The Physical Properties and Renneting Characteristics of the Synthetic Membrane on the Fat Globules of Microfluidized Milk

The Physical Properties and Renneting Characteristics of the Synthetic Membrane on the Fat Globules of Microfluidized Milk SUSAN M. TOSH and DOUGLAS G. DALGLEISH1,2 Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1

ABSTRACT The effect of particle size on the renneting properties of the synthetic fat globules that were formed in full fat homogenized milk were investigated using microfluidization. A decrease in the average hydrodynamic diameter of the fat globules from 390 to 313 nm had no apparent effect on the casein load on the synthetic membrane of the fat globules. However, fat globules with smaller diameters decreased the rennet coagulation time and the curd firming rate. The integrity of the curd microstructure also showed signs of deterioration as the diameters of the fat globules were decreased. Rennet curd was also produced from milk after the cream was separated from the milk, washed, and emulsified with whey proteins by microfluidization before being remixed with the skim milk. The resulting curd showed no significant change in coagulation time or firming rate as the fat globule diameter varied from 265 to 640 nm. There was also no significant change in the microstructure of the curds. We concluded that the changes in the properties of curd made from microfluidized milk were caused by restructuring in the micellar casein during microfluidization rather than by the change in size of the fat globules. ( Key words: milk, microfluidization, rennet, microstructure) Abbreviation key: CFR = curd firming rate, PCS = photon correlation spectroscopy, RCT = rennet coagulation time, WPI = whey protein isolate. INTRODUCTION There is considerable interest in developing new cheese products and more efficient processing methods. Cheese products that have reduced contents

Received October 21, 1997. Accepted March 25, 1998. 1Corresponding author. 2Present address: CIRDC, 15 avenue Galile ´ e, 92350 Le PlessisRobinson, France. 1998 J Dairy Sci 81:1840–1847

of fat and cholesterol are of interest because of perceived health benefits, although textural, flavor, and production problems have occurred with reduced fat cheeses. Cheddar cheese that is made with a 17% fat content becomes more elastic, dense, crumbly, and firm than full fat Cheddar cheese ( 4 ) . The concentration of milk before cheese making is also of interest because processing the smaller volume of milk could result in higher throughputs. Retention of whey proteins in cheese is also potentially beneficial because it can increase yields. However, cheeses made from concentrated milk also have textural problems, being harder and more crumbly than traditional Cheddar cheese ( 6 ) . Homogenization of the milk used for cheese making has been suggested as a method of counteracting these effects (4, 6 ) because homogenization results in cheeses that are higher in moisture and softer in texture (8, 14). Efforts to use homogenization for Cheddar cheese made from lowfat or ultrafiltered milk have met with limited success (4, 6); moisture content did increase and the texture was softer and less elastic, but the texture was significantly different from that of the control cheeses. Although homogenization shows promise as a process for altering the texture of cheese formulations, before the method can be successfully used, the effects of homogenization on the physicochemical properties of micellar casein as it adsorbs to the fatserum interface and the consequences for development of curd structure need to be understood better. It is known, for example, that the aggregation rates of particles in skim milk and in homogenized milks differ (18). Recently, a novel technique of homogenization, microfluidization, has been developed and is based on the use of the Microfluidizer (model 110S; Microfluidics, Inc., Newton, MA). The equipment configuration is different from a valve homogenizer and has been shown to produce emulsions with a smaller mean droplet diameter and a narrower size distribution than do valve homogenizers (15, 24). The microfluidization of milk has been shown to result in a Cheddar cheese that is whiter, softer, and stickier than the

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control cheese (10). Fat retention was better, and moisture content was higher in Cheddar cheese made from microfluidized milk than in Cheddar cheese made from untreated milk. However, the detrimental effects on texture were found to increase as the microfluidization pressure was increased. In the present work, whole milks were homogenized using a Microfluidizer under conditions that would yield a range of diameters of the synthetic fat globules. The casein load on the fat globule membrane was measured with SDS-PAGE. Measurements of fluid consistency were taken as a rennet curd developed in the microfluidized milks, which allowed us to observe the effect of microfluidization on the rennet coagulation time ( RCT) and the curd firming rate ( CFR) . The microstructure of the curds was examined by scanning electron microscopy on cryofixed samples. In homogenized and microfluidized milks, the synthetic fat globule membrane is made up mainly of caseins, which are also the proteins that interact when milk is renneted so that the curd is formed of caseins and fat globules bound together. Alternatively, it is possible to produce small fat globules that do not interact with the casein by homogenizing washed cream with whey protein isolate and then adding it back to the skim milk. The behavior of this reconstituted milk was compared with that of microfluidized whole milk. MATERIALS AND METHODS Sample Preparation Raw milk was collected from the bulk tank at the Dairy Research Station of the University of Guelph (Guelph, ON, Canada). The milk was heated at 60°C for 30 min to inactivate lipase and to reduce the microbial load, and then the milk was tempered at 40°C for 60 min before further processing. Milk was then homogenized with the Microfluidizer and collected. The diameters of the fat globules in the milk were varied by changes in the pressure in the Microfluidizer (14 to 35 MPa) and in the number of passes through the equipment (two to five times). The changes produced in the fat globule size were consistent with previous results (2, 15, 17, 24). Heattreated (30 min at 60°C), unhomogenized, full fat milk was used as a control in the experiments. To produce reconstituted milk, whole milk was cooled to room temperature after heat treatment (60°C for 30 min), and the cream was separated by centrifugation at 959 × g for 30 min. The skim milk was filtered through a glass fiber filter to remove any

remaining fat and was concentrated to 75% of its original volume by ultrafiltration (Amicon, Mississauga, ON, Canada) using a PM 10 membrane with a nominal cutoff of 10,000 Da. The cream layer was washed by recentrifugation with an equal volume of distilled water to remove any remaining casein. The fat content of the washed cream was determined using the Babcock method (11). An emulsion was made using the washed cream, the permeate from the ultrafiltration step, and whey protein isolate ( WPI; Protose Separations, Inc., Teeswater, ON, Canada). The proportions of these components were calculated to obtain 16% (vol/vol) fat and 2% (wt/vol) WPI in the final emulsion. The mixture was heated to 40°C and passed through the Microfluidizer. The pressure and number of passes were varied to produce a range of mean fat globule diameters that were comparable with those in the microfluidized milks. The emulsified fat was then recombined with the concentrated skim milk to restore the casein and fat concentrations to original levels. Measurement of the Mean Diameters of the Fat Globules Samples of microfluidized milk ( 5 ml ) were diluted into 2.5 ml of a calcium buffer ( 5 mM CaCl2, 50 mM NaCl, and 20 mM imidazole; pH 7.0), which had been filtered through a 0.22-mm filter (Millipore, Canada, Ltd., Mississauga, ON, Canada). The mean hydrodynamic diameters of the particles were measured using photon correlation spectroscopy ( PCS; Malvern Instruments Ltd., Southboro, MA) using a 4700 optical system with 7032 autocorrelator and a 30-mW He-Ne laser ( l = 632.8 nm). The measurements were taken at a light scattering angle of 90°. The fat globules in the control milk were too large to be measured conveniently by dynamic light scattering. Casein Load on the Synthetic Fat Globule Membrane As described previously, SDS-PAGE was used to separate the proteins on the synthetic fat globule membrane in microfluidized milk (21). The milk was centrifuged at 86,400 × g for 1 h at 25°C, and the cream layer was collected and washed by resuspension in ultrafiltration permeate from milk. The cream was collected and spread on filter paper to remove the excess aqueous phase. Approximately 10 mg of the washed cream were weighed out and used for the analysis, which was run on a 20% homogeneous Phastgel (Pharmacia Biotech, Baie d’Urfe´, QC, Journal of Dairy Science Vol. 81, No. 7, 1998

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Canada) as described previously ( 7 ) . The gels were scanned at a wavelength of 633 nm on an Ultroscan densitometer (Pharmacia Biotech). The total area under the peaks, which represented the caseins, was calculated. Standard solutions of casein were run to construct a calibration curve for each gel. The specific surface area of the synthetic fat globules was determined by integrated light scattering using a Mastersizer X (Malvern Instruments Inc.). The cream from microfluidized milk was dispersed in EDTA buffer ( 3 mM EDTA, 50 mM NaCl, and 20 mM imidazole; pH 7.0). From the pattern of light scattering, the distribution of particle sizes was calculated using software provided with the instrument, and, from this measurement, the total surface area of the particles was calculated. Increase in Consistency During Gelation The consistency (i.e., apparent viscosity) of the milk was measured with a Nametre dynamic viscometer (Nametre Co., Edison, NJ) using the technique developed by Sharma et al. (22). After being tempered in a water bath at 30°C for 1 h, 0.4 ml of a 5% rennet solution (Chr. Hansen Laboratories, Reading, United Kingdom) was added to 100 ml of milk, and consistency measurements were taken over a period of 1 h at 30°C. The CFR was defined as the slope of a line drawn by linear regression through the graphed results of measurements during the secondary stage of renneting when the apparent viscosity was increasing linearly. The RCT was defined as the point at which lines intersected when drawn through a plot of the consistency of milk during the primary and secondary stages of renneting. Microstructure of Rennet Gels To investigate differences in the curd structure, rennet gels of the same consistency were prepared and examined by scanning electron microscopy following the technique of Xu et al. (29). When a consistency of 30 mPa·s was reached during renneting, two 10- to 20-ml samples of the coagulating milk were placed on brass sample holders and cryofixed (EMScope, Ashford, United Kingdom) by submersion in nitrogen slush at –210°C. The samples were fractured, and ice was removed from the surface by sublimation at –80°C after which the samples were sputter-coated with 30 nm of gold in the presence of argon. The samples were photographed on a scanning electron microscope (Hitachi, Tokyo, Japan). ConcenJournal of Dairy Science Vol. 81, No. 7, 1998

tric sets of photos were taken on each of the paired samples. Three replicates of each sample were prepared and photographed. Image analysis involved manually counting the number of voids in 57- × 62-mm photographs (1000× magnification) from each of the samples and calculating the number of voids in a 100-mm × 100-mm sample of the fixed gel. Statistical Analysis Statistical analyses were performed using the general linear models procedure of SAS (19). A completely randomized block design was used with replicates of each sample. An ANOVA was used to test for fixed effects using a planned and protected least significant difference test adjusted for unbalanced data. RESULTS SDS-PAGE and Surface Protein Load The SDS-PAGE of the proteins adsorbed to the fat globules in microfluidized milk showed bands from the four caseins, but no bands from whey proteins were apparent. The proportions of the different caseins were the same as in the skim milk standards. The casein loads on the fat globules in microfluidized milk were independent of the mean particle diameter over the range examined (Table 1), and the mean casein load was 7.2 mg·m–2 of surface area of the fat. This value was considerably less than the 17.3 and 10.6 to 13.1 mg·m–2 reported by Dalgleish and Robson ( 1 ) and by McCrae et al. (12), respectively, for homogenized milk produced by a valve homogenizer but is somewhat larger than the estimate of about 6 mg m–2 given by Sharma et al. ( 2 3 ) for recombined milk and is much greater than the value of 3 mg·m–2 that is typical of monolayer coverage by caseinate or whey protein (5, 7). However, using electron microscopy ( 2 ) , we have shown that the structures of the particles in these microfluidized milks are not simple; indeed, the concept of even coverage of the fat by the protein is not well supported, because ratios of fat to protein differ widely among the particles, and the centrifugation conditions used to harvest the fat droplets may strongly affect the results. Consistency of Microfluidized Milk During Renneting The plots of consistency measured against time by the Nametre viscometer clearly showed the two stages of the renneting process (Figure 1). In both

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RENNETING OF MICROFLUIDIZED MILK TABLE 1. Casein load on the synthetic fat globule membrane in microfluidized milk on globules of different sizes.1

TABLE 2. Effect of fat globule diameter on the renneting characteristics of control, reconstituted, and microfluidized milks.

Mean diameter of the fat globules

Type of milk

Mean diameter of fat globules

RCT1

CFR

Control Reconstituted Microfluidized Microfluidized Microfluidized Microfluidized

(nm) . . .2 265–640 412 388 364 359

(min) 26.9a 25.6b 24.1c 22.0d 21.0de 20.8e

(mPa) 22.4b 25.0a 12.1c 10.9d 10.2d 8.6e

(mg·m –2)

(nm) 390 337 319 313 1Values

Protein load 7.4 7.6 6.8 6.9

for the load are not different ( P > 0.10).

microfluidized and reconstituted milks, viscosity decreased slightly during the primary stage, which is consistent with results of research on skim milk (20, 22). Decreased viscosity is caused by the decrease in the size of the casein micelles (or the microfluidized fat globules) as the macropeptides are removed from the k-CN (27). No corresponding minimum viscosity was observed in unhomogenized control milk. The RCT of microfluidized milks were within the range 20.8 to 24.1 min and were significantly shorter than the RCT of the control (Table 2). Microfluidization at the lowest pressure gave fat globules with a

a,b,c,d,eMeans within the same column with no common superscript letter are different ( P < 0.05). 1RCT = Rennet clotting time; CFR = curd-firming rate. 2Not measured.

mean diameter of 412 nm and decreased the RCT by 2.8 min; a further decrease in the mean diameter of the fat globules from 412 to 359 nm caused the RCT to decrease an additional 3.3 min. The largest difference in RCT was observed when the size of the fat globules in the microfluidized milks was reduced from 412 to 388 nm. Smaller differences in the RCT were observed as the mean diameter of fat globules in the samples was decreased to 364 and 359 nm. In contrast, the RCT of the reconstituted milk was slightly shorter than that of the control milk but was independent of the diameter of the fat globules in the range from 640 to 265 nm and was significantly longer than the RCT for all of the microfluidized whole milks. The range of CFR for microfluidized milks was 8.6 to 12.1 mPa, which is much lower than the average CFR of 22.4 mPa in the control milk (Table 2). A decrease in the diameter of the fat globules from 412 to 359 nm, caused by increased microfluidization pressure caused a significant decrease of 3.5 mPa. Over the range studied, the relationship between CFR and fat globule size was linear, but no significant change was found in the CFR of the reconstituted milk samples with fat globule diameters from 640 to 265 nm. The CFR of the renneted reconstituted milk samples, 25.0 mPa, was somewhat higher than that of the control milk and more than double the CFR of any microfluidized milk. Qualitative Assessment of the Curds

Figure 1. The increase in consistency of microfluidized ( ÿ) , reconstituted ( ⁄) , and control ( o) milk samples during renneting. The mean fat globule diameters in the particular samples of microfluidized and filled milks were 396 and 315 nm, respectively.

Gels made from the control milk were firm and broke into large, well-defined pieces; gels made from microfluidized milk were more fragile. A general deterioration in the integrity of the structure of the gel occurred as the fat globule size decreased. When the microfluidization pressure was lowest (mean fat Journal of Dairy Science Vol. 81, No. 7, 1998

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Figure 2. The microstructure of rennet gels as viewed by cryoscanning electron microscopy. Unhomogenized milk control ( a ) and microfluidized milks with mean fat globule sizes of 412 nm ( b ) , 364 nm ( c ) , and 353 nm ( d ) . Particles marked g are fat globules.

Figure 3. The microstructure of rennet gels as viewed by cryoscanning electron microscopy. Unhomogenized milk control ( a ) and reconstituted milks with mean fat globule sizes of 293 nm ( b ) , 344 nm ( c ) , and 330 nm ( d ) . Particles marked g are fat globules.

globule diameter, 412 nm), the pieces were large, although the fracture planes were not as clean as in the control milk. In the microfluidized milk with the smallest fat globules (359 nm), the rennet gel shattered as soon as it was disturbed. Despite its higher CFR and final curd consistency, the curd made from reconstituted milk did not cut as cleanly as the curd made from the control milk. The curd broke into smaller fragments than the control but was not as fragile as the curd made from microfluidized milk. Curds made from filled milk samples with larger fat globules appeared to be less fragile than those containing smaller fat globules.

protein strands in the gels made from microfluidized milks with the largest mean diameter (412 nm) were bulky and of uneven thickness; the fracture plane was uneven (Figure 2). The network was more open than was that of the control (Table 3). The mean number of voids was 183 per 10,000 mm2, and apparently more strands ended in nodules that were not tied into the gel structure. As the microfluidization pressure was increased, the mean fat globule diameter decreased (364 nm), the strands of protein became thinner (Figure 2c), and the number of voids increased to 284 per 10,000 mm2, which is significantly more than that of any of the other samples. In the samples made from microfluidized milk that had the

Microstructure of Gels Made from Microfluidized Milk In the photomicrographs of the gels made from the control milk (Figures 2a and 3a), the native fat globules are distinctly visible in the voids of the gel network and range in diameter up to 10 mm. There was an average of 257 voids per 10,000 mm2 of gel surface. The protein strands in the control gel were smooth, fractured cleanly, and were fairly uniform in thickness. In contrast, fewer than 10 small fat globules were observed in the 18 samples of gels made from microfluidized milk (Figure 2), which confirmed that the fat globules in microfluidized milk were incorporated into the protein strands that make up the gel network. As shown by the electron microscope, the Journal of Dairy Science Vol. 81, No. 7, 1998

TABLE 3. Effect of microfluidization on the number of voids per 10,000 mm2 of rennet gel made from the control and microfluidized milks, as measured by scanning electron microscopy. Voids

Type of milk

Mean fat globule diameter

Microfluidized milk

Control Microfluidized Microfluidized Microfluidized

(nm) . . .1 412 364 353

257b 183c 284a 233c

Reconstituted milk

(no.) 284b 293a 344a 330a

a,b,c,d,eMeans within the same column with no common superscript letter differ ( P < 0.05). 1Not measured.

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smallest mean fat globule size (353 nm), the protein strands were thin and ragged in appearance (Figure 2d), and the number of voids was to 233 per 10,000 mm2. The fracture plane was noticeably jagged and uneven. The number of voids in this sample was significantly different from that of the control and the sample containing the fat globules with a mean diameter of 364 nm but was not significantly different from the microfluidized sample with the largest fat globules. There was no effect of fat globule size on the thickness or appearance of the strands of the curds made from the reconstituted milk samples with the different sizes of fat globules (Figure 3), nor was there a significant difference in the average number of voids per unit area. The microstructure of the curd made from reconstituted milk was finer than that of either the control or the microfluidized samples. The control samples had a mean of 284 voids per 10,000 mm2, and the reconstituted samples had a mean of 323 voids per 10,000 mm2. The strands in the reconstituted milk samples did not appear to be different from those in the control sample, but very small fat globules were visible on the surface of the protein strands. The number of voids in the microfluidized milk samples was significantly lower than that of the control at the highest and lowest pressures, and the sample produced at medium pressure had significantly more voids per unit area. The samples of reconstituted milk had significantly more voids per unit area than did the corresponding control samples. DISCUSSION These results show that the influence of changing the structures of casein micelles by adsorption to the fat-serum interface during microfluidization affects their behavior during the renneting of milk. The diameters of the fat globules, which are determined by the pressure in the Microfluidizer and the number times the milk is passed through the Microfluidizer, affect the physical properties of the microfluidized milk (16, 17). There are two different ways in which caseins can cover the new surface area that is created when the fat globules are disrupted in the Microfluidizer. On the first pass through the Microfluidizer, casein micelles that are free in solution partially break up as they adsorb to the fat-water interface. There is also some bridging of the fat globules by the casein micelles. On subsequent passes through the Microfluidizer, the fat globules are further reduced in size, and the bridges between the fat globules are

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broken; also, more casein micellar fragments adsorb to the additional, newly created interface. Some other complexes that are rich in casein are formed with clusters of very small (30 to 100 nm) fat globules (2, 24). In the range of fat globule sizes tested, no significant change was found in the apparent casein load after successive passes through the Microfluidizer, although the total surface area of the fat globules increased by approximately 14%. However, the surface coverage that measured 7.2 mg/m2 is lower than the surface coverage that was measured on fat globules in homogenized milk (1, 12) and is much lower than the theorized maximum load if the surface were completely covered by casein micelles (13). Thus, there is a significant amount of disruption of the casein micelles occurring at the fat-serum interface in the Microfluidizer. However, much of the original casein remains in a form that, in the electron microscope at least, resembles the original micellar particles. The protein load on the fat globules in the reconstituted milk was known from previous experiments ( 7 ) to be approximately 3 mg/m2 for such oil-in-water emulsions stabilized by WPI made in the microfluidizer. Similarly, a surface coverage of about 3 mg·m–2 has been found to be the maximum limit for a monolayer of sodium caseinate or individual caseins used to stabilize emulsions when protein is slightly in excess (3, 5, 7). From this result, the coverage on the synthetic fat globules in the microfluidized milk must be more than a simple monolayer. The extreme disruption of the casein micelles in this process is emphasized by the average particle sizes in Table 1; those sizes are the volume to surface sizes, and there are quite large populations of fat globules smaller than 0.3 mm in diameter, which are required to be covered by the partly disrupted casein micelles (24). When the control milk was treated with rennet, a gel structure was formed that had strands of proteins with fat globules trapped within the voids of the gel structure, but the fat was not an integral part of the gel; that is, the fat did not bind to the casein matrix but helped to form a filled gel (25). However, after the milk was microfluidized, some of the micellar casein material was adsorbed to the synthetic fat globules. Then, when the homogenized milk was treated with rennet, the fat globules became a part of the gel strand structure. In this type of gel, a mixed gel, the amount of solid material linked in the gel strands, rather than simply being trapped, is nearly tripled (2.5% casein plus 4.0% fat). Intuitively, we expected this process to increase the strength of the gel, but the opposite effect was observed (Table 2). The rate of increase in gel consistency and the conJournal of Dairy Science Vol. 81, No. 7, 1998

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sistency of the gel an hour after the addition of the rennet was lower in the microfluidized milk. An earlier comparison between filled and complex gels ( 2 6 ) found that the particles in both types of gels increased the elasticity of the gel. Particles that were interactive with the gelling agent were more effective in improving the elasticity of the gel than were noninteractive particles at the same concentration. The consistency of the gel, which is analogous to the apparent viscosity of a liquid, depends on internal friction, and the noninteractive particles in the voids of our filled gels did not resist the stress on the network from an external force such as the vibration of the ball on the viscometer. For small deformations, the filler particles were not affected by changes in the shape of the voids. However, the interactive particles in a complex gel should act with the protein particles to resist external stress. Thus, we would expect that the same concentration of filler particles should increase the firmness more in a complex gel than in a filled gel. The firmness of acid-set milk gels and heatset whey protein gels is indeed enhanced by reducing the diameter of the fat globules contained in them (9, 28). The characteristics of a rennet gel made from the control milk were consistent with the model of a filled gel. The native fat globules became entrapped in the gel network as it formed and the curd firmness increased more rapidly than if the voids of the gel were filled with liquid serum, which can flow through the gel network when pressure is applied. Hence, the gels made from whole milk had a higher CFR (22.4 mPa) than those made from skim milk (9.2 mPa). The gels made from reconstituted milk also behaved much like other filled gels. The RCT was the same as for the control milk, which suggests that the primary stages of rennet coagulation in the two milks were the same, and, thus, the enzyme activity and the initial formation of aggregates of micelles was independent of the size of fat globules at an equal concentration of fat. The CFR in reconstituted milk was slightly faster than in the control milk, perhaps because the smaller fat globules (300 to 600 nm) did not interfere with the arrangement of aggregates into a network as much as the larger fat globules ( 1 to 10 mm ) did in the control milk. Although the control and reconstituted milks behaved like other filled gels, the characteristics of microfluidized milk were not the same as those in other complex gels (9, 26, 28). The CFR of rennet gels made from microfluidized milks was much lower than the CFR of reconstituted milk and decreased as the fat globule diameter decreased. Because neither the size nor the placement of fat globules caused a decrease in firmness in other filled or complex gels, Journal of Dairy Science Vol. 81, No. 7, 1998

the dependence of CFR on the fat globule size in microfluidized milk must be caused by changes in the three-dimensional structure of the micelle during microfluidization. It appeared that disruption of the micelles during homogenization broke down their structure in such a way as to give weaker interactions between the casein-coated fat particles and between these fat particles and unadsorbed casein micelles. Although results using dynamic rheometry show that the curd formed from reconstituted milk was firmer than curd made from the control milk before the curd was disturbed by removing it from the beaker, the cutting characteristics were not as good. The curd was weaker, and the pieces of curd were more fragile. The large fat globules in the control milk, which entirely fill the voids between the casein strands, apparently stabilized the matrix against the stresses involved in cutting. In the photomicrographs of the samples of control and microfluidized milks after renneting (Figure 2), the difference in the gel types was clearly visible. The gels from control milk had dense protein strands with large, spherical fat globules in the voids formed by the protein strands. In the gels made from microfluidized milk, most of the fat globules were incorporated into the protein strands. The gel from microfluidized milk with the largest fat globules (Figure 2b) had thick, lumpy strands. The size of the voids was larger than in the control, and junction points in the matrix were fewer, which may be the reason that the gel was weaker than the control gel. When the diameter of the fat globules was decreased (Figure 2c), the size of the voids was slightly smaller than that of the control, but many strands ended in nodes that were not connected to the three-dimensional network. The protein strands were thinner than those in Figure 2b, and small holes were visible in the strands. The protein strands in the gel from microfluidized milk that had the smallest fat globules (Figure 2d) were full of irregularly shaped holes. Both the reduced number of junctions and the integrity of the protein strands contributed to the weakness of the gel in these samples. Because the whey proteins that form the synthetic fat globule membrane in the reconstituted milk do not react in rennet coagulation, the reconstituted milk forms a filled gel matrix similar to that of the control milk. In the photomicrographs (Figure 3), small fat globules are visible on the surface of the protein strands. Unfortunately, because of the method used, whether the fat globules attached themselves to the surface during the renneting reaction or migrated there as the ice front receded during the sublimation step of the preparation for the SEM cannot be determined.

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CONCLUSIONS By studying the effect of microfluidization conditions on the renneting characteristics of microfluidized and reconstituted milks, we have determined that changes in the curd are related to changes in the casein micelles that occur during microfluidization. Disrupted casein micelles that have adsorbed to the fat-serum interface interacted with free casein micelles and micellar material on other fat globules. Therefore, microfluidized milks formed complex gels, unlike control milk, which formed filled gels. The protein strands in the complex gel appeared to be weaker than those in gels made from the control or reconstituted milks. This weakness appears to be related to changes in the structure of the casein micelles as they adsorb to the fat globules and spread across the interface. The rennet curd that was most qualitatively similar to that of the control was the one made from the reconstituted milk with the largest fat globules. The curd that was least similar to the control curd was the one made from the microfluidized milk with the smallest fat globules. This curd was not suitable for making cheese. Therefore, reconstituted milks of the type used here should be the most suitable material for making novel cheese-type products based on homogenized milks. ACKNOWLEDGMENTS The authors thank A. Smith and M. Corredig for technical assistance. Funding for this research was provided by the Ontario Dairy Council and the National Science and Engineering Research Council of Canada. REFERENCES 1 Dalgleish, D. G., and E. W. Robson. 1985. Centrifugal fractionation of homogenized milks. J. Dairy Res. 52:539–546. 2 Dalgleish, D. G., S. M. Tosh, and S. West 1996. Beyond homogenization: the formation of very small emulsion droplets during the processing of milk by a Microfluidizer. Neth. Milk Dairy J. 50:135–148. 3 Dickinson, E., S. E. Rolfe, and D. G. Dalgleish. 1988. Competitive adsorption of as1-casein and b-casein in oil-in-water emulsions. Food Hydrocolloids 2:397–405. 4 Emmons, D. B., M. Kalab, and E. Larmond. 1980. Milk gel structure. X. Texture and microstructure in Cheddar cheese made from whole milk and from homogenized low-fat milk. J. Texture Stud. 11:15–34. 5 Fang, Y., and D. G. Dalgleish. 1993. Dimensions of the adsorbed layers in oil-in-water emulsions stabilized by caseins. J. Colloid Interface Sci. 156:329–334. 6 Green, M. L., R. J. Marshall, and F. A. Glover. 1983. Influence of homogenization of concentrated milks on the structure and properties of rennet curds. J. Dairy Res. 50:341–348. 7 Hunt, J. A., and D. G. Dalgleish. 1994. Adsorption behaviour of

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