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Coagulation of skim milk under high hydrostatic pressure with acidification by glucono-δ-lactone
International Dairy Journal 9 (1999) 487}492
Coagulation of skim milk under high hydrostatic pressure with acidi"cation by glucono-d-lactone Marcus Schwertfeger*, Wolfgang Buchheim Federal Dairy Research Centre, Institute of Process Engineering, Hermann-Weigmann-Str. 1, D-24103 Kiel, Germany Received 28 January 1999; accepted 24 May 1999
Abstract Water, protein-free milk serum and skim milk were acidi"ed under high hydrostatic pressure by hydrolysis of glucono-d-lactone under various experimental conditions concerning pressure (50 up to 200 MPa), reaction time (5}40 min), initial concentration (100 and 200 mmol l\) and temperature (25 and 353C). The reaction rates increased under pressure as determined by measurements of pH after decompression. In some experiments acidi"cation of skim milk resulted in formation of coagulated products. The structures of these coagula di!ered from gels formed by acidi"cation of milk under standard conditions. 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: High pressure; Glucono-d-lactone; Milk; Casein; Coagulation
1. Introduction Application of high hydrostatic pressure may induce changes in properties of biopolymer gels (Gekko, 1992), in emulsi"ng properties of milk proteins (Galazka, Ledward, Dickinson & Langley, 1995; Galazka, Dickinson & Ledward, 1996) or in swelling and gelling of starch dispersions (Thevelein, van Assche, Heremans & Gerlsma, 1981; Ezaki & Hayashi, 1992; Douzals, Marechal, Coquille & Gervais, 1996). It has been known for almost 30 years that casein micelles disintegrate under high hydrostatic pressure, apparently due to shifts in the mineral balance in milk by dissolution of the colloidal calcium phosphate (Schmidt & Buchheim, 1970; Schrader, Buchheim & Morr, 1997; Schrader & Buchheim, 1998). The rennet or acid coagulation of pressuretreated milk has been studied extensively (Johnston, Austin & Murphy, 1993; Johnston, Murphy & Birks, 1994). However, the coagulation of milk by acidi"cation under pressure has not been attempted until recently although the possibilities o!ered by pressure-induced acceleration of chemical reactions, protein unfolding and changes in
* Corresponding author. Tel.: #49-(0)431-609-2275; fax: #49(0)431-609-2309. E-mail address: [email protected] (M. Schwertfeger)
number of particles are promising (Schwertfeger & Buchheim, 1998). Microbiological acidi"cation of milk, e.g. by lactic acid producing cultures, may be hindered by the pressure-sensitivity of most vegetative bacteria (Smelt, 1998), at least at higher pressures. In this study, skim milk was acidi"ed under high pressures by hydrolysis of glucono-d-lactone (GDL). This compound decomposes gradually in aqueous media (Sawyer & Bagger, 1959; Skou & Jacobsen, 1982; Mitchell & Duke, 1970) and thus allows slow acidi"cation. Therefore, GDL is widely applied in the food industry. The properties of acid-set milk gels using GDL at standard conditions have been described (Lucey & Singh, 1998).
2. Materials and methods 2.1. Materials Skim milk was prepared by adding 10 g of low-heat skimmilk powder to 100 g of water at room temperature under vigorous stirring. After standing for 30 min, foam was removed by adding a few drops of n-octanol (Merck, Darmstadt, Germany). The milk was stored at room temperature for 8 h max. until use. Protein-free milk serum (UF permeate) was obtained by ultra"ltering 15 l of reconstituted skim milk prepared
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M. Schwertfeger, W. Buchheim / International Dairy Journal 9 (1999) 487}492
without added octanol in a pilot plant (Alfa Laval, Germany) with a membrane "lter (Romicon PM 10, Romicon Inc. Woburn, MA, USA, 10 kD molecular weight cut-o!) at 503C. Glucono-d-lactone was obtained from Jungbunzlauer (Ladenburg, Germany). Deionized water was used for all operations. The room temperature was about 25}273C.
The experiments were repeated two to six times as indicated in the "gures. No statistical calculations were deemed necessary.
2.2. Measurements of pH
Under pressure, the hydrolysis of GDL as determined by the change in pH was accelerated. A pressure of 50 MPa had no measurable in#uence on the reaction rate. However, higher pressures resulted in small (less than 0.3 units) but de"nite pH-shifts. All samples had a "nal pH of about 2.4, measured after 40 min pressurization time or after one hour at ambient pressure. Under pressure, lower pH values could be reached at shorter reaction times as compared with reaction under ambient pressure, e.g., at 100 MPa a pH of 2.6 was reached within 20 min instead of 40 min at ambient pressure (Fig. 1). An increase in temperature led to a further acceleration of the reaction (Fig. 2). The "nal pH did not drop
At ambient pressure and room temperature (standard temperature and pressure, STP) 0.891 g GDL was quickly dissolved in water, UF permeate or skim milk at 253C to obtain a "nal concentration of 100 mmol l\. Immediately after dissolution, the pH was measured with a pHmeter equipped with a glass electrode (WTW SenTix 97T and pH 325, WTW, Weinheim, Germany). The pH values were recorded continuously. The temperature was held at 253C by using a water bath. 2.3. High-pressure experiments Pressure experiments were carried out in a home-built laboratory-scale high-pressure system (pressure chamber 16 mm in diameter, total volume approximately 20 ml). Hydraulic oil (DEA Econa 46, DEA Mineraloel AG, Hamburg, Germany) was used as the pressure-transducing medium. The samples were placed in cap-closed polyethylene tubes (volume about 5 ml). The pressure vessel was thermostatically controlled at 253C. The pressure was generated by using a pneumatic pressure intensi"er. Compression and decompression were accomplished within 5 s. To avoid any substantial warm-up beyond the selected temperature due to adiabatic heating, the samples (water, permeate or skim milk) were kept at a temperature somewhat lower than the desired reaction temperature (203C for pressure experiments at 50 MPa, 17 and 143C for experiments at 100 and 200 MPa, respectively). These temperatures were determined in preliminary experiments (data not shown). Exactly 0.891 g GDL was dissolved quickly in these precooled samples resulting in an initial concentration of 100 mmol l\. The mixtures were "lled into the tubes, sealed and then placed into the pressure vessel. The selected pressure was reached within 90 s after starting solubilisation of GDL. Immediately after decompression, the samples were removed from the pressure vessel and the pH was measured. The structure of the samples was characterized visually. In some experiments, modi"ed experimental conditions were used, including a higher initial concentration of GDL or higher reaction temperature. For reactions performed at higher temperatures the thermostat was set to 353C and the samples were precooled to 303C.
3. Results 3.1. Hydrolysis of GDL in water
Fig. 1. Changes of pH after various pressure treatments of solutions of GDL in water (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
Fig. 2. Variations of pH after pressure treatment of aqueous solutions of GDL with di!erent initial concentrations at 100 MPa and di!erent temperatures.
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489
below 2.4 after pressure treatment at 100 MPa. By using a higher initial concentration of GDL (200 mmol l\), a lower pH of about 2.2 could be reached within 40 min at 100 MPa (Fig. 2). 3.2. Hydrolysis of GDL in milk serum The GDL-induced acidi"cation of milk serum was di!erent in comparison with water. For all experimental conditions, the resulting pH di!erence was considerably larger as compared with ambient pressure. The di!erence was about 1.0 units for pressure treatment at 200 MPa and up to 0.2 and 0.5 units after pressurization at 50 and 100 MPa, respectively. In all experiments a constant "nal pH has not been reached within a pressurization time of 40 min (Fig. 3).
Fig. 4. Variations of pH after pressure treatment of solutions of GDL in skim milk (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
3.3. Hydrolysis of GDL in skim milk The acidi"cation of skim milk was di!erent from that in water or milk serum. A constant pH was not reached within more than one hour at ambient pressure or within 40 min at pressures up to 200 MPa. After short times of pressurization the di!erences in pH between pressurized and non-pressurized samples were relatively small, reaching at most about 0.5 pH units. These di!erences increased further with reaction time for all pressures applied. The pH after a reaction time of 60 min changed to 5.3 after hydrolysis at ambient pressure and to 4.3 after hydrolysis at 200 MPa (Fig. 4). At a higher reaction temperature of 353C, a lower pH was reached. Depending on the reaction time the decrease in pH as compared with the corresponding pH at 253C was about 0.2 after 5 min and up to 0.4 after 40 min of pressurization. A larger decrease in pH was observed after pressure treatment with a higher initial concentration of GDL (c "200 mmol l\). In this experiment the
Fig. 3. Variations of pH after pressure treatment of solutions of GDL in milk serum (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
Fig. 5. Variations of pH after pressure treatment of solutions of GDL with di!erent initial concentrations in skim milk at 100 MPa and di!erent temperatures.
pH was lowered about 0.4 units after 5 min and up to 1.0 units after 40 min of pressurization at 100 MPa as compared with an initial concentration of 100 mmol l\ (Fig. 5). Some samples which were treated at higher pressures and/or for long reaction times (i.e. 200 MPa for 10 min and longer and 100 MPa for 40 min) were coagulated. These samples had a pH below 5.3. The structures of these aggregates, however, di!ered distinctly from the homogeneous gel which is known to be formed by acidi"cation of milk at ambient pressure. In these experiments #occules formed at 100 MPa after 40 min (Fig. 6A); coarse #occules formed at 200 MPa after 10 min; and a "ne stranded, coherent coagulum, showing strong whey separation when taken out of the tube (Fig. 6B) formed after treatment at 200 MPa for 20 min. It appears remarkable that the structure of the coagula did not re#ect a continuous aggregation process during pressure treatment. Floccules formed after short pressurization times
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Fig. 6. Structures of the coagula formed after acidi"cation with GDL for 40 min at 100 MPa (A) and 20 min at 200 MPa (B). Bar"5 mm.
transformed to stranded coherent coagula after longer times of pressure treatment.
4. Discussion By application of high hydrostatic pressure, all processes in a given system accompanied by a decrease in reaction volume are preferred. In the system studied here the following processes were occurring in parallel: Under high pressure the hydrolysis of GDL was accelerated, as indicated by a more rapid shift in pH after pressure treatment in comparison to the reaction under ambient pressure. Although a covalent bond is being broken by hydrolysis, the total reaction volume will be negative, because the liberated gluconic acid anions and hydrogen ions are strongly hydrated leading to a negative reaction volume caused by stronger ordering of water molecules around the ions. As a result the equilibrium of the reaction is shifted. Chemical reaction rates are increased under pressure if the volume of the activated state is smaller than the volume of the reactants, i.e. the activation volume D
Fig. 7. Rate of pH-shift in milk serum and water caused by pressureaccelerated acidi"cation at 253C and di!erent pressures. The values are calculated from the slope of a curve "t of the values displayed in Figs. 1 and 3 using a potential function (pH"at\@).
the milk salts in#uenced the reaction rate of GDL hydrolysis which was higher in milk serum than in water (Fig. 7.) because the reaction rate depends on the medium, especially regarding its bu!er concentration and pH (Sawyer & Bagger, 1959; Skou & Jacobsen, 1982). Under high hydrostatic pressure, skim milk constituents are also a!ected. The mineral equilibrium of the system is changed by dissolution of the colloidal calcium phosphate (CCP) located in the micelles and therefore the casein micelles disintegrate under high pressure leading to a decrease in particle diameter and an increase in the number of particles (Schmidt & Buchheim, 1970; Anema, Lee, Schrader & Buchheim, 1997).
M. Schwertfeger, W. Buchheim / International Dairy Journal 9 (1999) 487}492
Although skim milk is not a colloidal system exclusively stabilized by electrostatic interactions, the variations of colloidal stability are caused by changes in the charge located at the surface of the micelles or along the chains of the i-casein molecules of the protein particles. This is displayed by changes in the resulting particle charge being measured as the f-potential. A high fpotential indicates strong repulsive forces. Coagulation can occur if the f-potential is low enough. For casein dispersions it is known (Walstra, 1990) that the f-potential depends on the pH of the medium and has a minimum of a about !3 millivolts at a pH of about 5.2 and reaches zero at a pH of about 4.5. In these pH ranges the repulsive forces should be minimal. For the process of coagulation, the particles of the dispersion must be in close contact. It is necessary to overcome the repulsive forces between the particles caused by electrostatic or steric stabilization. In the presence of GDL the pH is lowered by highpressure treatment more rapidly than at ambient pressure. Consequently, the stabilizing electrostatic e!ects caused by pH-sensitive carboxyl and phosphoseryl groups are diminished and the colloidal stability decreases. At moderate pressures (50 and 100 MPa) at which the pH of about 5.2 is not reached within the observed reaction time no visible coagulation could be detected. However, the samples treated at a higher pressure of 200 MPa resulting in a pH drop below 5.2 exhibited distinct coagulation. Additionally, the process of nucleation at the beginning of the coagulation process depends on the number of particle collisions. By high pressure treatment the number of casein particles caused by dissolution of the CCP (Schmidt & Buchheim, 1970; Anema et al., 1997) increases. At moderate pressures, i.e. about 100 MPa, this increase is small and therefore the coagulation is slow. At higher pressures around 200 MPa, however, the change of pH is faster, the CCP is dissolved more rapidly by the shift of pH and the pressure-induced shift in mineral balance instantaneously leads to di!erent coagulation kinetics. A full description of coagulation combine the e!ects of lowered surface charge of the protein particles resulting in smaller forces acting repulsively and the increase in the number of particles. These e!ects must lead to an accelerated coagulation of milk by acidi"cation under high hydrostatic pressure but do not describe the di!erent mechanical properties of the formed coagula. The in#uence of whey protein denaturation may be neglected in these experiments. Whey protein denaturation is known to become noticable at pressures above 200 MPa (Schrader & Buchheim, 1998). However, the in#uence of changes of the water layer surrounding the protein particles may not be negligible. Around casein particles, the water structure is decomposed at pressures around 150 MPa (Ohmiya, Kajino, Shimizu & Gekko,
491
1989). At higher pressures, the water of the hydration sphere moves away from the protein surface into the bulk phase, where the volume of the water is smaller under pressure. This is caused by hydration of the ions formed by autodissociation of water molecules. Taking into account the concept of &distance of closest approach' well known in clay colloid chemistry (Overbeek, 1977; Lagaly, 1993) these changes in water structure around the particles are very important. When coagulation occurs at moderate pressures (around 100 MPa) the particles may be in contact but a layer of water molecules may remain between the casein particles preventing coagulation at the lowest point of the so-called primary minimum leading to a #u!y aggregate. When this water layer is removed at higher pressures (around 200 MPa) the interparticle distances in the coagulum may be smaller and the particle interactions will be stronger leading to di!erent mechanical properties of the formed coagula.
5. Conclusions Due to disaggregation of casein particles caused by pressure treatment in combination with the acceleration of the hydrolysis reaction of GDL the acidi"cation of milk under high pressure follows a di!erent mechanism compared with acidi"cation at standard conditions. Although the interactions of the processes under pressure are not completely understood, acidi"cation of skim milk under high pressure might be a tool to obtain new aggregate structures which can not be achieved by acidi"cation at ambient pressure. These high-pressure acidi"ed protein aggegates can be useful for development of new food additives or food systems, after further studies of structure and mechanical behaviour of the aggregates.
References Anema, S. G., Lee, S. K., Schrader, K., & Buchheim, W. (1997). E!ect of pH on the turbidity of pressure-treated calcium caseinate suspensions and skim milk. Milchwissenschaft, 52, 141}146. Asano, T., & Le Noble, J. (1978). Activation and reaction volume in solution. Chemical Reviews, 78, 407}489. Douzals, J. P., Marechal, P. A., Coquille, J. C., & Gervais, P. (1996). Microscopic study of starch gelatinization under high hydrostatic pressure. Journal of Agricultural and Biological Chemistry, 44, 1403}1408. Ezaki, S., & Hayashi, R. (1992). High pressure e!ects on starch: structural changes and retrogradation. In C. Balny, R. Hayashi, K. Heremans, & P. Masson, High pressure and biotechnology. Colloques INSERM, vol. 224 (pp. 163}165). Galazka, V. B., Dickinson, E., & Ledward, D. A. (1996). E!ect of high pressure on the emulsifying behaviour of b-lactoglobulin. Food Hydrocolloids, 10, 213}219. Galazka, V. B., Ledward, D. A., Dickinson, E., & Langley, K. R. (1995). High pressure e!ects on emulsifying behavior of whey protein concentrate. Journal of Food Science, 60, 1341}1343.
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Gekko, K. (1992). E!ects of pressure on the sol}gel transition of food macromolecules. In C. Balny, R. Hayashi, K. Heremans, & P. Masson, High pressure and biotechnology. Colloques INSERM, vol. 224 (pp. 105}113). Johnston, D. E., Austin, B. A., & Murphy, R. J. (1993). Properties of acid-set gels prepared from high pressure treated skim milk. Milchwissenschaft, 48, 206}209. Johnston, D. E., Murphy, R. J., & Birks, A. W. (1994). Stirred-style yoghurt-type product prepared from pressure-treated skim milk. High Pressure Research, 12, 215}219. Lagaly, G. (1993). Reaktionen der Tonminerale. In K. Jasmund, & G. Lagaly, Tonminerale und Tone (pp. 112}114). Darmstadt: Steinkop! Verlag. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529}542. Mitchell, R. E., & Duke, F. R. (1970). Kinetics and equilibrium constants of the gluconic acid}gluconolactone system. Annals of the New York Academy of Sciences, 172, 129}138. Ohmiya, K., Kajino, T., Shimizu, S., & Gekko, K. (1989). E!ect of pressure on the association states of enzyme treated caseins. Journal of Agricultural and Biological Chemistry, 53, 1}7. Overbeek, J. T. G. (1977). Recent developments in the understanding of colloid stability. Journal of Colloid and Interface Science, 58, 408}422. Sawyer, D. T., & Bagger, J. B. (1959). The lactone-acid salt equilibria for D-glucono-d-lactone and the hydrolysis kinetics for this lactone. Journal of the American Chemical Society, 81, 5302}5306.
Schmidt, D. G., & Buchheim, W. (1970). Elektronenmikroskopische Untersuchung der Feinstruktur von Caseinmizellen in Kuhmilch. Milchwissenschaft, 25, 596}600. Schrader, K., Buchheim, W., & Morr, C. V. (1997). High pressure e!ects on the colloidal calcium phosphate and the structural integrity of micellar casein in milk. I. High pressure dissolution of colloidal calcium phosphate in heated milk systems. Nahrung, 41, 133}138. Schrader, K., & Buchheim, W. (1998). High pressure e!ects on the colloidal calcium phosphate and the structural integrity of micellar casein in milk. II. Kinetics of the casein micelle disintegration and protein interactions in milk. Kieler Milchwirtschaftliche Forschungsberichte, 50, 79}88. Schwertfeger, M., & Buchheim, W. (1998). Acidi"cation of milk by glucono-d-lactone under high pressure. In N. S. Isaacs, High pressure food science, bioscience and chemistry (pp. 242}247). Cambridge: The Royal Society of Chemistry. Skou, E. M., & Jacobsen, T. (1982). The hydrolysis kinetics of gluconod-lactone in solutions without anion catalysis and a systematical error in weakly bu!ered pH-static kinetics. Acta Chemica Scandinavica, A36, 417}422. Smelt, J. P. P. M. (1998). Recent advances in the microbiology of high pressure processing. Trends in Food Science and Technology, 9, 152}158. Thevelein, J. M., van Assche, J. A., Heremans, K., & Gerlsma, S. Y. (1981). Gelatinisation temperature of starch, as in#uenced by high pressure. Carbohydrate Research, 93, 304}307. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965}1979.
Coagulation of skim milk under high hydrostatic pressure with acidi"cation by glucono-d-lactone Marcus Schwertfeger*, Wolfgang Buchheim Federal Dairy Research Centre, Institute of Process Engineering, Hermann-Weigmann-Str. 1, D-24103 Kiel, Germany Received 28 January 1999; accepted 24 May 1999
Abstract Water, protein-free milk serum and skim milk were acidi"ed under high hydrostatic pressure by hydrolysis of glucono-d-lactone under various experimental conditions concerning pressure (50 up to 200 MPa), reaction time (5}40 min), initial concentration (100 and 200 mmol l\) and temperature (25 and 353C). The reaction rates increased under pressure as determined by measurements of pH after decompression. In some experiments acidi"cation of skim milk resulted in formation of coagulated products. The structures of these coagula di!ered from gels formed by acidi"cation of milk under standard conditions. 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: High pressure; Glucono-d-lactone; Milk; Casein; Coagulation
1. Introduction Application of high hydrostatic pressure may induce changes in properties of biopolymer gels (Gekko, 1992), in emulsi"ng properties of milk proteins (Galazka, Ledward, Dickinson & Langley, 1995; Galazka, Dickinson & Ledward, 1996) or in swelling and gelling of starch dispersions (Thevelein, van Assche, Heremans & Gerlsma, 1981; Ezaki & Hayashi, 1992; Douzals, Marechal, Coquille & Gervais, 1996). It has been known for almost 30 years that casein micelles disintegrate under high hydrostatic pressure, apparently due to shifts in the mineral balance in milk by dissolution of the colloidal calcium phosphate (Schmidt & Buchheim, 1970; Schrader, Buchheim & Morr, 1997; Schrader & Buchheim, 1998). The rennet or acid coagulation of pressuretreated milk has been studied extensively (Johnston, Austin & Murphy, 1993; Johnston, Murphy & Birks, 1994). However, the coagulation of milk by acidi"cation under pressure has not been attempted until recently although the possibilities o!ered by pressure-induced acceleration of chemical reactions, protein unfolding and changes in
* Corresponding author. Tel.: #49-(0)431-609-2275; fax: #49(0)431-609-2309. E-mail address: [email protected] (M. Schwertfeger)
number of particles are promising (Schwertfeger & Buchheim, 1998). Microbiological acidi"cation of milk, e.g. by lactic acid producing cultures, may be hindered by the pressure-sensitivity of most vegetative bacteria (Smelt, 1998), at least at higher pressures. In this study, skim milk was acidi"ed under high pressures by hydrolysis of glucono-d-lactone (GDL). This compound decomposes gradually in aqueous media (Sawyer & Bagger, 1959; Skou & Jacobsen, 1982; Mitchell & Duke, 1970) and thus allows slow acidi"cation. Therefore, GDL is widely applied in the food industry. The properties of acid-set milk gels using GDL at standard conditions have been described (Lucey & Singh, 1998).
2. Materials and methods 2.1. Materials Skim milk was prepared by adding 10 g of low-heat skimmilk powder to 100 g of water at room temperature under vigorous stirring. After standing for 30 min, foam was removed by adding a few drops of n-octanol (Merck, Darmstadt, Germany). The milk was stored at room temperature for 8 h max. until use. Protein-free milk serum (UF permeate) was obtained by ultra"ltering 15 l of reconstituted skim milk prepared
0958-6946/99/$ - see front matter 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 1 9 - 3
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without added octanol in a pilot plant (Alfa Laval, Germany) with a membrane "lter (Romicon PM 10, Romicon Inc. Woburn, MA, USA, 10 kD molecular weight cut-o!) at 503C. Glucono-d-lactone was obtained from Jungbunzlauer (Ladenburg, Germany). Deionized water was used for all operations. The room temperature was about 25}273C.
The experiments were repeated two to six times as indicated in the "gures. No statistical calculations were deemed necessary.
2.2. Measurements of pH
Under pressure, the hydrolysis of GDL as determined by the change in pH was accelerated. A pressure of 50 MPa had no measurable in#uence on the reaction rate. However, higher pressures resulted in small (less than 0.3 units) but de"nite pH-shifts. All samples had a "nal pH of about 2.4, measured after 40 min pressurization time or after one hour at ambient pressure. Under pressure, lower pH values could be reached at shorter reaction times as compared with reaction under ambient pressure, e.g., at 100 MPa a pH of 2.6 was reached within 20 min instead of 40 min at ambient pressure (Fig. 1). An increase in temperature led to a further acceleration of the reaction (Fig. 2). The "nal pH did not drop
At ambient pressure and room temperature (standard temperature and pressure, STP) 0.891 g GDL was quickly dissolved in water, UF permeate or skim milk at 253C to obtain a "nal concentration of 100 mmol l\. Immediately after dissolution, the pH was measured with a pHmeter equipped with a glass electrode (WTW SenTix 97T and pH 325, WTW, Weinheim, Germany). The pH values were recorded continuously. The temperature was held at 253C by using a water bath. 2.3. High-pressure experiments Pressure experiments were carried out in a home-built laboratory-scale high-pressure system (pressure chamber 16 mm in diameter, total volume approximately 20 ml). Hydraulic oil (DEA Econa 46, DEA Mineraloel AG, Hamburg, Germany) was used as the pressure-transducing medium. The samples were placed in cap-closed polyethylene tubes (volume about 5 ml). The pressure vessel was thermostatically controlled at 253C. The pressure was generated by using a pneumatic pressure intensi"er. Compression and decompression were accomplished within 5 s. To avoid any substantial warm-up beyond the selected temperature due to adiabatic heating, the samples (water, permeate or skim milk) were kept at a temperature somewhat lower than the desired reaction temperature (203C for pressure experiments at 50 MPa, 17 and 143C for experiments at 100 and 200 MPa, respectively). These temperatures were determined in preliminary experiments (data not shown). Exactly 0.891 g GDL was dissolved quickly in these precooled samples resulting in an initial concentration of 100 mmol l\. The mixtures were "lled into the tubes, sealed and then placed into the pressure vessel. The selected pressure was reached within 90 s after starting solubilisation of GDL. Immediately after decompression, the samples were removed from the pressure vessel and the pH was measured. The structure of the samples was characterized visually. In some experiments, modi"ed experimental conditions were used, including a higher initial concentration of GDL or higher reaction temperature. For reactions performed at higher temperatures the thermostat was set to 353C and the samples were precooled to 303C.
3. Results 3.1. Hydrolysis of GDL in water
Fig. 1. Changes of pH after various pressure treatments of solutions of GDL in water (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
Fig. 2. Variations of pH after pressure treatment of aqueous solutions of GDL with di!erent initial concentrations at 100 MPa and di!erent temperatures.
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below 2.4 after pressure treatment at 100 MPa. By using a higher initial concentration of GDL (200 mmol l\), a lower pH of about 2.2 could be reached within 40 min at 100 MPa (Fig. 2). 3.2. Hydrolysis of GDL in milk serum The GDL-induced acidi"cation of milk serum was di!erent in comparison with water. For all experimental conditions, the resulting pH di!erence was considerably larger as compared with ambient pressure. The di!erence was about 1.0 units for pressure treatment at 200 MPa and up to 0.2 and 0.5 units after pressurization at 50 and 100 MPa, respectively. In all experiments a constant "nal pH has not been reached within a pressurization time of 40 min (Fig. 3).
Fig. 4. Variations of pH after pressure treatment of solutions of GDL in skim milk (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
3.3. Hydrolysis of GDL in skim milk The acidi"cation of skim milk was di!erent from that in water or milk serum. A constant pH was not reached within more than one hour at ambient pressure or within 40 min at pressures up to 200 MPa. After short times of pressurization the di!erences in pH between pressurized and non-pressurized samples were relatively small, reaching at most about 0.5 pH units. These di!erences increased further with reaction time for all pressures applied. The pH after a reaction time of 60 min changed to 5.3 after hydrolysis at ambient pressure and to 4.3 after hydrolysis at 200 MPa (Fig. 4). At a higher reaction temperature of 353C, a lower pH was reached. Depending on the reaction time the decrease in pH as compared with the corresponding pH at 253C was about 0.2 after 5 min and up to 0.4 after 40 min of pressurization. A larger decrease in pH was observed after pressure treatment with a higher initial concentration of GDL (c "200 mmol l\). In this experiment the
Fig. 3. Variations of pH after pressure treatment of solutions of GDL in milk serum (¹"253C, c (GDL)"100 mmol l\) in comparison to standard temperature and pressure (STP).
Fig. 5. Variations of pH after pressure treatment of solutions of GDL with di!erent initial concentrations in skim milk at 100 MPa and di!erent temperatures.
pH was lowered about 0.4 units after 5 min and up to 1.0 units after 40 min of pressurization at 100 MPa as compared with an initial concentration of 100 mmol l\ (Fig. 5). Some samples which were treated at higher pressures and/or for long reaction times (i.e. 200 MPa for 10 min and longer and 100 MPa for 40 min) were coagulated. These samples had a pH below 5.3. The structures of these aggregates, however, di!ered distinctly from the homogeneous gel which is known to be formed by acidi"cation of milk at ambient pressure. In these experiments #occules formed at 100 MPa after 40 min (Fig. 6A); coarse #occules formed at 200 MPa after 10 min; and a "ne stranded, coherent coagulum, showing strong whey separation when taken out of the tube (Fig. 6B) formed after treatment at 200 MPa for 20 min. It appears remarkable that the structure of the coagula did not re#ect a continuous aggregation process during pressure treatment. Floccules formed after short pressurization times
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Fig. 6. Structures of the coagula formed after acidi"cation with GDL for 40 min at 100 MPa (A) and 20 min at 200 MPa (B). Bar"5 mm.
transformed to stranded coherent coagula after longer times of pressure treatment.
4. Discussion By application of high hydrostatic pressure, all processes in a given system accompanied by a decrease in reaction volume are preferred. In the system studied here the following processes were occurring in parallel: Under high pressure the hydrolysis of GDL was accelerated, as indicated by a more rapid shift in pH after pressure treatment in comparison to the reaction under ambient pressure. Although a covalent bond is being broken by hydrolysis, the total reaction volume will be negative, because the liberated gluconic acid anions and hydrogen ions are strongly hydrated leading to a negative reaction volume caused by stronger ordering of water molecules around the ions. As a result the equilibrium of the reaction is shifted. Chemical reaction rates are increased under pressure if the volume of the activated state is smaller than the volume of the reactants, i.e. the activation volume D
Fig. 7. Rate of pH-shift in milk serum and water caused by pressureaccelerated acidi"cation at 253C and di!erent pressures. The values are calculated from the slope of a curve "t of the values displayed in Figs. 1 and 3 using a potential function (pH"at\@).
the milk salts in#uenced the reaction rate of GDL hydrolysis which was higher in milk serum than in water (Fig. 7.) because the reaction rate depends on the medium, especially regarding its bu!er concentration and pH (Sawyer & Bagger, 1959; Skou & Jacobsen, 1982). Under high hydrostatic pressure, skim milk constituents are also a!ected. The mineral equilibrium of the system is changed by dissolution of the colloidal calcium phosphate (CCP) located in the micelles and therefore the casein micelles disintegrate under high pressure leading to a decrease in particle diameter and an increase in the number of particles (Schmidt & Buchheim, 1970; Anema, Lee, Schrader & Buchheim, 1997).
M. Schwertfeger, W. Buchheim / International Dairy Journal 9 (1999) 487}492
Although skim milk is not a colloidal system exclusively stabilized by electrostatic interactions, the variations of colloidal stability are caused by changes in the charge located at the surface of the micelles or along the chains of the i-casein molecules of the protein particles. This is displayed by changes in the resulting particle charge being measured as the f-potential. A high fpotential indicates strong repulsive forces. Coagulation can occur if the f-potential is low enough. For casein dispersions it is known (Walstra, 1990) that the f-potential depends on the pH of the medium and has a minimum of a about !3 millivolts at a pH of about 5.2 and reaches zero at a pH of about 4.5. In these pH ranges the repulsive forces should be minimal. For the process of coagulation, the particles of the dispersion must be in close contact. It is necessary to overcome the repulsive forces between the particles caused by electrostatic or steric stabilization. In the presence of GDL the pH is lowered by highpressure treatment more rapidly than at ambient pressure. Consequently, the stabilizing electrostatic e!ects caused by pH-sensitive carboxyl and phosphoseryl groups are diminished and the colloidal stability decreases. At moderate pressures (50 and 100 MPa) at which the pH of about 5.2 is not reached within the observed reaction time no visible coagulation could be detected. However, the samples treated at a higher pressure of 200 MPa resulting in a pH drop below 5.2 exhibited distinct coagulation. Additionally, the process of nucleation at the beginning of the coagulation process depends on the number of particle collisions. By high pressure treatment the number of casein particles caused by dissolution of the CCP (Schmidt & Buchheim, 1970; Anema et al., 1997) increases. At moderate pressures, i.e. about 100 MPa, this increase is small and therefore the coagulation is slow. At higher pressures around 200 MPa, however, the change of pH is faster, the CCP is dissolved more rapidly by the shift of pH and the pressure-induced shift in mineral balance instantaneously leads to di!erent coagulation kinetics. A full description of coagulation combine the e!ects of lowered surface charge of the protein particles resulting in smaller forces acting repulsively and the increase in the number of particles. These e!ects must lead to an accelerated coagulation of milk by acidi"cation under high hydrostatic pressure but do not describe the di!erent mechanical properties of the formed coagula. The in#uence of whey protein denaturation may be neglected in these experiments. Whey protein denaturation is known to become noticable at pressures above 200 MPa (Schrader & Buchheim, 1998). However, the in#uence of changes of the water layer surrounding the protein particles may not be negligible. Around casein particles, the water structure is decomposed at pressures around 150 MPa (Ohmiya, Kajino, Shimizu & Gekko,
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1989). At higher pressures, the water of the hydration sphere moves away from the protein surface into the bulk phase, where the volume of the water is smaller under pressure. This is caused by hydration of the ions formed by autodissociation of water molecules. Taking into account the concept of &distance of closest approach' well known in clay colloid chemistry (Overbeek, 1977; Lagaly, 1993) these changes in water structure around the particles are very important. When coagulation occurs at moderate pressures (around 100 MPa) the particles may be in contact but a layer of water molecules may remain between the casein particles preventing coagulation at the lowest point of the so-called primary minimum leading to a #u!y aggregate. When this water layer is removed at higher pressures (around 200 MPa) the interparticle distances in the coagulum may be smaller and the particle interactions will be stronger leading to di!erent mechanical properties of the formed coagula.
5. Conclusions Due to disaggregation of casein particles caused by pressure treatment in combination with the acceleration of the hydrolysis reaction of GDL the acidi"cation of milk under high pressure follows a di!erent mechanism compared with acidi"cation at standard conditions. Although the interactions of the processes under pressure are not completely understood, acidi"cation of skim milk under high pressure might be a tool to obtain new aggregate structures which can not be achieved by acidi"cation at ambient pressure. These high-pressure acidi"ed protein aggegates can be useful for development of new food additives or food systems, after further studies of structure and mechanical behaviour of the aggregates.
References Anema, S. G., Lee, S. K., Schrader, K., & Buchheim, W. (1997). E!ect of pH on the turbidity of pressure-treated calcium caseinate suspensions and skim milk. Milchwissenschaft, 52, 141}146. Asano, T., & Le Noble, J. (1978). Activation and reaction volume in solution. Chemical Reviews, 78, 407}489. Douzals, J. P., Marechal, P. A., Coquille, J. C., & Gervais, P. (1996). Microscopic study of starch gelatinization under high hydrostatic pressure. Journal of Agricultural and Biological Chemistry, 44, 1403}1408. Ezaki, S., & Hayashi, R. (1992). High pressure e!ects on starch: structural changes and retrogradation. In C. Balny, R. Hayashi, K. Heremans, & P. Masson, High pressure and biotechnology. Colloques INSERM, vol. 224 (pp. 163}165). Galazka, V. B., Dickinson, E., & Ledward, D. A. (1996). E!ect of high pressure on the emulsifying behaviour of b-lactoglobulin. Food Hydrocolloids, 10, 213}219. Galazka, V. B., Ledward, D. A., Dickinson, E., & Langley, K. R. (1995). High pressure e!ects on emulsifying behavior of whey protein concentrate. Journal of Food Science, 60, 1341}1343.
492
M. Schwertfeger, W. Buchheim / International Dairy Journal 9 (1999) 487}492
Gekko, K. (1992). E!ects of pressure on the sol}gel transition of food macromolecules. In C. Balny, R. Hayashi, K. Heremans, & P. Masson, High pressure and biotechnology. Colloques INSERM, vol. 224 (pp. 105}113). Johnston, D. E., Austin, B. A., & Murphy, R. J. (1993). Properties of acid-set gels prepared from high pressure treated skim milk. Milchwissenschaft, 48, 206}209. Johnston, D. E., Murphy, R. J., & Birks, A. W. (1994). Stirred-style yoghurt-type product prepared from pressure-treated skim milk. High Pressure Research, 12, 215}219. Lagaly, G. (1993). Reaktionen der Tonminerale. In K. Jasmund, & G. Lagaly, Tonminerale und Tone (pp. 112}114). Darmstadt: Steinkop! Verlag. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529}542. Mitchell, R. E., & Duke, F. R. (1970). Kinetics and equilibrium constants of the gluconic acid}gluconolactone system. Annals of the New York Academy of Sciences, 172, 129}138. Ohmiya, K., Kajino, T., Shimizu, S., & Gekko, K. (1989). E!ect of pressure on the association states of enzyme treated caseins. Journal of Agricultural and Biological Chemistry, 53, 1}7. Overbeek, J. T. G. (1977). Recent developments in the understanding of colloid stability. Journal of Colloid and Interface Science, 58, 408}422. Sawyer, D. T., & Bagger, J. B. (1959). The lactone-acid salt equilibria for D-glucono-d-lactone and the hydrolysis kinetics for this lactone. Journal of the American Chemical Society, 81, 5302}5306.
Schmidt, D. G., & Buchheim, W. (1970). Elektronenmikroskopische Untersuchung der Feinstruktur von Caseinmizellen in Kuhmilch. Milchwissenschaft, 25, 596}600. Schrader, K., Buchheim, W., & Morr, C. V. (1997). High pressure e!ects on the colloidal calcium phosphate and the structural integrity of micellar casein in milk. I. High pressure dissolution of colloidal calcium phosphate in heated milk systems. Nahrung, 41, 133}138. Schrader, K., & Buchheim, W. (1998). High pressure e!ects on the colloidal calcium phosphate and the structural integrity of micellar casein in milk. II. Kinetics of the casein micelle disintegration and protein interactions in milk. Kieler Milchwirtschaftliche Forschungsberichte, 50, 79}88. Schwertfeger, M., & Buchheim, W. (1998). Acidi"cation of milk by glucono-d-lactone under high pressure. In N. S. Isaacs, High pressure food science, bioscience and chemistry (pp. 242}247). Cambridge: The Royal Society of Chemistry. Skou, E. M., & Jacobsen, T. (1982). The hydrolysis kinetics of gluconod-lactone in solutions without anion catalysis and a systematical error in weakly bu!ered pH-static kinetics. Acta Chemica Scandinavica, A36, 417}422. Smelt, J. P. P. M. (1998). Recent advances in the microbiology of high pressure processing. Trends in Food Science and Technology, 9, 152}158. Thevelein, J. M., van Assche, J. A., Heremans, K., & Gerlsma, S. Y. (1981). Gelatinisation temperature of starch, as in#uenced by high pressure. Carbohydrate Research, 93, 304}307. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965}1979.