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Effects of High Pressures Combined with Moderate Temperatures on the Rennet Coagulation Properties of Milk
PII : S0958-6946(98)00093-4
Int. Dairy Journal 8 (1998) 623—627 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$ — see front matter
Effects of High Pressures Combined with Moderate Temperatures on the Rennet Coagulation Properties of Milk Rosina Lo´ pez-Fandin o* and Agustı´ n Olano Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain (Received 7 April 1998; accepted 25 June 1998) ABSTRACT The effects of milk pressurization (100—400 MPa) at different temperatures (25—60°C) on the denaturation of whey proteins, rennet coagulation time, wet cheese yield and enzymatic phase of coagulation have been investigated. The results indicated that there is a synergistic effect between pressure and temperature on the biochemical properties of milk. Pressurization at 40°C delayed coagulation time and impaired gel strength, although protein and water retention in the curd were improved as compared with milk pressurized at 25°C. The use of temperatures higher than 40°C adversely affected milk coagulation. The combined application of high pressures and moderate temperatures was found to increase the extent of b-lactoglobulin denaturation over treatments at 25°C, and pressurization at 50°C induced denaturation of a-lactalbumin. Enhanced whey protein denaturation is supposed to be responsible for hindering the enzymatic hydrolysis of i-casein, in particular that of the glycosylated forms. 1998 Elsevier Science Ltd. All rights reserved Keywords: high pressure; temperature; milk; rennet coagulation
INTRODUCTION
reconstituted milk powder such as hydrophobicity and average diameter of particles (Gaucheron et al., 1997). This work was conducted to investigate the effects of the combined application of high pressures (100— 400 MPa) and temperatures (25—60°C) on the rennet coagulation properties of milk.
High-pressure treatments are increasingly being studied as an alternative to thermal treatments for food preservation. These processes not only inactivate microorganisms but also affect enzyme activities and influence the structure of biopolymers, therefore they can also be used in the preparation of foods with better functional properties and in the development of new products (Farr, 1990). High-pressure treatment of milk was found to improve its microbiological quality and technological properties for cheesemaking, such as coagulation time, cheese yield, gel strength and resistance to syneresis (Desobry-Banon et al., 1994; Johnston et al., 1993; Lo´pezFandin o et al., 1996; Patterson et al., 1995), although it may give rise to texture defects in cheese due to excess moisture (Drake et al., 1997). Several studies have proved that moderate heating (40—60°C) enhanced the efficiency of pressure inactivation of microorganisms (Carlez et al., 1993; Gervilla et al., 1997) and increased the degree of enzyme inactivation (Seyderhelm et al., 1996). The combined use of high pressures and moderate temperatures can lead to efficient microbial inactivation comparable to that with thermal pasteurization and pressurization at room temperature, respectively (Cheftel, 1995). From a physicochemical point of view, it was shown that changes in the temperature of pressurization influence certain characteristics of
MATERIALS AND METHODS High pressure processing Freshly drawn bovine milk was obtained from a local dairy farm, 0.2 g L\ sodium azide was added and it was stored for maximally 3 h before use. Samples of 300 mL were poured into polyethylene bottles, a lid was placed avoiding headspace, and the bottles were vacuum-sealed in polyethylene bags before being pressurized. Treatments were carried out at pressures from 100 to 400 MPa and temperatures from 25 to 60°C using a 900 HP equipment (Eurotherm Automation, Lyon, France). The pressure was raised to the desired value at a rate of 2.5 MPa s\, maintained for 15 min, and released at the same rate. The temperature of the hydrostatic fluid medium was measured with a thermocouple, and, before pressure processing, it was controlled by circulating water through a jacket surrounding the pressure vessel. Before processing, samples were preequilibrated for 10 min at the selected temperature and the temperature increase, as a result of the pressure treatment, was 2°C 100 MPa\. Control samples were obtained by keeping the milk for the same period of time at
*Corresponding author. Tel.:#34 91 5622900; fax:#34 91 5644853; e-mail: [email protected]. 623
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each processing temperature but without pressure treatment. High-pressure treatments were performed in triplicate with milk from different batches and all the analytical determinations were performed at least in duplicate. Denaturation of whey proteins Whey proteins soluble at pH 4.6 were separated from raw and pressurized milk samples by adjusting the pH with 2 M HCl and determined by RP-HPLC (Resmini et al., 1989). Analyses were performed on a PLRP 8 km column (300 A> , 150;4.6 mm; Polymer Laboratories, Church Stretton, UK) with a linear binary gradient. Solvent A was 0.1% (w/v) trifluoroacetic acid (TFA; Pierce, Rockford, IL, USA) in HPLC grade water (LabScan Ltd., Dublin, Ireland), and solvent B consisted of 0.1% (w/v) TFA in acetonitrile (HPLC grade; LabScan Ltd.). The wavelength was set at 205 nm and the flow was 1 mL min\. Denaturation of a-lactalbumin (a-La) and b-lactoglobulin (b-Lg) following the pressure treatments were expressed as percentage of the contents of the corresponding raw milk. Coagulation properties of milk A Formagraph (N. Foss and Co., Hiller+d, Denmark) was used to measure the rennet coagulation time and the curd firming time (McMahon and Brown, 1982). Rennet (85% chymosin and 15% bovine pepsin, strength 1 : 10,000, Chr. Hansen’s Lab. Copenhagen, Denmark) was diluted (0.3% w/v) in acetate buffer (0.2 M, pH 5.4), and 200 kL were added to 10 mL of milk that had been preequilibrated to 36°C for 30 min.
5°C) and filtered through a Durapore 0.45 km filter (Millipore Corp., Bedford, MA, USA) before injection. RP-HPLC separations were performed on a Beckman Ultrapore RPSC column (75;4.6 mm) as described previously (Lo´pez-Fandin o et al., 1993). Solvent A was 0.1% TFA (Merck, Darmstadt, Germany) in Milli-Q water (Millipore Corp.) and solvent B was 0.1% TFA in acetonitrile (HPLC grade from Scharlau, Barcelona, Spain). SE-HPLC separations were performed on a Beckman Spherogel-TSK 2000 SW column (300;7.5 mm) with 0.1 M K HPO —KH PO (pH 6.0) buffer contain ing 21.41 g L\ of Na SO filtered through a 0.45 km Millipore filter (van Hooydonk and Olieman, 1982). In both cases a flow rate of 1 mL min\ was employed with detection at 210 nm. Analysis of the variance of the data was carried out using the Statgraphics Statistical System.
RESULTS AND DISCUSSION Whey protein denaturation Denaturation of whey proteins following the pressure—temperature treatments is shown in Fig. 1. Control samples which were heat-treated but not pressurized presented the same levels of pH 4.6 soluble b-Lg and a-La. Pressures up to 100 MPa did not induce b-Lg denaturation at any of the temperatures assayed, but at higher pressures denaturation increased with increasing temperature, reaching more than 93% at 300 MPa and 50—60°C and at 400 MPa and 40°C. Denaturation of
Cheese yield Wet cheese yields were estimated by centrifugation which provided a good assessment of fresh cheese yield unadjusted for moisture content (Macheboeuf et al., 1993). Milk (30 mL) prewarmed to 30°C was treated with 600 kL of the rennet solution described above and stirred for 1 min. After 40 min at 30°C, the curd was cut and, 10 min later, centrifuged at 15000 g for 15 min at 5°C. The curd was then separated from the whey and weighed. Primary phase of rennin action The extent of the enzymatic reaction was determined on the basis of the release of caseinomacropeptide (CMP) soluble in 4% trichloroacetic acid (TCA), as analysed by reversed-phase (RP) HPLC and size-exclusion (SE) HPLC. To milk (30 mL) that had been prewarmed at 30°C, 750 kL of a 0.04% (w/v) crystalline chymosin solution (EC 3.2.23.4; Sigma Chemical Co., St. Louis, MO, USA; activity approximately 23.6 units mg\ of protein) was added. This concentration of coagulant was lower than that used to determine the rennet coagulation properties of milk, because a slow release of CMP was necessary to accurately determine the initial rate of the enzymatic phase of coagulation (Lo´pez-Fandin o et al., 1997). Samples were incubated at 30°C, and 2 mL aliquots were withdrawn from the reaction mixtures at intervals. The reaction was stopped by vigorously vortex-mixing into 4 ml of 6% (w/v) TCA. After 30 min at 30°C, the samples were centrifuged (4500 g for 20 min at
Fig. 1. Effect of pressurization at 25 (—䉬—), 40 (—䊐—), 50 (—䉭—) and 60°C (—;—) for 15 min on the percentage of denaturation of b-Lg (a) and a-La (b). The means of three independent experiments are shown. The error bars ($SD) are included in the symbols.
Pressurization of milk at different temperatures
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b-Lg, in milk treated at pressures over 300 MPa at room temperature, was responsible for increased cheese yields due to enhanced protein incorporation and moisture retention (Drake et al., 1997; Lo´pez-Fandin o et al., 1996). As previously observed (Lo´pez-Fandin o et al., 1996), a-La was resistant to pressurization up to 400 MPa at room temperature, but considerable denaturation took place at 300 MPa at temperatures of 50—60°C, reaching almost 60% after treatments at 400 MPa and 60°C. The stability of a-La towards high pressure is higher than that of b-Lg. It has been reported that, at room temperature, the structural change of a-La was practically reversible up to 400 MPa, whereas that of b-Lg lost reversibility at 150 MPa or lower, probably due to differences in the secondary structure, in the number of disulfide bonds and Ca> binding sites (Tanaka et al., 1996). Felipe et al. (1997), studying caprine milk, also found that pressurization at 50°C induced denaturation of a-La and increased the extent of b-Lg denaturation over treatments at 25°C. Cheesemaking properties Figure 2 illustrates the effects of pressurization of milk at different temperatures on the rennet coagulation time and cheese yield estimated by centrifugation. As expected, heating at 25—60°C for 15 min at atmospheric pressure did not have any effect in both properties. Treatments at 100 MPa significantly (P(0.05) reduced the coagulation time as compared to that of the unpressurized samples and no differences were observed among the various temperatures studied (Fig. 2a). Treatments at 60°C at pressures higher than 100 MPa hindered rennet coagulation, while milk pressurized at 50°C and 200 MPa took very long to coagulate and, over 200 MPa, failed to coagulate in less than 30 min. At 40°C and pressures over 100 MPa, the rennet coagulation times were significantly longer (P(0.05) than those of milks pressurized at 25°C. The curd firming times (results not shown) followed a trend similar to the rennet coagulation times, except that samples processed at 40°C at 300 and 400 MPa did not achieve a gel firmness sufficient for cutting (the arms of firmness versus time diagram produced by the Formagraph did not reach a separation of 20 mm). Cheese yields only differed statistically from that of the unpressurized samples at 300 and 400 MPa (Fig. 2b). The curds obtained from milk treated at 400 MPa and 40°C weighed significantly more than those from milk submitted at the same pressure at 25°C. However, despite this positive effect on wet cheese yield, from the present results on rennet coagulation time and curd firming rate, it can be deduced that, unlike pressurization at 25°C, the use of temperatures of 40°C or higher did not improve the cheesemaking properties of milk. Hydrolysis of j-casein by rennet In an attempt to understand the combined effects of temperature and pressure on the rennet coagulation properties of milk, the enzymatic phase of coagulation was determined in milk samples pressurized at 300 MPa at 25, 40 and 50°C. Those conditions were chosen because they yielded very different degrees of b-Lg denaturation (Fig. 1a) and coagulation behaviours (Fig. 2a).
Fig. 2. Effect of pressurization at 25 (—䉬—), 40 (—䊐—), 50 (—䉭—) and 60°C (—;—) for 15 min on the coagulation time (r) (a), and wet weight cheese yield (b). The means ($SD) of three independent experiments are shown.
The measurement of the release of caseinomacropeptides soluble in 4% TCA by RP-HPLC revealed that, although the rate of i-casein hydrolysis was slightly faster in the unpressurized milk, it proceeded to the same extent in all cases and no differences were observed among the various temperatures that could explain the dissimilar coagulation properties (Fig. 3a). However, more severe pressure conditions assayed in a previous study (400 MPa, 30 min, 25°C) reduced the rate of formation and final level of CMP, indicating that the primary phase of the coagulation process was inhibited by the pressure (Lo´pez-Fandin o et al., 1997). It has been reported that UHT treatment caused a 40% reduction in glycosylated CMP release but led to identical final contents of carbohydrate free CMP, suggesting that the b-Lg-i-casein complexes which hinder rennet coagulation in heated milk were only formed with the glycosylated forms of i-casein (Ferron-Baumy et al., 1992). Since the RP-HPLC method selected only allowed a proper determination of A and B genetic variants of CMP without sugar side chains (Lo´pez-Fandin o et al., 1993), we then tried a SE-HPLC method which did not discriminate between the genetic variants and the nonglycosylated and glycosylated forms soluble in 4% TCA (Olieman and van Riel, 1989). The results obtained showed that pressurization at 300 MPa affected the kinetics of i-casein hydrolysis and decreased the final CMP released by 15%, in the case of milk pressurized at 25°C, and around 40%, in the case of milk pressurized at 40 and 50°C. This indicates that pressurization rendered
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milk as compared with pressurization at 25°C. Increasing the temperature of pressurization to 40°C delayed the coagulation time and impaired gel strength, although protein and water retention in the curd were improved as compared with milk pressurized at 25°C. Pressurization at temperatures higher than 40°C affected coagulation adversely or prevented milk from coagulating at all. At least part of the effects observed could be attributable to a lower extent of enzymatic hydrolysis of icasein, in particular of the glycosylated forms, that might result from pressure-induced associations with b-Lg. aLa, which was resistant to pressures below 450 MPa applied at room temperature, denatured significantly when pressures of 300 and 400 MPa were used in combination with temperatures of 40 and 50°C.
ACKNOWLEDGEMENTS The authors acknowledge Ms. C. Talavera for skilful technical assistance. This work was supported by the projects 06G/053/96 (Comunidad de Madrid) and ALI97—0759 (Comisio´n Interministerial de Ciencia y Tecnologı´ a).
REFERENCES Fig. 3. Release of caseinomacropeptides (CMP) soluble in 4% TCA, from raw milk (—䊊—) and milks pressurized at 300 MPa for 30 min at 25 (—䉬—), 40 (—䊐—) and 50°C (—䉭—), incubated with chymosin at 30°C, as determined by RP-HPLC (a), and SEHPLC (b). Some variation in the levels of CMP were found among the three replicates conducted with milk from different batches, therefore means from a single experiment are represented, although the results were coincident in the three of them.
glycosylated i-casein less susceptible to the action of the enzyme, maybe through a complex formation with denatured b-Lg, as it has been found for heated milk (Ferron-Baumy et al., 1992). Nevertheless, it should be noticed that milk pressurized at 400 MPa and 25°C underwent a 75% denaturation of b-Lg (Fig. 1a), but its rennet coagulation time was similar to that of the unpressurized milk (Fig. 2a). In addition, no differences were found in the primary phase of coagulation between samples pressurized at 300 MPa for 15 min at 40 and 50°C (Fig. 3b), despite they exhibited very different coagulation properties (Fig. 2a). These findings support previous observations on the lack of correlation between b-Lg denaturation and the rate of i-casein hydrolysis and the coagulation time of pressurized milk (Lo´pez-Fandin o et al., 1996, 1997).
CONCLUSIONS The present results show that there is a synergistic effect between pressure and temperature on the biochemical properties of milk. The combined use of high pressures (100—400 MPa) and temperatures of 40°C and higher did not improve the cheesemaking properties of
Carlez, A., Rosec, J. P., Richard, N. and Cheftel, J. C. (1993) High pressure inactivation of Citrobacter freundii, Pseudomonas fluorescens and ¸isteria innocua in inoculated minced beef. ¸ebesnmittel ¼issenschaft und ¹echnologie 26, 357—363. Cheftel, J. C. (1995) Review: high-pressure, microbial inactivation and food preservation. Food Science and ¹echnology International 1, 75—90. Desobry-Banon, S., Richard, F. and Hardy, J. (1994) Study of acid and rennet coagulation of high pressurized milk. Journal of Dairy Science 77, 3267—3274. Drake, M. A., Harrison, S. L., Asplund, M., Barbosa-Canovas, G. and Swanson, B. G. (1997) High pressure treatment of milk and effects on microbiological and sensory quality of Cheddar Cheese. Journal of Food Science 62, 843—845, 860. Farr, D. (1990) High pressure technology in the food industry. ¹rends in Food Science and ¹echnology 1(7), 14—16. Felipe, X., Capellas, M. and Law, A. J. R. (1997) Comparison of the effects of high pressure treatments and heat pasteurization on the whey proteins in goat’s milk. Journal of Agricultural and Food Chemistry 45, 627—631. Ferron-Baumy, C., Molle´, D., Garric, G. and Maubois, J. L. (1992) Characterization of caseinomacropeptides released from renneted raw and UHT treated milks. ¸e ¸ait 72, 165—173. Gaucheron, F., Famelart, M. H., Mariette, F., Raulot, K., Michel, F. and Le Graet, Y. (1997) Combined effects of temperature and high-pressure treatments on physicochemical characteristics of skim milk. Food Chemistry 59, 439—447. Gervilla, R., Capellas, M., Ferragut, V. and Guamis, B. (1997) Effect of high hydrostatic pressure on ¸isteria innocua 910 CECT inoculated into ewe’s milk. Journal of Food Protection 60, 33—37. Johnston, D. E., Austin, B. A. and Murphy, R. J. (1993) Properties of acid-set gels prepared from high pressure treated skim milk. Milchwissenschaft 48, 206—209. Lo´pez-Fandin o, R., Acedo, M. I. and Ramos, M. (1993) Comparative study by HPLC of caseinomacropeptides from cow’s, ewe’s and goat’s milk. Journal of Dairy Research 60, 117—121.
Pressurization of milk at different temperatures Lo´pez-Fandin o, R., Carrascosa, A. V. and Olano, A. (1996) The effects of high pressure on whey protein denaturation and the cheesemaking properties of raw milk. Journal of Dairy Science 79, 929—936. Lo´pez-Fandin o, R., Ramos, M. and Olano, A. (1997) Rennet coagulation of milk submitted to high pressure. Journal of Agricultural and Food Chemistry 45, 3233—3237. Macheboeuf, D., Coulon, J. B. and D’hour, P. (1993) Effect of breed, protein genetic variants and feeding on cow’s milk coagulation properties. Journal of Dairy Research 60, 43—54. McMahon, D. J. and Brown, R. J. (1982) Evaluation of Formagraph for comparing rennet solutions. Journal of Dairy Science 65, 1639—1642. Patterson, M. F., Quinn, M., Simpson, R. and Gilmour, A. (1995) Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods. Journal of Food Protection 58, 524—529. Olieman, C. and van Riel, J. A. M. (1989) Detection of rennet whey solids in skim milk powder and buttermilk powder
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with reversed-phase HPLC. Netherlands Milk and Dairy Journal 43, 171—184. Resmini, P., Pellegrino, L., Andreini, R. and Prati, F. (1989) Evaluation of milk whey proteins by reversed-phase HPLC. Scienza e ¹ecnica ¸attiero-Casearia 40, 7—23. Seyderhelm, I., Boguslawski, S., Michaelis, G. and Knorr, D. (1996) Pressure induced inactivation of selected food enzymes. Journal of Food Science 61, 308—310. Statgraphics Version S Reference Manual. STSC, Inc.: Rockville, MD, 1991. Tanaka, N., Koyasu, A., Kobayashi, I. and Kunugi, S. (1996) Pressure-induced change in proteins studied through chemical modifications. International Journal of Biological Macromolecules 18, 275—280. Van Hooydonk, A. C. M. and Olieman, C. A. (1982) A rapid and sensitive high-performance liquid chromatography method of following the action of chymosin in milk. Netherlands Milk and Dairy Journal 36, 153—158.
Int. Dairy Journal 8 (1998) 623—627 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$ — see front matter
Effects of High Pressures Combined with Moderate Temperatures on the Rennet Coagulation Properties of Milk Rosina Lo´ pez-Fandin o* and Agustı´ n Olano Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain (Received 7 April 1998; accepted 25 June 1998) ABSTRACT The effects of milk pressurization (100—400 MPa) at different temperatures (25—60°C) on the denaturation of whey proteins, rennet coagulation time, wet cheese yield and enzymatic phase of coagulation have been investigated. The results indicated that there is a synergistic effect between pressure and temperature on the biochemical properties of milk. Pressurization at 40°C delayed coagulation time and impaired gel strength, although protein and water retention in the curd were improved as compared with milk pressurized at 25°C. The use of temperatures higher than 40°C adversely affected milk coagulation. The combined application of high pressures and moderate temperatures was found to increase the extent of b-lactoglobulin denaturation over treatments at 25°C, and pressurization at 50°C induced denaturation of a-lactalbumin. Enhanced whey protein denaturation is supposed to be responsible for hindering the enzymatic hydrolysis of i-casein, in particular that of the glycosylated forms. 1998 Elsevier Science Ltd. All rights reserved Keywords: high pressure; temperature; milk; rennet coagulation
INTRODUCTION
reconstituted milk powder such as hydrophobicity and average diameter of particles (Gaucheron et al., 1997). This work was conducted to investigate the effects of the combined application of high pressures (100— 400 MPa) and temperatures (25—60°C) on the rennet coagulation properties of milk.
High-pressure treatments are increasingly being studied as an alternative to thermal treatments for food preservation. These processes not only inactivate microorganisms but also affect enzyme activities and influence the structure of biopolymers, therefore they can also be used in the preparation of foods with better functional properties and in the development of new products (Farr, 1990). High-pressure treatment of milk was found to improve its microbiological quality and technological properties for cheesemaking, such as coagulation time, cheese yield, gel strength and resistance to syneresis (Desobry-Banon et al., 1994; Johnston et al., 1993; Lo´pezFandin o et al., 1996; Patterson et al., 1995), although it may give rise to texture defects in cheese due to excess moisture (Drake et al., 1997). Several studies have proved that moderate heating (40—60°C) enhanced the efficiency of pressure inactivation of microorganisms (Carlez et al., 1993; Gervilla et al., 1997) and increased the degree of enzyme inactivation (Seyderhelm et al., 1996). The combined use of high pressures and moderate temperatures can lead to efficient microbial inactivation comparable to that with thermal pasteurization and pressurization at room temperature, respectively (Cheftel, 1995). From a physicochemical point of view, it was shown that changes in the temperature of pressurization influence certain characteristics of
MATERIALS AND METHODS High pressure processing Freshly drawn bovine milk was obtained from a local dairy farm, 0.2 g L\ sodium azide was added and it was stored for maximally 3 h before use. Samples of 300 mL were poured into polyethylene bottles, a lid was placed avoiding headspace, and the bottles were vacuum-sealed in polyethylene bags before being pressurized. Treatments were carried out at pressures from 100 to 400 MPa and temperatures from 25 to 60°C using a 900 HP equipment (Eurotherm Automation, Lyon, France). The pressure was raised to the desired value at a rate of 2.5 MPa s\, maintained for 15 min, and released at the same rate. The temperature of the hydrostatic fluid medium was measured with a thermocouple, and, before pressure processing, it was controlled by circulating water through a jacket surrounding the pressure vessel. Before processing, samples were preequilibrated for 10 min at the selected temperature and the temperature increase, as a result of the pressure treatment, was 2°C 100 MPa\. Control samples were obtained by keeping the milk for the same period of time at
*Corresponding author. Tel.:#34 91 5622900; fax:#34 91 5644853; e-mail: [email protected]. 623
624
R. Lo& pez-Fandin o, A. Olano
each processing temperature but without pressure treatment. High-pressure treatments were performed in triplicate with milk from different batches and all the analytical determinations were performed at least in duplicate. Denaturation of whey proteins Whey proteins soluble at pH 4.6 were separated from raw and pressurized milk samples by adjusting the pH with 2 M HCl and determined by RP-HPLC (Resmini et al., 1989). Analyses were performed on a PLRP 8 km column (300 A> , 150;4.6 mm; Polymer Laboratories, Church Stretton, UK) with a linear binary gradient. Solvent A was 0.1% (w/v) trifluoroacetic acid (TFA; Pierce, Rockford, IL, USA) in HPLC grade water (LabScan Ltd., Dublin, Ireland), and solvent B consisted of 0.1% (w/v) TFA in acetonitrile (HPLC grade; LabScan Ltd.). The wavelength was set at 205 nm and the flow was 1 mL min\. Denaturation of a-lactalbumin (a-La) and b-lactoglobulin (b-Lg) following the pressure treatments were expressed as percentage of the contents of the corresponding raw milk. Coagulation properties of milk A Formagraph (N. Foss and Co., Hiller+d, Denmark) was used to measure the rennet coagulation time and the curd firming time (McMahon and Brown, 1982). Rennet (85% chymosin and 15% bovine pepsin, strength 1 : 10,000, Chr. Hansen’s Lab. Copenhagen, Denmark) was diluted (0.3% w/v) in acetate buffer (0.2 M, pH 5.4), and 200 kL were added to 10 mL of milk that had been preequilibrated to 36°C for 30 min.
5°C) and filtered through a Durapore 0.45 km filter (Millipore Corp., Bedford, MA, USA) before injection. RP-HPLC separations were performed on a Beckman Ultrapore RPSC column (75;4.6 mm) as described previously (Lo´pez-Fandin o et al., 1993). Solvent A was 0.1% TFA (Merck, Darmstadt, Germany) in Milli-Q water (Millipore Corp.) and solvent B was 0.1% TFA in acetonitrile (HPLC grade from Scharlau, Barcelona, Spain). SE-HPLC separations were performed on a Beckman Spherogel-TSK 2000 SW column (300;7.5 mm) with 0.1 M K HPO —KH PO (pH 6.0) buffer contain ing 21.41 g L\ of Na SO filtered through a 0.45 km Millipore filter (van Hooydonk and Olieman, 1982). In both cases a flow rate of 1 mL min\ was employed with detection at 210 nm. Analysis of the variance of the data was carried out using the Statgraphics Statistical System.
RESULTS AND DISCUSSION Whey protein denaturation Denaturation of whey proteins following the pressure—temperature treatments is shown in Fig. 1. Control samples which were heat-treated but not pressurized presented the same levels of pH 4.6 soluble b-Lg and a-La. Pressures up to 100 MPa did not induce b-Lg denaturation at any of the temperatures assayed, but at higher pressures denaturation increased with increasing temperature, reaching more than 93% at 300 MPa and 50—60°C and at 400 MPa and 40°C. Denaturation of
Cheese yield Wet cheese yields were estimated by centrifugation which provided a good assessment of fresh cheese yield unadjusted for moisture content (Macheboeuf et al., 1993). Milk (30 mL) prewarmed to 30°C was treated with 600 kL of the rennet solution described above and stirred for 1 min. After 40 min at 30°C, the curd was cut and, 10 min later, centrifuged at 15000 g for 15 min at 5°C. The curd was then separated from the whey and weighed. Primary phase of rennin action The extent of the enzymatic reaction was determined on the basis of the release of caseinomacropeptide (CMP) soluble in 4% trichloroacetic acid (TCA), as analysed by reversed-phase (RP) HPLC and size-exclusion (SE) HPLC. To milk (30 mL) that had been prewarmed at 30°C, 750 kL of a 0.04% (w/v) crystalline chymosin solution (EC 3.2.23.4; Sigma Chemical Co., St. Louis, MO, USA; activity approximately 23.6 units mg\ of protein) was added. This concentration of coagulant was lower than that used to determine the rennet coagulation properties of milk, because a slow release of CMP was necessary to accurately determine the initial rate of the enzymatic phase of coagulation (Lo´pez-Fandin o et al., 1997). Samples were incubated at 30°C, and 2 mL aliquots were withdrawn from the reaction mixtures at intervals. The reaction was stopped by vigorously vortex-mixing into 4 ml of 6% (w/v) TCA. After 30 min at 30°C, the samples were centrifuged (4500 g for 20 min at
Fig. 1. Effect of pressurization at 25 (—䉬—), 40 (—䊐—), 50 (—䉭—) and 60°C (—;—) for 15 min on the percentage of denaturation of b-Lg (a) and a-La (b). The means of three independent experiments are shown. The error bars ($SD) are included in the symbols.
Pressurization of milk at different temperatures
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b-Lg, in milk treated at pressures over 300 MPa at room temperature, was responsible for increased cheese yields due to enhanced protein incorporation and moisture retention (Drake et al., 1997; Lo´pez-Fandin o et al., 1996). As previously observed (Lo´pez-Fandin o et al., 1996), a-La was resistant to pressurization up to 400 MPa at room temperature, but considerable denaturation took place at 300 MPa at temperatures of 50—60°C, reaching almost 60% after treatments at 400 MPa and 60°C. The stability of a-La towards high pressure is higher than that of b-Lg. It has been reported that, at room temperature, the structural change of a-La was practically reversible up to 400 MPa, whereas that of b-Lg lost reversibility at 150 MPa or lower, probably due to differences in the secondary structure, in the number of disulfide bonds and Ca> binding sites (Tanaka et al., 1996). Felipe et al. (1997), studying caprine milk, also found that pressurization at 50°C induced denaturation of a-La and increased the extent of b-Lg denaturation over treatments at 25°C. Cheesemaking properties Figure 2 illustrates the effects of pressurization of milk at different temperatures on the rennet coagulation time and cheese yield estimated by centrifugation. As expected, heating at 25—60°C for 15 min at atmospheric pressure did not have any effect in both properties. Treatments at 100 MPa significantly (P(0.05) reduced the coagulation time as compared to that of the unpressurized samples and no differences were observed among the various temperatures studied (Fig. 2a). Treatments at 60°C at pressures higher than 100 MPa hindered rennet coagulation, while milk pressurized at 50°C and 200 MPa took very long to coagulate and, over 200 MPa, failed to coagulate in less than 30 min. At 40°C and pressures over 100 MPa, the rennet coagulation times were significantly longer (P(0.05) than those of milks pressurized at 25°C. The curd firming times (results not shown) followed a trend similar to the rennet coagulation times, except that samples processed at 40°C at 300 and 400 MPa did not achieve a gel firmness sufficient for cutting (the arms of firmness versus time diagram produced by the Formagraph did not reach a separation of 20 mm). Cheese yields only differed statistically from that of the unpressurized samples at 300 and 400 MPa (Fig. 2b). The curds obtained from milk treated at 400 MPa and 40°C weighed significantly more than those from milk submitted at the same pressure at 25°C. However, despite this positive effect on wet cheese yield, from the present results on rennet coagulation time and curd firming rate, it can be deduced that, unlike pressurization at 25°C, the use of temperatures of 40°C or higher did not improve the cheesemaking properties of milk. Hydrolysis of j-casein by rennet In an attempt to understand the combined effects of temperature and pressure on the rennet coagulation properties of milk, the enzymatic phase of coagulation was determined in milk samples pressurized at 300 MPa at 25, 40 and 50°C. Those conditions were chosen because they yielded very different degrees of b-Lg denaturation (Fig. 1a) and coagulation behaviours (Fig. 2a).
Fig. 2. Effect of pressurization at 25 (—䉬—), 40 (—䊐—), 50 (—䉭—) and 60°C (—;—) for 15 min on the coagulation time (r) (a), and wet weight cheese yield (b). The means ($SD) of three independent experiments are shown.
The measurement of the release of caseinomacropeptides soluble in 4% TCA by RP-HPLC revealed that, although the rate of i-casein hydrolysis was slightly faster in the unpressurized milk, it proceeded to the same extent in all cases and no differences were observed among the various temperatures that could explain the dissimilar coagulation properties (Fig. 3a). However, more severe pressure conditions assayed in a previous study (400 MPa, 30 min, 25°C) reduced the rate of formation and final level of CMP, indicating that the primary phase of the coagulation process was inhibited by the pressure (Lo´pez-Fandin o et al., 1997). It has been reported that UHT treatment caused a 40% reduction in glycosylated CMP release but led to identical final contents of carbohydrate free CMP, suggesting that the b-Lg-i-casein complexes which hinder rennet coagulation in heated milk were only formed with the glycosylated forms of i-casein (Ferron-Baumy et al., 1992). Since the RP-HPLC method selected only allowed a proper determination of A and B genetic variants of CMP without sugar side chains (Lo´pez-Fandin o et al., 1993), we then tried a SE-HPLC method which did not discriminate between the genetic variants and the nonglycosylated and glycosylated forms soluble in 4% TCA (Olieman and van Riel, 1989). The results obtained showed that pressurization at 300 MPa affected the kinetics of i-casein hydrolysis and decreased the final CMP released by 15%, in the case of milk pressurized at 25°C, and around 40%, in the case of milk pressurized at 40 and 50°C. This indicates that pressurization rendered
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milk as compared with pressurization at 25°C. Increasing the temperature of pressurization to 40°C delayed the coagulation time and impaired gel strength, although protein and water retention in the curd were improved as compared with milk pressurized at 25°C. Pressurization at temperatures higher than 40°C affected coagulation adversely or prevented milk from coagulating at all. At least part of the effects observed could be attributable to a lower extent of enzymatic hydrolysis of icasein, in particular of the glycosylated forms, that might result from pressure-induced associations with b-Lg. aLa, which was resistant to pressures below 450 MPa applied at room temperature, denatured significantly when pressures of 300 and 400 MPa were used in combination with temperatures of 40 and 50°C.
ACKNOWLEDGEMENTS The authors acknowledge Ms. C. Talavera for skilful technical assistance. This work was supported by the projects 06G/053/96 (Comunidad de Madrid) and ALI97—0759 (Comisio´n Interministerial de Ciencia y Tecnologı´ a).
REFERENCES Fig. 3. Release of caseinomacropeptides (CMP) soluble in 4% TCA, from raw milk (—䊊—) and milks pressurized at 300 MPa for 30 min at 25 (—䉬—), 40 (—䊐—) and 50°C (—䉭—), incubated with chymosin at 30°C, as determined by RP-HPLC (a), and SEHPLC (b). Some variation in the levels of CMP were found among the three replicates conducted with milk from different batches, therefore means from a single experiment are represented, although the results were coincident in the three of them.
glycosylated i-casein less susceptible to the action of the enzyme, maybe through a complex formation with denatured b-Lg, as it has been found for heated milk (Ferron-Baumy et al., 1992). Nevertheless, it should be noticed that milk pressurized at 400 MPa and 25°C underwent a 75% denaturation of b-Lg (Fig. 1a), but its rennet coagulation time was similar to that of the unpressurized milk (Fig. 2a). In addition, no differences were found in the primary phase of coagulation between samples pressurized at 300 MPa for 15 min at 40 and 50°C (Fig. 3b), despite they exhibited very different coagulation properties (Fig. 2a). These findings support previous observations on the lack of correlation between b-Lg denaturation and the rate of i-casein hydrolysis and the coagulation time of pressurized milk (Lo´pez-Fandin o et al., 1996, 1997).
CONCLUSIONS The present results show that there is a synergistic effect between pressure and temperature on the biochemical properties of milk. The combined use of high pressures (100—400 MPa) and temperatures of 40°C and higher did not improve the cheesemaking properties of
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