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Effect of adding CaCl2 or whey protein to cow's milk on Mozzarella cheese

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Conference Paper · November 2008

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The 4th Arab Mansoura Conference of Food and Dairy Science & Technology. Faculty of
Agriculture, Mansoura University, Egypt, 10-14 November 2008

Effect of adding CaCl2 or whey protein to cow's milk on Mozzarella

cheese properties
El-Tahra, M. A. Ammar*, Ismail, M. M.**1 and El-Metwally, R. I.**

* Dairy Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.

** Dairy Technology Department, Animal Production Research Institute, Agriculture
Research Center, Giza, Egypt.
1
** Corresponding author email: abo-omar98@hotmail.com

SUMMARY
Mozzarella cheese was produced from cow’s milk after increasing its calcium
content to 0.175 or 0.200% by adding CaCl2. Also, the cheese was made from cow's
milk (4 or 2% fat) mixed with 0.5 and 1% whey protein. The resultant cheese was
examined for chemical, rheological and organoleptic properties when fresh and during
storage period at 5°C for 28 days.
Results showed that addition of CaCl2 to cow’s milk decreased RCT while
increased curd tension and curd syneresis. As a result of adding whey protein to cow’s
milk, rennet coagulation time (RCT) and curd tension decreased and curd syneresis
increased.
Whey of Mozzarella cheese made from cow's milk fortified with CaCl2 had
higher TS and calcium contents than that of cheese made from cow's milk alone,
whereas the values of acidity, pH, fat, TN and ash were similar in both types of
cheese whey. Adding whey protein to cheese milk increased acidity, TS, TN, ash and
calcium contents of cheese whey.
Adding CaCl2 to cow's milk increased the Mozzarella cheese yield, TS, fat,
Fat/DM, ash, salt, salt in moisture, calcium, TVFA and oiling-off values while
decreased acidity, WSN, WSN/TN, NPN, NPN/TN, meltability values and the
organoleptic evolution scores of resultant cheese. TN values were similar in both
cheeses without and with adding CaCl2 to cow's milk.
Addition of 0.5 or 1% whey protein to cheese milk increased the yield, acidity,
TS, fat, Fat/DM, ash, salt, salt in moisture, calcium, TVFA and TN, TN/DM WSN,
WSN/TN, NPN, NPN/TN, the meltability, oiling-off values and the organoleptic
evolution scores of Mozzarella cheese.
The microstructure of cheeses made from cow's milk contained 0.200% calcium
showed a dense protein matrix compared with that made from cow's milk contained
0.145%. Similar microstructure was observed in scanning electron micrographs for
control and cheeses with 0.5 or 1% whey protein.
Data of pizza baking test reveled that control and 0.175% calcium milk cheese
were similar in appearance whereas the 0.200% calcium milk cheese had lower
suitable appearance. Adding 0.5 or 1% whey protein to cow's milk improved the
meltability, flow, shred fusion, and reduced browning and blistering of Mozzarella
cheese.
Key ward: Mozzarella cheese – Cow's milk – CaCl2 -Whey protein.

1
 INTRODUCTION
Mozzarella cheese is originally made from water buffalo’s milk in Italy.
Nowadays, it is made all over Europe and USA from cow’s milk. Recently,
Mozzarella cheese of satisfactory quality has been made from buffalo’s and cow’s
milk or their mixtures (Kindstedt, 1993). In Middle East (especially in Syria)
Mozzarella cheese known as (El-Medaffarah) is mainly made from ewe‫ۥ‬s milk and
sometimes goat‫ۥ‬s milk may be added (Darwish 1977).
Calcium plays an important role during manufacturing and also in deciding
functionality of cheese. Calcium helps to form a network structure during coagulation
of milk, provides linkages within and among casein micelles and affects almost all
aspects of cheese manufacture (Joshi et al., 2003). The majority (63.7%) of total
calcium in milk is present as a colloidal calcium phosphate (CCP). This CCP
functions as a bridge to bind submicelles in the casein micelles.
On the other hand, whey protein is a pure, natural, high quality co-product of the
cheese making process. Whey protein is more soluble than casein and also has a
higher quality rating. It is often referred to as the "Gold Standard" of protein as it is
the most nutritious protein available. During the last 15-20 years the value of whey
protein has become more widely known, especially in the area of sports nutrition.
So, this study was conducted to investigate the effect of adding CaCl2 or whey
protein to cow's milk on some chemical and rheological properties of Mozzarella
cheese.
Materials and Methods
Materials:
Fresh cow's milks were obtained from El-Serw Animal Production Research
Station, Animal Production Research Institute, Agriculture Research Center. Whey
protein was prepared in El-Serw Animal Production Research Station from Ras
cheese whey. Yoghurt starter culture (Yo-Fex,YC-350,DVS) consists of
Streptococcus saliverus ssp. thermophilus and L. delbrueckii ssp. bulgaricus were
obtained from Chr. Hansen’s Lab A/S Copenhagen, Denmark. Liquid calf rennet was
obtained from local market and was added to milk at a ratio of 30 mL 100 kg-1milk.
Dry coarse commercial food grade salt was obtained from El-Nasr Company of
Alexandria. All chemicals were of laboratory grade.
Methods:
Mozzarella cheese manufacture:
Preparation of cheese milk and whey protein:
Fresh morning cow’s milk was standardized to 2% fat. As followed in many
laboratories of Ras cheese production in Dakahlia and Domait Governorates, Ras
cheese whey was heated to 95°C for 10 min. Because of high acidity of the whey,
acidification step was not required. The whey drained in cloth bag as Labneh
manufacture and after 3 hours the bags were put in wooden frames and pressed
overnight. Following early day, whey protein was taken out and kept in refrigerator at
5°C tile using.
Cheese manufacture:
Mozzarella cheese was made as described by Kosikowski (1982). Seven
treatments of Mozzarella cheese were manufactured as fallow:
◘ Mozzarella cheese made from whole cow’s milk (4% Fat) (Treatment A)
◘ Mozzarella cheese made from whole cow’s milk (4% Fat) and 0.175% Ca
(Treatment B)
◘ Mozzarella cheese made from whole cow’s milk (4% Fat) and 0.200% Ca
(Treatment C)

2
◘ Mozzarella cheese made from whole cow’s milk (4% Fat) and 0.5% whey protein
(Treatment D)
◘ Mozzarella cheese made from whole cow’s milk (4% Fat) and 1.0% whey protein
(Treatment E)
◘ Mozzarella cheese made from standardized cow’s milk (2% Fat) and 0.5% whey
protein (Treatment F)
◘ Mozzarella cheese made from standardized cow’s milk (2% Fat) and 1.0% whey
protein (Treatment G)
Figure (1) describes Mozzarella cheese manufacture.
The resultant cheese were stored at 5°C and analyzed when fresh and after 7, 14, 21
and 28 days of storage period.
Methods of analysis:
Milk and whey samples were analyzed for titratable acidity (TA), total solids
(TS), fat, total protein and ash contents according to Ling (1963). The pH values were
estimated using a pH meter type CG 710. The curd tension was determined using the
method of Chandrasekhara et al., (1957).The rennet coagulation time (RCT) was
determined according to Davies and White (1958) whereas the curd syneresis was
measured as given by Mehanna and Mehanna (1989). Actual cheese yield was
determined by dividing the weight of cheese by the weight of milk used to make
cheese, multiplied by 100. Adjusted to 55% moisture and 1.5% salt cheese yield was
calculated by Metzger et al., (2000) formula:
Adjusted yield = (actual yield × (100 − actual moisture + actual salt)) / (100 − (55 +
1.5))
Cheese was analyzed for total solids (TS), titratable acidity (TA), pH, fat, total
nitrogen (TN), water soluble nitrogen (WSN), Non-protein-nitrogen (NPN) and ash
contents according to Ling (1963). Salt contents of Mozzarella cheese were estimated
using Volhard method according to Richardson (1985). Calcium was determined
using calcin as indicator by the method described by Graham et al., (1962) and as
modified by Abdel-Kader (1993). Total volatile fatty acids (TVFA) was determined
as described by Kosikowski (1978), and expressed as ml of 0.1N NaOH, 100 g-1
cheese. Meltability was determined as described by Olson and Price (1985) with the
modification of Rayan et al., (1980) while oiling-off was measured as described by
Ghosh and Singh (1992).
The cheese microstructure was evaluated in fresh Mozzarella cheese using the
thin-sectioning technique according to Taneya et al., (1980). Small pieces (1 mm3
each)of cheese were prefixed for 2 hrs in a 2% glutaraldehyde solution (pH 6.8)
buffered with sodium cacodylate, followed by a postfixation for 2 hrs in a mixture of
osmium tetroxide and 2%tannic acid at pH 6.0 (cacodylate buffer). The fixed samples
were dehydrated stepwise in ethanol, and then transferred into acetone. Samples were
dried in critical point dryer and coated with thin film of gold. Samples were viewed
using a scanning electron microscope (Jeol JSM-T20). Micrographs were taken in
several locations on each sample, and those shown here demonstrate representative
characteristics of each treatment.
The cheese samples were scored for flavor (50 points), body and texture (35
points) and appearance and color (15 points) by ten panelists in El-Serw Animal
Production Research Station. Pizza bake test was occurred as follow: shredded
Mozzarella cheese (300 g) was used as a topping on a pizza base covered with a thin
layer of tomato paste. One-half of each pizza was topped with fresh cheese shreds
(150 g) and the other half was covered with cheese stored for 28 days. Pizzas were
baked at about 260°C for 7 min using home conventional oven. The appearance of the

3
Figure (1). Flow sheet of Mozzarella cheese manufacture
Fresh cow’s milk

Whole cow’s milk Standardized cow’s milk

(2%Fat)
Whole Whole Whole cow’s
cow’s milk cow’s milk and milk and 0.200% Heating at 55°C/2min Heating at 55°C/2min
(Treat. A) 0.175% CaCl2 CaCl2 (Treat. C)
(Treat. B)
Adding 0.5% Adding 1.0% Adding 0.5% Adding 1.0%
whey protein whey protein whey protein whey protein
and blending and blending and blending and blending
at 2000rpm at 2000rpm at 2000rpm at 2000rpm
/5min (Treat. D) /5min(Treat. E) /5min(Treat. F) /5min (Treat. G)

Culture adding (1%) (preripening 33°C for 15 min)

Renneting at 35°C (40-50min)

Cutting the curd 1x1x1cm - Stirring the curd

Scalding to 40-45°C through 50 min.

Drainage-off the whey (pH 6.3-6.1)

Cheddaring (30-60min)

Cutting of the curd (pH 5.4-5.3)

Kneading of the curd (hot water at 75-85°C for 4-5 min- cheese 58-65°C)

Molding of the cheese (Braiding- Rectongular- Ball- Loaf.....etc.)

Brining the cheese – Immersing in 25% salt cold brine solution for 1 h.

Packing and storage (At 5±1°C for 28 days)

4
 cheese on the pizza was then evaluated for shred fusion and melt, color, and extent of
blistering.
The obtained results were statistically analyzed using a software package (SAS,
1991) based on analysis of variance. When F-test was significant, least significant
difference (LSD) was calculated according to Duncan (1955) for the comparison
between means. The data presented, in the Tables, are the mean (± standard deviation)
of 3 experiments.
RESULTS AND DISCUSSION
Chemical composition of milk and whey protein used in Mozzarella cheese
manufacture
As shown in the Table 1, acidity, pH, TS, fat, total protein ash contents of cow's
milk contained 0.145 (control- Treatment A), 0.175 (Treatment B) and 0.200%
calcium (Treatment C) were similar. From the other side, adding 0.5 or 1% whey
protein to whole cow' milk (4% fat) or standardized cow's milk ( 2% fat) increased the
acidity, TS, total protein, ash and calcium contents and decreased the pH values.
Some rheological properties of cheese milk
Results in Table 2 illustrated the effect of increasing calcium content of cow's
milk to 0.175 and 0.200% using CaCl2 on RCT, curd tension and curd syneresis. The
average rennet coagulation time of cow's milk was 231 sec., while addition of CaCl2
to raise calcium level of cow's milk to 0.200% decreased the RCT to 195 sec. This
may be due to reduced soluble casein concentrations. Also, the addition of calcium
chloride to cow's milk increased curd tension and increasing the concentration of
CaCl2, increased the curd tension values. Increasing of curd tension values of milk by
adding CaCl2 may be due to the effect of CaCl2 on precipitation of paracasein, which
resulted in the firmness of the curd (Ammar, 1999). Addition of CaCl2 to raise
calcium content of cow's milk to 0.175 and 0.200 % increased the amount of whey
excluded from the curd.
On the other hand, the effect of adding different concentrations of whey protein
(0.5 and 1%) to cow's milk on RCT, curd syneresis was presented in Table 2.
Addition of whey protein to cow's milk lowered the RCT values. Replacement of cow'
milk by whey protein decreased the curd tension and as ratios of whey protein added
to milk increased, also the decreasing rates of curd tension increased. On contrary,
adding whey protein to cow's milk increased curd syneresis and at higher levels of
whey protein led to more curd syneresis.

Table (1). Chemical composition of milk and whey protein (WP) used in
Mozzarella cheese manufacture
Treatment pH Total
Acidity TS Fat Ash Salt Ca
values Protein
% % % % % %
%
Whole
0.18 6.60 12.38 4.0 3.73 0.64 - 0.145
cow's Milk (Control)
WCM * (0.175%Ca) 0.18 6.61 12.38 4.0 3.72 0.67 - 0.175
WCM (0.200%Ca) 0.18 6.61 12.39 4.0 3.74 0.69 - 0.200
WCM + 0.5% WP 0.19 6.57 12.81 4.1 3.86 0.73 - 0.145
WCM+ 1% WP 0.20 6.52 13.42 4.0 4.06 0.77 - 0.149
PSCM**+ 0.5% WP 0.19 6.58 11.02 2.0 3.98 0.70 - 0.150
PSCM + 1% WP 0.20 6.50 11.88 2.0 4.11 0.75 - 0.150
Whey Protein 0.30 5.15 23.6 0.8 14.4 7.81 6.93 0.179
* WCM: whole cow's milk
**PSCM: part skimmed cow's milk

5
Table (2). Effect of adding CaCl2 or whey protein (WP) on RCT, curd tension
and curd syneresis of cow's milk (CM)
Curd Curd syneresis (gm/15 gm of curd)*
Treatments RCT(sec.) tension Time (min)
(gm) 10 30 60 120
Control 231 37.11 3.41 5.94 6.82 7.69
0.175 % Ca 202 41.73 4.03 6.28 7.36 8.54
0.200 % Ca 195 43.22 4.39 6.43 7.49 8.62
CM+0.5%WP 163 28.38 4.21 6.64 8.09 8.89
CM + 1%WP 139 24.86 4.50 6.92 8.47 9.39
*Whey excluded (grams) from 15 gm of curd kept at room temperature after 10, 30, 60 and 120min

Chemical composition of Mozzarella cheese whey

Results in Table 3 included the chemical composition of whey extracted from
various treatments of Mozzarella cheese. Increasing calcium content of cow's milk to
0.175 or 0.200% (Treatments B and C) significantly increased (P<0.001) TS and
calcium contents of cheese whey whereas, there is no significant differences in
acidity, pH, fat, TN and ash values of whey between control (treatment A) and
treatments B and C.
As shown in the above table, whey protein Mozzarella cheese whey had the
highest acidity percentages and the lowest pH values. This is may be due to the higher
acidity of whey protein. On the other hand, the addition of why protein to cow's milk
increased TS, total nitrogen, ash and calcium contents of extracted whey. Obtained
results of the acidity, pH, TS, fat and TN contents of Mozzarella cheese whey were
within the range obtained by El-Zoghby (1994) who used different milks in
Mozzarella cheese making.

Table (3). Chemical composition of Mozzarella cheese whey

Treatments Acidity % pH TS % Fat % TN % Ash % Ca %
A 0.28 5.45 8.95 0.6 0.18 0.57 0.148
B 0.27 5.50 9.11 0.5 0.19 0.56 0.153
C 0.27 5.57 9.30 0.6 0.19 0.58 0.157
D 0.29 5.43 9.08 0.5 0.19 0.60 0.150
E 0.30 5.40 9.78 0.6 0.18 0.63 0.151
F 0.30 5.41 9.23 0.6 0.20 0.59 0.155
G 0.32 5.35 9.66 0.6 0.20 0.62 0.158

Yield and chemical composition of Mozzarella cheese:

From Table 4, it could be concluded that, addition of calcium chloride to cow's
milk to raising its calcium ratios to 0.175 and 0.200% (Treatments B and C) increased
both actual and adjusted yields of fresh Mozzarella cheese as compared with control
(Treatment A).
Ammar (1999) stated that adding 0.01% calcium chloride to fresh or cooled
buffaloe's milk increased the yield of resultant Domiati cheese.
On the other hand, adding 0.5 or 1% whey protein to cow's milk increased the
yield of Mozzarella cheese.
As it is expected, reduction of fat content of cow's milk from 4 to 2%
(Treatments F and G) decreased the actual and adjusted yields of cheese. Rudan et
al., (1999) found that the actual and theoretical yields significantly decreased as the
fat content in the cheese decreased.
Statistical analysis of variance (Table 8) showed that the differences in yield
values between treatments were highly significant (P<0.001).

6
 Results of acidity and pH values were tabulated in Table (4). Acidity of
Mozzarella cheese made from cow's milk without adding CaCl2 (Control A) was
slightly higher than that cheese made form cow's milk fortified with CaCl2
(Treatments B and C) for fresh cheese and within refrigerated storage. Addition of 0.5
and 1% whey protein to cow's milk raised the level of acidity and lowered pH values
of Mozzarella cheese at zero time and during ripening period. pH values had an
opposite trend of acidity of the resultant cheese during the storage period for all
treatments. The acidity values significantly increased (P<0.001) while pH values
significantly decreased (P<0.001) with progressing storage period in all samples.
These results are in agreement with El-Batawy et al., (2004).
Changes in total solids (TS), fat and Fat/DM contents of different cheese
treatments during storage period were illustrated in Table 4. It was noticed that TS, fat
and Fat/DM values of Mozzarella cheese gradually increased during the storage
period. This increasing of these contents is due to the moisture evaporation. Similar
results of TS and fat increasing in Mozzarella cheese during storage period were
reported by Abd El-Hamid et al., (2006) and Ismail et al., (2007). Adding of CaCl2
to cow's milk increased TS, fat and Fat/DM contents of resultant Mozzarella cheese.
From the present table, it could be seen that whey protein Mozzarella cheese
(Treatments D and E) had higher TS and fat contents than that of control cheese
(Treatment A) during the storage period. Similar results were found by Punidadas et
al., (2007). Because treatments F and G made from part skimmed cow's milk (2% fat)
mixed with 0.5 or 1% whey protein, thus the total solids, fat and Fat/DM values were
lower in these treatments as compared with treatments D and E which made from
whole cow's milk and same ratios of whey protein.
It is clear from the results in Table 4 that in all treatments, there were a slight
increase in ash, salt, salt in moisture and calcium contents of the resultant cheese
during storage due to loss in moisture content. Calcium chloride cheese (Treatments B
and C) had higher ash, salt and salt in moisture values than that of control cheese
(Treatment A).
It was not surprising that increasing calcium content of cow's milk to 0.175 and
0.200% by addition of CaCl2 significantly raised calcium content of fresh Mozzarella
cheese and during storage period and an increase in CaCl2 concentration added to
milk caused a proportional increase in the calcium values of the cheese. Mcmahon et
al., (2005) showed that fortification of cheese milk with 14 g of CaCl2 in the low pH
cheeses increased calcium content in the finished Mozzarella cheese to 0.6%. This is
similar to the level of calcium found in commercially manufactured low moisture,
part-skim Mozzarella cheese (USDA, 1980).
Substitution of part of the cow's milk used for manufacturing of Mozzarella
cheese with whey protein decreased the calcium content of cheese. On the other hand,
cheese made from 2% fat cow's milk and whey protein had lower calcium content
than that of cheese made from 4% fat cow's milk and whey protein. Conversely,
Rudan et al., (1999) found that the calcium content of Mozzarella cheese increased
progressively as fat content was reduced, with low fat containing 50% more calcium
per gram than low-moisture, part-skim Mozzarella.
Total nitrogen (TN) values were similar in the cheese made without or with
adding calcium chloride to cow's milk (Table 5). With regard to water soluble
nitrogen (WSN), samples B (0.175% calcium cow's milk) had WSN values lower than
that of samples A and C (control and 0.200% calcium cow's milk respectively). Also,

7
Table (4). Effect of increasing of calcium concentration or adding whey protein to cow's milk on yield and some chemical
properties of Mozzarella cheese
Storage Actual Adjusted TS Fat F/DM Ash Salt Salt in Ca
Treatments Acidity % pH
Period (days) Yield % Yield* % % % % % % moisture % %
0 9.15 11.59 0.81 5.44 53.44 22.7 42.47 3.42 1.70 3.53 0.610
7 0.87 5.36 53.86 23.2 43.07 3.78 1.77 3.69 0.637
A 14 0.91 5.31 54.24 23.7 43.69 3.94 1.84 3.86 0.651
21 0.94 5.27 54.52 24.1 44.20 4.12 1.91 4.03 0.670
28 0.97 5.21 54.83 24.6 44.86 4.26 2.06 4.36 0.679
0 9.25 11.76 0.79 5.48 53.52 22.9 42.78 3.50 1.78 3.68 0.635
7 0.84 5.41 53.96 23.6 43.73 3.83 1.90 3.96 0.659
B 14 0.89 5.33 54.36 24.0 44.15 3.96 1.99 4.17 0.679
21 0.92 5.28 54.63 24.4 44.88 4.20 2.06 4.34 0.691
28 0.94 5.25 54.96 24.8 45.12 4.31 2.10 4.45 0.705
0 10.97 13.99 0.78 5.50 53.64 23.3 43.43 3.56 1.86 3.85 0.643
7 0.84 5.42 53.98 23.8 44.09 3.91 2.02 4.20 0.670
C 14 0.88 5.35 54.43 24.3 44.64 4.18 2.10 4.41 0.692
21 0.91 5.31 54.70 24.7 45.15 4.38 2.14 4.50 0.710
28 0.92 5.27 55.06 25.1 45.58 4.54 2.19 4.64 0.723
0 11.29 14.27 0.91 5.33 53.54 22.9 42.77 3.46 1.76 3.62 0.603
7 0.98 5.23 53.90 23.7 43.97 3.66 1.85 3.83 0.629
D 14 1.03 5.17 54.60 24.5 44.87 3.97 1.93 4.02 0.649
21 1.07 5.13 54.83 24.8 45.23 4.16 2.03 4.25 0.670
28 1.11 5.08 55.11 25.0 45.36 4.35 2.07 4.37 0.678
0 11.56 14.62 0.94 5.26 53.66 23.1 43.05 3.51 1.82 3.74 0.596
7 1.01 5.20 54.13 23.8 43.97 3.82 1.93 3.99 0.624
E 14 1.04 5.13 54.71 24.7 45.15 3.97 1.99 4.13 0.640
21 1.09 5.10 54.96 25.1 45.67 4.20 2.07 4.32 0.663
28 1.15 5.06 55.22 25.3 45.82 4.40 2.15 4.51 0.672
0 8.91 10.52 0.93 5.24 49.56 17.5 35.31 3.40 1.78 3.41 0.592
7 0.99 5.18 50.10 17.9 35.72 3.78 1.92 3.70 0.620
14 1.05 5.11 50.39 18.6 36.91 3.90 2.01 3.89 0.633
F
21 1.09 5.07 50.76 19.0 37.43 4.09 2.05 3.99 0.657
28 1.13 5.04 51.06 19.4 37.99 4.25 2.10 4.11 0.669
0 9.03 10.83 0.96 5.23 50.32 17.7 35.17 3.43 1.86 3.60 0.594
7 1.05 5.15 50.74 18.2 35.86 3.80 1.97 3.84 0.615
G 14 1.09 5.09 50.98 18.7 36.68 3.96 2.08 4.07 0.630
21 1.12 5.06 51.46 19.1 37.11 4.11 2.14 4.22 0.655
28 1.15 5.01 51.76 19.5 37.67 4.28 2.22 4.39 0.665
* moisture and salt adjusted cheese yield.

8
Table (5). Effect of increasing of calcium concentration or adding whey protein to cow's milk on TN and some ripening indices of
Mozzarella cheese
Treatments Storage TN TN/DM WSN WSN/TN NPN NPN/TN TVFA*
period(days) % % % % % %
0 3.65 6.83 0.272 7.45 0.146 4.01 8.48
7 3.78 7.01 0.290 7.67 0.166 4.39 10.14
A 14 3.90 7.19 0.302 7.74 0.181 4.64 13.77
21 3.95 7.24 0.317 8.02 0.192 4.86 16.01
28 4.02 7.33 0.341 8.48 0.210 5.22 20.21
0 3.62 6.76 0.260 7.18 0.140 3.86 8.62
7 3.75 6.94 0.281 7.49 0.161 4.29 10.20
B 14 3.88 7.14 0.294 7.51 0.175 4.47 14.00
21 3.94 7.21 0.305 7.74 0.189 4.79 16.62
28 4.00 7.27 0.324 8.10 0.205 5.12 21.41
0 3.63 6.76 0.268 7.38 0.138 3.80 8.80
7 3.77 6.98 0.290 7.69 0.157 4.16 10.40
C 14 3.89 7.14 0.300 7.71 0.171 4.39 14.20
21 3.94 7.20 0.314 7.96 0.183 4.64 16.82
28 4.01 7.28 0.335 8.35 0.201 5.01 21.60
0 3.71 6.96 0.280 7.54 0.153 4.12 8.50
7 3.82 7.17 0.296 7.74 0.172 4.50 10.20
D 14 3.93 7.28 0.311 7.91 0.190 4.83 14.10
21 3.99 7.34 0.325 8.14 0.214 5.36 16.10
28 4.00 7.37 0.350 8.62 0.227 5.59 20.28
0 3.75 7.05 0.287 7.65 0.160 4.26 8.60
7 3.86 7.20 0.299 7.74 0.181 4.68 10.22
E 14 3.95 7.32 0.317 8.02 0.202 5.15 14.20
21 4.01 7.39 0.330 8.22 0.222 5.53 16.20
28 4.09 7.50 0.358 8.75 0.243 5.94 20.40
0 3.75 7.56 0.284 7.57 0.157 4.18 6.28
7 3.84 7.66 0.300 7.81 0.176 4.58 8.47
14 3.92 7.77 0.318 7.82 0.198 5.05 10.40
F
21 4.01 7.89 0.327 8.17 0.219 5.46 13.62
28 4.07 7.97 0.355 8.72 0.235 5.77 16.80
0 3.82 7.59 0.293 7.67 0.170 4.45 6.40
7 3.93 7.74 0.314 7.98 0.197 5.01 8.60
G 14 4.00 7.84 0.334 8.35 0.218 5.45 12.80
21 4.04 7.85 0.352 8.71 0.239 5.91 14.00
28 4.10 7.92 0.376 9.17 0.358 8.73 17.02
* expressed as ml 0.1 NaOH 100 g -1 cheese.

9
increasing calcium contents of cow's milk to 0.175 and 0.200% decreased non-
protein- nitrogen (NPN) values of the resultant Mozzarella cheese. Results of TN/DM,
WSN/TN and NPN/TN were lower in the Mozzarella cheese made by adding CaCl2 to
cow's milk comparing with that of control cheese. These results agreed with Ammar
(1999) who stated that calcium chloride treatment of milk decreased the TN/DM, SN,
SN/TN, NPN and NPN/TN% of the resultant Domiati cheese during ripening period,
compared with the corresponding cheese without adding CaCl2 .
As expected, addition of 0.5 or 1% whey protein to 4 or 2% fat cow's milk
increased TN, TN/DM, WSN, WSN/TN, NPN and NPN/TN values of fresh
Mozzarella cheese and during storage period. The increasing in the above values was
more noticeable in cheese made from 2% fat cow's milk than that made from 4%fat
milk. As it is observed from Table 5, TN, TN/DM, WSN, WSN/TN, NPN and
NPN/TN values increased gradually during storage period in all cheese treatments.
These results were confirmed by those obtained by El-Safty et al., (2004).
TVFA values of Mozzarella cheese at zero time and during storage period were
tabulated in Table 5. As storage time increased, TVFA contents significantly (P<
0.001) increased in all cheese treatments. Adding calcium chloride to cow's milk
increased TVFA of Mozzarella cheese. Replacement of cheese milk by 0.5 or 1%
whey protein increased TVFA contents of fresh cheese and during ripening period.
Meltability and oiling off of Mozzarella cheese:
Meltability is defined as the ease with which cheese flows or spreads upon heating
(Muthukumarappan et al., 1999). As the calcium added to cheese milk was raised
from 0.145% in the control to 0.175% in B treatment and 0.200% in C treatment, a
decrease (P < 0.001) in meltability of the Mozzarella cheeses was observed (Table 6).
It is generally accepted that reduced calcium cheese has higher meltability. Caseins
are better able to emulsify fat in curd with reduced calcium (McMahon et al., 1993).
Such emulsified fat has fewer tendencies to exude and coalesce from the cheese upon
melting. This will result in higher melting; a desirable characteristic in Mozzarella
cheese .Also, Paulson et al., (1998) found that increasing calcium content resulted in
decreased melting of nonfat Mozzarella cheese. Joshi et al., (2004) stated that the low
calcium cheeses had almost 2.6 times greater melt area compared with the control.
These results establish the impact of calcium reduction on meltability of cheese.
Reducing the calcium causes increased inter action of proteins with surrounding
serum, causing more hydration of proteins and better melting of the cheese.
Data in Table 6 cleared that addition of 0.5 or 1% whey protein to cow's milk
increased the meltability of Mozzarella cheese. As known, meltability increase with
increasing of fat content of cheese, thus sample F (2% fat cow's milk and 0.5% whey
protein) had lower meltability values than that of sample A (4% fat cow's milk).
However, adding 1% whey protein to reduced fat milk (2%) increased the meltability
of resultant Mozzarella cheese (sample G). This means that, substitution of 2% fat
milk with 1% whey protein improved the meltability of cheese. This may be attributed
to the increasing of cheese acidity as a result of adding whey protein which lowered
the calcium content of cheese. Reducing the calcium causes increased inter action of
proteins with surrounding serum, causing more hydration of proteins and better
melting of the cheese (Joshi et al., 2004).
Increasing calcium content of cow's milk to 0.175% (Treatment B) increased
oiling-off values of fresh Mozzarella cheese by 18.18% (Table 6) while raising milk
calcium percentage to 0.200% (Treatment C) increased it further (22.73%). Addition
of 0.5 or 1% whey protein to cow's milk increased results of oiling-off of fresh
Mozzarella cheese and within storage period.
The higher oiling-off values of treatments D and E (4% fat milk and 0.5 and
1% whey protein) than of treatments F and G (2% fat milk and 0.5 and 1% whey
11
protein) was undoubtedly related to higher fat content in the former. A direct
relationship between oiling-off and fat contents in commercial Mozzarella cheeses
was reported by Kindstedt and Rippe (1990) and Kindstedt et al.,(1992). The
oiling-off values of all samples cheeses increased (P < 0.001) during storage. Tunick
(1994) found that refrigerated storage of Mozzarella cheese increased free oil because
of proteolysis.

Table (6). Effect of increasing of calcium concentration or adding whey protein

to cow's milk on some rheological properties of Mozzarella cheese
Properties Treatments
Storage
period(days) G H I J K L M
0 60 53 51 65 73 57 62
Meltability 7 96 89 85 102 112 94 98
(mm) 14 117 109 104 121 130 112 120
21 144 135 130 150 156 144 144
28 166 159 156 170 177 163 170
0 2.2 2.6 2.7 2.5 2.5 1.6 1.8
7 3.0 3.2 3.2 3.1 3.2 2.1 2.4
Oiling off *
14 3.5 3.8 4.0 3.8 3.9 2.6 3.0
%
21 4.1 4.3 4.4 4.3 4.4 3.1 3.3
28 4.8 4.9 5.1 4.9 4.9 3.5 3.7
*area of fat diffusions
Scanning Electron Microscopy (SEM)
The changes of microstructure of Mozzarella cheese as a result of increasing
calcium levels of cow's milk or adding whey protein were visualized by scanning
electron microscopy (Figure 2). SEM was just done for A, C, E, F and G fresh
samples.
The microstructure of cheeses made from cow's milk contained 0.200% calcium
(Treatment C) showed a dense protein matrix compared with that made from cow's
milk contained 0.145% (control –A). On the other hand, similar microstructure was
observed in scanning electron micrographs for control and cheeses with 0.5 or 1%
whey protein.
Organoleptic properties
Table 7 shows the organoleptic evaluation of various treatments of resultant
Mozzarella cheese. As ripening advanced, the colour & appearance, body & texture
and flavour properties of cheese were significantly improved (P < 0.001). The high
calcium content cheese treatments (Treatments B and C) had lower scores of sensory
evolution than that of control cheese (Treatment A). Addition of 0.5 or 1% whey
protein to both 4 and 2% fat cow's milk improved the organoleptic properties of
Mozzarella cheese.
Pizza baking
Data of pizza baking test of fresh and 28 days old Mozzarella cheese made from
cow's milk contained 0.145, 0.175 and 0.200% calcium or made from cow's milk
mixed with 0.5 and 1% whey protein shown in Figure 3.
It could be seen that, at 28 days all 7 batches of cheese showed the minimum
appearance of the browning colour. This may be attributed to decomposition of
lactose to lactic acid which prevents Millard reaction. Also, the meltability of all
cheese samples improved at the end of storage period.
Samples of A and B (control and 0.175% calcium milk) were similar in
appearance whereas the treatment I had lower suitable appearance at zero time or after
28 days of cold storage.

11
 Results from figure 3 showed that adding 0.5 or 1% whey protein to 4 and 2% fat
cow's milk (Samples D, E, F and G) improved the meltability, flow, shred fusion, and
reduced browning and blistering of fresh Mozzarella cheese and within storage period
at 5°C.

Treatment G Treatment I

Treatment K Treatment L

Treatment M

Figure (2). Scanning electron micrographs of Mozzarella cheese made from

cow's milk fortified with CaCl2 or whey protein

12
Table (7). Sensory evaluation of Mozzarella cheese
Treatments Storage Color & Body Texture Flavour Total
period Appearance (35) (50) (100)
(days) (15)
0 10 28 39 77
7 11 29 39 79
G 14 12 30 41 83
21 12 31 43 86
28 13 32 43 88
0 8 29 39 76
7 9 31 40 80
H 14 10 32 42 84
21 11 32 44 87
28 11 32 44 87
0 8 28 37 73
7 9 28 38 75
I 14 10 29 40 79
21 11 31 42 84
28 13 31 42 86
0 8 29 38 75
7 10 30 41 81
J 14 11 31 42 84
21 12 32 42 86
28 12 34 43 89
0 9 28 39 76
7 9 29 40 78
K 14 11 31 42 84
21 12 32 43 87
28 13 33 44 90
0 8 26 38 72
7 10 27 40 77
L 14 11 29 41 81
21 12 30 42 84
28 12 31 43 86
0 9 27 39 75
7 9 29 42 80
M 14 11 31 43 84
21 12 33 45 89
28 13 33 45 90

13
 Treatment G Treatment H

Treatment I Treatment J

Treatment K Treatment L

Treatment M

Figure (3). Pizza baking of Mozzarella cheese made from cow's milk fortified
with CaCl2 or whey protein

14
 Table (8). Statistical analysis of Mozzarella cheese treatments
Effect of cheese treatments
Analysis
G H I J K L M LSD
Actual Yield % 9.15 e 9.25 d 10.97c 11.29b 11.56a 8.91 g 9.04 f 0.066***
Adjusted Yield% 11.59 e 11.76 d 13.99 c 14.28 b 14.63 a 10.53 g 10.84 f 0.083***
Whey acidity% 0.285a 0.27 a 0.265 a 0.295 a
0.30 a
0.30 a
0.32 a 0.059
Whey pH 5.45bc 5.50 b 5.57 a 5.43 c 5.40cd 5.41cd 5.35 d 0.066**
Whey TS% 8.96 e 9.11 d 9.30c 9.08d 9.78a 9.23c 9.66b 0.076***
Whey Fat% 0.6 a 0.5 a 0.61 a 0.5 a
0.61 a
0.6 a
0.6 a 0.202
Whey TN% 0.175a 0.19 a 0.19 a 0.19 a 0.18 a 0.20 a 0.20 a 0.053
Whey Ash% 0.57ab 0.56 b 0.59ab 0.60ab 0.63 a 0.59ab 0.62ab 0.064*
Whey Ca% 0.148d 0.153abc 0.157ab 0.150cd 0.152bcd 0.155abc 0.159 0.006***
Cheese acidity% 0.90c 0.87cd 0.86d 1.02 b
1.04 b
1.04b 1.08a 0.026***
Cheese pH 5.32b 5.32a 5.37a 5.19c 5.15d 5.13ed 5.11e 0.026**
Cheese TS% 54.18e 54.29d 54.36c 54.40b 54.54a 50.38g 51.05f 0.034***
Cheese Fat% 23.67d 23.95c 24.24b 24.18b 24.40a 18.48f 18.64e 0.078***
Cheese Ash % 3.91c 3.96b 4.12a 3.92 c
3.98 b
3.89 c
3.92c 0.042***
Cheese Salt% 1.86d 1.97b 2.06 a 1.93c 1.99 b 1.98 b 2.06a 0.031***
Salt in moisture 3.89d 4.12b 4.32a 4.02c 4.14b 3.88d 4.03c 0.074***
Cheese Ca% 0.649c 0.649b 0.688a 0.646d 0.639e 0.635f 0.632f 0.003***
Cheese TN% 3.86d 3.86 d 3.85 d 3.90 c 3.94b 3.92bc 3.98a 0.029***
Cheese WSN% 0.305d 0.293 e 0.302d 0.313c 0.318b 0.317b 0.334a 0.003***
Cheese NPN% 0.179e 0.174f 0.170g 0.191d 0.202b 0.197c 0.237a 0.003***
Cheese TVFA 13.73e 14.17b 14.37a 13.84d 13.92c 11.12 g 11.77f 0.049***
Meltability(mm) 110.6e 109.1e 105.5f 121.7b 129.7a 114.0d 118.8c 2.704***
Oiling off % 3.52c 3.76 b 3.88a 3.72 b 3.78b 2.58e 2.84 d 0.081***
Appearance& color 11.8 a 10.6b 10.2b 10.5b 10.7b 10.4 b 11.1 b 0.989*
Body & Texture 31.2ab 30.1bcd 29.3 cd 32.1a 30.9 ab 28.8 d 30.6bc 1.32***
Flavour 42.1ab 41.0bcd 39.8 d 41.3bc 41.4 bc 40.7cd 42.8 a 1.33***
Effect of storage time (days)
0 7 14 21 28 LSD
Cheese acidity% 0.87e 0.94d 0.98c 1.02 b 1.05a 0.022***
Cheese pH 5.35 a 5.28b 5.21c 5.17 d 5.13e 0.022***
e d c b
Cheese TS% 52.42 52.83 53.18 53.52 53.85a 0.029***
Cheese Fat% 21.45e 22.03d 22.64 c 23.03b 23.39a 0.066***
Cheese Ash % 3.47e 3.80d 3.98 c 4.18b 4.34a 0.036***
Cheese Salt% 1.79e 1.91d 1.99c 2.06 b 2.13a 0.026***
Salt in moisture 3.68 e 3.89d 4.08c 4.24b 4.41a 0.062***
Cheese Ca% 0.611e 0.637d 0.654c 0.674b 0.685a 0.003***
Cheese TN% 3.71e 3.82d 3.93c 3.98b 4.05a 0.025***
Cheese WSN% 0.278e 0.296d 0.311c 0.324b 0.349a 0.003***
Cheese NPN% 0.152e 0.173d 0.191c 0.209b 0.240a 0.003***
Cheese TVFA 7.96 e 9.75d 13.36c 15.63b 19.68a 0.042***
Meltability(mm) 63.64e 101.57 d 120.14 c 142.86b 168.71a 2.285***
Oiling off % 2.27e 2.89 d 3.52c 3.99b 4.55a 0.069***
Appearance &color 8.64 d 9.85c 11.07 b 11.43 b 12.79a 0.836***
Body & Texture 27.78 d 29.07c 30.5b 32.0 a 32.78 a 1.116***
Flavour 38.07 d 40.14c 41.57b 43.14a 43.57a 1.127***
Significant different at p > (*0.05, **0.01, ***0.001). For each effect the different letters in the means the multiple
comparison are different from each. Letters a is the highest means followed by b, c …..etc.

15
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