Effects of Lactobacillus feed supplementation on cholesterol, fat content and fatty acid composition of the liver, muscle and carcass of broiler chickens
Effects of Lactobacillus feed supplementation on cholesterol, fat content and fatty acid composition of the liver, muscle and carcass of broiler chickens
Ramasamy KALAVATHY 3
Norhani ABDULLAH 1
Syed JALALUDIN 2
Michael C.V.L. WONG 0
Yin Wan HO 3
0 Biotechnology Research Institute , Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah
1 Department of Biochemistry and Microbiology, Universiti Putra Malaysia , 43400 UPM, Serdang, Selangor , Malaysia
2 Department of Animal Science, Universiti Putra Malaysia , 43400 UPM, Serdang, Selangor , Malaysia
3 Institute of Bioscience, Universiti Putra Malaysia , 43400 UPM, Serdang, Selangor , Malaysia
- An experiment was conducted to study the effects of feed supplementation with a mixture of Lactobacillus cultures (LC) on cholesterol, fat and fatty acid composition in the liver, muscle and carcass of broiler chickens. One hundred and thirty-six, one-day-old male broiler chicks (Avian-43) were assigned randomly to two dietary treatments: (i) a basal diet (control), and (ii) a basal diet + 0.1% LC. The cholesterol contents of the carcass and liver but not the muscle, were significantly (P < 0.05) lower in LC-fed broilers. The fat contents of the liver, muscle and carcass were also significantly (P < 0.05) lower in the LC-fed broilers when compared to the control broilers. Supplementation of LC in the broiler diets significantly (P < 0.05) reduced the oleic acid (C18:1) levels of the liver, muscle and carcass but the arachidonic acid (C20:4) level was significantly (P < 0.05) increased in the liver only. Supplementation of LC also increased the total polyunsaturated fatty acids (PUFA) in the liver. The results of the present study indicate that LC reduces the fat content of the liver, muscle and carcass of broiler chickens, but it has very little potential to modify the fatty acid composition.
dans les aliments a réduit significativement (P < 0,05) la teneur en acide oléique (C18:1) dans le foie,
le muscle et la carcasse mais celle en acide arachidonique (C20:4) n’était significativement augmentée
(P < 0,05) que dans le foie. La supplémentation en LC a également augmenté la teneur du foie en
acides gras polyinsaturés (PUFA). Les résultats de la présente étude suggèrent que LC réduit la teneur
en graisse du foie, du muscle et de la carcasse des poulets de chair mais a une action limitée sur la
composition en acides gras.
lactobacille / cholestérol / graisse / acides gras / poulet de chair
Probiotics or direct-fed microbials are
live microbial supplements which
beneficially affect the health of the host animal by
improving its intestinal microbial balance
]. In recent times, with the renewal of
interest in the use of probiotics,
development of more effective probiotic
preparations, which comply with the various
probiotic characteristics, has been found to
improve performance in poultry [
Several of our studies have consistently
shown that the growth performances of
broilers supplemented with a probiotic
containing 12 Lactobacillus cultures are
significantly improved [
]. The present
study was conducted to determine the
effects of this Lactobacillus probiotic on
cholesterol, fat content and fatty acid
composition of the liver, muscle and carcass of
2. MATERIALS AND METHODS
2.1. Chickens and diets
One hundred and thirty-six, one-day-old
Avian-43 male broiler chicks were obtained
from a commercial hatchery. The chicks
were weighed individually, wing banded
and assigned randomly to two dietary
treatments. Each dietary treatment had four
replicate cages with 17 chicks per cage and the
cages were randomized with respect to the
dietary treatments. Each cage (0.9 m length
× 0.6 m width × 0.5 m height) was provided
with a self-feeder and waterer. Feed was
provided ad libitum in a mash form
throughout the experimental period.
]. The 12 Lactobacillus cultures (which
belong to four species) and the method of
preparation as a feed supplement were the
same as those described by Jin et al. [
]. The viability of LC was checked
biweekly to ensure that the viability of the
cultures remained at 1 × 109 viable cells per
gram. The viable LC cells were
incorporated into the basal diet daily at feeding
time. The experiment was carried out for
2.2. Sampling procedure
At the end of the experiment (42 days of
age), eight chickens (2 chickens per cage)
from each treatment (4 cages) were randomly
selected, weighed and sacrificed. The
carcasses were opened and the livers and the
breast muscles (without skin) were removed
and frozen at –20 ºC. The remaining
carcasses were eviscerated, weighed, and
frozen at –20 ºC until being used for analyses.
The frozen carcasses were thawed for 24 h
at 4 ºC prior to autoclaving. Each carcass was
autoclaved for 1 h [
], and then
homogenized in a Waring commercial blender1. The
homogenate was freeze-dried2 to a constant
weight and re-ground with a domestic
blender3 to give a homogenous consistency
prior to chemical analysis.
2.3. Chemical analysis
Fat, cholesterol and fatty acid composi
tion of the carcass, liver and muscle
samples were determined. Fat contents of the
carcass, liver and muscle samples were
measured by extraction in a Soxhlet
apparatus4 with petroleum ether [
Total lipids were extracted from the
carcass (about 1 g), liver (about 2 g) and muscle
(about 4 g) with 40 mL
chloroform:methanol (2:1, vol/vol) in a 50 mL ground-glass
1 Waring Products Division, New Hartford,
2 Labconco Corporation Kansas City, Missouri
3 Braun GmbH, Kronberg, Germany.
4 Soxtect System HT1043 Extraction Unit.
extraction flask according to the method of
Folch et al. [
]. In order to determine the
cholesterol content, the lipids were further
saponified with ethanolic KOH to remove
the fatty acids. Cholesterol was then extracted
with n-hexane and measured using the
method of Rudel and Morris [
]. For fatty
acid determination, extracted lipids were
transmethylated with BF3 and methanolic
]. Fatty acid profiles were
determined by gas chromatography5. Aliquots
of 2 μL were injected into a fused-silica
capillary column (30 m × 250 μm, inside
diameter)6. The injector temperature was
programmed at 220 ºC and the detector at
220 ºC. The column temperature was set at
the range of 100–190 ºC with temperature
programming at the rate of 5 ºC·min–1
increments for optimal separation. The
identification of peaks was made by comparison
with the retention time of the authentic fatty
acid methyl ester standard7. Quantification
was made using an internal standard
(heneicosanoic acid; C21:0)8 added to the initial
sample prior to transmethylation. An HP
Chemstation software9 was used to
integrate peak areas and fatty acid values were
expressed as weight percentages.
2.4. Statistical analysis
Treatment effects were compared using
analysis of variance and treatment means
were separated using the least significance
difference. Computation was done by using
the SAS program [
The average body weights of the control
and LC-fed chickens sampled for the
analysis of the body composition were 2195 g
and 2296 g respectively. The cholesterol
contents in the carcass and liver of LC-fed
broilers were significantly (P < 0.05) reduced
5, 9 Hewlett Packard Co., Wilmington, DE
6 Supelco Park, Bellefonte, PA.
7, 8 Sigma-Aldrich, St. Louis, M063178.
by 13 and 19%, respectively, when compared
to the control broilers (Tab. II). However,
the cholesterol content of the muscle was
not affected by the supplementation of LC
to the broilers. The fat contents in the
carcass, liver and muscle were significantly
(P < 0.05) decreased in LC-supplemented
broilers as compared to the control broilers
(Tab. II). The fat contents in the liver and
muscle were not as high as that in the carcass.
The major fatty acid compositions of the
total lipids from the carcass, muscle and
liver of broilers are presented in Table III.
There were significantly (P < 0.05) lower
levels of oleic acid (C18:1) in the carcass,
muscle and liver but significantly (P < 0.05)
higher levels of arachidonic acid (C20:4) in
the liver of the LC-supplemented broilers
when compared to the control broilers.
Broilers supplemented with LC also had
significantly (P < 0.05) lower levels of total
monounsaturated fatty acids (MUFA) in
the carcass and liver than the control birds.
However, there were no significant
differences in the total saturated fatty acids (SFA)
in the carcass, liver and muscle of the
control and LC-fed broilers. The level of total
polyunsaturated fatty acids (PUFA) was
significantly (P < 0.05) higher in the liver
of broilers supplemented with LC but there
were no significant differences in the levels
Means within a row for carcass, liver or muscle, with no common letters differ significantly (P < 0.05).
1 Control = basal diet; LC = basal diet + 0.1% mixture of 12 Lactobacillus cultures.
SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.
of PUFA in the carcass and muscle between
the treatments. The lowest level of PUFA
was observed in the carcass. The
supplementation of LC in broiler diets did not
affect the levels of linolenic acid (C18:3) or
the saturated fatty acids (C14:0, C16:0 and
C18:0) in the liver, muscle and carcass.
In this study, broilers fed LC were found
to have lower cholesterol contents in the
carcass and liver. This result lends support
to the finding of our earlier study in which
LC-fed broilers had significantly lower
serum cholesterol level than the control
]. Significant reductions of
cholesterol in the liver and carcass of broilers
fed Bacillus subtilis have been reported by
Santoso et al. [
]. Other workers have also
reported reductions of cholesterol in the
muscle and liver of lambs fed L.
], and in the liver of rats fed a
mixture of bacteria and yeast [
]. Based on the
studies to date, the mechanism(s)
responsible for the cholesterol-lowering effect of
probiotics remains unclear, but it has been
suggested that the effect could be obtained
through retarded cholesterol synthesis and
increased degradation of cholesterol [
The hepatic synthesis of bile acids from
cholesterol is the major route of cholesterol
]. Certain lactic acid bacteria
have the ability to produce bile salt
hydrolase enzyme, which deconjugates bile salts
], resulting in greater fecal excretion of
bile acids [
]. To re-establish the
enterohepatic circulation of biliary acids, the liver
would partition more cholesterol into the
bile and less into the bloodstream and this
may cause a loss of cholesterol from the
]. The 12 Lactobacillus cultures
(LC) used as a probiotic in the present study
have been found to produce bile salt
hydrolase and exhibit deconjugating activity of
bile salts (unpublished data), which could
have contributed to an increase in excretion
of cholesterol and reduced cholesterol in
Supplementation of LC to broilers
significantly reduced the fat contents of the
liver, muscle and carcass. Santoso et al. [
also found that supplementation of B.
subtilis in broiler diets significantly (P < 0.05)
reduced carcass fat deposition and
triglyceride levels in the serum, liver and carcass,
and suggested that B. subtilis could
significantly decrease the activity of acetyl-Co A
carboxylase which catalyses the
rate-limiting step in fatty acid biosynthesis.
In the present study, supplementation of
LC was more effective in reducing the
monoenoic acid (oleic acid) than the
saturated fatty acids. This suggests that
supplementation of LC may reduce the synthesis
or absorption of oleic acid. A reduced level
of oleic acid has also been observed in the
liver of rats fed a mixture of probiotics [
Davis and Boogaerts [
] demonstrated that
oleic acid is the most potent fatty acid in
enhancing triglyceride synthesis in the
cells. However, the mechanism by which
oleic acid activates the triglyceride
secretion remains unclear. Among the different
tissues examined, only the liver PUFA was
significantly (P < 0.05) improved with the
supplementation of LC. The increased level
of arachidonic acid (C20:4) in LC-fed
broilers observed in this study was in agreement
with the findings of Fukushima and Nakano
] and Fukushima et al.  in which
significant increased levels of arachidonic acid
(C20:4) were found in the liver and serum of
rats supplemented with a mixture of
probiotics. Fukushima et al. [
] suggested that
the increased levels of arachidonic acid
were due to the Δ6-desaturase activity in the
liver microsomes of the probiotic-fed
animals. Supplementation of LC in broiler
diets had no significant effects on the fatty
acid composition of muscle tissues, with
the exception of a significantly (P < 0.05)
lower level of oleic acid. The change in the
composition of fatty acids in muscle tissue
lipid was less susceptible than that in the
liver or carcass.
In conclusion, the results from the present
study demonstrated that LC supplementation
to broilers reduced the fat contents in the
carcass, muscle and liver, and cholesterol in
the carcass and liver. Supplementation of
LC also increased the total PUFA in the
liver but has very little effect on the fatty
acid composition of the muscle.
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