J. Dairy Sci.

90:13–23
© American Dairy Science Association, 2007.

Effects of Ultra-High Pressure Homogenization

on the Cheese-Making Properties of Milk
A. Zamora, V. Ferragut, P. D. Jaramillo, B. Guamis, and A. J. Trujillo1
Centre Especial de Recerca Planta Tecnologia dels Aliments (CERPTA), Departament de Ciència Animal i dels Aliments, Facultat de
Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

ABSTRACT namely longer coagulation times and weaker gels

(Guinee et al., 1997; Singh and Waungana, 2001), and
The effects of single- or 2-stage ultra-high pressure on curd syneresis; that is, higher moisture content
homogenization (UHPH; 100 to 330 MPa) at an inlet (Walstra et al., 1985; Pearse and Mackinlay, 1989;
temperature of 30°C on the cheese-making properties of Rynne et al., 2004). Nevertheless, the effect of high heat
bovine milk were investigated. Effects were compared treatment has received considerable attention owing to
with those from raw, heat-pasteurized (72°C for 15 s), its potential for improving cheese yield through incorpo-
and conventional homogenized–pasteurized (15 + 3 ration of whey proteins into cheese curd (Lucey, 1995;
MPa, 72°C for 15 s) treatments. Rennet coagulation Singh and Waungana, 2001).
time, rate of curd firming, curd firmness, wet yield, and Conventional homogenization, developed by Gaulin
moisture content of curds were assessed. Results of in 1899, has been widely adopted by the dairy industry.
particle size and distribution of milk, whey composition, Homogenization is usually performed at 60°C, and the
and gel microstructure observed by confocal laser scan- milk is processed to break milk fat globules into fine
ning microscopy were analyzed to understand the effect lipid droplets, preventing cream separation, thereby
of UHPH. Single-stage UHPH at 200 and 300 MPa increasing stability and shelf life of milk emulsion. Two-
enhanced rennet coagulation properties. However, stage homogenization is commonly used, in which the
these properties were negatively affected by the use of primary stage reduces the size of fat globules and the
the UHPH secondary stage. Increasing the pressure led secondary stage disrupts clusters that may be formed.
to higher yields and moisture content of curds. The Although homogenization of whole milk has detrimen-
improvement in the cheese-making properties of milk tal effects on curd forming properties (Emmons et al.,
by UHPH could be explained by changes to the protein– 1980) and curd syneresis (Humbert et al., 1980; Green
fat structures due to the combined effect of heat and et al., 1983), it improves rennet action (Humbert et al.,
homogenization. 1980; Robson and Dalgleish, 1984) and increases cheese
Key words: ultra-high pressure homogenization, milk, yield due to better fat recovery (Jana and Upadhyay,
rennet coagulation, cheese-making properties 1992).
In recent years, homogenization equipment design
INTRODUCTION has been modified to achieve far greater pressures. Al-
though the principle of ultra-high pressure homogeni-
Research on technological processes on food is focused zation (UHPH) is similar to that of conventional ball-
on 2 main goals: improving safety and quality of final and-seat homogenizers, current developments in the
products, and changing the characteristics of raw mate- design (e.g., the Stansted valve) allow homogenization
rials to obtain value-added products. However, process- at pressures of up to 350 MPa. Forces encountered dur-
induced modifications can have both beneficial and det- ing UHPH include cavitation, friction, turbulence, high
rimental effects on technological aspects. velocity, and shear (Floury et al., 2004a,b), and result
In dairy processes, thermal treatment of milk aims in the heating of the homogenized liquid (Floury et al.,
at increasing shelf life and improving food safety of the 2000; Hayes and Kelly, 2003a; Thiebaud et al., 2003).
final product. Milk for cheese manufacture is generally Applications of high-pressure homogenization are
pasteurized at 72°C typically for 15 to 35 s. Higher mainly found in the pharmaceutical and biotechnology
temperatures have adverse effects on curd formation, sectors where the technique is used to emulsify, dis-
perse, and mix (Floury et al., 2000). However, there
has been increasing interest in its application in food
technology. Reports on the effect of UHPH on some
Received July 20, 2006.
Accepted August 31, 2006. pathogenic and spoilage microorganisms in model and
1
Corresponding author: [email protected] real food systems have proved its efficiency in reducing

13
14 ZAMORA ET AL.

microbial counts (Hayes and Kelly, 2003a; Thiebaud treatments of cheese milk in different cheese varieties
et al., 2003; Hayes et al., 2005; Briñez et al., 2006). (fresh or ripened). Two-stage homogenization and pas-
Moreover, studies with whole and skimmed milk have teurization of raw milk were performed with a Niro
shown that UHPH produces fine emulsion particles Soavi homogenizer (model X68P Matr. 2123, Niro
(Hayes and Kelly, 2003a; Thiebaud et al., 2003; Hayes Soavi, Parma, Italy) and a Finamat heat exchanger
et al., 2005), modifies protein structure and characteris- (model 6500/010, GEA Finnah GmbH, Ahaus, Ger-
tics (Hayes and Kelly, 2003a; Hayes et al., 2005; Sandra many), respectively.
and Dalgleish, 2005), and inactivates enzymes (Hayes The complete experiment was repeated on 3 indepen-
and Kelly, 2003b; Datta et al., 2005), all of which could dent occasions.
have indirect effects on the coagulation properties of
milk and the microstructural properties of cheese. Particle Size and Distribution
At the present time, data to describe the technologi-
cal aptitude of milk treated by UHPH are scarce. The The particle size distribution in milk samples was
aim of this work was to determine the effect of UHPH determined using a Beckman Coulter laser diffraction
treatment on the cheese-making properties of milk by particle size analyzer (LS 13 320 series, Beckman
comparing this new technology with pasteurization Coulter, Fullerton, CA). Milk samples were diluted in
and conventional homogenization-pasteurization distilled water until an appropriated obscuration was
treatments. obtained in the diffractometer cell. An optical model
based on the Mie theory of light scattering by spherical
MATERIALS AND METHODS particles was applied by using the following conditions:
real refractive index, 1.471; refractive index of fluid
Supply and Treatment of Milk (water), 1.332; imaginary refractive index, 0; pump
Raw whole bovine milk was obtained from a local speed, 21%. The diameter below which 90% of the vol-
dairy farm (S.A.T. Can Badó, Roca del Vallès, Spain). ume of particles are found (Dv0.9), the diameter below
Milk was standardized at 3.5 ± 0.2% fat and kept over- which 50% of the volume of particles are found (Dv0.5),
night at 4°C. Before all treatments, the milk was the volume-weighted mean diameter [D(4,3)], and the
warmed to approximately 20°C. surface-weighted mean diameter [D(3,2)] were de-
Ultra-high pressure homogenization was carried out termined.
by subjecting milk to single- or 2-stage UHPH (100,
200, and 300 MPa on the primary valve and 30 MPa Rennet Coagulation Properties
on the secondary valve) using a Stansted high-pressure
homogenizer (model FPG11300, Stansted Fluid Power Milk was warmed to 32°C, and recombinant rennet
Ltd., Essex, UK) at an inlet temperature of 30 ± 1°C. (chymosin with a declared activity of 180 International
This homogenizer comprises a high pressure valve Milk Clotting Units/mL, Maxiren 180, DSM Food Spe-
made of ceramics able to support 350 MPa and a second cialties, Seclin Cedex, France) was added at 0.074%
pneumatic valve able to support up to 50 MPa located (vol/vol). Coagulation was carried out at 32°C for 30
behind the first one. The high-pressure system con- min. Rennet coagulation properties [rennet coagulation
sisted of 2 intensifiers driven by a hydraulic pump. The time (RCT), rate of curd firming (RCF), and curd firm-
flow rate of milk in the homogenizer was 120 L/h. The ness at 30 min (CF)] were assessed in triplicate by the
inlet temperature of milk was kept at 30°C by a heat Optigraph system (Ysebaert Inc., Frepillon, France).
exchanger located behind the feeding tank. Tempera- This device passes an infrared beam through a sam-
ture thermocouples and pressure gauges placed at the pling tube containing milk. A sensor on the other side
2 valves measured temperature and pressure changes measures the amount of light absorbed by the milk as
during processing. Throughout the experiment, the it coagulates; the changes are analyzed in real time by
range of milk temperature was 33 to 41°C at the pri- a computer that converts them into directly usable data.
mary valve and 54 to 94°C at the secondary valve. To
minimize temperature retention after treatment, 2 spi- Evaluation of Yield and Moisture Content of Curds
ral-type heat exchangers (Garvı́a S.A., Barcelona,
Spain) located behind the second valve were used. The The potential yield of cheese curd was estimated in
outlet temperature of milk never exceeded 40°C. quadruplicate as described by Macheboeuf et al. (1993).
Milks from UHPH treatments were compared with Milk samples (270 mL) were warmed to 32°C and re-
raw and heat-treated milks. Pasteurized milk (72°C for combinant rennet (chymosin with a declared activity
15 s) and homogenized–pasteurized milk (15 MPa + 3 of 180 International Milk Clotting Units/mL, Maxiren
MPa at 57 to 60°C, 72°C for 15 s) were chosen as typical 180, DSM Food Specialties) at 0.074% (vol/vol) was

Journal of Dairy Science Vol. 90 No. 1, 2007

 ULTRA-HIGH PRESSURE HOMOGENIZATION 15

added. Portions of the renneted milks (30 g) were trans- were dissolved in ethanol at a concentration of 2 and
ferred into centrifuge tubes and allowed to coagulate 1 mg/mL, respectively. Milks (10 mL) warmed at 32°C
at 32°C for 30 min. The coagulum was centrifuged at were dyed with 2 drops of FITC and 3 drops of Nile
13,000 × g for 15 min at 10°C. Wet yield of curds, ex- red. Recombinant rennet (Maxiren 180, DSM Food Spe-
pressed as grams of retained curd per one hundred cialties) at a concentration of 0.074% (vol/vol) was added
grams of milk, was determined by weighing the ob- to the dyed milks. Then, 3 to 4 drops of the labeled
tained pellets. renneted milks were transferred to microscope slides
Curds were analyzed in duplicate for TS content with concave cavities, covered with a coverslip, sealed to
(IDF, 1987) to calculate their moisture content (100 prevent evaporation, and incubated in a temperature-
− TS) and the yield of total curd solids (wet yield × controlled incubator at 30°C for 30 min. The prepara-
TS/100). tions were cooled and kept at 4°C for a maximum of 3 h.
The confocal microscope (Leica TCS SP2 AOBS, Hei-
Whey Composition: Total N, Whey Proteins, delberg, Germany) was equipped with an oil-coupled
and Minerals Content Leica objective with a 63× augmentation and a numeri-
cal aperture of 1.4. Fluorescence from the samples was
The total N content of whey was analyzed in duplicate excited with the 488 nm line of an argon laser. Images
by the Dumas combustion method (IDF, 2002). were acquired in 2 channels simultaneously (501 to 549
Reversed-phase HPLC analysis of rennet whey was nm and 574 to 626 nm) as 1,024 × 1,024 pixel slices in
performed using an automated system (LCM1, Waters the horizontal x–y plane along the z plane at constant
Corporation, Milford, MA). Separations were carried gain and offset. Three-dimensional images were ob-
out in a 250- × 4.6-mm column packed with C8-bonded tained by the average projection of 4 slices with Leica
silica gel with a particle diameter of 5 ␮m and pore software.
width of 3,000 nm (Tracer Excel, Teknokroma, Sant
Cugat del Vallès, Spain) at a constant temperature of Statistical Analysis
40°C, following the method of Resmini et al. (1989).
Residual levels of α-LA and β-LG were measured as Data were processed by ANOVA using the GLM pro-
total area of the respective peaks. cedure of Statgraphics (Statgraphics, Inc., Chicago, IL).
Calcium, Mg, and P in whey were determined in trip- Tukey’s test was used for comparison of sample data.
licate by inductively coupled plasma optical emission Evaluations were based on a significance level of P <
spectroscopy with a Perkin-Elmer inductively coupled 0.05.
plasma spectroscopy unit (model 4300, Perkin-Elmer,
Shelton, CT) with axial plasma viewing. The spectros- RESULTS
copy operating conditions were as follows: power = 1.3
Particle Size and Distribution
kW; argon plasma flow rate = 15 L/min; argon auxiliary
flow rate = 0.2 L/min; argon nebulizer flow rate = 0.74 Four parameters [Dv0.9, Dv0.5, D(4,3), and D(3,2)]
L/min; sample uptake rate = 1.5 mL/min; wavelengths as well as the distribution patterns were taken into
(nm) for Ca, P, and Mg = 317.925, 213.611, and 285.213, consideration to see the effects of UHPH on particle
respectively. Whey samples of 1 mL were transferred to size and distribution (Table 1).
a 25-mL volumetric flask and nitric acid and deionized The size distribution of particles in raw milk was
water were added to reach a final concentration of 0.2% characterized by a main peak at 3.8 ␮m and a second
(vol/vol) nitric acid. Standard solutions from 1 mg/mL lower peak around 0.2 ␮m, which corresponded to fat
stock solution of Ca, P, and Mg were used to prepare globules and casein micelle particles, respectively. Pas-
the calibration curves. teurized milk showed a similar pattern. As expected,
the size distribution of homogenized milks changed
Confocal Laser Scanning Microscopy markedly; their main peaks were between 0.1 and 0.3
of Rennet Gels ␮m for UHPH-treated milks, and approximately 0.4
␮m for homogenized–pasteurized milk. Samples under-
Confocal laser scanning microscopy observations going UHPH treatment at 330 MPa showed a second
were performed in fluorescence mode essentially as Mi- peak, lower but much wider at 4.6 ␮m with a shoulder
chalski et al. (2002) described. The protein matrix of at approximately 11.9 ␮m.
renneted milks was stained by the fluorescent dye, flu- Significant differences (P < 0.05) between pasteurized
orescein isothiocyanate (FITC; Fluka, Steinheim, Ger- and raw milks were found for Dv0.5 and D(3,2). Homog-
many), and the fat globules were stained by Nile red enized–pasteurized samples showed values between
(Sigma, Steinheim, Germany). The FITC and Nile red those of pasteurized and raw milks, on one side, and

Journal of Dairy Science Vol. 90 No. 1, 2007

16 ZAMORA ET AL.

Table 1. Particle size (␮m) of raw, pasteurized, homogenized-pasteurized, and ultra-high pressure homoge-
nized (UHPH) milks1
Treatment2 Dv0.9 Dv0.5 D(4,3) D(3,2)

Raw 5.07 ± 0.03 b

3.11 ± 0.04 b
2.90 ± 0.05 a
0.63 ± 0.03a
Pasteurized 5.16 ± 0.04b 3.16 ± 0.01a 2.94 ± 0.01a 0.59 ± 0.01b
Homogenized–pasteurized 1.10 ± 0.03c 0.39 ± 0.00c 0.50 ± 0.01c 0.32 ± 0.01c
UHPH (MPa)
100 0.80 ± 0.03d 0.34 ± 0.00d 0.44 ± 0.01cd 0.28 ± 0.01d
130 0.68 ± 0.01de 0.31 ± 0.01e 0.40 ± 0.01d 0.25 ± 0.01e
200 0.43 ± 0.01f 0.21 ± 0.02g 0.25 ± 0.02e 0.19 ± 0.02g
230 0.49 ± 0.01ef 0.25 ± 0.01f 0.31 ± 0.03e 0.21 ± 0.01f
300 0.29 ± 0.01f 0.15 ± 0.00h 0.17 ± 0.01f 0.13 ± 0.00h
330 5.82 ± 0.35a 0.25 ± 0.01f 2.01 ± 0.14b 0.22 ± 0.01f
a–h
Values without common superscripts were significantly different (P < 0.05).
1
Mean value ± standard error. Particle size parameters: Dv0.9 and Dv0.5 = the diameters below which
90% and 50% of the volume of particles are found, respectively; D(4,3) = the volume-weighted mean diameter;
and D(3,2) = the surface-weighted mean diameter.
2
Pasteurized (72°C for 15 s); homogenized–pasteurized (15 + 3 MPa at 57 to 60°C, 72°C for 15 s); UHPH
treatments at 130, 230, and 330 MPa made using 2-stage homogenization: 100, 200, and 300 MPa in first
valve and 30 MPa in second valve.

those of UHPH-treated milks, on the other. Increasing increased RCF compared with raw milk. However, only
the pressure of UHPH significantly decreased all 4 pa- the UHPH treatments at 200 and 300 MPa resulted in
rameters, except for UHPH treatment at 330 MPa. Two- significantly higher CF than raw milk.
stage homogenization did not affect either Dv0.9 or
D(4,3) at pressures below 300 MPa. However, above 100 Curd Yield and Moisture Content
MPa, the 2-stage homogenized samples showed higher
Dv0.5 and D(3,2) values than their counterparts treated Wet yields and moisture content of the curds obtained
by single-stage homogenization. Milk samples UHPH- from the UHPH-treated milks were significantly
treated at 330 MPa showed a Dv0.9 value significantly greater than those of homogenized–pasteurized, pas-
higher than that of raw milk, and a D(4,3) value closer teurized, and raw milks (Table 3). Increasing the pres-
to that of raw milk than to UHPH-treated milks. sure from 100 to 300 MPa increased wet curd yield
by approximately 33 to 65%, and moisture content by
Rennet Coagulation Properties approximately 11 to 18% compared with raw milk. Sam-
ples treated at 330 MPa showed significantly lower val-
Pasteurization did not affect pH, but homogenized– ues than samples treated at 300 MPa.
pasteurized milk showed significantly lower pH than Conventional pasteurization and homogenization–
that of raw milk. The influence of UHPH on the pH pasteurization increased the yield of total curd solids by
highly depended on the applied pressure; below 300 approximately 7% compared with raw milk. For UHPH-
MPa, the values were significantly lower (Table 2). treated milks, the increases were approximately 4, 6,
Rennet coagulation times were very much dependent 7, 8, 10, and 11% at 130, 100, 200, 230, 330, and 300
on the treatment, although 2-stage homogenization did MPa, respectively.
not seem to affect it. Samples treated at 100 to 130
MPa had significantly lower RCT than raw milk. In the Whey Composition
case of milk treated at 200 to 230 MPa, RCT were also
significantly lower than that of raw milk but similar to All treatments significantly (P < 0.05) decreased the
that obtained with homogenized–pasteurized milk. On amount of total N in whey (Table 4). The UHPH samples
the other hand, the RCT of the samples treated at 300 showed a decrease of approximately 2 to 22% correlated
to 330 MPa were similar to those obtained with pasteur- to the increase of pressure. Above 200 MPa, the effect
ized and raw milks. of UHPH was much higher than those of the pasteuriza-
Two-stage homogenization in all UHPH treatments tion and homogenization–pasteurization treatments,
significantly (P < 0.05) diminished both RCF and CF although the 2-stage homogenized samples did not dif-
in relation to their homologues treated by single stage. fer from their single-stage homologues. The same re-
The values of RCF were either significantly lower or sults were observed for β-LG content of whey (Table 4).
similar to those of raw milk. The UHPH treatments at For α-LA, although no statistical differences were found
200 and 300 MPa and heat pasteurization significantly between whey from raw, pasteurized, and homoge-

Journal of Dairy Science Vol. 90 No. 1, 2007

 ULTRA-HIGH PRESSURE HOMOGENIZATION 17
Table 2. pH and rennet coagulation properties [rennet coagulation time (RCT), rate of curd firming (RCF),
and curd firmness (CF)] of raw, pasteurized, homogenized–pasteurized, and ultra-high pressure homogenized
(UHPH) milks1

Treatment2 pH RCT (min) RCF (mA/min) CF (mA)

Raw 6.75 ± 0.01 a
7.43 ± 0.13 a
1.37 ± 0.04 d
13.33 ± 0.27c
Pasteurized 6.75 ± 0.01a 7.66 ± 0.15a 1.47 ± 0.03c 13.80 ± 0.24bc
Homogenized–pasteurized 6.72 ± 0.01b 6.91 ± 0.15b 0.99 ± 0.04f 10.21 ± 0.16e
UHPH (MPa)
100 6.52 ± 0.03e 5.44 ± 0.27c 1.07 ± 0.10e 11.51 ± 0.71d
130 6.51 ± 0.03f 5.18 ± 0.27c 0.77 ± 0.07g 10.32 ± 0.54e
200 6.68 ± 0.02d 6.91 ± 0.29b 1.84 ± 0.07a 15.00 ± 0.31a
230 6.69 ± 0.01c 7.02 ± 0.09b 1.32 ± 0.05d 11.99 ± 0.27d
300 6.75 ± 0.01a 7.69 ± 0.09a 1.72 ± 0.02b 14.46 ± 0.16ab
330 6.74 ± 0.00a 7.68 ± 0.11a 1.32 ± 0.05d 11.75 ± 0.33d

Values without common superscripts were significantly different (P < 0.05).

a–g

Mean value ± standard error.

1
2
Pasteurized (72°C for 15 s); homogenized–pasteurized (15 + 3 MPa at 57 to 60°C, 72°C for 15 s); UHPH
treatments at 130, 230, and 330 MPa made using 2-stage homogenization: 100, 200, and 300 MPa in first
valve and 30 MPa in second valve.

nized–pasteurized milks, all UHPH treatments showed and homogenized–pasteurized samples showed lower
a significant decrease of α-LA in whey compared with amounts of Ca and Mg and similar amounts of P com-
raw milk. Denaturation of β-LG was more important pared with raw milk.
than that of α-LA; levels were obtained of up to approxi-
mately 35% for β-LG at 300 and 330 MPa, and around Confocal Laser Scanning Microscopy
12% for α-LA at 300 MPa. of Rennet Gels
Only the whey obtained from UHPH treatments per-
formed at 100 to 130 MPa and 300 to 330 MPa had Confocal micrographs of rennet gels revealed the ex-
significantly higher or lower amounts, respectively, of istence of visual differences between treatments in the
all 3 mineral salts (Ca, P, and Mg) compared with raw proteinaceous matrix and fat globule size as well as at
milk (Table 5). Whey from milk UHPH-treated at 200 their interaction (Figure 1).
MPa had higher amounts of Ca and P and a lower Rennet gels from pasteurized milk were similar to
amount of Mg than that of raw milk. Whey from treated those obtained from raw milk. The micrographs showed
milk at 230 MPa presented similar amounts of Ca and a porous structure of the casein network with native
Mg and higher amounts of P than raw milk. Pasteurized milk fat globules mainly located in the serum pores of
the gels (results not shown).
When milk was homogenized–pasteurized, the ren-
Table 3. Wet yield and moisture content of curds from raw, pasteur- net gels presented open matrices; serum pores were
ized, homogenized–pasteurized, and ultra-high pressure homoge- large, irregular, and delimited by thick and lumpy
nized (UHPH) milks1
strands. Nile red fluorescence revealed that fat globules
Moisture had different locations depending on their size (Figure
Treatment2 Wet yield (%) content (%)
1a). The smallest fat globules (<0.5 ␮m) became part
Raw 21.38 ± 0.38h 64.76 ± 0.22h of the proteinaceous network, explaining the thickness
Pasteurized 22.00 ± 0.58i 63.45 ± 0.39i of the strands. The gels presented many strands that
Homogenized–pasteurized 26.68 ± 0.37g 69.86 ± 0.19g
UHPH (MPa) ended with midsized fat globules (∼1 ␮m). Larger fat
100 28.39 ± 0.57f 71.92 ± 0.22f globules (∼1.5 to 2 ␮m), which accounted for a very
130 29.01 ± 0.51e 73.03 ± 0.15e small number, were retained in the serum pores.
200 31.47 ± 0.76d 74.35 ± 0.24d
230 32.76 ± 0.57c 75.10 ± 0.16c Although UHPH treatments at 100 MPa showed a
300 35.35 ± 0.71a 76.42 ± 0.09a greater amount of smaller fat globules and few larger
330 34.33 ± 0.64b 75.95 ± 0.09b globules (∼4 to 6 ␮m), the general aspect of the matrix
a–i
Values without common superscripts were significantly different was rather similar to that of homogenized–pasteurized
(P < 0.05). milks (Figure 1b). The second stage at 100 MPa reduced
1
Mean value ± standard error. the size of the largest fat globules (∼3 ␮m). Moreover,
2
Pasteurized (72°C for 15 s); homogenized–pasteurized (15 + 3 MPa the structure of the gel was smoother with smaller
at 57 to 60°C, 72°C for 15 s); UHPH treatments at 130, 230, and 330
MPa made using 2-stage homogenization: 100, 200, and 300 MPa in pores. However, the smallest fat globules were also em-
first valve and 30 MPa in second valve. bedded within the proteinaceous network.

Journal of Dairy Science Vol. 90 No. 1, 2007

18 ZAMORA ET AL.

Table 4. Total N content and residual α-LA and β-LG (measured as total area ×105 of the respective peaks)
in whey of raw, pasteurized, homogenized–pasteurized, and ultra-high pressure homogenized (UHPH) milks1
Treatment2 Total N (%) α-LA β-LG

Raw 0.143 ± 0.004 a

41.09 ± 1.55 a
107.71 ± 5.99a
Pasteurized 0.135 ± 0.005c 40.52 ± 1.52ab 102.76 ± 5.66b
Homogenized–pasteurized 0.133 ± 0.004d 39.88 ± 1.50abc 94.65 ± 4.82c
UHPH (MPa)
100 0.140 ± 0.004b 38.41 ± 0.56cd 103.16 ± 3.04b
130 0.137 ± 0.003c 38.43 ± 0.39bcd 103.88 ± 4.06b
200 0.125 ± 0.001e 37.55 ± 0.29de 85.65 ± 1.60d
230 0.126 ± 0.004e 36.75 ± 0.72de 86.25 ± 1.30d
300 0.112 ± 0.003f 36.08 ± 0.22e 70.04 ± 2.07e
330 0.112 ± 0.002f 36.86 ± 0.42de 69.28 ± 1.86e
Values without common superscripts were significantly different (P < 0.05).
a–f
1
Mean value ± standard error.
2
Pasteurized (72°C for 15 s); homogenized–pasteurized (15 + 3 MPa at 57 to 60°C, 72°C for 15 s); UHPH
treatments at 130, 230, and 330 MPa made using 2-stage homogenization: 100, 200, and 300 MPa in first
valve and 30 MPa in second valve.

Micrographs of gels from single-stage treatments rounding a large number of noncoated, midsized fat
above 200 MPa showed that Nile red fluorescence at globules (∼2 ␮m; Figures 1g and h).
the level of the proteinaceous network was markedly
weaker (Figures 1c and d). Rennet gels from milk
DISCUSSION
treated at 200 MPa revealed tight matrices. It should
be mentioned that micrographs from UHPH-treated Effects of Heat and Conventional
milk at 300 MPa not only had lower levels of Nile red Homogenization Treatments
fluorescence but also lower FITC fluorescence (Figure
1d). Mild heat treatments are considered to have no or
The second stage above 200 MPa provoked the coat- little effect on the whey proteins of milk, although there
ing of midsized fat globules; this phenomenon was more are reports that heat pasteurization (72°C for 20 s or
visible as the pressure at the first valve was increased. 73°C for 15 s) could cause denaturation of approxi-
Moreover, the proteinaceous matrices, which were more mately 7% of the whey protein fraction of milk (Jelen
lax than those of rennet gels from single-stage treated and Rattray, 1995). Our results showed that pasteur-
milks, were strongly stained by Nile red (Figures 1e ization heat treatment of 72°C for 15 s was sufficient
and f). Two-stage UHPH treatment at 300 MPa pro- to reduce ∼5% of the total N of whey, with levels of
voked the formation of spherical protein aggregates sur- residual β-LG and α-LA being ∼5 and 1.4% lower, re-

Table 5. Whey composition in minerals of raw, pasteurized, homogenized–pasteurized, and ultra-high

pressure homogenized (UHPH) milks1

Minerals (mg/L)
Treatment2 Ca P Mg

Raw 377.33 ± 9.17d 435.07 ± 3.12e 88.24 ± 1.66b

Pasteurized 371.64 ± 8.98e 433.75 ± 1.63e 87.23 ± 1.62cd
Homogenized–pasteurized 372.23 ± 9.16e 436.17 ± 2.65de 86.91 ± 1.65d
UHPH (MPa)
100 393.12 ± 7.61b 461.73 ± 3.10b 89.29 ± 1.21a
130 404.56 ± 6.33a 471.16 ± 4.79a 90.04 ± 1.17a
200 386.20 ± 5.38c 447.15 ± 4.36c 87.19 ± 1.28cd
230 381.21 ± 12.47d 442.48 ± 5.68cd 87.91 ± 1.72bc
300 356.10 ± 15.68g 414.91 ± 7.09f 83.52 ± 1.64e
330 361.33 ± 9.95f 419.60 ± 3.73f 83.36 ± 1.21e

Values without common superscripts were significantly different (P < 0.05).

a–f

1
Mean value ± standard error.
2
Pasteurized (72°C for 15 s); homogenized–pasteurized (15 + 3 MPa at 57 to 60°C, 72°C for 15 s); UHPH
treatments at 130, 230, and 330 MPa made using 2-stage homogenization: 100, 200, and 300 MPa in first
valve and 30 MPa in second valve.

Journal of Dairy Science Vol. 90 No. 1, 2007

 ULTRA-HIGH PRESSURE HOMOGENIZATION 19

Figure 1. Confocal laser scanning micrographs of rennet curds from a) homogenized–pasteurized milk; ultra-high pressure homogenized
(UHPH) milk at b) 100 MPa; c) 200 MPa; and d) 300 MPa; Nile red fluorescence (fat) from milk UHPH-treated at e) 200 MPa, and f) 230
MPa; and aggregates of fat globules dyed with g) Nile red and fluorescein isothiocyanate, and h) Nile red. Color images are available online
at http://jds.fass.org/.

Journal of Dairy Science Vol. 90 No. 1, 2007

20 ZAMORA ET AL.

spectively, in whey from pasteurized milk compared It has been reported that homogenization processes
with untreated milk. do not affect the distribution of calcium in milk (Robson
Furthermore, heating has a marked effect on the milk and Dalgleish, 1984). In our study, the amount of Ca,
salts equilibrium and their interaction with casein. It P, and Mg in whey of homogenized-pasteurized milks
is generally agreed that heating leads to a decrease did not differ from those of pasteurized milks.
in diffusible calcium and inorganic phosphate, due to Homogenized-pasteurized milks presented lower
precipitation of calcium phosphate, which may be re- RCT than raw and pasteurized milks, results that have
versed depending on the intensity of the treatment also been observed by other authors (Robson and Dal-
(Gaucheron, 2005). Under our experimental conditions, gleish, 1984; Ghosh et al., 1994; Guinee et al., 1997).
the decrease of Ca, P, and Mg in whey from pasteurized The lower RCT of homogenized milks could be explained
milk suggests a mineral transfer from soluble to colloi- by the fact that most κ-casein is located on the micelle
dal phase of milk. surface. As the casein enrobes the fat globules, the κ-
Milk pasteurization has only minor effects on the casein level is effectively diluted and a smaller critical
formation and physical properties of rennet-induced level of κ-casein hydrolysis is required to start coagula-
milk gels (Lucey, 1995). In our study, pasteurized milk tion (Guinee et al., 1997). Furthermore, homogeniza-
had no significantly different RCT and CF in relation tion increases the surface area of casein by a spreading
to untreated milk. However, more severe heating condi- process, κ-casein being more available for chymosin ac-
tions impair renneting milk properties (Dalgleish and tion, and thus, reducing RCT (Ghosh et al., 1994).
Banks, 1991; Guinee et al., 1996, 1997; Singh and In the current study, the CF of homogenized milks
Waungana, 2001). The causes have been broadly inves- was reduced by approximately 23% compared with un-
tigated even though they are not yet fully understood. treated milk. The weaker gels from homogenized–pas-
Both the enzymatic and nonenzymatic phases of rennet teurized milks could be attributed, according to differ-
clotting are delayed and the RCT is longer than that ent authors (Humbert et al., 1980; Robson and Dal-
of unheated milk. The strength of renneted milk gels gleish, 1984; Ghosh et al., 1994), to a greater dispersion
is also adversely affected in heated milk. It has been of fat in the curd, to a reduced number of casein particles
established that when heated, β-LG and κ-casein form available to form a strong network because some of
a complex by sulfydryl–disulfide interchange at the mi- the casein is tied to the surface of the new formed fat
celle surfaces that reduces the accessibility of the ren- globules, or to the small fat globules that are entrapped
net to the κ-casein and provides steric hindrance to in the gel disrupting the continuity of gel structure
close approach and fusion of paracasein micelles. More- and acting as weak centers in the gel. Confocal laser
over, heat induces the deposition of calcium phosphate scanning microscopy revealed fat globules embedded
and the consequent reduction in native calcium phos- within the protein matrix resulting in thick and lumpy
phate, which is important for cross-linking paracas- strands and a concomitant coarser texture.
ein micelles. Compared with single pasteurization, the homogeni-
Incorporation of denatured whey protein in the curd zation–pasteurization treatment produced higher (P <
from pasteurized milk did not increase the moisture 0.05) amounts of denatured β-LG and significantly in-
content of the curd compared with raw milk. However, creased the moisture content of the curd. Homogeniza-
the yield of TS of the curd from pasteurized milk was tion of milk resulting in slower whey drainage of the
∼7% higher than that from raw milk, which is probably curd has been observed by several researchers (Hum-
due to the incorporation of denatured whey proteins bert et al., 1980; Green et al., 1983; Ghosh et al., 1994).
into the curd. According to Lau et al. (1990) pasteuriza- The effects of homogenization on the moisture content
tion (63°C for 30 min) has little effect on fat recovery of the curd have been attributed to the higher incorpora-
in cheese but N recovery is improved, and approxi- tion of denatured β-LG and the alteration in the pro-
mately 5% of the whey proteins are associated with tein–fat structure of the curd.
casein micelles after pasteurization, resulting in an in-
creased theoretical cheese yield. Effects of UHPH Treatments
During conventional homogenization, the fat globule
size is reduced, the fat surface area increases markedly, In accordance with Hayes and Kelly (2003a), smaller
and a new adsorbed layer consisting of milk proteins fat globules were obtained by applying pressures below
(mainly casein micelles and casein subunits and whey 200 MPa at the second valve. Thus, the second valve
proteins) is formed around the fat globules (Cano-Ruiz would act as the secondary stage of a normal homoge-
and Richter, 1997). Our results showed a marked reduc- nizer; that is, stopping or decreasing coalescence. Many
tion of both volume- and surface-weighted mean diame- studies have shown that above a critical pressure, there
ters, from 2.9 to 0.5 ␮m and 0.6 to 0.3 ␮m, respectively. is an increased susceptibility of fat globule coalescence

Journal of Dairy Science Vol. 90 No. 1, 2007

 ULTRA-HIGH PRESSURE HOMOGENIZATION 21

(Floury et al., 2000, 2004b; Desrumaux and Marcand, associated to casein micelles are dissolved
2002). Above 200 MPa, the secondary stage not only (Gaucheron, 2005).
increased the average size of particles but also widened Single-stage UHPH above 200 MPa produced the
the distribution; that is, higher heterogeneity, com- smallest particles with the narrowest distributions. A
pared with single-stage homogenization. This may be further reduction of fat globule size and the increase
due to partial agglomeration of very small, insuffi- in interfacial fat surface would lead to a higher adsorp-
ciently coated globules that collide within the second tion of casein and whey proteins to the newly formed
valve. Thiebaud et al. (2003) detected very small fat fat globules. Sandra and Dalgleish (2005) reported a
globules (40- to 60-nm droplets) in single-stage UHPH- decreased micelle average size in skimmed milk by in-
treated milk at 200 and 300 MPa, and the impact forces creasing UHPH pressure. They suggested that UHPH
that act on the droplets as the result of a collision have would not cause complete disruption of the casein mi-
been determined as sufficient to cause disruption of the celles but rather dissociate parts of their surfaces. The
interfacial membranes (Floury et al., 2000). A broaden- obtained particle distributions corroborated that casein
ing of the size distributions was observed for single- micelle fragments, rather than intact casein micelles,
stage UHPH of warmed milk (Thiebaud et al., 2003), would surround fat globules (Hayes et al., 2005). Thus,
and model oil-in-water emulsions (Floury et al., 2000) very small fat globules would behave as casein micelles
at 300 MPa. The formation of large particles was attrib- rather than embedded fat globules observed in normal
uted to unfolding and aggregation of whey proteins of homogenization or lower UHPH pressures. Such struc-
the newly created droplets. In our case, 2-stage UHPH- tures could enhance gel firmness and rate of aggrega-
treated milks at 330 MPa showed much broader size tion by increasing the amount of particle associations;
distributions. Confocal laser scanning microscopy of hence, leading to the higher RCF and CF values ob-
rennet gels revealed that this phenomenon was due to served for milk UHPH-treated at 200 and 300 MPa.
the aggregation of well-defined small fat globules Confocal micrographs of rennet milks treated at 200
within dense proteinaceous structures (Figures 1g and 300 MPa showed lower levels of Nile red fluores-
and h). cence at the level of the proteinaceous network (Figures
Milks UHPH-treated below 200 MPa showed similar 1c and d). This could be explained by the fact that more
than 50% of their particles were beyond the resolution
gel strength compared with homogenized–pasteurized
threshold (0.23 ␮m/pixel).
milk. In fact, the protein matrices of the rennet gels
In early studies on UHPH, no denaturation of whey
observed by confocal microscopy were very similar to
proteins was reported (Hayes and Kelly, 2003a; Sandra
those of homogenized–pasteurized gels; that is, thick
and Dalgleish, 2005). However, the temperature of the
and lumpy strands giving a rough texture to the matrix
process in these studies never exceeded 55°C. Our tem-
(Figures 1a and b). However, their coagulation times
perature values during UHPH treatments were much
were lower than those of homogenized–pasteurized
higher (from ∼55 to 95°C), presumably because of differ-
milks. This decrease could be attributed to the lower
ent experimental designs (i.e., a much higher flow rate
pH of UHPH-treated milks, which would enhance chy-
and relatively larger volumes of milk being processed).
mosin performance. At these pressures, the tempera-
If only heat effect is considered, at ∼65°C, whey proteins
ture during processing never exceeded 60°C. Thus, the start to denature and interact with casein micelles
decrease in pH could be attributed to the action of resid- (Singh, 1993). However, in UHPH, simultaneous heat-
ual indigenous lipoprotein lipase after UHPH treat- ing and homogenization processes exist. In fact, Hayes
ment. Treatment with UHPH increased the interfacial et al. (2005), treating warmed milk up to 250 MPa
fat surface by decreasing the average size of particles, that reached 83.6°C, suggested that the physical forces
which would lead to a greater potential for lipolysis to experienced by whole milk during UHPH also dena-
occur (Hayes and Kelly, 2003a; Hayes et al., 2005). tured β-LG. Our results showed that the amount of
Mineral equilibrium in milk is very dependent on denatured β-LG was much greater (17%) for UHPH-
physicochemical parameters; that is, pH and tempera- treated at 200 MPa, which reached approximately 75°C
ture. The distribution of ions between the different frac- for a very short time (∼0.7 s), than for pasteurized milk
tions of milk (diffusible and nondiffusible) is defined by at 72°C for 15 s. Such results corroborate the idea that
the balance between these factors. The pH of milks not only heat but also homogenization forces induce the
treated at 100 to 130 MPa could be, to some extent, denaturation of whey proteins. Increasing the pressure
responsible for the higher amounts of minerals in their led to higher recovery of N in curd with lower levels of
whey. As pH decreases, the acido-basic groups of milk residual β-LG and α-LA in the whey.
constituents become more protonated; hence, micellar As previously mentioned, both the pH of the milk and
calcium phosphate and the small amount of magnesium the temperature reached during the treatment affected

Journal of Dairy Science Vol. 90 No. 1, 2007

22 ZAMORA ET AL.

the mineral equilibrium. Moreover, UHPH produces and cross-linking of casein by denatured whey proteins,
partial disruption of casein micelles (Sandra and Dal- leads to higher volume of the network relative to that
gleish, 2005) that could lead to a transfer of calcium and of the interstices, and thus a reduction of the relative
inorganic phosphate from the micellar to the diffusible ease of movement of the strands in the protein network
fraction. The balance between these factors could ex- (Green et al., 1983; Lucey et al., 2001).
plain the differences observed between the treatments As already stated, 2-stage UHPH above 200 MPa led
at 200 and 300 MPa. The fact that whey from milk to greater average particle size and higher heterogene-
treated at 200 MPa showed higher amounts of calcium ity than single-stage treatments. The obtained rennet
than whey from homogenized–pasteurized milk could gels showed similar firmness to those of homogenized–
be explained by both the release due to disruption of pasteurized and 100 to 130 MPa UHPH-treated milks.
casein micelles and its slightly lower pH value. In con- Confocal microscopy revealed a higher number of fat
trast, the amount of minerals in whey from milk UHPH- globules embedded within the proteinaceous matrix
treated at 300 MPa, which was lower than those of giving a rougher texture to the gels than in single-stage
the other treatments, suggests a mineral transfer from UHPH (Figures 1e and f). These results corroborate the
soluble to colloidal phase due to heat during UHPH hypothesis that 1) embedded fat globules, which lead
treatment. to thicker strands and a concomitant coarser matrix,
As pressure was increased, the RCT of milks was are responsible for weaker gels, and 2) the presence
prolonged. The differences between RCT at 200 and of very small fat globules behaving as casein micelles
300 MPa could be explained by the relative effect of the results in stronger rennet gels.
following factors: 1) the spreading of κ-casein; that is,
higher availability and lower critical level for chymosin
CONCLUSIONS
action; 2) denaturation of β-LG; that is, steric hin-
drance; 3) the pH of milk; and 4) changes in the concen- The results of this study show that UHPH treatment
tration of calcium between soluble and colloidal phases. of milk reduced fat globule size, increased the wet yield
Increasing UHPH pressure led to a higher recovery of curd and its moisture content, and decreased the
of N with lower levels of residual β-LG and α-LA in protein content of whey. The rennet coagulation proper-
whey, and higher TS yield and moisture content of ties were enhanced by single-stage UHPH at 200 and
curds. The observed differences between treatments 300 MPa. However, taking curd firmness into account,
could be explained by variations in 1) the association the application of a secondary stage produced weaker
of denatured whey proteins to the surface of casein gels similar to those obtained by conventional homoge-
micelles, 2) the reduction of fat globule size, 3) the incor- nization–pasteurization. The improvement of cheese-
poration of denatured whey proteins and casein micelle making properties of milk by UHPH could be attributed
fragments at the fat globule membrane, and 4) the mi- to the combined effect of homogenization (i.e., reduction
crostructure of the resulting gels. The association of of particle size) and heat (i.e., denaturation of whey
whey proteins at the micelle surface by heat sterically
proteins) on the protein–fat structures of the milk.
impedes the fusion of rennet-altered micelles resulting
in less shrinkage of the paracasein network (Singh and
Waungana, 2001). Moreover, the incorporation of dena- ACKNOWLEDGMENTS
tured whey proteins into the gel matrix increases the
The authors acknowledge the Ministerio de Educa-
water-binding capacity of the paracasein-whey network
ción y Ciencia (AGL2004-01943) and the Commission
(Singh and Waungana, 2001). The reduction of fat glob-
of the European Communities (EU project 512626) for
ule size implies a dispersion of fat into an increased
the financial support given to this investigation. Anna
number of smaller globules. The newly built surfaces
Zamora acknowledges the predoctoral fellowship from
are modified by the presence of adhering casein parti-
the Ministerio de Educación y Ciencia and the techno-
cles and become part of the paracasein network, thus
hindering shrinkage of the network (Walstra et al., logical support from J. M. Quevedo.
1985). The water-holding capacity of curds is directly
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