Effect of Processing Methods on Cholesterol Contents and Cholesterol Oxides Formation in Some Dairy Products

Saudi Journal of Biological Sciences 15 (1) 35-45 June 2008
ISSN 1319-562X
The Official Journal of the Saudi Biological Society
htt:www.saudibiosoc.com
Effect of Processing Methods on Cholesterol Contents and Cholesterol
Oxides Formation in Some Dairy Products
Meshref. A. Al-Rowaily
Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Al-Jouf University,
Saudi Arabia.
Abstract
The effects of pasteurization, boiling, microwaving, processing and storage of milk and some locally produced dairy products on
cholesterol contents and cholesterol oxides formation were studied and evaluated. The 7-ketocholesterol were not detected (ND)
in all raw milk samples. On the contrary, heating of milk led to formation of cholesterol oxidation products (COPs), mostly, 7-
ketocholesterol in different quantities. No significant effect of heating of milk on cholesterol level was observed with the exception
of the ultra-high temperature (UHT) milk prepared from milk powder heated at 140 ± 1.0°C for 4 sec showed the highest value
of 7-ketocholesterol (80.97 mgg-1), followed by microwave heated milk for 5 min (31.29 mgg-1), whereas the lowest value was in
milk pasteurized at 85 ± 1.0°C for 16 sec (3.125 mgg-1). Commercial storage showed no significant effect on cholesterol and 7-
ketocholestrol but lowered cholesterol concentration and increased 7-ketocholestrol level of UHT reconstituted milk. Cholesterol
content of both yogurt and labaneh strained by centrifugal separator showed significant decrease while 7-ketochostrol level was
increased significantly with refrigerated storage. The findings are discussed in the context with the results of previous similar
studies.
Key words: Fat oxidation; cholesterol; ultra-high temperature; pasteurization; 7-ketocholestrol; microwaving; milk products.
Introduction
preparation (boiling and microwaving) or processing, which
Dairy products are an important group in human nutrition.
may include moderate or severe heat treatments that can
They are consumed as such or used in preparation of many
lead to undesirable changes in lipids or proteins. Cooking
food items such as pastries, pies, cakes,…etc to provide
and reheating of foods by microwave ovens are widely
specific functional properties (e.g., texture, crust color,
used in food preparation in millions of kitchens throughout
flavor,…etc.). Milk lipids may undergo chemical and
the world. Food heating by microwave results from the
physical changes during processing and storage such as
conversion of microwave energy into heat by friction of
autoxidation and formation of trans fatty acids (Semma,
water molecules vibration due to rapid fluctuation in the
2002). Cholesterol oxidation products (COPs) have been
electromagnetic field (Potter and Hotchkiss, 1996; decareau,
found in many foods due to the autoxidation of cholesterol
1992). The trend of using the microwave oven in food
in presence of light, heat and pro-oxidants (Kummar and
processing could be attributed to the speed of heating and
Singhal, 1991). Many of them showed some biological
energy saving. Although microwave oven is widely used
activities such as enzymatic inhibition of cholesterol
as a means of food preparation, insufficient information is
biosynthesis, mutagenicity and atherosclerosis (Kummar
available on the consequences of microwave heating on
and Singhal, 1991; Tavani et al, 1997). Milk and milk
the composition and nutritional quality of the food. Some
products usually undergo different changes during their
studies revealed that microwave heating affect fat oxidation
Saudi Journal of Biological Sciences Vol. 15, No (1) June, 2008
35

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Meshref. A. Al-Rowaily
and fatty acid isomer formations (Albi et al, 1997).
Yogurt Production
Variations in heating treatments may have different
Set yogurt was produced from milk pasteurized at 95 ± 1.0°C
effects on COPs in dairy products. Therefore, the objective
for 5 minutes (tube pasteurization), 95 ± 1.0°C for 15 (plate
of this study is to evaluate the effect of the conventional
pasteurization) or 85-90°C for 2 min (batch pasteurization).
heating methods of milk, processing of milk into boiled
The milk was cooled to 45±1.0°C and inoculated with 2-3%
white brined cheese, yogurt and labaneh, microwave heating
freeze-dried mixed starter culture (Danisco, Denmark), then
of milk and white brined cheese and storage conditions
filled into plastic containers of different sizes, incubated at
of pasteurized milk, yogurt, labaneh and cheese on their
42±1.0°C up to 2 to 2.5 hr. When the desired acidity of
cholesterol contents and cholesterol oxides formation.
0.7% or a pH of 4.5-4.6 was reached, the yogurt was cooled
at 4±1.0°C (Smit, 2003).
Materials and Methods
Raw Cow’s milk used in the study was obtained from
Labaneh Production
the bulk tank of three dairy plants: Danish Jordan Dairy
Labaneh was produced by the following two methods
Company (DJD), Jordan University Dairy Plant (JUDP)
(Ozer, 2006):
and Al-Sanabel Dairy Co. (SDC). The milk was produced
by Cow’s Breeder Society, Jordan University farm and
Traditional Method (cloth sacks): The set yogurt after
from Haj Mustafa Farm, respectively. Ewe’s milk used for
cooling was stirred and then poured in a cloth sacks
white-brined cheese production was obtained from SDC
overnight to drain off the whey for 12 to 24 hrs. The drained
and yogurt and labaneh were from DJD and JUDP. The
yogurt labaneh was salted with NaCl 1%, blended, filled
UHT (at 140±1.0°) and Pasteurized (at 85.0±1.0°C) milk
into suitable plastic containers and refrigerated at 4°C. The
were obtained from DJD Co.
produced labaneh was of 23-25 percent total soluble solids
and a pH of 5 to 5.5.
Heat Treatments of Milk and Milk Products
The raw cow’s milk obtained from the three selected
Separator method (centrifugal separator): Cream
sources was subjected to different heat treatments as shown
–separated milk was pasteurized at 83 to 85°C for 16
in Table 1.
Table 1. Cow’s milk and milk products produced by different heat treatments.
Milk
Heating Treatment
Product
Producer
Source
Type
Temperature (°C)
Time
85
16sec
Pasteurized milk
95
5 min
Yogurt
Tube pasteurization
82
16 sec
labaneh
140
4 sec
UHT
Lab scale pasteurization
63±1.0
30 min
Pasteurized milk
Lab scale boiling
97.5±1.0
5 min
Boiled milk
CBS
DJD
Microwave boiling
96.8±1.0
5 min
Microwave boiled milk
Plate pasteurization
95
15 min
Yogurt
95
15 min
labaneh
Lab scale pasteurization
63±1.0
30 min
Pasteurized milk
Lab scale boiling
97.5±1.0
5 min
Boiled milk
HMDF
SDC
Microwave boiling
96.8±1.0
5 min
Microwave boiled milk
Batch pasteurization
85-90
2 min
Yogurt
85-90
2 min
Labaneh
Lab scale pasteurization
63±1.0
30 min
Pasteurized milk
Lab scale boiling
97.5±1.0
5 min
Boiled milk
JUDF
JUDP
Microwave boiling
96.8±1.0
5 min
Microwave boiled milk
36
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Effect of Processing Methods on Cholesterol Contents
sec, NaCl 1% was added, the pasteurized skim milk was
Milk Fat Extraction and Analysis
inoculated with ca 0.002% (w/v) powder freeze dried
Lipids were extracted from the milk and milk products
mixed starter culture (Danisco, Denmark) and kept for 15-
samples with chloroform and methanol as described by
17 hr at 42-44°C. When the pH of the yogurt reached 4.5
Bligh and Dyer (1959) with some modifications regarding
to 4.6, the product was stirred and the whey was separated
sample weight, solvent volume and centrifugation speed
via separator at 40°C (Ozer, 2006). The produced labaneh
and time. Approximately 70 g of cheese, yogurt or labaneh
was filled into suitable plastic containers and stored at 4°C
and 100 ml of fluid milk products were homogenized with
while the concentrated skim yogurt and the cream (40%
100 ml methanol and 100 ml chloroform using a Hamilton
butter) were mixed to have a total solid not less than 23%
Beach Scovel homogenizer (NSF, USA) for 2 min at a
and 10% fat.
medium speed. Chloroform (100 ml) was added and the
mixture was rehomogenized for an additional 2 min. The
Cheese Production
homogenate was centrifuged at 4000 rpm for 20 min using
The brined white cheese ( Nabulsi) was produced according
Haeraeus centrifuge (Haeraeus Christ, GmbH, Osterode/
to the method described by Humeid and Tukan (1986) and
Harz, OJ3, Germany). The upper layer (methanol and
Humeid et al. (1990). Cheese samples were subjected to the
water layer) was removed through aspiration. The middle
following heat treatments as follows:
and the lower layer (chloroform layer and precipitated
protein layer) were filtered through a filter paper to separate
Microwave heat treatments: Two desalted, grated cheese precipitate particles. The chloroform-lipid extracts were
samples of approximately 200 g each were heated in a
again filtered through anhydrous sodium sulfate (Na SO )
2
4
microwave oven (Galanz, 800 Watts, WD800B, Korea)
and the Na SO was rinsed 3 times with 30 ml chloroform
2
4
at 80% power. The first one was heated at 96.3 ± 1.0°C
10 ml each. The lipid extracts were dried under nitrogen
until browning (ca. 10 min), while the second was filled in
using rotoevaporator (LABOROTA, 4001 WB, Heidolph,
a polyethylene bag, placed in a Pyrex saucepan, filled with
Germany) with 150 rpm at 50°C and stored for analysis
distilled water and boiled while floating in the microwave
in 5 ml vials (brown glass) under nitrogen at –18°C. The
oven at 96.3 ± 1.0°C for 5 min.
lipid samples were then used for the analysis of cholesterol
content and cholesterol oxidation products (COPs) mainly
Conventional heat treatments (gas cooker): Desalted 7-ketocholesterol.
grated cheese sample (ca. 100g) was placed in polyethylene
bags in Pyrex saucepan, covered with distilled water and
Cholesterol and cholesterol oxides determination:
boiled on a gas cooker for 5 min at 95.5±1.0°C.
Cholesterol, 7-ketocholesterol and 5?-cholestane standards
were obtained from SIGMA, Inc., Ethyl acetate was
UHT Reconstituted Milk: UHT milk sample prepared HPLC grade (J.T. Baker Chemical Co. Phillibsburg, N.J.),
from powder milk (reconstituted) produced by Kuwaiti
potassium hydroxide was from GCC Laboratory Reagent
Danish Dairy Co., Kuwait (KDD) were purchased from the
(85%), anhydrous sodium sulfate from SDS (fine chemical
local market for comparison (production date 09/09/02 and
limited, Boisar), and methanol HPLC grade from Lab
expired on 09/03/03).
Scan, UK. Chloroform was from GCC (Gainland chemical
Co., UK), pyridine (analytical reagent grade) from CBH,
Storage of Milk and Milk Products
Chlorotrimethyl silane (CH ) SiCl was obtained from
3 3
The milk and milk products used in the present study;
Fluka (Switzerland) and Hexamethyl disilazane C H NSi
6 19
2
(pasteurized milk, UHT milk, yogurt and labaneh) were
was from Janssen, US. Accurately 60 to 200 mg of the
stored at 5.0 ± 1.0°C and analyzed after a storage period
lipid extract (3.8) was weighed into a 25 ml screw capped
of 3, 5, 7, 15 days for pasteurized milk, UHT milk, yogurt
test tube; 10 ml of 1M KOH in methanol and 20 µl of 5?-
and labaneh, respectively, as indicated on the package lable
cholestane solution (4µg/µl) as internal standard (IS) were
(commercial shelf life). On the other hand, the produced
added to the sample. The mixture was shaken until its free
white brined cheese (Nabulsi) was evaluated after one month
of dispersed fat particles and placed in a shaking water
of storage in tins at room temperature at 18 ± 1.0°C.
bath (Memmert, Germany) set at 27°C for 18 to 24 hr. Ten
Saudi Journal of Biological Sciences Vol. 15, No (1) June, 2008
37

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Meshref. A. Al-Rowaily
milliliters of distilled water were added to the saponified
5?-cholestane (Fig. 1). This concentration agreed with the
mixture, which was transferred to 50 ml separatory funnel
results obtained by Regueirio and Maraschellio (1997) who
fitted with a Teflon cap. Unsaponifiables were extracted three
found values ranging between 0.1 to 1 µg g-1 for cholesterol
times with 10, 5, 5 ml of diethyl ether (98%, Laboratory
oxides. The linearity of the calibration curves are shown
grade, GCC, England), and the pooled diethyl ether extracts
in Figure (3-1). It is obvious that the relationship is linear
were washed once with 5ml of 0.5M KOH and 5 times with
with R2 of 0.9991, 0.9994 and 0.9913 for 5??cholestane,
5 ml distilled water. The ether extract was dried and filtered
cholesterol and 7-ketocholesterol, respectively.
using Whatman No.1 filter paper and dried over anhydrous
The recoveries percentage of cholesterol and 7-
sodium sulfate (Na SO ) . The Na SO and the filter paper
ketocholeaterol as shown in Table 2, were 99.5 and 95.2
2
4
2
4
were washed twice with 5 ml diethyl ether to minimize the
respectively.
losses due to the transfer steps. The combined filtrates were
6000
concentrated under nitrogen in dark to about 1 ml and dried
under nitrogen (ultra pure) after being transferred into a 5-
5000
y = 15.831x-1.27
ml vial and stored at -18°C (Vlenzuela et al., 2003).
R2 = 0.9991
5 Cholestane
y = 8.5426x+20.59
4000
R2 = 0.9997
Trimethylsilylation of cholesterol and cholesterol oxides
( Cholesterol)
The trimethylsilyl derivatives (TMS) of cholesterol and
3000
cholesterol oxides was carried out according to the method
2000
used by Pie et al. (1990) with some modification regarding
Peak Area
the derivatization condition (time and temperature). The
y = 7.6546x+34.05
1000
R2 = 0.9966
dried nonsaponifiables extracts were dissolved in 100 µl
7-Ketocholesterol
pyridine (CBH, Nottengham, UK) and mixed for 30 sec
0
by vortex mixer. A 100 µl of each hexamethyldisilazane
0
100
200
300
400
-1000
(Janssen, Belgium) and trimethylsilylchloride (Fluka,
Concentration ugg-1
Switzerland) was added and mixed for another 20 sec. The
vial was placed in a water bath for 40 min, and then cooled
Figure 1: Calibration curves of the 5? cholestane, cholesterol
and 7-ketocholesterol.
to room temperature. The mixture dissolved in 2ml distilled
water and extracted 4 times with 1ml hexane (GC grade,
Table 2: Recoveries of cholesterol and 7-ketocholesterol. ( addition of
200 ug)
Lab Scan, Dublin). The hexane layer was evaporated under
extra pure nitrogen gas. The derivatized (TMS) cholesterol
Determination
Cholesterol
CL1
CL
7-ketocholesterol
and cholesterol oxides were redissolved in 100 µl of hexane
Recovery
(%)
(GC grade).
Recovery (%)
1
97.9
195 ±
96.5
193
4.9
±
Recovery determination: Quantitative recovery of
3.1
cholesterol, 5?cholestane and 7-ketocholesterol was
2
96.0
192 ±
99.0
198
1.8
±
determined for the above mentioned procedure using 10g
2.8
starch sample previously washed with chloroform and
3
108.0
216 ±
96.3
192
1.9
±
precisely spiked with 100 µl of 5?-cholestane (4µg/µl),
3.02
7-?etocholesterol and cholesterol (2µg/µl) using 100 µl
4
96.0
192 ±
89.0
178
1.3
±
Hamilton syringe (Hamilton, USA). The nonsaponifiables
8.6
in the spiked and nonspiked starch samples were extracted
Mean
99.5
95.2
and analyzed by the same procedure followed in sample
Recovery
Standard
analysis.
deviation (SD)
5.6
4.3
The detection limits and calibration curves of cholesterol,
Coefficient
5.6
4.5
of Variability
7-ketocholesterol and 5?-cholestane were found to be
(CV)
about 1 µg g-1 for each cholesterol, 7-ketocholesterol and
1 Confidence limits are calculated at 95% confidence level. (p>0.05).
38
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Effect of Processing Methods on Cholesterol Contents
Gas chromatographic analysis: The derivatized sterols lowered the cholesterol concentration. For example, the
(trimethylsilylated cholesterol and cholesterol oxides)
cholesterol percentages of raw, pasteurized milk (95 ±1.0°C
were analyzed on gas chromatograph (Varian 3700)
for15 min), microwave heated and UHT milk were 0.293,
supplied with an split-splitless injector port and flame
0.283, 0.275, and 0.285%, respectively. The insignificant
ionization detector. A TRB-5 (95% dimethyl-5% diphenyl
decrease in cholesterol content of the heat treated milk
polysiloxan) capillary column (25m x 0.25mm i.d.; phase
samples compared to that of raw milk is probably due to
thickness, 0.25µm ; Teknokroma, Barcelona, Spain). The
oxidation of cholesterol and formation of cholesterol oxides
GC conditions used were; 280°C oven temperature, 300°C
and to inactivation of heat labile antioxidants (Regueiro
injector port temperature, and 310°C detector temperature.
et al, 1997, Lehtinen et al., 2003)). The decrease in the
One microliter of the derivatized sample was injected at
cholesterol content of the reconstituted UHT compared to
a split ratio of 50:50 into the capillary column. Flow rate
that of the fresh raw milk may be due to the fact that this
was set at 1.4 ml/min of N carrier gas. The cholesterol
product contains stabilizers and emulsifiers, as indicated on
2
oxidation products COPs peaks were-identified compared
the label of the package (carageenan, guar gum, vegetable
with the retention time of the reference standard. The COPs
mono and diglycerides).
content of milk and milk products samples was determined
These compounds are probably able to bind (complex)
using the internal standard techniques (IS) of 5?-cholestane
some lipid components such as cholesterol and cholesterol
and the units of measurement were expressed as µg/g for
oxides thus lowering their availability for solvent
the COPs and as percent (%) for the cholesterol (Lin et al,
(chloroform) that is used to extract the milk fat (Valerizuela
1995, Sander et al, 1988).
et al., 2003). Furthermore, the significant (p<0.05)
decrease in cholesterol contents of UHT milk prepared
Statistical Analysis of Experimental Data
from reconstituted milk powder compared to that of UHT
Experiments were conducted using completely random
prepared from fresh cows milk (Table 3) could be partially
design to find the effect of different treatments. Data were
explained by the increase in the level of 7-ketocholesterol.
analyzed using the analysis of variance (ANOVA) procedure
The values of 7-ketocholesterol for UHT reconstituted milk
of SAS institute Inc., (Konda and Rajurkar, 2005) version
was 80.97 µgg-1 compared to 8.708 µgg-1 found in UHT
seven software. Duncan’s multiple range test was applied to
from fresh cow milk. The high level of cholesterol oxides
determine significance among different treatments.
is probably due to the drying process including exposure to

heat and oxygen (Rodriguez et al., 1997).
Results and Discussion
The concentrations of 7-ketocholesterol of milk
A-Effect of heating and processing of milk on cholesterol presented in Table 3 showed that 7-ketocholesterol is not
content and 7-ketocholesterol formation
detected in raw milk and is formed upon all types of heat
Heating of milk showed the following effects on cholesterol
treatments and during storage. The concentration of this
content and cholesterol oxide (7-ketocholesterol)
indicator of cholesterol oxidation ranged between 3.125
formation:
ugg-1 in milk pasteurized at 85±1.0°C for 16 sec and 132.965
ugg-1 in UHT prepared from reconstituted milk powder at
Effect of pasteurization and boiling: The effects 140±1.0°C and stored for 5 days. The results showed that 7-
of different heating methods of milk on cholesterol
ketocholesterol was significantly affected (p<0.05) by both
and 7-ketocholesterol levels are shown in Table 3. A
heat treatment and refrigerated storage of the previously
chromatogram of cholesterol and cholesterol oxidation
heated milk products. As shown in Table 3, the range of
products of milk sample is given in Figure 2. In general,
7-ketocholesterol content in raw and heated milk samples
no significant effect of heating of milk on cholesterol level
before storage was < 1.0 (ND) to 34.538 µgg-1 and 5.52 to
was observed with the exception of the UHT milk prepared
132.965 µg g-1 after refrigerated storage for 3 and 5 days for
from reconstituted powdered milk before and after storage.
the pasteurized and UHT milk respectively.
Along with that, commercial storage of milk had also no
significant effect (p>0.05) with the exception of UHT
Effect of microwave heating: Microwave heating of raw
reconstituted milk where storage significantly (p<0.05)
cows milk samples show a significant (p<0.05) increase in
Saudi Journal of Biological Sciences Vol. 15, No (1) June, 2008
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Meshref. A. Al-Rowaily
Table 3: Effect of heat treatment of and refrigerated storage of milk on the formation of 7-ketocholesterol 1.
Treatment
Cholesterol
7-Ketocholesterol
(%)
(µgg-1 fat)
Raw cow’s milk
0.293a ± 0.017
ND
Milk Pasteurized at 85 ± 1.0°C for 16 sec.
0.291 a ± 0.01
3.125e ± 0.806
Milk pasteurized at 85 ± 1.0°C for 16 sec and stored for 3 days2.
0.290 a ± 0.058
5.520e ± 0.186
Milk pasteurized at 63 ± 1.0°C for 30 min.
0.284 a ± 0.009
9.383e ± 1.005
Milk pasteurized at 95°C for 5 min.
0.290 a ± 0.023
11.733e ± 8.119
Milk pasteurized at 95 ± 1.0°C for 15 min.
0.283 a ± 0.043
16.328 de ± 1.717
Milk pasteurized at 85-90°C for 2 min.
0.292 a ± 0.023
3.142 e ± 0.694
Milk boiled at 96.3 ± 1.0°C for 5min.
0.283 a ± 0.017
15.363 e ± 1.922
Milk boiled in microwave oven at (80% power) 95.8 ± 1.0°C for 5min.
0.275a ± 0.036
31.029 c ± 1.089
Milk heated at 140 ± 1.0°C for 4 sec (UHT)3.
0.285 a ± 0.006
8.708e ± 1.399
Milk heated at 140 ± 1.0°C for 4 sec (UHT) and stored for 5 days.
0.283 a ± 0.009
34.538c ± 1.094
Reconstituted milk powder (UHT) 4.
0.260 b ± 0.006
80.97 b ± 1.232
Reconstituted milk powder (UHT) and stored for 5 days.
0.205 c ± 0.016
132.965a ± 1.523
Values represent means ± SD (n = 4). Means values in the same column with different superscript letters are significantly different( p ? 0.05) according to
(ANOVA) Duncan’s multiple range test. 2 Commercial refrigeration at 5 .0 ± 1.0°C. 3 Ultra high pasteurization temperature of fresh cow’s milk provided by
Danish Jordan Dairy Company (DJD). 4 Milk prepared from cow’s milk powder after reconstitution KDD brand name (Kuwaiti Danish Dairy Company),
purchased from local market.
7-ketocholesterol compared to those heated by conventional
prevailing aerobic condition (Kumar and Singhal 1991).
methods (boiling and/or tube, plate or batch pasteurization)
These results are consistent with those obtained by Nourooz-
(Table 3). The values of 7-ketocholesterol of the pasteurized
Zadeh and Appelqvist (1988) who studied the formation of
(85±1.0°C for 16 sec), boiled (96.3±1.0°C for 5min), UHT
COPs during storage of milk powder for one year in paper
and microwave heated milk samples were 3.125, 15.363,
cans at about 20°C and found 7-ketocholestrol to be 5.6-
8.708 and 31.029 µgg-1, respectively. Microwave heating
9.2 ugg-1. The formation of COPs as a result of heating
seems to be highly detrimental to quality compared to the
is expected because heating positively increases lipid
other heating method due to its unique heating mechanism.
oxidation in the presence of air, prooxidant and radicals that
These results were in agreement with those obtained
enhance the formation of cholesterol oxides (Kumar and
by others where heating of oil samples and edible fat in
Singhal 1991, Morgan and Armstrong 1992).
microwave oven enhances lipid oxidation (Albi et al.,
1997 a&b; Yoshida and Kajimoto, 1994; Yoshida et al.,
B-Effect of heating and processing of yogurt and labaneh
1991&1992).
on cholesterol content and 7-ketocholesterol formation
Cholesterol and 7-ketocholesterol content of yogurt and
Effect of storage: Refrigerated storage of pasteurized milk labaneh are shown in Table 4. The results indicate that
for three days showed no significant effect on cholesterol
processing of yogurt and labaneh had no significant effect
or 7-ketocholesterol content, whereas, UHT heated milk
on cholesterol content. On the contrary, processing steps
prepared from recostitueted milk powder as well as its
produced significant (p<0.05) increase in 7-ketocholesterol
storage for five days had significantly (p<0.05) lowered
levels compared with those values of the raw or pasteurized
cholesterol but increased 7-ketocholesterol levels as shown
milk. Fore example, the values of 7-ketocholesterol content
in Figure 3. The values of the 7-ketocholesterol for UHT
in the fat extracted from raw milk, yogurt (produced from
milk were 8.708 and 34.538 ugg-1 before and after storage,
milk pasteurized at 80-90°C for 2 min and labaneh were
respectively. The increase in the 7-ketocholesterol content
ND (not detected), 3.78 and 5.16 ugg-1, respectively.
of UHT upon storage can be seen as continuation of the
Refrigerated storage of yogurt or labaneh did not produce
oxidation process that was indicated by heating under the
significant changes in cholesterol levels with the exception
40
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Effect of Processing Methods on Cholesterol Contents
Fig. 2. (a)- Derivatives for standard mix of cholesterol and 7-ketocholesterol, including the internal standard, 5a-cholestane. (b)-
chromatogram of COPs isolated from UHT reconstituted milk. GC conditions were as described under material and methods.
compared with those of fresh products. For example, the
values of 7-ketocholesterol in yogurt and labaneh were
3.78 and 5.11 ugg-1 in fresh product compared to 5.16 and
6.55 ugg-1 after storage, respectively. The increase in 7-
ketocholesterol content of labaneh or yogurt after storage is
expected since almost aerobic condition is prevailing during
fermentation. The time elapsed between pasteurization and
analysis of the extracted fat did not exceed 24 hr in which the
oxidation process could not produce significant difference
of the values. In contrast prolonged storage of yogurt or
labaneh for 7 or 15 days resulted in a significant increase
in 7-ketocholesterol content. It is worth mentioning that the
samples analyzed after storage period were taken from the
same cups that were opened at the beginning of the analyses
Figure 3. Effect of heat treatment and refrigerated storage on 7-
and stored after reclosure in refrigerator (at 5 ± 1.0ºC). This
ketocholesterol content of milk( RM; raw cows milk, PM; pasteurized
milk at 85°C for 16 sec, PMA; pasteurized milk stored for 3 days, MCBM;
implies that either yogurt or labaneh were exposed to air
microwave boiled milk, UHT; ultra-high-temperature and UHTA; ultra high
temperature milk stored for 5 days).
diffusion during storage, which might lead to pronounced
of labaneh produced mechanically using a centrifugal
aerobic condition that in turn facilitates oxidation.
separator, where the cholesterol content decreased
Cholesterol content of labaneh strained by centrifugal
significantly (p<0.05) after 15 days storage. On the other
separator showed significant (p<0.05) decrease in
hand, 7-ketocholesterol content increased significantly
cholesterol content after refrigerated storage for 15 days
with storage of both yogurt and labaneh for 7 or 15 days,
compared with that of the labaneh strained in cloth. The
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41

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Meshref. A. Al-Rowaily
Table 4: Effect of different heat treatments, processing and refrigerated storage of milk, yogurt and labaneh on their
cholesterol content and 7-ketocholesterol formation.
Treatment
Cholesterol
7-Ketocholes-terol
(%)
(µgg-1 fat)
Raw cows milk2.
0.293 a ± 0.017
ND
Milk pasteurized at 85-90°C for 2 min 3.
0.292 a ± 0.023
3.14 d ± 0.694
Pasteurized milk4 at 85± 1.0°C for 16 sec.
0.291a ± 0.01
3.125 d ± 0.806
Yogurt( produced from the milk pasteurized at 85-90°C for 2 min).
0.286 a ± 0.013
3.78 cd ± 0.664
Yogurt after 7 days storage5.
0.281 a ± 0.011
5.11 b ± 0.324
Yagurt produced from milk pasteurized at 85± 1.0°C for 16 sec.
0.281a ± 0.013
4.18 bc ± 0.806
Yagurt produced from milk pasteurized at 85± 1.0°C for 16 sec. After 7 days storage
0.282a ± 0.023
6.35 a ± 0.15
Labaneh produced by Separator 6.
0.276 a ± 0.024
5.03 b ± 0.181
Labaneh produced by modern method after 15 days storage.
0.18 b ± 0.040
5.108 b± 0.205
Labaneh produced by conventional method 7.
0.288 a ± 0.010
5.16 b ± 0.119
Labaneh produced by conventional method after 15 days storage.
0.283 a ± 0.011
6.55 b a ± 0.443
1Values represent means ± SD (n =4). Means with different letters within a column are significantly different (p ? 0.05)according to
(ANOVA) Duncan’s multiple range test. 2, 3,4 milk used for yogurt production and Labaneh. 5 Commercial storage at 5 ± 1.0ºC for 7
and 15 days for yogurt and labaneh respectively. 6 Straining by separator. 7 Straining in cloth.
cholesterol values for labaneh were 0.276 and 0.18% before
decrease in cholesterol content. Fore example, the averaged
and after storage, respectively. The decrease in cholesterol
cholesterol percentages for the cheese samples heated in a
percentage could be due to the additional heat treatment
microwave oven for 5 and 10 min were 0.278 and 0.265
of the cream before mixing with the concentrated yogurt
%, respectively. This is obviously due to the formation of
(Rodriguez et al, 1997). Another explanation is the possible
cholesterol oxides as will be explained below.
decrease in the extractability of cholesterol as a result of
Processing steps and reheating of the cheese by
complex formation between cholesterol as lipid component
conventional heating method (boiling on gas cooker) had
and protein or carbohydrate (Min and Steenson, 1998). It is
no significant effect on oxidation of cholesterol as shown in
also worth to notice that, the cream added to the concentrated
Table 5. However, reboiling of cheese using a microwave
yogurt was mixed with stabilizers and emulsifiers such as
oven for 5 min had significantly increase 7-ketocholesterol
guar gum and carageenan. These additives may interact
content with clear effect of the heating duration.
with cholesterol during storage and made it unextractable
Storage of the salted boiled cheese in polyethylene bags
by chloroform.
placed in a Pyrex saucepan at (5±1.0°C) had no significant
effect on the 7-ketocholesterol levels. On the other hand,
C- Effect of heating, processing and storage of white the insignificant effect of processing on 7-ketocholesterol is
brined (Nabulsi) cheese on cholesterol level and 7-
in agreement with those obtained by Al-Ismail and Humeid
ketocholesterol formation
(2002) in Nabulsi cheese.
Cholesterol and 7-ketocholesterol levels of processed
The effect of microwaving on cholesterol oxidation of
cheese are presented in Table 5. The results indicate that
milk, is shown in table 5.
boiling of cheese pieces in brine (17% NaCl) for 15 min
It can be concluded from the above findings that
caused a significant reduction (p<0.05) of the cholesterol
cholesterol oxides, in particular 7-ketocholesterol, which
content by 6%. Reboiling of the desalted and grated cheese
are considered carcinogenic were not detected in fresh
samples for 5 minutes or its microwaving did not produce
milk, while all of the applied heating treatments led to
any significant (p>0.05) changes in cholesterol content
the formation of cholesterol oxides at different levels.
(Table 5). However, heating of cheese for longer period
Conventional heating of milk (pasteurization and boiling)
(10 min) in a microwave resulted in a significant (p>0.05)
caused formation of these oxides with significant differences.
42
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Effect of Processing Methods on Cholesterol Contents
Table 5: Effect of microwave heating, processing steps and storage of one month of white brined cheese on cholesterol content and 7-
ketocholesterol formation1.
Treatment
Cholesterol
7-Ketocholesterl
(%)
(µgg-1 fat)
Raw ewe milk
0.302 a ± 0.028
1.023 d ± 0.554
Fresh curd
0.296 a ± 0.008
1.132 d ± 1.640
Boiled cheese curd 2.
0.283 b ± 0.009
4.025 d± 0.306
Boiled cheese stored for one-month3.
0.282 b ± 0.010
2.355 d ± 1.414
Boiled cheese reheated on gas cooker 4 at 94.3±1.0°C for 5min
.
0.279 bc ± 0.031
8.17 d± 1.153
Boiled cheese reheated on gas cooker after storage at 94.3
±1.0°C for 5min.
0.275 bcd ± 0.013
9.835 d ± 1.793
Boiled cheese reheated in microwave oven5 at 94.3±1.0°C for
5min.
0.280b± 0.015
24.94 c ± 2.130
Boiled cheese reheated in microwave oven within aliquid at
94.3±1.0°C for 5min.
0.278 bc ± 0.018
27.705 c ± 1.383
Boiled cheese reheated in microwave oven after storage. at
94.3±1.0°C for 5min.
0.282 b ± 0.018
23.084c ± 1.361
Boiled cheese reheated in microwave oven within a liquid after
storage. at 94.3±1.0°C for 5min.
0.276 bc ± 0.010
24.053c ± 1.206
Boiled cheese reheated in microwave oven at 94.3±1.0°C for
10 min.
0.267 d ± 0.009
103.698 b ± 1.439
Boiled cheese reheated in microwave oven at 94.3±1.0°C for 10
min after storage.
0.265 d ± 0.006
115.308 a ± 2.493

Values represent means ± SD (n =4), means with different letters within a column are significantly different ( p ? 0.05) according to (ANOVA) Duncan’s
multiple range test. 2 white brined cheese pieces boiled in brine (17% NaCl) at 94.3 ± 1.0°C measured at the centre of the cheese pieces for 5min. 3 White
brined boiled cheese pieces stored in tins at room temperature of 18 ± 1.0°C for one month. 4 Desalted grated white-brined boiled cheese reheated in a liquid
medium (distilled water). 5 Desalted grated white-brined boiled cheese reheated in a liquid medium (distilled water) in microwave oven at 80% power.
Flash pasteurization gave the lowest level followed by low
which were not extractable with organic solvent.
temperature long time pasteurization without significant
differences between them. Processing steps of milk,
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