Effect of Milk Preacidification on Low Fat Mozzarella Cheese.
I. Composition and Yield1
ABSTRACT
The effect of milk preacidification with acetic or citric
acid on the composition and yield of low fat Mozzarella
cheese was determined. Two cheese manufacturing tri-
als were conducted. In trial 1, three vats (230 kg of
milk per vat) of cheese were made in 1 d using no
preacidification (control) and preacidification to pH 6.0
and pH 5.8 with citric acid. In trial 2, four vats (230
kg of milk per vat) of cheese were made in 1 d using
no preacidification (control), preacidification to pH 6.0
and 5.8 with acetic acid, and preacidification to pH 5.8
with citric acid. Cheese manufacture was repeated on
three different days in trial 1 and four different days
in trial 2 using a randomized-complete block design.
Preacidification to pH 5.8 with citric acid decreased
cheese calcium more than preacidification to pH 5.8
with acetic acid. Preacidification with citric acid in trial
1 decreased protein recovery in the cheese, and there
was a trend for decreased protein recovery in the cheese
for trial 2. Differences in fat recovery due to preacidifi-
cation varied, sometimes being lower than the control
other times being higher than the control. The reduc-
tion in calcium and protein recovery in the cheese
caused by preacidification lowered composition ad-
justed cheese yield and yield efficiency. Yield efficiency
was reduced by about 2.5 and 5.5%, respectively, with
preacidification to pH 6.0 and 5.8 with citric acid. Yield
efficiency was reduced by about 2.2 and 3.4%, respec-
tively, with preacidification to pH 6.0 and pH 5.8 with
acetic acid.
(Key words: low fat, Mozzarella cheese, preacidifica-
tion, yield)
Received December 12, 1998.
Accepted November 11, 1999.
Corresponding author: D. M. Barbano; e-mail: DMB37@
cornell.edu.
1
Use of names, names of ingredients, and identification of specific
models of equipment is for scientific clarity and does not constitute
an endorsement of product by authors, Cornell University, University
of Vermont, or the Northeast Dairy Foods Research Center.
2
Department of Nutrition and Food Sciences, University of Ver-
mont, Burlington 05405.
2000 J Dairy Sci 83:648–658 648
L. E. Metzger, D. M. Barbano,
M. A. Rudan, and P. S. Kindstedt2
Northeast Dairy Foods Research Center,
Department of Food Science,
Cornell University, Ithaca, NY 14853
INTRODUCTION
Extensive research has been done to improve the
functional characteristics of fat free and low and re-
duced fat Mozzarella cheeses (15, 16, 18, 19, 20). Of
particular importance was the discovery that the melt-
ing and browning characteristics of fat free, low, and
reduced fat Mozzarella cheese can be controlled by pre-
venting surface drying during baking with the use of a
hydrophobic surface coating (18). Although this tech-
nique improves the melting and browning characteris-
tics of lower fat Mozzarella cheeses, the unmelted and
melted functional properties and postmelt texture of
fat free and low fat Mozzarella cheese remain inferior
to low moisture part skim Mozzarella (17, 18).
Calcium plays an integral role in cheese texture by
cross-linking protein (13). As a result, the amount of
calcium in cheese has an effect on texture (8, 24, 31).
When the same cheese manufacturing procedure is
used for both low moisture part skim and low fat Mozza-
rella, the total calcium content increases from 0.67 to
1.00% as fat content is reduced from 26.7 to 4.1% (21).
If the total calcium content of cheese is reduced, then
the amount of cross-linking between casein polymers is
reduced and the cheese becomes softer (24). The calcium
content of Mozzarella cheese can be manipulated by
modifying cheese manufacturing conditions. When the
whey is drained from the curd, micellar calcium is re-
tained in the cheese, while most of the nonmicellar
calcium is lost in the whey. Decreasing the pH of the
curd at draining increases the level of nonmicellar cal-
cium and lowers the calcium content of cheese (9, 29).
In addition, if the pH of milk is lowered by acid addition
prior to rennet coagulation, a portion of the micellar
calcium is solubilized (7, 26) and this will lower the
calcium content of cheese. Keller et. al (11) determined
that the type of acid used to manufacture direct acid
Mozzarella cheese had an effect on cheese calcium
content.
In addition to calcium, a portion of the micellar casein
is disassociated when the pH of milk is reduced (6, 12,
23, 26). Temperature of acidification and pH affect the
amount of casein disassociated from the micelle struc-
ture during acidification (6, 26). At pH 6.6 at 20 and
 PREACIDIFICATION AND MOZZARELLA YIELD
30°C, 7% of the casein in milk is disassociated, while
15% is disassociated at 4°C. After acidification to pH
5.8, 7, 15, and 30% of the casein is disassociated at 30,
20, and 4°C, respectively (6). Thus, at low temperature
(i.e., 4°C), casein and calcium tend to dissociate from
the casein micelles. Reduction of the pH of cold milk
causes more calcium and casein to dissociate. Increas-
ing the temperature of acidified milk prior to rennet
addition should cause some of the casein to go back into
the micelles because of increased hydrophobic at-
traction among caseins. As was the case with nonmicel-
lar calcium, the nonmicellar (disassociated) casein may
also be lost in the whey during cheese manufacture. The
increase in nonmicellar casein and calcium produced
by acidification may decrease the protein and calcium
recovery in the cheese, which would cause a reduction
in cheese yield. The amount of these losses has not
been documented. In addition, cheese yield may also
be affected if acidification influences fat recovery.
In light of the potential benefit of reducing the cal-
cium content of low fat Mozzarella cheese on unmelted
and melted functional properties and reducing the ex-
cessively chewy postmelt texture, the objective of this
research was to determine if the level and type of acid
used to preacidify milk, with the goal of reducing cheese
calcium content and reducing post bake chewiness, af-
fects the fat, protein, and calcium recovery in low fat
Mozzarella cheese. The effect of milk preacidification
on cheese yield and cheese yield efficiency was also de-
termined.
MATERIALS AND METHODS
Milk Standardization and Cheese Manufacturing
Two cheese making trials were conducted. In each
trial, raw cream (40% fat) and skim milk (< 0.1% fat)
were obtained from the Cornell University dairy plant.
The skim milk was standardized to 0.5% fat after the
fat content of the cream [(1) method number 33.3.18,
995.18] and skim milk [(14) method number 15.8 B]
were determined by Babcock. The standardized milk
was HTST pasteurized (model University Pilot Plant;
PMS Processing Machinery and Supply Co, Philadel-
phia, PA) at 72°C for 16 s, cooled to 4°C, and stored at
4°C overnight until used for cheese manufacturing the
following day.
In trial 1, three treatments were employed: control
(no preacidification), pH 6.0 citric (preacidification to
pH 6.0 with citric acid), and pH 5.8 citric (preacidifica-
tion to pH 5.8 with citric acid). The pH 6.0 citric and pH
5.8 citric treatments were preacidified by the addition of
a 50% (wt/wt) aqueous solution of food grade citric acid
(anhydrous granular, Harrmann & Reiner Corp., Elk-
art, IN) at a rate of 1.44 and 2.02 ml/kg of milk, respec-
649
tively. In trial 2, four treatments were employed: control
(no preacidification), pH 6.0 acetic (preacidification to
pH 6.0 with acetic acid), and pH 5.8 acetic (preacidifica-
tion to pH 5.8 with acetic acid), and pH 5.8 citric (pre-
acidification to pH 5.8 with citric acid). The pH 6.0
acetic and pH 5.8 acetic were preacidified to pH 6.0 and
pH 5.8 by the addition of a 50% (vol/vol) solution of
nonfood grade acetic acid (glacial acetic acid, Mallinck-
rodt Baker, Inc., Paris, KY) at a rate of 1.25 and 1.82
ml/kg of milk, respectively. As in trial 1, the pH 5.8
citric treatment was preacidified with a 50% solution
of citric acid at a rate of 2.02 ml/kg of milk. The reported
pH of preacidification in trial 1 and trial 2 is the pH at
38°C as described in the materials and methods section
of this study.
For each treatment, 230 kg of 4°C milk were trans-
ferred to a cheese vat (model 4MX; Kussel Equipment
Co., Watertown, WI). The acid was added to the milk
slowly and with gentle agitation for the preacidified
treatments. The cheese was manufactured using a no-
brine, stirred curd Mozzarella manufacturing proce-
dure (4). The milk was heated in the vat to 38°C and
a direct-to-vat Thermococcus C120 (Streptococcus ssp.
thermophilus) and Thermorod R160 (Lactobacilus del-
brueckii ssp. bulgaricus) starter cultures (Rhone-
Poulenc, Madison, WI) were added in a 1:1 ratio. In
trial 1, each culture was added at a rate of 0.40, 0.35,
and 0.30 ml/kg of milk and a 60, 45, and 30 min ripening
time were used for the control, pH 6.0 citric, and pH 5.8
citric treatments, respectively. In trial 2, each starter
culture was added at a rate of 0.40 ml/kg of milk and
a ripening time of 60 min was used for all treatments.
Following ripening, double strength chymosin derived
by fermentation (Chymax, Chr. Hansen Inc., Milwau-
kee, WI) was added at a rate of 0.10 ml/kg of milk. Prior
to addition, the chymosin was diluted in 200 ml of water
reduced in hardness by reverse osmosis. A 30-min pe-
riod from chymosin addition to cutting was used for the
control treatments in trials 1 and 2, while 15 min was
used for all of the preacidification treatments in trials
1 and 2. The lower pH at chymosin addition in the
preacidification treatments reduced the coagulation
time (27). The coagulum was cut with 1.2-cm wire
cheese knives and allowed to heal for 5 min. In the
control treatment in trials 1 and 2, the curd was stirred
until the whey pH reached approximately 6.20, at which
time the whey was drained. In the preacidification
treatments the curd was stirred for 30 min prior to
whey drainage. The pH of the whey at draining for trial
1 was 6.21, 5.95, and 5.79 for the control, pH 6.0 citric,
and pH 5.8 citric treatments, while in trial 2, the pH
of the whey at draining was 6.22, 5.90, 5.72, and 5.71
for the control, pH 6.0 acetic, pH 5.8 acetic, and pH 5.8
citric treatments, respectively. The dry curd was stirred
Journal of Dairy Science Vol. 83, No. 4, 2000
650 METZGER ET AL.
and maintained at 38°C until the desired cheese pH for
salting was reached. In trial 1, all treatments were
salted at a cheese pH of 5.50, while in trial 2 the control
was salted at pH 5.50 and the curd made by preacidifi-
cation was salted at pH 5.35. The salt was applied in
three equal portions (total of 2.3%, wt/wt) with a 5-min
interval between applications.
After salting the curd was stretched with a twin-
screw, pilot-scale Mozzarella mixer (model 640; Stain-
less Steel Fabricating, Columbus, WI). The salted curd
was fed into the mixer at the rate of 2 kg/min and was
stretched in 6% salt water solution at 57°C in trial 1
and 65°C in trial 2. A screw speed of 12 rpm was used
in both trials 1 and 2. The cheese was extruded into
stainless steel cylinders (7.5 cm diameter × 30 cm long).
The cylinders were filled with about 1.4 kg of cheese
and were capped and placed in an ice water bath for
60 min. The cheese was removed from the cylinders and
vacuum packaged (Multi Vac model 160; Koch, Kansas
City, MO) in a barrier bag (model B150; Cryovac, Dun-
can, SC). Eight cylinders of cheese were obtained from
each treatment and were stored at 4°C until analysis.
Chemical Analysis
Titratable acidity [(14); method number 15.3.B] of
the milk and whey, and pH (Xerolyt electrode, model
HA405; Ingold Electrode, Willmington, MA, and Accu-
met pH meter, model 915; Fisher Scientific, Fair Lawn,
NJ) of the milk, whey, and cheese were determined
during cheese manufacturing. Temperature of the milk,
whey, and cheese during measurement was 38°C. The
electrode was immersed in 3 M KCL storage solution
at 38°C between pH measurement to improve stability.
Reference solutions (Fisher Scientific, Fair Lawn, NJ)
for pH 4 (SB101-500) and pH 7 (SB107-500) were tested
at 38°C for calibration of the pH meter. The actual pH
of the reference buffers was calculated for 38°C based
on the temperature coefficients recommended by the
manufacturer. After cheese manufacture, the fat con-
tent of the milk, whey, and stretching water was deter-
mined by ether extraction [(1); method number 33.2.26,
989.05]; total nitrogen was determined by Kjeldahl [(1);
method number 33.2.11, 991.20].
Calcium was determined by an atomic absorption
spectroscopy procedure adapted from Brooks et al. (5).
A sample size of 1.5, 0.75, 0.75, and 5 g was used,
respectively, for the cheese, milk, whey, and stretching
water. The cheese, milk, and whey were mixed with 45,
29.25, and 29.25 g of 12% (wt/vol) trichloroacetic acid
(Mallinckrodt Baker Inc., Paris, KY), respectively, and
the stretching water was mixed with 10 g of distilled
water and 15 g of 24% (wt/vol) trichloroacetic acid. The
cheese and trichloroacetic acid mixture was blended for
Journal of Dairy Science Vol. 83, No. 4, 2000
30 s with an Omni mixer-homogenizer (model 17105;
Omni International, Waterbury, CT) while the milk,
whey, and stretching water were inverted several
times. After 30 min, all samples were filtered (What-
man 541; Whatman International Ltd., Maidstone,
England). For the cheese, 1 g of filtrate was added to
9 g of 12% trichloroacetic acid, 9.6 g of distilled water,
and 0.4 g of a 5% (wt/vol) solution of lanthanum oxide
(Sigma Chemical Co., St. Louis, MO). For the milk,
whey, and stretching water 10 g of filtrate was added
to 9.6 g of distilled water and 0.4 g of 5% lanthanum
oxide. The final concentration of trichloroacetic acid
and lanthanum oxide in all cheese, milk, whey, and
stretching water samples was 6% (wt/vol) and 0.02%
(wt/vol), respectively. Each sample was aspirated into
an atomic absorption spectrophotometer (model 2380;
Perkin Elmer Corp., Norwalk, CT) fitted with a calcium
lamp (0303-6017 Perkin Elmer Corp., Norwalk, CT) for
calcium determination. The atomic absorption spectro-
photometer was calibrated with reference standards
prepared from calcium reference solution (SC191-500,
Fisher Scientific, Fair Lawn, NJ). The appropriate
amount of calcium reference solution, trichloroacetic
acid, 5% lanthanum oxide, and distilled water were
mixed in a volumetric flask to obtain reference stan-
dards containing 0, 4, 6, 8, 10, 12, and 16 mg/kg of
calcium. All reference standards contained 6% (wt/vol)
trichloroacetic acid and (0.02% wt/vol) lanthanum
oxide.
To prepare the cheese for analysis, samples were
ground in a blender (model 31BL92; Waring, New Hart-
ford, CT) to a particle size of 2 to 3 mm. The ground
cheese particles were packed into 50-ml plastic snap-
lid vials (leaving no head space) and stored at 4°C.
Fat content was determined by ether extraction [(1);
method number 33.2.26, 989.05] with 1 g of cheese and
9 ml of distilled water instead of a 10-ml milk sample
and 3 ml of ammonium hydroxide instead of 1.5 ml;
total nitrogen was determined by Kjeldahl using a 1-g
sample size [(1); method number 33.2.11; 991.20]; salt
was determined by the Volhard test [(14); method num-
ber 15.5.B.); and moisture was determined gravimetri-
cally by drying 2 g of cheese at 100°C in a forced air
oven (model OV-490A-2; Blue M, Blue Island, IL) for
24 h.
Calculation of Recoveries and Yield
The weight of milk, whey, stretching water, and
cheese for each vat was determined to the nearest gram
(model PE 24; Mettler Instrument Co., Highstone, NJ).
The actual percentages of calcium, fat, and protein re-
covered in the cheese, whey, and stretching water were
calculated by dividing the weight of calcium, fat, or
 PREACIDIFICATION AND MOZZARELLA YIELD
protein present in each product by the total weight of
calcium, fat, or protein in the milk and then multipling
by 100. Next, the mean actual total recovery of fat,
protein, and calcium were compared among treatments
within trial, to determine if there were any significant
differences in total recovery among treatments within
trial. If there were no significant differences in total
recovery due to treatment, then the actual calcium, fat,
and protein recoveries were adjusted by dividing the
actual recovery by the mean total recovery on each
cheese making day, multiplied by 100 as described pre-
viously (20, 21). This adjustment of recovery brings the
mean value within trial 1 and the mean value within
trial 2 to 100%. This allows relative comparison of re-
sults among different trials and different studies.
Actual cheese yield was determined by dividing the
weight of cheese by the weight of milk used to make
cheese, multiplied by 100. Moisture and salt adjusted
cheese yield was calculated by the following formula:
adjusted yield = ((actual yield × (100 − actual moisture
+ actual salt)))/(100 − (55 + 1.5)). Theoretical yield was
calculated with the modified Van Slyke and Barbano
cheese yield formula (2). The original Van Slyke for-
mula (28) was based on Cheddar cheese yield and was
modified for Mozzarella cheese (3). The modifications
included changing the assumed fat recovery from 0.93
to 0.85 and changing the constant factor from 1.09 to
1.13. The Barbano formula for theoretical yield includes
terms for fat, casein, and calcium phosphate recovery
and a term for whey solids retained in the water phase
of the cheese (2). The Barbano cheese formula is as
follows:
yield = ( A + B + C)/1 − ((target cheese moisture
+ target cheese salt)/100)
where, A is the milk fat recovered in the cheese, B is
the milk casein plus calcium phosphate recovered in
the cheese, and C is the other milk solids recovered
in the cheese (i.e., nonfat, noncasein, noncalcium phos-
phate milk solids recovered.
A = (percentage fat recovery in the cheese) ×
(percent fat in the milk)
B = (percent casein in the milk − 0.1) × (cal-
cium phosphate retention factor)
C = [(((A+B)/(1 − actual cheese moisture per-
cent/100)) − (A + B) × (separated whey sol-
ids percent/100)] × (solute exclusion fac-
tor)
Theoretical yield using the Barbano formula was calcu-
lated in two ways: 1) without (Barbano-1) and 2) with
(Barbano-2) adjustment of the calcium phosphate re-
651
tention factor. A calcium phosphate retention factor of
1.092 was used for all treatments for the Barbano-1
theoretical yield formula. In the Barbano-2 theoretical
yield formula the calcium phosphate retention factor
was adjusted to account for the observed differences in
the calcium content of the cheeses. Values of 1.092,
1.077, 1.062, 1.084, and 1.076 were used for the control,
pH 6.0 citric, pH 5.8 citric, pH 6.0 acetic, and pH 5.8
acetic treatments, respectively. Values of 0.85 and
0.8534 were used for the fat retention factor and solute
exclusion factor, respectively, in both of the Barbano-
1 and Barbano-2 theoretical yield formulae. The values
used for the fat retention factor and solute exclusion
factor were determined in a previous study of low fat
Mozzarella (21). The yield efficiency was determined
by dividing the moisture and salt adjusted yields by
the theoretical yield using the three different formulae,
multiplied by 100. The efficiency with the Barbano-2
formula accounts for changes in yield as a result of
calcium reduction (due to different pH at whey drain-
ing), while yield efficiency with the modified Van Slyke
and Barbano-1 formulae does not.
Statistical Analysis
A 3 × 3 and 4 × 4 randomized complete block design
was used for trial 1 and trial 2, respectively. In trial 1
the three treatments (control, pH 6.0 citric, and pH
5.8 citric) were made from the same standardized and
pasteurized milk on each day of cheese making; this
was repeated three times on different days. In trial 2,
the four treatments (control, pH 6.0 acetic, pH 5.8 ace-
tic, and pH 5.8 citric) were made from the same stan-
dardized and pasteurized milk on each day of cheese
making; this was repeated four times on different days.
In both trials, treatment and day of cheese manufacture
were analyzed as class variables. The PROC GLM pro-
cedure of SAS was used for all data analysis (22). If the
F-test for the statistical model was significant (P < 0.05),
the treatment means were compared using least sig-
nificant difference test.
RESULTS AND DISCUSSION
Composition of Milk, Cheese, Whey,
and Stretching Water
The milk used in trial 1 contained 0.54% fat (SD =
0.02), 3.22% crude protein (SD = 0.04), 0.114% calcium
(SD = 0.0006), 0.22% nonprotein nitrogen (SD = 0.003),
2.47% casein (SD = 0.03), 82.29% casein as a percentage
of true protein (SD = 0.28), and 9.35% total solids (SD
= 0.06). The milk used in trial 2 contained 0.53% fat
(SD = 0.026), 3.16% crude protein (SD = 0.024), 0.117%
calcium (SD = 0.0015), 0.21% nonprotein nitrogen (SD
Journal of Dairy Science Vol. 83, No. 4, 2000
652 METZGER ET AL.

Table 1. Mean (N = 3) chemical composition of cheese, whey and stretching water for trial 1 (preacidification
with citric acid).
Treatment
pH 6.0 pH 5.8
Citric Citric
Component Control acid acid SEM F test LSD1
(%)
Cheese
Moisture 55.77 55.36 56.74 0.292 0.16 NS2
Fat 6.27 6.22 5.86 0.142 0.29 NS
Protein 32.15 32.69 32.33 0.181 0.20 NS
Salt 1.42 1.52 1.50 0.048 0.23 NS
Calcium 0.947a 0.759b 0.596c 0.014 <0.01 0.053
Whey
Fat 0.068 0.079 0.114 0.008 0.08 NS
Protein 0.935 0.951 0.958 0.007 0.02 0.029
Calcium 0.046c 0.063b 0.075a 0.001 <0.01 0.002
Total solids 6.56c 6.81b 6.91a 0.014 <0.01 0.055
Stretching water
Fat 0.068 0.040 0.045 0.010 0.42 NS
Protein 0.078 0.077 0.117 0.007 0.07 NS
Calcium 0.0160a 0.0098b 0.0097b 0.001 <0.01 0.0028
Means within the same row not sharing common superscripts are different (P < 0.05).
a,b,c
1
Least significant difference (P = 0.05).
2
NS = Not significant.

= 0.008), 2.44% casein (SD = 0.009), 82.24% casein as
a percentage of true protein (SD = 0.41), and 9.35%
total solids (SD = 0.03). The composition of the cheese,
whey, and stretching water is shown in Table 1 for trial
1 and in Table 2 for trial 2. In both trials the calcium
content of the cheese decreased (P < 0.05) and the cal-
cium content of the whey increased (P < 0.05) with all
levels of preacidification.
Preacidification to pH 5.8 decreased (P < 0.05) cheese
calcium content and increased (P < 0.05) whey calcium
content more than preacidification pH 6.0 with citric
acid in trial 1 and acetic acid in trial 2. However, in

Table 2. Mean (N = 4) chemical composition of cheese, whey and stretching water for trial 2 (preacidification
with acetic and citric acid).

Treatment
pH 6.0 pH 5.8 pH 5.8
Acetic Acetic Citric
Component Control acid acid acid SEM F test LSD1
(%)
Cheese
Moisture 55.10 53.58 53.34 55.30 0.384 0.09 NS3
Fat 6.03c 6.51c 6.55a 6.19b 0.035 <0.01 0.111
Protein 32.32b 33.80a 33.52a 33.03ab 0.349 0.04 1.120
Salt 1.31 1.29 1.23 1.48 0.072 0.39 NS
Calcium 0.976a 0.872b 0.754c 0.578d 0.013 <0.01 0.043
Whey
Fat 0.073a 0.064b 0.061b 0.079a 0.003 <0.01 0.007
Protein 0.890 0.890 0.908 0.911 0.010 0.30 NS
Calcium 0.046d 0.058c 0.067b 0.080a 0.001 <0.01 0.001
Total solids 6.60d 6.74c 6.80b 6.90a 0.001 <0.01 0.020
Stretching water
Fat 0.050 0.051 0.051 0.061 0.005 0.49 NS
Protein 0.058 0.085 0.077 0.123 0.013 0.10 NS
Calcium 0.0117a 0.0110ab 0.0099b 0.0102b 0.001 0.01 0.0014
Means within same row not sharing common superscripts are different (P < 0.05).
a,b,c,d
1
Least significant difference (P = 0.05).
2
NS = Not significant.

Journal of Dairy Science Vol. 83, No. 4, 2000

 PREACIDIFICATION AND MOZZARELLA YIELD
trial 2 preacidification with citric acid to pH 5.8 caused
a larger decrease (P < 0.05) in cheese calcium and a
larger increase (P < 0.05) in calcium in whey than pre-
acidification to pH 5.8 with acetic acid. As the calcium
content of the curd decreased, the curd became softer
and stretched more easily.
The observed decrease in cheese calcium content and
increase in whey calcium content with preacidification
was expected. The addition of acid to milk causes an
increase in nonmicellar calcium (6, 26). During cheese
manufacture, the micellar calcium in milk is retained
in the cheese, while the nonmicellar calcium is lost in
the whey. Therefore, as a result of preacidification, the
high level of nonmicellar calcium caused the observed
decrease in cheese calcium content and increase in
whey calcium content. Increased calcium content, de-
creased pH, and increased acidity of the whey resulting
from preacidification may have impacts on whey pro-
cessing and whey product functionality.
The molar concentration (0.00858 M) of acetic acid
in the preacidified cheese milk for the pH 5.8 acetic
treatment was 2.6 times the molar concentration
(0.00331 M) of citric acid in the preacidified cheese milk
for the pH 5.8 citric treatment. Acetic acid has one
ionizable carboxyl group and citric acid has three ioniz-
able carboxyl groups. At pH 5.8 the single ionizable
carboxyl groups in acetic acid is completely disassoci-
ated while with citric acid two of the ionizable carboxyl
groups are completely disassociated and one is partially
disassociated (25). As a result the normalities of acetic
and citric acid in the preacidified cheese milk were simi-
lar in the pH 5.8 acetic and pH 5.8 citric treatments.
Therefore, the difference in calcium content of the
cheese and whey between the pH 5.8 acetic and pH
5.8 citric treatments may be a result of differences in
binding affinity of calcium for acetic or citric acid. The
binding affinity of acetic acid for calcium is less than
the binding affinity of phosphate for calcium, and the
binding affinity of citric acid for calcium is greater than
the binding affinity of phosphate for calcium (10).
Therefore, in preacidification with citric acid versus
acetic acid, citric acid will remove a larger amount of
calcium from the micelles than acetic acid at the same
whey pH and the calcium plus citric acid will be lost
in the whey.
The calcium content of the stretching water was lower
(P < 0.05) in the pH 6.0 citric and pH 5.8 citric treat-
ments than the control in trial 1. In trial 2 the pH 5.8
acetic and pH 5.8 citric treatments were lower than for
the control and pH 6.0 acetic treatments (Table 1). The
treatments with the lowest cheese calcium content also
had the lowest stretching water calcium content.
Although there were substantial (P < 0.05) differ-
ences in cheese calcium content within trials 1 and 2, no
653
significant (P > 0.05) differences in cheese fat, protein,
moisture, and salt within trial 1 (Table 1) and cheese
moisture and salt within trial 2 (Table 2) were detected.
There were small but significant (P < 0.05) differences
among treatments in cheese fat and protein in trial 2.
The total solids content of the whey increased signifi-
cantly (P < 0.05) with all levels of preacidification in
both trials 1 and 2. The fat content of the whey was
not affected (P > 0.05) by preacidification with citric
acid in trials 1 or 2, while preacidification with acetic
acid decreased (P < 0.05) the fat content of the whey in
the pH 6.0 acetic and pH 5.8 acetic treatments in trial
2 (Table 2). The protein content of the whey was not
affected (P > 0.05) by preacidification in trial 1 or trial
2. The protein and fat contents of the stretching water
were not affected (P > 0.05) by preacidification in ei-
ther trial.
Fat, Protein, and Calcium Recovery
The mean actual total fat, protein, and calcium recov-
eries for each preacidification treatment within trial 1
were 100.14, 99.68, and 98.10% for fat; 100.82, 100.63,
and 100.53% for protein; and 100.31, 100.05, and
99.34% for calcium, respectively, for the control, pH 6.0
citric and pH 5.8 citric treatments. In trial 2, the mean
actual total fat, protein, and calcium recoveries for each
preacidification treatment were 98.35, 98.30, 97.85, and
97.75% for fat; 101.00, 100.75, 100.59, and 100.28% for
protein; and 99.05, 98.75, 98.74, and 99.02% for cal-
cium, respectively, for the control, pH 6.0 acetic, pH 5.8
acetic, and pH 5.8 citric treatments. There were no
significant (P > 0.05) differences in actual total fat, pro-
tein, or calcium recovery among the treatments in trials
1 and 2. Therefore, the individual fat, protein, and cal-
cium recoveries in the cheese, whey, and stretching
water were adjusted by dividing the actual recovery for
each treatment by the mean actual total recovery on
each cheese making day, multiplied by 100, and are
shown in Table 3 for trial 1 and in Table 4 for trial 2.
Preacidification to pH 5.8 with citric acid in trial 1
decreased (P < 0.05) fat recovery in the cheese in trial
1 and the same trend for pH 5.8 with citric acid was
observed in trial 2, but the difference was not signifi-
cant. Additionally, in trial 2 preacidification with acetic
acid to pH 5.8 increased (P < 0.05) fat recovery in the
cheese. In trial 1, a trend (P = 0.07) of increased fat
loss in the whey was observed with citric acid, while
in trial 2 preacidification to pH 5.8 with acetic acid
decreased (P < 0.05) fat loss in the whey. The fat loss
in the stretching water was not significantly (P > 0.05)
affected by treatment in trial 1 or trial 2. The opposite
effect that was observed with cheese fat recovery with
acetic acid versus citric acid was not expected. The ef-
Journal of Dairy Science Vol. 83, No. 4, 2000
654 METZGER ET AL.

Table 3. Adjusted mean (N = 3) fat, protein, calcium recoveries in the cheese, whey, and stretching water
for trial 1 (preacidification with citric acid).
Treatment
pH 6.0 pH 5.8
Citric Citric
Component Control acid acid SEM F test LSD1
(%)
Fat recovery
Cheese 86.64a 85.53a 77.98b 1.352 0.05 5.32
Whey 11.42 13.85 19.22 1.286 0.07 NS2
Stretching water 2.32 1.46 1.59 0.333 0.43 NS
Protein recovery
Cheese 73.75a 72.57b 72.26b 0.260 0.05 1.02
Whey 25.96 26.86 27.01 0.228 0.06 NS
Stretching water 0.45 0.44 0.69 0.047 0.07 NS
Calcium recovery
Cheese 61.63a 47.93b 37.83c 0.404 <0.01 1.59
Whey 36.13a 50.62b 59.97c 0.530 <0.01 2.08
Stretching water 2.64 1.60 1.63 0.192 0.08 NS

Means within same row not sharing common superscripts are different (P < 0.05).
a,b,c
1
Least significant difference (P = 0.05).
2
NS = Not significant.

fects of preacidification on cheese fat retention may be
related to modifications in protein-protein interactions
as a result of calcium reduction (27) which may effect
gel structure during set.
Preacidification with citric acid to pH 6.0 and pH 5.8
significantly (P < 0.05) decreased protein recovery in
the cheese in trial 1 (Table 3). In trial 2, a similar trend
(P = 0.10) occurred with both acetic and citric acid (Table
4). The protein recovery in the whey and stretching
water was not significantly (P > 0.05) affected by treat-
ment in trial 1 or 2. However, there was a trend of
increased protein loss in the whey observed with in-
creasing amount of preacidification. This may be re-
lated to the effects of acid addition on micelle structure
and resulting solubilization of casein (6, 26) prior to
cutting the curd. Some of the solubilized casein may be
lost in the whey at draining and could be responsible for
the trend for decreased protein recovery in the cheese.
The purpose of acidification of milk prior to cheese
making is to reduce the calcium content of cheese. Milk

Table 4. Adjusted mean (N = 4) fat, protein, and calcium recoveries in the cheese, whey, and stretching
water for trial 2 (preacidification with acetic and citric acid).

Treatment
pH 6.0 pH 5.8 pH 5.8
Acetic Acetic Citric
Component Control acid acid acid SEM F test LSD1
(%)
Fat recovery
Cheese 85.17bc 86.58ab 87.54a 83.75c 0.529 0.01 1.69
Whey 12.89a 11.25b 10.74b 14.00a 0.404 <0.01 1.29
Stretching water 2.28 1.99 1.82 1.98 0.203 0.32 NS3
Protein recovery
Cheese 74.35 73.50 73.37 72.94 0.305 0.10 NS
Whey 25.63 25.63 26.25 26.37 0.270 0.16 NS
Stretching water 0.37 0.50 0.46 0.63 0.066 0.18 NS
Calcium recovery
Cheese 61.68a 51.94b 45.16c 35.04d 0.512 <0.01 1.64
Whey 36.54d 46.20c 53.08b 63.54a 0.279 <0.01 0.89
Stretching water 1.93a 1.72b 1.60b 1.57b 0.061 <0.01 0.196
Means within same row not sharing common superscripts are different (P < 0.05).
a,b,c,d
1
Least significant difference (P = 0.05).
2
NS = Not significant.

Journal of Dairy Science Vol. 83, No. 4, 2000

 PREACIDIFICATION AND MOZZARELLA YIELD
acidification may also have impacts on proteolysis and
other changes in the cheese during refrigerated storage.
Large decreases in recovery of calcium in the cheese
were expected. Calcium recovery in the cheese de-
creased (P < 0.05) and calcium loss in the whey in-
creased (P < 0.05) with all levels of preacidification in
trials 1 and 2 (Tables 3 and 4). Preacidification with
citric acid to pH 5.8 decreased (P < 0.05) calcium recov-
ery in the cheese and increased (P < 0.05) calcium loss
in the whey, more than preacidification to pH 6.0 in
trial 1. In trial 2, preacidification with acetic acid pro-
gressively decreased (P < 0.05) recovery of calcium in
the cheese and increased (P < 0.05) the loss of calcium
in the whey. Additionally, in trial 2 the pH 5.8 citric
treatment had a lower (P < 0.05) calcium recovery in
the cheese and higher (P < 0.05) calcium loss in the
whey than the pH 5.8 acetic treatment. Thus, given the
same pH of preacidification, the citric acid shifts more
calcium from the cheese into the whey than acetic acid.
There was a trend (P = 0.08) for lower loss of calcium
in the stretching water with preacidification in trial 1.
In trial 2, all preacidification treatments had lower (P
< 0.05) loss of calcium in stretching water than the
control. The lower calcium loss in stretching water is
consistent with the lower calcium content of the cheese
in the preacidified treatments.
Yield and Yield Efficiency
The potential benefits of improved functionality of
low fat Mozzarella cheese that may be produced by
preacidification of milk could be less attractive if they
are accompanied by large decreases in cheese yield effi-
ciency. The modified Van Slyke theoretical yield for-
mula (28) using a constant 1.13 value for the retention
of nonfat, noncasein, milk solids plus salt makes the
assumption that the pH of whey at draining is constant
and that retention of calcium in the matrix of the curd
and the solids content of the aqueous phase of the cheese
are constant for all treatments. Thus, it would be ex-
pected that the modified Van Slyke formula using a
constant would overpredict cheese yield when milk is
preacidified. The Barbano yield formula (3, 20, 21), in-
corporates a compensation for variation in the solids
content of the whey at draining and a factor for the
retention of calcium phosphate. Different independent
factors can influence these two variables. For example,
when milk for cheese making is fortified with NDM
or condensed skim, the solids content of the whey at
draining may be elevated, which causes an increase in
the solids content in the water phase of the cheese, but
no change in the ratio of calcium to protein in the cheese
(30). In contrast, changing the pH at whey draining
changes the ratio of calcium to protein in the cheese
655
matrix (Tables 1 and 2). In this paper, two versions (1
and 2) of the Barbano yield formula are used: Barbano-1
used a constant value of 1.092 for the calcium phosphate
retention factor to calculate theoretical yield; Barbano-
2 used values for the calcium phosphate retention factor
for each treatment calculated from cheese calcium data
in the present study. Thus, changes in cheese yield
efficiency calculated using the Barbano-1 formula re-
flect changes due to losses of fat, protein, and calcium
phosphate caused by milk preacidification, while
changes in cheese yield efficiency calculated using the
Barbano-2 formula reflect changes due to fat and pro-
tein losses only.
Cheese yields and cheese yield efficiencies are shown
in Tables 5 and 6 for trials 1 and 2, respectively. Pre-
acidification did not affect (P > 0.05) actual yield in trial
1. However, preacidification decreased (P < 0.05) actual
yield in trial 2. Moisture and salt adjusted cheese yields
decreased (P < 0.05) with all levels of preacidification
in both trials 1 and 2. Preacidification to pH 5.8 de-
creased (P < 0.05) moisture and salt adjusted cheese
yield more than preacidification to pH 6.0 with both
citric acid (trial 1) and acetic acid (trial 2). In trial 2,
preacidification to pH 5.8 with citric acid decreased (P
< 0.05) moisture and salt adjusted cheese yield more
than preacidification to pH 5.8 with acetic acid. The
progressive reduction in moisture and salt adjusted
cheese yield with increasing preacidification was
caused by several factors. As mentioned previously, pre-
acidification resulted in a substantial reduction in cal-
cium recovery in the cheese (Tables 3 and 4) and a
tendency for decreased protein recovery in the cheese.
This would decrease moisture and salt adjusted yield.
Since the same milk was used for all treatments
within each trial, theoretical yield using the Van Slyke
formula was not affected (P > 0.05) by preacidification in
either trial. Differences in theoretical yields calculated
among treatments in both trials with the Barbano-1
formula were small. Theoretical yield using the Bar-
bano-2 formula decreased (P < 0.05) with all levels of
preacidification in trials 1 and 2, as expected because
the calcium phosphate retention factor was adjusted by
treatment to account for the observed reductions in
calcium retention in the cheese.
Yield efficiency (composition adjusted yield/theoreti-
cal yield) determined with the Van Slyke theoretical
yield formula and the Barbano-1 and Barbano-2 pre-
dicted yield formula was significantly (P < 0.05) affected
by preacidification in trials 1 and 2. In trial 1, the con-
trol had a significantly (P < 0.05) higher yield efficiency
than the pH 6.0 citric and pH 5.8 citric treatments
using the Van Slyke formula and Barbano-1 formula.
However, only the pH 5.8 citric treatment was signifi-
cantly different from the control in trial 1 using the
Journal of Dairy Science Vol. 83, No. 4, 2000
656 METZGER ET AL.

Table 5. Mean (N = 3) actual, moisture and salt adjusted, and theoretical (modified Van Slyke, Barbano-
1 and Barbano-2) cheese yields and yield efficiencies for trial 1 (preacidification with citric acid).
Treatment
pH 6.0 pH 5.8
Citric Citric
Component Control acid acid SEM F test LSD1
(kg/100 kg)
Yield
Actual 7.45 7.22 7.24 0.051 0.14 NS2
Adjusted to 55%
moisture and
1.5% salt 7.33a 7.15b 6.95c 0.027 <0.01 0.104
Van Slyke 7.10 7.10 7.10 ... ... ...
Barbano-1 7.48 7.50 7.54 0.018 <0.01 0.072
Barbano-2 7.48a 7.41b 7.37b 0.018 <0.01 0.071
(%)
Efficiency
Van Slyke 103.3a 100.8b 97.7c 0.490 <0.01 1.60
Barbano-1 97.9a 95.43b 92.1c 0.408 0.01 1.60
Barbano-2 97.9a 96.6a 94.3b 0.416 <0.01 1.63
Means within same row not sharing common superscripts are different (P < 0.05).
a,b,c
1
Least significant difference (P = 0.05).
2
NS = Not significant.

Barbano-2 formula. In trial 2, the control treatment
had a significantly (P < 0.05) higher yield efficiency
than the remaining treatments using the Van Slyke,
Barbano-1, and Barbano-2 theoretical yield formulas.
The yield efficiency of all the treatments in trials 1 and
2 were higher with the Van Slyke formula than with
the Barbano-1 or Barbano-2 formulas. The modified
Van Slyke formula works well on full fat Mozzarella,
but progressively underestimates theoretical yield on
lower fat Mozzarella (21). Thus, the yield efficiencies
for the control in both trials were >100%. The yield
efficiency estimated with the modified Van Slyke for-
mula progressively decreases with increasing level of
preacidification because the modified Van Slyke for-
mula using a fixed 1.13 factor can not compensate for
differences in calcium loss into the whey. The yield
efficiency for the control using the Barbano formula for
trials 1 and 2 were 97.9 and 99.7%, respectively. Why
was the yield efficiency using the Barbano theoretical
yield formula lower in trial 1 than trial 2 for the control?

Table 6. Mean (N = 4) actual, moisture and salt adjusted, and theoretical (modified Van Slyke, Barbano-
1, and Barbano-2) cheese yields and yield efficiencies for trial 2 (preacidification with acetic and citric acid).

Treatment
pH 6.0 pH 5.8 pH 5.8
Acetic Acetic Citric
Component Control acid acid acid SEM F test LSD1
(kg/100 kg)
Yield
Actual 7.34a 6.91b 6.95b 7.03b 0.059 <0.01 0.187
Adjusted to
55% moisture
and 1.5% salt 7.35a 7.17b 7.10c 6.99d 0.020 <0.01 0.065
Van Slyke 7.00 7.00 7.00 7.00 ... ... ...
Barbano-1 7.37b 7.35b 7.37b 7.40a 0.007 <0.01 0.023
Barbano-2 7.37a 7.31b 7.28c 7.22d 0.008 <0.01 0.024
(%)
Efficiency
Van Slyke 105.1a 102.4b 101.4c 99.8d 0.287 <0.01 0.921
Barbano-1 99.7a 97.5b 96.3c 94.4d 0.303 <0.01 0.970
Barbano-2 99.7a 98.1b 97.4bc 96.7c 0.325 <0.01 1.040

Means within same row not sharing common superscripts are different (P < 0.05).
a,b,c,d
1
Least significant difference (P = 0.05).

Journal of Dairy Science Vol. 83, No. 4, 2000

 PREACIDIFICATION AND MOZZARELLA YIELD
Figure 1. Control cheese yield efficiency minus treatment cheese
yield efficiency (yield loss, %) using the Barbano-1 formula (total
percentage yield loss) and Barbano-2 formula (yield loss due to fat
and protein only) for trials 1 and 2.
The Barbano theoretical yield formula uses standard
values for cheese fat and protein recovery. However,
differences in cheese fat and protein recovery (Tables
3 and 4) between the control in trials 1 and 2 were
observed. In trial 2, the cheese fat and protein recovery
were close to the standard values in the Barbano theo-
retical yield formula, while in trial 1 the cheese fat
recovery was higher than the standard value and the
protein recovery was lower than the standard value.
The difference between the values produced by the
Barbano-1 and Barbano-2 theoretical yield formulae
allows determination of how much of the reduction in
yield efficiency, caused by preacidification, resulted
from calcium reduction versus changes in fat and pro-
tein recovery in the cheese. The reductions in yield
efficiency calculated with the Barbano-1 and Barbano-
2 formulae are shown in Figure 1. In trial 1, when
the pH 6.0 citric and pH 5.8 citric treatments were
compared to the control, yield efficiencies using the Bar-
bano-1 formula were lower by 2.5 and 5.8%, respec-
tively, and using the Barbano-2 formula were lower by
1.3 and 3.6%, respectively. In trial 2, when the pH 6.0
acetic, pH 5.8 acetic, and pH 5.8 citric treatments were
compared to the control, yield efficiencies using the Bar-
bano-1 formula were lower by 2.2, 3.4, and 5.2%, respec-
tively, and using the Barbano-2 formula were lower by
1.6, 2.3, and 3.0%, respectively. Preacidification to pH
6.0 and pH 5.8 with citric acid resulted in a total yield
efficiency reduction of about 2.5 and 5.5%, respectively,
while preacidification pH 6.0 and 5.8 with acetic acid
resulted in a total yield efficiency reduction of about
2.7 and 3.7%, respectively. The portion of the decrease
in total yield efficiency caused by fat and protein losses
657
(reduction in yield efficiency using Barbano-2 formula
divided by reduction in yield efficiency using Barbano-
1 formula) was 52 to 62% with citric acid and 68 to 73%
with acetic acid.
CONCLUSIONS
All levels of preacidification reduced the calcium con-
tent of low fat Mozzarella cheese. Preacidification with
citric acid versus acetic acid, at equal pH values, re-
duced cheese calcium more. Preacidification affected
calcium, protein, and fat recovery in the cheese. Cheese
yield efficiency decreased by about 3.4% for acetic acid
and about 5.5% for citric acid when milk was preacidi-
fied to pH 5.8. A large portion (> 50%) of the decrease
in cheese yield efficiency was caused by losses of fat
and protein in whey. The potential improvements in
cheese functional properties as a result of calcium re-
duction need to be balanced against the yield reduction
caused by preacidification.
ACKNOWLEDGMENTS
The authors thank Joanna Lynch, Maureen Chap-
man, Laura Landolf, and Pat Wood for technical sup-
port and the Northeast Dairy Foods Research Center
and Dairy Management Inc. (Rosemont, IL) for finan-
cial support.
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