our results are not in contradiction with the fact of the importance of hydrophobic effect in casein / casein interactions. Extremely high water content in casein micelles, submicelles and other milk-derived protein aggregates [12] presumably plays its indispensable role in the architecture and functionality of these structures. Our work shows that a change of water should be taken into consideration when studying transformation of milk structures.
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References [1] S.I. Aksyonov, Water and its Role in the Regulation of Biological Processes, Nauka, Moscow, 1990, p. 117. [2] W. Yan-Zhen, A.C. Kumbharkhane, M. Sadeghi, J.T. Sage, W.D. Tian, P.M. Champion, S. Sridhar, M.J. McDonald, J. Phys. Chem. 98 (1994) 6644. [3] Yu.I. Khurgin, V.A. Kudryashova, V.A. Zavizion, O.V. Betski, in: W. Coffey (Ed.), Adv. Chem. Phys., Phys. Series, vol. 87, Wiley, New York, 1994, p. 483. [4] M.M. Vorob’ev, in: Z. Berk, R.B. Leslie, P.J. Lillford, S. Mizrahi (Eds.), Water Science for Food, Health, Agricul-
[5] [6] [7] [8] [9] [10] [11] [12]
ture and Environment, Technomic, Lancaster, 2001, p. 59. M.M. Vorob’ev, Z. Naturforsch. 52c (1997) 227. J. Janin, J. Mol. Biol. 105 (1976) 13. M. Ide, Y. Maeda, H. Kitano, J. Phys. Chem. B 101 (1997) 7022. D. Hecht, L. Tadesse, L. Walters, J. Am. Chem. Soc. 115 (1993) 3336. D.S. Horne, Int. Dairy J. 8 (1998) 171. D.G. Dalgleish, A.J.R. Law, J. Dairy Res. 55 (1988) 529. D.G. Dalgleish, A.J.R. Law, J. Dairy Res. 56 (1989) 727. P.X. Qi, E.M. Brown, H.M. Farrell, Trends Food Sci. Technol. 12 (2001) 339.
Bound water measurements for aqueous protein solutions and food gels Mikhail Vorob’ev Institute of Elementoorganic Compounds, Russian Academy of Science, 28 ul. Vavilova, Moscow 117813, Russia Received 21 August 2002; received in revised form 22 October 2002; accepted 14 January 2003
Abstract The role of aqueous media in the stabilization of globular proteins and formation of gels was studied by absorption millimeter spectroscopy. This method allowed to measure bound water, the fraction of water which had decreased rotational mobility owing to the presence of solute. Hydration data for globular proteins were compared with data obtained previously for low-weight molecules and groups. It was found that rotational mobility of water molecules in the hydration shells of various kinds of solutes (groups) decreased in the following order: water structure breaking compounds /polar groups /unfolded proteins /globular proteins /non-polar groups. Time courses of the storage modulus were determined for the chemical acidification by glucono-d-lactone (GDL) of milk samples prepared from skimmed milk powder (SMP). Gelation of unheated milk was a monotonous process that started at pH 4.9. Heattreated milk from SMP (16 and 14 g per 100 ml) acidified by GDL (3 g per 100 ml) at 43 8C gave non-monotonous kinetics of gelation with two phases corresponding to the mechanisms induced by denatured whey proteins at pH /5 and by casein /casein interactions at pH 4.8 /4.9. For heat-treated milk, measurement of bound water gave two stages of decrease in water mobility. Additional hydration of SMP during acidification gave 0.15 /0.2 g and 0.8 g bound H2O per gram of SMP for unheated and heat-treated milk, respectively. # 2003 Elsevier B.V. All rights reserved. Keywords: Water; Protein hydration; Millimeter spectroscopy; Milk gels; GDL
1. Introduction Presence of other molecules in water leads to modification of water structure. An important problem is to estimate the amount of modified water in the hydration shell of solute. This water known as bound water exhibits properties which are different from those of bulk water. A decrease
E-mail address: [email protected] (M. Vorob’ev).
of water mobility is one of the most significant properties of bound water, which can be determined by several experimental techniques. It was shown by various relaxation methods including dielectric relaxation spectroscopy and NMR that the reorientational mobility of water molecules in a hydration shell is significantly lower than mobility of bulk water [1,2]. A change of rotational mobility of H2O molecules influenced by solute can be studied by far-infrared and millimeter spectroscopies [3]. In the millimeter
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00050-X
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M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
range of electromagnetic waves (1 /10 mm wavelength, 30 /300 GHz), the absorption of solute is negligible and, consequently, the absorption of water solution is almost completely determined by the fraction of freely rotating water molecules [3,4]. In this range of electromagnetic waves, a coefficient of absorption is significantly lower for ‘‘bound’’ water than for bulk water. Therefore, the deviation (absorption deficit) of the experimental absorption of solution from the theoretical contribution to the absorption of the aqueous phase is determined by the solute-induced alteration in adjacent water. Absorption millimeter spectroscopy (AMS) is able to display formation of Hbonds between water and polar groups of solute or between water molecules themselves (hydrophobic hydration) because the lifetime of corresponding water associates is comparable with the characteristic time of AMS method (10 ps) [4,5]. A lot of physicochemical principles were used for the studies of water structure. Meanwhile, a problem of the selection of appropriate methods that are able to measure hydration effects in various aqueous systems including low-molecular solutes, proteins, protein gels, etc. still exists. The aim of this work was to measure bound water by AMS for different systems from protein solutions to milk gels and to compare hydration of these systems to the data obtained previously for amino acids [5].
(DL30; Haake, Germany) for 20 min. Heat-treated samples were rapidly cooled by immersing vessels containing samples in cold water. Glucono-dlactone (GDL) was supplied by Sigma (St. Louis). 2.2. Acidification and characterization of milk gels
2. Materials and methods
Small deformation oscillatory measurements of storage modulus (G ?) and loss modulus (G ??) were made using cup-and-bob geometry (inner diameter 25 mm; outer diameter 28 mm) on a straincontrolled Bohlin VOR rheometer with 17.52 g cm torsion bar. For characterization of the time course of gel formation, the rheometer was preset at the gelation temperature of 43 8C. Reconstituted milk (15 ml) was incubated at 43 8C in closed vessel for 30 min, then 450 mg of GDL (for experiments with the concentration of 3%, 3 g per 100 ml) was introduced into this vessel. GDL was mixed intensively for 1 min with a magnetic stirrer. The mixture was kept at rest for 1 min before a 13 ml sample was loaded into the rheometer cup. After loading, the exposed surface was covered by silicon oil to prevent evaporation. A further 3 min was allowed for sample equilibration before the test was started. G ? and G ?? were recorded over time at a fixed frequency of 1 rad s 1 and at a strain of 0.5% for 3 h of gelation with interval 1 /5 min. Loss angle (d) was determined as arctg(G ??/ G ?). Acidification dependencies were monitored using a pH electrode which was inserted into the acidified milk samples (GDL concentrations from 1.8 to 3.0%, w/v) by a digital pH-meter (HI32200).
2.1. Materials
2.3. Bound water measurements
Globular proteins were supplied by Sigma (St. Louis, MO) and used without additional purification. Protein concentrations were determined by microbiuret method. Reconstituted milk was prepared by dissolving skimmed milk powder (SMP), kindly supplied by Glanbia (Dungarvan, Ireland), in the demineralized water with addition of NaN3 (0.02 g per 100 ml). Aqueous solutions of SMP (16 and 14%, 16 and 14 g per 100 ml) were stirred overnight at room temperature. Heat treatments were performed at 80 8C in a circulating water bath
Electromagnetic radiation in the millimeter range (10 mm wavelength, 31 GHz) was generated by a stabilized generator G4-156 (RAS, Russia, Moscow). The waveguides were used for canalization of radiation into measuring line with a waveguide resonator at the end. Introducing the protein solutions or acidified milk into waveguide resonator leads to change of the parameters of dielectric resonance including amplitude and resonance frequency [4]. Absorption of electromagnetic radiation a is /log(I/I0), where I0 is the intensity of the initial radiation and I the intensity
M. Vorob’ev / Colloids and Surfaces B: Biointerfaces 31 (2003) 133 /140
of radiation after passing through the solution. The hydration effect was calculated as the difference (da ) between the theoretical contribution of the aqueous component k1C1 and absorption of the solution aexp using the equation: da k1 C1 aexp ;
(1)
where C1 and k1 are the molar concentration of water component and the extinction coefficient of pure water, respectively [4]. The validity of the Lambert /Beer law in the millimeter range allows the hydration number to be calculated: N
da k1 C2
;
(2)
where C2 is the molar concentration of solute. The quantity of bound water in grams of bound H2O (N /18) was determined for milk gels as a function of gelation time.
135
3. Results and discussion Positive hydration as a surface phenomenon implies an increase in hydration parameter with increase of accessible surface area (ASA) of molecules. Bound water, the number (hydration number, N ) of water molecules which lose their rotational mobility, was measured by AMS method for 1% (w/v) aqueous solutions of following globular proteins: bovine serum albumin, ovalbumin, pepsin, chymotrypsinogen, chymotrypsin, trypsin, soybean trypsin inhibitor, blactoglobulin, a-lactalbumin, lysozyme and ribo˚ 2) nuclease. For these globular proteins, ASA (A was calculated by empirical formula, ASA / 11.12M2/3, where M is the molecular mass of protein in Da and 11.12 is an empirical coefficient according to Janin [6]. Dependence of N on ASA for globular proteins (Fig. 1) can be treated as a proportional one with a slope in the interval of ˚ 2. This parameter represents bound 0.04 /0.06 A ˚ 2 of ASA (specific hydration water per 1 A
˚ 2) of globular proteins: (1) bovine serum albumin, (2) ovalbumin, (3) Fig. 1. Relationship between hydration numbers N and ASAs (A pepsin, (4) chymotrypsinogen, (5) chymotrypsin, (6) trypsin, (7) soybean trypsin inhibitor, (8) b-lactoglobulin, (9) a-lactalbumin, (10) lysozyme and (11) ribonuclease.
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number, n /N /ASA) characterizing average hydration which takes place at the surface of globular proteins. Previously, the values of N were determined for protein a-amino acids, that allowed to build up a hydrophobicity scale on the basis of water rotational mobility in the hydration shell [5]. These values were shown to decrease in the following order: Ile (8.2), Leu (7.4), Trp (7.1), Phe (6.5), Val (6.2), Tyr (5.4), Met (3.9), Lys (3.4), Arg (2.5), Thr (2.2), Pro (2.2), Ala (1.9), Gln (1.9), His (1.9), Asp (1.4), Glu (1.4), Ser (0.8), Asn (0.8), and Gly (/1.3). Hydrophobic amino acids Ile, Leu, Trp, Phe, Val and Tyr have highest N [5]. Thus, AMS is sensitive to hydrophobic hydration characterizing relatively weak additional stabilization of H-bonds in the water near non-polar groups. Hydrophobic hydration can also be depicted as an increase of the degree of water /water hydrogen bonding [7] or an increase of the size of water clathrates around hydrophobic groups [8]. An increase of water vibrations is accompanied by decrease in water rotational mobility. Use of AMS method makes it convenient to evaluate hydrophobic hydration in terms of the rotational mobility and thereafter hydration number. For low-weight amphiphilic molecules like alcohols and amino acids, hydration number N was represented as an additive sum of DNnp and DNp, the contributions of non-polar and polar groups, respectively. Hydrophobic part of hydration is linear function of ASA of aliphatic groups with ˚ 2 [5]. slope nnp /DNnp/ASAnp /0.0819/0.003 A Contribution of methylene group to hydration (hydrophobic hydration) is DNnp /nnp ASAnp / 2.6, where ASAnp for the CH2 group is accepted ˚ 2. This constitutes 2.6 /18 g bound to be 32 A water per 16 g of solute (weight of CH2 group), i.e. 2.9 g of bound H2O per 1 g of non-polar groups. The proportionality of hydrophobic hydration to ASA of non-polar groups, which we used for calculations, can be registered not only by millimeter spectroscopy method but also by other methods including calorimetric, ultrasonic, etc. Contributions of single hydrophilic groups (OH, CONH2, COOH) to the total hydration of small amphiphilic molecules, as determined by AMS, were lower than hydrophobic hydration of CH2
group [4]. Meanwhile, it is difficult to evaluate contributions of hydrophobic and hydrophilic parts to the total hydration of polymers because of the presence of intramolecular interactions, which make polymer hydration to be a nonadditive phenomenon in principle. Normalization of the hydration parameters either to 1 union of surface area or weight depends on the solute and task. We concluded that hydration effects of organic molecules and proteins with known ASA can be compared between themselves by means of n [4]. Oppositely, hydration effects of gels with unknown ASA are easy to evaluate and compare in grams of bound water per gram of dry matter. Estimation of this parameter for globular proteins gave 0.3 g bound water per gram of protein [3,4]. Hydration data for proteins (Fig. 1) and our previous studies show that rotational mobility of water molecules in the hydration shells for the various kinds of solutes (groups) decreases in the following order: water structure breaking compounds (negative values of n )B/polar groups ˚ 2) B/ (/0) B/unfolded proteins (0.015 /0.025 A 2 ˚ globular proteins (0.04/0.06 A )B/non-polar ˚ 2). groups (0.081 A According to the structural data for globular proteins, ASA of non-polar groups constitutes about 50% of total ASA of groups located at the protein surface. The same parameter for unfolded polypeptide chain in the assumption of the absence of intramolecular association and for natural occurring of amino acid residues was estimated to be approximately 50% of total surface. Specific hydration of these non-polar groups constitutes ˚ 2. Thus, hydrophobic 0.081 /0.5 :/0.04 A groups of globular proteins give main contribution to the hindering of water rotational mobility. Time courses of the storage modulus (G ?), pH and bound water were determined during milk gelation at slow acidification by GDL. Time courses of G ? during chemical acidification by GDL at 43 8C are presented in Fig. 2 for both unheated and heat-treated milk prepared from SMP. Dependencies of G ? on pH are shown in Fig. 3 for heat-treated milk (16% SMP; 3, 2.4 and 1.8% of GDL) and for unheated milk (16% SMP; 3% GDL). As can be seen from Fig. 2, the heat
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Fig. 2. Time courses of storage modulus (G ?) for milk acidification with GDL (3%). Unheated milk was made from (k) 14% and (I) 16% SMP. Heat-treated milk was made from (m) 14% SMP and (j) 16% SMP by heating at 85 8C for 20 min.
Fig. 3. Graph showing storage modulus (G ?) as a function of pH change during acidification of milk. Unheated milk was acidified by (k) 3% GDL. Heat-treated milk (20 min at 85 8C) was acidified by (') 3%, (m) 2.4% and (") 1.8% GDL. The pH interval 4.8 /4.9 corresponds to the onset of gelation for unheated milk and to the beginning of the basic gelation process for heat-treated milk.
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treatment promotes the gelation process faster by shortening gel-point time. Gelation of unheated milk is a monotonous process that starts at pH 4.9 (Fig. 3). Heat-treated milk gives non-monotonous kinetics of gelation with two phases, first of which is induced by denatured whey proteins at pH/5 including, probably, an interaction of heated blactoglobulin with k-casein (Figs. 2 and 3). After first stage of gelation (pre-gelation), some retardation of the process takes place (Fig. 2) producing a characteristic shoulder on the kinetic curves which is dependent on the temperature of heat treatment (these data are not presented here). Gel-point time and height of the shoulder were found to exhibit sharp change in the temperature interval of denaturation of whey proteins (70 /80 8C). The remarkable observation for heat-treated milk is that the pH interval of the onset of basic gelation is approximately the same for all samples in spite of different concentrations of GDL used (Fig. 3). This interval is 4.8 /4.9, which also coincides with the pH of gelation onset for unheated milk (Fig. 3), and is in the area of
isoelectric point of a- and b-caseins. Thus, this finding confirms our suggestion that after the shoulder appearance, the gelation is controlled by casein /casein interactions (basic gelation process). Specific value of hydration, the quantity of bound water in grams of H2O per 1 g of SMP, is presented together with G ? and loss angle d (Fig. 4 for unheated milk and Fig. 5 for heat-treated milk). Before gelation, hydration of SMP constitutes around 0.6 g bound H2O per 1 g of SMP. Additional hydration of SMP on gel formation gives 0.15 /0.2 g and 0.8 g bound H2O per gram of SMP for unheated and heat-treated milk, respectively. For unheated milk, as shown in Fig. 4, the onset of the increase of bound water is accompanied by the onset of decrease in d . For heat-treated milk, in which pre-gelation and basic gelation were observed by rheology, two stages of the alteration of water state were also found (Fig. 5). In this case, the change of bound water corresponding to the secondary increase in G ? and to the simultaneous
Fig. 4. Time courses of (m) bound water (g H2O per 100 g SMP), (I) storage modulus, G ? (Pa) and (') loss angle, d (8) for the acidification of unheated milk. Concentration of SMP was 16%. The acidification was performed with 3% GDL for all treatments. Increase of bound water as a result of gelation is indicated by arrows.
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Fig. 5. Time courses of (m) bound water (g H2O per 100 g SMP), (I) storage modulus, G ? (Pa) and (') loss angle, d (8) for the acidification of heat-treated milk. Concentration of SMP was 16%. The acidification was performed with 3% GDL. Increase of bound water as a result of gelation is indicated by arrows.
decrease in d is significantly high (0.8 g H2O). For example, this value is higher than bound water for globular proteins (0.3 g bound H2O per gram or proteins). Thus, water molecules with additional stabilization of H-bonds and respective loss in rotational mobility are involved in the interactions of denatured whey proteins with other proteins of the system. Possible mechanism of the stabilization of hydrogen bonds could be similar to the hydrophobic hydration mechanism. This could be if an additional number of non-polar amino acid residues become more acceptable for water as a result of the destabilization of casein micelle, but an interaction was not enough to minimize contact with water of these non-polar groups. The increase of bound water during both gelation of unheated milk and pre-gelation of heat-treated milk by 0.15 /0.2 g bound H2O per gram of SMP is comparable by magnitude with the contribution of hydrophobic hydration to the total hydration of proteins, as estimated by AMS. The increase of bound water by 0.8 g bound H2O per gram of SMP, as determined for the overall acidification of
heat-treated milk, is too high to be explained by the mechanism of hydrophobic hydration. Other possible mechanism of the water structure stabilization includes the cooperative H-bonding of water to the various H-bonding sites of proteins. Water bridges can be stabilized owing to the limited mobility of the milk proteins in gel. Horne [9] suggested that the positive drive to association of caseins comes from interaction between hydrophobic sequences of the molecules. This hypothesis is supported by Dalgleish and Law [10,11]. They reported that colloidal calcium phosphate is almost totally solubilized at low pH (below /5.3), but casein micelles remain intact. Our results are not in contradiction with the fact of the importance of hydrophobic effect in casein / casein interactions. Extremely high water content in casein micelles, submicelles and other milk-derived protein aggregates [12] presumably plays its indispensable role in the architecture and functionality of these structures. Our work shows that a change of water should be taken into consideration when studying transformation of milk structures.
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